Quorum sensing involvement in T4 bacteriophage infection

CONTENT

摘要

Abstract

List of Figures

Acronyms and Abbreviations

Chapter No: 1 Introduction
1.1 Quorum Sensing
1.1.1 Quorum Sensing in Bacteria
1.1.2 Quorum Sensing in Plant
1.1.3 Quorum Sensing in Parasite
1.2 Different Quorum Signals System in Bacteria
1.2.1 Mechanisms of Quorum Sensing
1.2.2 Quorum-Sensing Signals
1.2.3 Autoinducer-1 or AI-1
1.2.4 Autoinducer-2 or AI-2
1.2.5 Autoinducer-3 or AI-3
1.2.6 Autoinducing Peptides
1.3 Escherichia coli and T4 Bacteriophage in Natural Environment
1.3.1 Escherichia coli in Natural Environment
1.3.2 Escherichia coli in Soil Environment
1.3.3 Escherichia coli in Tropical Fresh Water Environment
1.3.4 T4 Bacteriophage in Natural Environment
1.3.5 Interaction between Phage and Hosts in their Physical Environment
1.4 Escherichia coli as a Model Organism
1.4.1 Life cycle of Escherichia coli
1.4.2 Escherichia coli Transmission
1.4.3 Escherichia coli Genera History
1.4.4 Escherichia coli Phenotypic Diversity
1.4.5 Escherichia coli Strains
1.4.6 DNA Sequence of Escherichia coli
1.4.7 Escherichia coli Proteins
1.4.8 Escherichia coli as a Model Organism
1.4.9 Nonpathogenic Escherichia coli Advantages
1.4.10 Role of Escherichia coli in Research Field
1.5 T4 Bacteriophage as a Model Organism
1.5.1 Infection and Life cycle of T4 Bacteriophage
1.5.2 T4 Bacteriophage Unique Feature
1.5.3 Bacteriophage Classification
1.5.4 Lysis from within" and "Lysis from without
1.5.5 T4 Bacteriophage as Model System
1.5.6 T4 Bacteriophage Therapy
1.5.7 T4 Bacteriophage Lysins as Antimicrobials
1.5.8 T4 Bacteriophage Display
1.5.9 Function of T4 Bacteriophage in Vaccines
1.5.10 T4 Bacteriophage for Detection of Pathogens
1.6 Goal of our Study

Chapter No: 2 The Effect of Different pfu, Temperature and pH on T4 Bacteriophage Infection Activity and Production
2.1 Material and Methods
2.1.1 Bacteria and Bacteriophage
2.1.2 Culture Media
2.1.3 Semi Solid Media
2.1.4 Plaque Count Assay
2.1.5 Plating of T4 Bacteriophage
2.1.6 Dilution of T4 Bacteriophage Stock
2.1.7 Media pH
2.1.8 Sterilization of Instruments and Media
2.1.9 Statistical Analysis
2.2 Result
2.2.1 Effect of Different pfu of T4 Bacteriophage on its host interaction
2.2.2 Effect of Temperature on T4 Bacteriophage and its host interaction
2.2.3 Effect of pH on T4 Bacteriophage and its host interaction
2.3 Discussion

Chapter No: 3 Various Culture Media Effect on T4 Bacteriophage Infection Activity and Production
3.1 Material and Methods
3.1.1 Bacteria and Bacteriophage
3.1.2 Culture Media
3.1.2.1 Luria-Bertani Media
3.1.2.2 Luria-Bertani pulse 0.0% Glucose Media
3.1.2.3 Minimal Media
3.1.2.4 Nutrient Media
3.1.3 Semi Solid Media
3.1.4 Plaque Count Assay
3.1.5 Plating of T4 Bacteriophage
3.1.6 Sterilization of Instruments and Media
3.1.7 Statistical Analysis
3.2 Result
3.3 Discussion

Chapter No: 4 Effect of Different Phase Escherichia coli on T4 Bacteriophage Infection Activity and Production
4.1 Material and Methods
4.1.1 Bacteria and Bacteriophage
4.1.2 Culture Media
4.1.3 Semi Solid Media
4.1.4 Plaque Count Assay
4.1.5 Plating of T4 Bacteriophage
4.1.6 Sterilization of Instruments and Media
4.1.7 Statistical Analysis
4.2 Result
4.3 Discussion

Chapter No: 5 Lysis Inhibition Effect T4 Bacteriophage Plaque Burst Size
5.1 Material and Methods
5.1.1 Bacteria and Bacteriophage
5.1.2 Culture Media
5.1.3 Semi Solid Media
5.1.4 Plaque Count Assay
5.1.5 Plating of T4 Bacteriophage
5.1.6 Sterilization of Instruments and Media
5.2 Result
5.3 Discussion

Chapter No: 6 Quorum Sensing Molecule Acyl-Homoserine Lactones and Indole Effect on T4 Bacteriophage Infection and Production
6.1 Material and Methods
6.1.1 Bacteria and Bacteriophage
6.1.2 Luria-Bertani Media
6.1.3 Semi Solid Media
6.1.4 Chemicals
6.1.5 Plaque Count Assay
6.1.6 Plating of T4 Bacteriophage
6.1.7 Plaque Size Determination
6.1.8 Sterilization of Instrument and Media
6.1.9 Statistical Analysis
6.2 Result
6.2.1 Quorum Sensing Molecule AHL Effect on T4 Bacteriophage Infection and Production
6.2.2 Quorum Sensing Molecule Indole Effect on T4 Bacteriophage Infection and Production
6.2.3 The AHL and Indole Effect on Diluted T4 Bacteriophage Production and Lysis Activity
6.2.4 Quorum Sensing Molecules AHL and Indole Effect on the Plaque size of T4 bacteriophage
6.3 Discussion

Chapter No: 7 Separation and Purification of two new Quorum Sensing Molecules W1 and W2
7.1 Material and Methods
7.1.1 Bacteria and Bacteriophage
7.1.2 Minimal Media
7.1.3 Semi Solid Media
7.1.4 Plaque Count Assay
7.1.5 Plating of T4 Bacteriophage
7.1.6 0.1M Tris-Base Buffer
7.1.7 0.1M Hcl Buffer
7.1.8 0.05M Tris-Hcl Buffer (100ml)
7.1.9 0.05M Tris-Hcl with 2M Nacl Buffer (100ml)
7.1.10 20% Ethanol/ Alcohol
7.1.11 Acetonitrile
7.1.12 Cutt-off membrane (MWCO 9 and 5 KDa)
7.1.13 HiPrep QFF Column and its Preparation
7.1.14 Dialysis Tube
7.1.15 Zorbax semi-preparative C18 (9.4 × 250 mm) column
7.1.16 Sterilization of Instruments and Media
7.1.17 Protocol for Purification of W1 and W2 Quorum Sensing Molecules
7.1.18 Spot Test
7.1.19 Dialysis Process
7.1.20 Ion-Exchange Chromatography
7.1.21 High-Performance Liquid Chromatography (HPLC)
7.2 Result
7.2.1 Fast Protein Liquid Chromatography ( FPLC)
7.2.2 High-Performance Liquid Chromatography (HPLC)
7.2.3 Time-of-Flight Mass Spectrometry (TOF MS)
7.2.4 Quorum Sensing Molecules W1 and W2 Effect on T4 Bacteriophage Infection and Production
7.2.5 To check the effect of Different condition on W1 and W2 Quorum Sensing Molecules Efficiency
7.3 Discussion

Chapter No: 8 Summery and Outlook

Acknowledgment

References

Papers Details

Annexure-1: Instruments

Annexure-2: Columns and Dialysis Tube/Bag

Annexure-3: FPLC and HPLC Procedure

Annexure-4: Sterilization of Instruments

Annexure-5: Recipes of Media

LIST OF FIGURES

Figure- 1.2.1: Mechanisms of quorum sensing

Figure-1.2.4: Schemes of autoinducer-2 response

Figure- 1.2.5: Autoinducer-3 or AI-3

Figure-1.2.6: Autoinducer peptides or AIP

Figure-1.3.1: Escherichia coli in natural environments

Figure-1.4: Structure of Escherichia coli under microscope

Figure-1.4.1: Life cycle of Escherichia coli

Figure-1.5: Schematic of T4 bacteriophage

Figure-1.5.1: Infection and life cycle of T4 bacteriophage

Figure-2.2.1: Effect of different pfu of T4 bacteriophage on its host interaction

Figure-2.2.2: Effect of temperature on T4 bacteriophage lysis activity

Figure-2.2.3: Effect of pH on T4 bacteriophage infection activity

Figure- 3.2: Various media effect on T4 bacteriophage lysis and production

Figure-4.1.1: E. coli growth curve

Figure-4.2: Different phase E. coli effect on T4 phage infection activity

Figure-5.2 A: Effect of lysis inhibition on T4 phage burst size

Figure-5.2 B: Effect of lysis inhibition on T4 phage burst size

Figure-5.2 C: Plaque after lysis inhibition

Figure-6.1.4 A: General chemical structure of AHL

Figure-6.1.4 B: General chemical structure of indole

Figure-6.2.1: Effect of AHL on the infection activity of T4 bacteriophage

Figure-6.2.2: Effect of indole on the infection activity of T4 bacteriophage

Figure-6.2.3: AHL and indole effect the lysis activity of diluted T4 phage

Figure-6.2.4: AHL and indole effect on plaques size of T4 bacteriophage

Figure-7.2.1: W1 and W2 peaks separated by FPLC

Figure-7.2.2: W1 and W2 peaks separated by HPLC

Figure-7.2.3A: W1 molecular mass after TOF MS

Figure-7.2.3B: W1 molecular mass after TOF MS

Figure-7.2.4: W1 and W2 effect of T4 bacteriophage lysis activity

Figure-7.2.5: Effect of different condition on W1 and W2 activity

ACRONYMS AND ABBREVIATIONS

Abbildung in dieser Leseprobe nicht enthalten

摘要

长期以来，人们一直认为由于细菌以单细胞形式存在，彼此之间没有信息交流和协作分工。近年的大量研究表明，为了适应复杂多变的外界环境，细菌也可以通过细胞内或细胞间的信息交流来协调群体的行为，使其能够与多细胞一样，行使单个细胞无法完成的功能。群体感应（Quorum Sensing，QS）是微生物间通过分泌、释放一些特定的信号分子，并感知其浓度变化，当浓度达到一定阀值时，便能启动或是抑制一系列基因的表达，以此来监测菌群密度、调控菌群生理功能，从而适应周围环境的一种信号交流机制。

群体感应首先在海洋细菌费氏弧菌(Vibrio fischeri)中发现，现在被证实存在于众多的微生物群体中。群体感应不仅与致病菌的毒力因子合成、生物膜形成有关，而且与生物发光、芽孢形成、运动性、抗生素的生成等多种细菌群体行为相关。很多细菌群体行为都与人类生活关系密切。因此，群体感应的研究在医疗、环境、农业、食品、发酵等领域受到广泛关注。

目前，在铜绿假单胞菌、大肠杆菌、金黄色葡萄球菌及费氏弧菌等许多细菌中都发现了群体感应系统，而弧菌及假单胞菌的群体感应现象研究的最为清楚。已报道的细菌群体感应系统有三种类型：第一类是以酰基高丝氨酸内酯AHL (AI-1) 作为信号分子的革兰氏阴性菌群体感应系统；第二类是以寡肽类分子作为信号分子的革兰氏阳性细菌的群体感应系统；第三类是以DPD类衍生物（AI-2）为信号分子的细菌群体感应系统，这类信号分子在革兰氏阳性和阴性菌种均存在，可用于进行不同细菌种群间的信息交流。

大肠杆菌、T4噬菌体作为模式生物，广泛应用于分子生物学等研究领域。研究发现，在大肠杆菌中存在着两套群体感应信号系统：以AI-1为信号分子的群体感应系统一，以及以AI-2为信号分子的群体感应系统二。大肠杆菌利用这两套系统对种群的群体行为进行调控。在所有已知的烈性噬菌体的生活周期中，噬菌体都是通过控制宿主的代谢系统来实现对宿主的“接管”，并利用宿主细胞中的核糖体、蛋白质合成时所需的各种因子等来实现其自身的生长和增值。细胞内成熟子代噬菌体颗粒的释放引发了宿主细胞的破裂死亡，从而间接控制了其宿主细菌的种群密度。

群体感应是细菌间信息交流的重要方式并控制着细菌的种群密度；噬菌体也是控制细菌种群规模的重要生物因子，它们二者均依赖于细菌的种群密度，在这一点上它们天然存在一定的相关性。为了探索群体感应与噬菌体感染存在的相互关系及揭示两者相互作用机制，本研究以T4噬菌体及其宿主大肠杆菌BL21为研究材料，分析了影响细菌种群密度的生理生化因子（包括温度、pH、不同的培养基类型）、宿主不同生长时期以及人为加入的已知信号分子（AHL、Indole）对噬菌体感染裂解活性的影响。

培养温度对T4噬菌体裂解活性的影响实验：分别选用4℃、15℃、25℃、30℃、37℃、41℃、45℃、55℃、70℃这几个温度梯度。实验表明：当培养温度为4℃时，大肠杆菌生长极为缓慢，噬菌体无裂解现象；当外界培养温度为15℃、25℃、30℃时，相同的培养时间内，大肠杆菌种群密度随温度的升高，呈现逐步增长的趋势，并均伴随有噬菌体感染裂解情况的发生，且感染裂解活性随温度的升高而增强。当温度为37℃时，相同时间内大肠杆菌种群密度的增长达到最大，此时，噬菌体裂解活性最强。随着温度的进一步的升高，大肠杆菌种群密度增长变慢，噬菌体裂解活性也随着下降，41℃为T4噬菌体发生裂解的最高温度，当培养培养温度超过41℃时，平板内无T4噬菌体裂解现象的发生。

不同营养成分的培养基对噬菌体裂解活性影响实验：实验表明，大肠杆菌种群密度的增长与培养基中的营养丰富程度有直接的关系，营养成分越丰富，一定时间内细菌种群密度的增长越快。本研究共选用了营养丰富程度高低依次排列的四种不同类型的培养基：LB+葡萄糖培养基(LBG）、LB培养基（LB）、营养培养基（NM）、基础培养基（MM），通过选用这四种不同的培养基分别对噬菌体的感染裂解活性进行测试。37℃培养时，LB+葡萄糖培养基细菌种群密度最高，T4噬菌体裂解活性也最高，而选用基础培养基（MM）时，细菌种群密度最低，噬菌体裂解活性也最低。说明了T4噬菌体的感染裂解活性与大肠杆菌种群密度呈正相关。

pH对T4噬菌体感染裂解活性影响实验：实验表明，大肠杆菌适应力强，能在一个较为广谱的pH范围内生长繁殖并维持一个相对稳定的种群密度。本研究选用的pH范围为4-10，通过观察平板上是否出现噬菌斑来判断T4噬菌体的裂解。结果表明，在pH为4-10时，噬菌体均可裂解，无显著差异。

宿主不同生长时期对噬菌体感染裂解活性影响实验：实验表明，当细菌培养至对数生长中后期时，其分泌并积累在细胞外的信号分子浓度达到最大，极易突破阀值从而通过主动/被动运输方式进入细胞，继而引发一系列的细菌群体行为。在这一过程中，随着信号分子进入细胞内与受体结合，胞外的信号分子浓度逐渐降低。本研究分别取培养至对数期、稳定期、衰亡期这三个不同生长阶段的大肠杆菌，在相同条件下对其进行裂解实验。37℃培养大肠杆菌处于对数生长期时，T4噬菌体的感染裂解活性最高，而当大肠杆菌处于稳定期或死亡期时，T4噬菌体的裂解活性较对数生长期有所降低。

细菌信号分子（AHL、Indole）对噬菌体感染裂解活性影响实验：大肠杆菌具有不完整的以AI-1为信号分子的群体感应系统一，以及完整的以AI-2为信号分子的群体感应系统二。不完整的群体感应系统一是指大肠杆菌中仅有能够与其他细菌的AI-1信号分子结合的受体。本研究通过人为加入信号分子来探讨信号分子对噬菌体感染裂解活性的影响。通过实验表明，一定浓度的AHL类信号分子对T4噬菌体的裂解具有显著的促进作用，能提高感染率一倍左右，并使单个噬菌斑直径增大近一倍；而吲哚的作用则正相反，它显著抑制了噬菌体的裂解活性，使噬菌体感染率下降了50%。

新群体感应信号分子的分离鉴定实验：本研究采用浓缩（5K蛋白浓缩管）、透析（100Da）、蛋白纯化(AKTA)、分离纯化（HPLC）及物质结构的分析（TOF-MASS）等实验技术对T4噬菌体裂解液进行了分离和鉴定工作，最终分离得到了两组具有活性的组分（W1/W2）。通过相关实验表明，两组成分均能促进T4噬菌体的裂解。进一步的研究表明两个组分对低温（-20℃、液氮）、有机溶剂（氯仿）不敏感，对高温（煮沸）敏感性较高。然而，由于样品的复杂性，至今未能将它们彻底分离纯化和进行鉴定。

通过研究，首次证明T4噬菌体的感染裂解活性对影响大肠杆菌种群密度的生理生化因子（温度、pH、不同的培养基类型）及宿主不同的生长时期具有高度的依赖性；不同类型的信号分子对噬菌体的感染裂解活性有着不同的影响，且T4噬菌体裂解中存在能提高其裂解活性的信号分子。本研究初步证实了大肠杆菌群体感应系统与T4噬菌体的感染存在的相互关系这一现象，这为揭示噬菌体与宿主相互作用深层机制提供了新的视角。

关键词： 群体感应，大肠杆菌、T4噬菌体、信号分子

ABSTRACT

Quorum sensing (QS) is an important way to communicate information between bacteria. Bacteria used different quorum sensing molecule for communication and these signals molecules are called autoinducer (AI). These autoinducers are produced and released by the quorum sensing bacteria to levels dominating the increasing cell-population density. The attainment of minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Both Gram-positive and Gram-negative bacteria are capable of using quorum sensing communication circuits for regulating a diverse array of physiological activities. These activities include symbiosis, competence, virulence, conjugation, antibiotic production, sporulation, motility and biofilm formation. The Gram-negative bacteria use acylated homoserine lactones as autoinducers, while Gram-positive bacteria use processed oligo-peptides to communicate. In the field of quorum sensing revealed, cell to cell communication via autoinducers both within and between bacterial species. The establishment of enormous data in this field suggests autoinducers acquiring specific responses from host organisms. Despite the difference in chemical signals, signal relay mechanisms and the target genes controlled by the bacterial quorum sensing systems, the ability to communicate with one another allows bacteria to coordinate the gene expression as well as the behavior of the entire community. This process presumably confers upon the bacteria some of the qualities of higher organisms. The evolution of quorum sensing systems in bacteria thus could have been one of the early steps in the development of multicellularity.

Quorum sensing phenomenon was first discovered in bacteria but now different researcher reported quorum sensing between bacteria and plants, between bacteria and parasite, and recently quorum sensing between bacteria and fungi was also reported. Due to such wide role of quorum sensing in microbiology research, attract many researches to explore it in details and now quorum sensing has been burning topic for researchers.

Escherichia coli (E. coli) and T4 bacteriophage were widely used as a model organism in molecular biology research field. In order to conduct the study, the E. coli BL21 strain was used as the primary host and T4 bacteriophage as predator. The influence of different things on T4 bacteriophage and E. coli interaction mechanisms were studied. The first time the effect of quorum sensing molecules on T4 bacteriophage infection was studied. In this research work we found that different physiological and biochemical conditions, different media, different growth conditions of hosts, signaling molecules (AHL, Indole) and W1/W2 substance extracted from T4 phage lysate have effect on T4 bacteriophage infection and on its plaque size. In this research the following experimental techniques were used e.g dialysis, protein purification, analysis of the structure and double agar overlay plate method was used to checked lytic activity.

The bacteriophage rate of interaction and adsorption to its host directly depended on physical-chemical factors (phage pfu, pH, temperature). Results obtained during the study showed that T4 bacteriophage performed infection activity from 10-1 to 10-7 dilutions, while at dilutions 10-8 to 10-10; T4 bacteriophage did no lysis activity. Study results also indicated that the yield of T4 bacteriophage is highly dependent upon temperature. The low temperatures of 4°C did not permit T4 bacteriophage to perform infection activity on E. coli BL21. While at 15°C, 25°C and 30°C there has been infection activity but with little delay. Similarly the study showed that at thermophilic temperature 41°C, T4 bacteriophage developed and performed lysis on its host bacteria. During study it was also observed that at temperature regimes of 45°C, 55°C and 70°C, the T4 bacteriophage was completely inactive. Present study results indicated that the ideal temperature for interaction and bacteriolytic activity of T4 bacteriophage against E. coli BL21 was 37°C. Present study results also indicated that T4 bacteriophage was stable in the pH range from 4 to 10.

The bacteriophage rate of interaction and adsorption to its host directly depended on media and culture condition. In our present study we observed that the maximum growth and infection activity of T4 bacteriophage was on luria-bertani (LB) and nutrient media (NM). Moreover the T4 bacteriophage production and lysis was also good in luria-bertani plus glucose (LBG) media but when compared with its production in luria-bertani (LB) and nutrient media it was found to be less than these medium. Our study results also showed that in minimal media (MM) rate of growth and infection activity of T4 bacteriophage was lowest as compared to other mentioned medium.

This study characterizes the influences of well-defined physiological conditions on Escherichia coli growth and its interactions with T4 bacteriophage. In our present study we observed that the maximum growth and infection activity of T4 bacteriophage was in stationary phase. T4 bacteriophage production and infection was also good in log phase but in lag phase and death phase production and infection activity was less as compared to other mentioned phase.

The present study firstly explains the basic biology of lysis inhibition and also describes lysis of T4-infected cells at high culture densities. Secondly we presented that when the lysis inhibition is initiated there is sudden increase in the size of plaque which is caused by adsorption of T4 bacteriophage (secondary adsorption). It is a novel mechanism through which the lytic nature of T4 phage particle has evolved and our study provides logical arguments to conclude that lysis inhibition increase the plaque burst size.

The present study was an effort to look at the effect of quorum sensing molecules which are acyl-homoserine lactones and indole on production and infection activity of T4 bacteriophage. A predator/prey system model was employed to conduct the experiment. Results obtained during the study showed that acyl-homoserine lactones (AHL), which are essential signaling molecules of quorum sensing in many gram-negative bacteria, can increase T4 phage production and infection activity against E. coli BL21 (DE3). In addition, our study showed that indole which can also act as a signaling molecule and is a common diagnostic marker for the identification of Escherichia coli reduced the lysis activity and production of T4 bacteriophage.

Our study also showed that AHL increased the lysis activity by 54.4% in diluted T4 bacteriophage while indole decreased the lysis activity by 27.4% in diluted bacteriophage. The effect of AHL and indole on plaque size was checked and result showed that AHL increased the plaque size by 100% as compared to the control and indole reduced the plaque size as compared to the control. The plaque size of AHL was 10mm and indole was 5mm while the size of control plaque was 6mm after 16 hours.

In this study we identified and separated two new quorum sensing molecules which are W1 and W2. We have demonstrated that W1 and W2 can increased the lysis activity and production of T4 phage.effects of -20 oC, liquid nitrogen and chloroform on W1 and W2 molecules. The result showed that W1 and W2 still have activity after treated with them but after boiling W1 and W2 lost activity.

KEY WORDS: T4 Bacteriophage, Escherichia coli , Quorum Sensing, AHL, Indole

CHAPTER No: 1 INTRODUCTION

1.1 QUORUM SENSING:

Quorum sensing is a mechanism of cell-to-cell signaling involving the production of hormone like compounds called autoinducers. Through the accumulation of these autoinducers, the organisms “sense” their own population as well as the population of other organisms in a given environment [1].

Cellular signaling and communication are vital for the appropriate development and growth of every multicellular living organism. Because of the universal significance of cell communication it is quite evident that many of its fundamental aspects have been evolutionarily preserved between animals, plants and unicellular eukaryotes despite of the fact that more than 1 billion years ago these kingdoms diverged [1].

However, once it was thought that the ability to coordinate cellular behavior is restricted to eukaryotic organisms and there is only indirect bacterial perception of neighboring bacteria. But now due to the research done in the past 2 decades has revealed that bacteria also uses sophisticated communication systems in order to coordinate a variety of biological activities which include growth in biofilm communities and virulence factors production [2].

1.1.1 Quorum Sensing in Bacteria:

In bacteria quorum sensing (QS) is cell–cell communication process that involves the production, detection, and response to extracellular signaling molecules called autoinducers. Autoinducers accumulate in the environment as the bacterial population density increases, and bacteria monitor this information to track changes in their cell numbers and collectively alter gene expression. QS controls genes that direct activities that are beneficial when performed by groups of bacteria acting in synchrony. Processes controlled by QS include bioluminescence, sporulation, competence, antibiotic production, biofilm formation, and virulence factor secretion [3].

Despite differences in regulatory components and molecular mechanisms, all known QS systems depend on three basic principles. First, the members of the community produce autoinducers, which are the signaling molecules. At low cell density, autoinducers diffuse away, and, therefore, are present at concentrations below the threshold required for detection. At high cell density, the cumulative production of autoinducers leads to a local high concentration, enabling detection and response. Second, autoinducers are detected by receptors that exist in the cytoplasm or in the membrane. Third, in addition to activating expression of genes necessary for cooperative behaviors, detection of autoinducers results in activation of autoinducers production. This feed-forward auto induction loop presumably promotes synchrony in the population [4].

Signaling molecules involved in bacterial quorum sensing are divided into two groups. One group includes the fatty acid derivatives exploited by Gram-negative bacteria and the second group peptide derivatives are typically used by Gram-positive bacteria [5].

1.1.2 Quorum Sensing in Plant:

Bacterial infection of plants often depends on the exchange of quorum sensing signals between nearby bacterial cells. It is now evident that plants, in turn, 'listen' to these bacterial signals and respond in sophisticated ways to the information. Plants also secrete compounds that mimic the bacterial signals and thereby confuse quorum sensing regulation in bacteria [6].

Its reported that plants also respond to the bacterial AHLs signals, as has more importantly, different plants were found to participate in the signaling cascade by the production of AHL mimics (black rectangles and triangles) that can positively or negatively affect bacterial QS, possibly via effects on bacterial AHL synthesis or secretion [7].

1.1.3 Quorum Sensing in Parasite:

Recently, it was found that quorum sensing signal molecules acylated homoserine lactones produced by bacterium P. aeruginosa can attract nematodes to word its self [8].

1.2 DIFFERENT QUORUM SIGNALS SYSTEMS IN BACTERIA:

In the past 30 years quorum sensing, which is the process of communication between bacterial cells, is one of the very significant discoveries in microbiology [5]. Signaling molecules involved in bacterial quorum sensing are divided into two groups. One group includes the fatty acid derivatives exploited by Gram-negative bacteria and the second group peptide derivatives are typically used by Gram-positive bacteria. Quorum sensing is ubiquitous in many known species of bacteria. Gram-negative bacteria which are pathogenic in human and plants including the genera Pseudomonas, Brucella, Erwinia, Ralstonia,Vibrio, Agrobacterium, Enterobacter, Serratia, Yersinia, Bukholderia and Vibrio. These genera utilize the mechanism of quorum-sensing in order to regulate synthesis of virulence factors [9]. Bacteria included in genera Enterococcus, Bacillus, Streptococcus , Streptomyces and Staphylococcus make use of this mechanism for the production of antimicrobial peptides or exotoxin, formation of biofilms and development of genetic competence [10].

Quorum sensing is used by the Rhizobiumgenus for nitrogen fixation. The symbiosome development and nodulation that is essential for nitrogen fixation is regulated by complex quorum-sensing systems in these symbiotic bacteria [11]. In extremophiles quorum sensing has also been described such as in the haloalkaliphilic archeon Natronococcus occultus and in Halomonas bacterial genus [12], in Acidithiobacillus ferrooxidanss [13] and in the hyperthermophilic bacterium Thermotoga maritima [14].

Bacterial community utilizes the system of interspecies quorum sensing in order to determine that how many of other and their own species are present in an area. Quorum sensing molecules are secreted by the bacteria which are increased in proportion to cell number. When these molecules hit a certain concentration, they activate the transcription of some specific genes for example virulence factors transcription. It has been revealed that bacteria not only interact with members of their own species via quorum sensing but also gather information about other species and there is a kind of some universal molecule which permits them to do so. This universal molecule is named as autoinducer 2 or AI-2 [15].

1.2.1 Mechanisms of Quorum Sensing:

The mechanism of quorum sensing is divided into 4 steps: (1) small biochemical signaling molecules production by the bacterial (2) signal molecules release in the surrounding environment, either passively or actively (3) specific receptors recognize these signaling molecules when concentration of these molecules exceeds a certain threshold, thus leading to (4) gene regulation changes (Fig-1.2.1) [16].

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Figure-1.2.1: Mechanism of quorum sensing. In the figure two pathway are explained. In one pathway autoinducer directly enter to cell and activate target gene while in other pathway autoinducer combined with membrane receptor. Then formed a complex and enter into cell and activate the target gene

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Figure- 1.9.1: Mechanisms of quorum sensing

1.2.2 Quorum-Sensing Signals:

Quorum sensing signaling systems are broadly classified into four main groups. Gram-negative bacteria utilizes two of these systems which involve autoinducer-1 (AI-1) and autoinducer-3 (AI-3), while on the other hand the third type of signaling system is used by Gram-positive cells involving the autoinducing polypeptide (AIP) system. These cell to cell signaling are principally involved in intra species communication [17][18].

However the fourth system is found in both Gram negative and Gram-positive bacteria using autoinducer-2 (AI-2). Quorum sensing which involves AI-2 is used mainly for interspecies communication [19].

1.2.3 Autoinducer-1 or AI-1:

Investigating the phenomenon of bioluminescence, the LuxIR system was discovered for the first time in Vibrio fisheri and presently the LuxI/LuxR system is considered to be the model system and is the base of other quorum-sensing systems [20].

It is composed of LuxI which manufactures an N-AHL known as AI-1 and LuxR which is a transcription factor accountable for scheming gene expression in the company of the autoinducer. Autoinducers are synthesized by LuxI and its homologues by transfer of a fatty acid chain to SAM from an acylated acyl carrier protein (ACP) which results in the release of methyl thioadenosine and AHL [21].

LuxI homologues synthesize AHL composed of diverse fatty acid moieties which recognize specific ACPs by the enzyme synthases and result in intrageneric and intraspecies signaling. After synthesis, AI-1 is released into the adjacent environment by diffusion across the bacterial cell membrane. Environmental concentration of AI-1 rises with the increase in population. When there is high population density, AI-1 diffuses back into the cell because its local concentration is very high. Within the cell it binds to Lux resulting in the activation of the transcription of the luxCDABEGH operon located within the promoter region [22]. Luciferase is the product of this operon that catalyzes a chemical reaction resulting in luminescence. Most of the Gram-negative bacteria have homologous LuxI/LuxR systems having the ability to produce specific AHLs. These signaling processes govern the expression of the virulence factors in many opportunistic pathogens like Serratia marcescens and Pseudomonas aeruginosa. P. aeruginosa have two systems which are homologous to LuxI/Lux. These systems not only control extracellular enzymes production and biofilm formation but also transcription of another quorum-sensing system i.e RhlI/RhlR thus adding further in the level of control by AHL signaling mechanism [23].

Earlier it was thought that bacteria utilizes quorum sensing systems to regulate density of their population, however, studies on Salmonella Typhimurium and E. coli showed that this does not happen all the time. For example, E. coli and S. Typhimurium do not possess a LuxI homologue so they are not able to produce any AI- 1. However, they are capable of encoding a LuxR homologue called SdiA which when over expressed have a negative effect on those genes that are involved in cellular attachment in enterohemorrhagic E. coli (EHEC) [24], while SdiA positively regulates numerous genes situated on the virulent plasmid of S. Typhimurium for example rck which is a protein concerned with the evasion of the immune response of host [25]. Although the exact role of SdiA in pathogenesis is unclear, this protein allows EHEC and S. Typhimurium to alter gene expression in response to the presence of AI-1 produced by other bacteria [26].

1.2.4 Autoinducer-2 or AI-2:

Most of the Gram-negative and Gram-positive bacteria use systems of quorum sensing that recognize an extracellular signal called as AI-2. It is synthesized from a SAM metabolism by- product. AI-2 synthesis from protein LuxS, a synthase encoded by luxS genes, in V. harveyi is good example. A series of steps are involved in synthesis of AI-2 from SAM by LuxS including conversion of ribosehomocysteine into 4,5-dihydroxy-2,3- pentanedione (DPD) i.e a compound which cyclizes into numerous furanones in water presence and homocysteine [19]. Two separate AI-2-binding proteins cocrystalize to determine the structure of the AI-2 signals. These include a BAI-2 and R-THMF (Fig-1.2.4) [27].

Bacterium releases AI-2 which accumulates in the cellular environment and can be detected by two different mechanisms. V. harveyi detects the form BAI-2. The presence of periplasmic AI-2 is detected by binding of the signaling molecule with LuxP which is a specific autoinducer binding protein. A phosphotransfer cascade is initiated when AI-2/LuxP complex interacts with LuxQ, a sensor kinase, thus resulting in luciferase production and luminescence. The cascade of LuxP/LuxQ has been recognized in Vibrio spp only. AI-2 is managed by a separate mechanism in S. Typhimurium and E. coli. In contrast to LuxP/LuxQ system, AI-2 is transported into the cytoplasm through Lsr (LuxS regulated) system of cell that initiates a cellular response. LsrB, a periplasmic protein, recognizes the signal and binds to the R-THMF which is a form of AI-2. After binding, the Lsr ABC transporter that consists of LsrC and LsrA transports AI-2 into the cell where it is phosphorylated by LsrK. AI-2 in its phosphorylated form interacts with the LsrR that is a transcriptional repressor, in order to alleviate repression of the operon lsr which may up regulate other operons (Fig-1.2.4) [28]. In Gram-negative and Gram-positive bacteria a wide range of LuxS/AI-2 systems have been found which has proposed the idea that the AI-2 system is utilized for cross-species signaling process by organisms that live in mixed-species communities like biofilms [29].

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Figure-1.2.4: Schemes of autoinducer 2 response. In Gram-negative and Gram-positive bacteria a wide range of LuxS/AI-2 systems have been found which has proposed the idea that the AI-2 system is utilized for cross-species signaling

1.2.5 Autoinducer-3 or AI-3:

AI-3 was firstly described as a compound employed by the QseC system and is found in spent media which activates genes expression that are concerned in EHEC attachment to eukaryotic cells and subsequent rearrangement of actin (Fig-1.2.5) [30].

But still the synthesis and structure of this signaling molecule is not well known. LuxS was supposed to be involved in the AI-3 production because of the fact that its synthesis was impaired in mutants of Luxs. However, further studies revealed that impairment of AI-3 production in LuxS mutants was because of oxaloacetate used as a metionine precursor instead of SAM. The use of L-aspartate in the growth medium decreased the demand of oxaloacetate thus restored AI-3 production but had zero effect on production of AI-2 [31].

Many studies also revealed that a number of bacteria including nonpathogenic Enterobacter cloacae and E. coli and also pathogenic bacteria like Klebsiella, Shigella and Salmonella species can produce AI-3. Thus it is suggested that AI-3 might correspond to another cross-species signal. While the AI-3 has not been detected in Gram-positive bacteria till date. AI-3 is detected by using a 2-component system which comprises response regulator QseB and sensor kinase QseC. QseC autophosphorylate in the presence of periplasmic AI-3 and then phosphate is transferred to QseB that results in the up regulation of master flagellar regulator gene flhDC which are those genes responsible for biosynthesis of flagella and its motility [32]. AI-3 presence is also related to the formation of EHEC’s effacing and attaching lesions. This is accomplished by 5 different loci’s up regulation in enterocyte effacement (LEE) operons that are present within the chromosome of EHEC [30]. However, the cascade accountable for regulation of these genes is still not clear completely, but most likely it involves QseA which is a regulator of LysR family and is influenced due to cell to cell signaling process. It is also involved in the frank up regulation of LEE genes (Fig-1.2.5) [30].

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Figure- 1.2.5: Autoinducer type 3; EHEC, enterohemorrhagic Escherichia coli; LEE, locus for enterocyte effacement; flh, flagella regulon; P, promoter site; P, phosphorylated; Qse, AI-3 system in enteric bacteria

1.2.6 Autoinducing Peptides:

Autoinducing peptides responsible for cell to cell signaling are present absolutely in Gram positive bacteria. Prototypic Agr system is the base of these signaling processes which was described in S. aureus for the first time. The Gram-positive bacteria utilize a polypeptide signal in place of a smaller molecule. These polypeptides work as an autoinducer for organism that synthesizes and produces it while inhibits other organisms. This polypeptide signal is known as AIP and determined by the gene agrD. After its translation, N-terminal signal sequence targets the AgrD propeptide to the membrane. After reaching the membrane, C-terminus of the propeptide is cleaved by AgrB that is a membrane-bound endopeptidase. Signal peptidase i.e. SpsB removes the propeptide’s N-terminus which also includes the signal sequence. Lastly, a thiolactone ring having a free N-terminal tail is formed when the C-terminus of processed polypeptide is linked covalently to a centrally located cysteine. Both of these structures are essential for appropriate performance of the AIP in many cases. Signal receptor that is AgrC recognizes AIP when it is released in the environment. AgrC consist of a transmembrane N-terminal domain in order to recognize specific AIPs and also a C-terminal histidine kinase domain that phosphorylates a response regulator called AgrA in the presence of the correct AIP. Phosphorylated AgrA initiates transcription of selective genes after binding to direct repeats that are present in the promoter regions. One feature that is specific to the AIP/Agr system is the reality that an AIP which is produced by one strain of Staphylococcus will interfere with Agr system of another strain. This double role as an inhibitor and activator is associated to the AIP and AgrC interaction. The cyclic structure of AIP is necessary in order to interact with AgrC, while it is the N-terminal tail that is accountable for activation of AgrC. In fact removal of this tail results in the formation of a universal inhibitor which binds to AgrC but is not able to activate the Agr system (Fig- 1.2.6) [33]. In many Gram-positive bacteria Agr system has associated to pathogenesis. AIP-directed regulation of genes by AgrA results in the making and release of many toxins by S. aureus, such as beta-hemolysins, alpha-hemolysins, delta-hemolysins, serine proteases and toxic shock syndrome toxin 1 [34]. Another Gram-positive Enterococcus faecalis that take advantage from the use of AIP signal sensing. This organism uses a 2-component system homologous to the Agr system of Staphylococcus to sense the presence of AIP. When AIP is detected, the cell produces and releases 2 extracellular proteases, gelatinase and SprE [35].

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Figure-1.2.6: Autoinducing Peptides system. The agr locus. The various components are shown, indicating their functions in the signaling network. This signaling system is a doubly auto-catalytic circuit, as AgrC activation induces transcription of more receptor, along with more of the propeptide inducer, AgrD, processed by AgrB to form the pheromone AIPs. The end result of the pathway is activation of RNAIII transcription by the response regulator, AgrA, which leads to downstream activation and repression of virulence-associated factors via transcriptional and translational regulation by this unique RNA molecule

1.3 ESCHERICHIA COLI AND T4 BACTERIOPHAGE IN NATURAL ENVIRONMENT:

1.3.1 Escherichia coli in Natural Environment:

Escherichia coli is a member of the coliform group of bacteria that is naturally found in the intestines of humans and warm-blooded animals. However Escherichia coli populations can also survive and grow in open environments such as soils and water environments [36]. In tropical ecosystems nutrients for Escherichia coli is maintained at high concentrations and together with constant warm air, soil, and water temperatures; this provides an ideal habitat for survival, growth, and proliferation of Escherichia coli (Fig-1.3.1).

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Figure-1.3.1: Free-living E. coli populations exist in tropical environments in the absence of human or animal contamination. In tropical environment nutrients for E. coli is too much for its survival and growth

1.3.2 Escherichia coli in Soil Environment:

The growth of E. coli in soils environment been extensively documented and the organism is considered to be an established part of the soil biota within these regions [37]. The integration of E. coli as a part of the original micro flora in soils of tropical and subtropical regions may be attributable to the nutrient-rich nature and warm temperatures of these habitats [38], joint with the metabolic versatility of the organism and its simple nutritional necessities [39]. In addition to tropical and subtropical regions, the occurrence of autochthonous E. coli populations in the cooler soils of temperate and northern temperate regions has also been reported [40], with one report on an alpine soil [41] and, most newly, a report on a maritime temperate grassland soil [42]. The growth of E. coli within soils can act as a reservoir for the further pollution of bodies of water [43], compromising the indicator position of E. coli within these regions. As such, an understanding of the ecological characteristics of E. coli in soil is dangerous to its validation as an indicator organism. With respect to the input of pathogenic E. coli into the environment, this knowledge becomes essential for assessing the potential health risk to human and animal hosts from agricultural activities such as land spreading of manures and slurries [44].

It has been recommended that E. coli can maintain autochthonous populations within soils in temperate regions, wherever good conditions exist [39]. The phenotypic traits of the organism (including its metabolic diversity and its ability to grow both aerobically and anaerobically in a broad temperature range) may assist the persistence, colonization, and growth of E. coli when conditions allow. The challenging nature of the soil environment and the disparity of conditions between the primary host and the secondary habitat raises the question of how these E. coli populations survive and compete for niche space among the highly competitive and diverse coexisting populations of the indigenous micro flora [39]. There is some evidence that naturalized E. coli may form genetically distinct populations in the environment [45]. This suggests that autochthonous E. coli populations in soil may have increased environmental fitness, facilitating their residence in soil [46]. Little is known, however, of the physiology of these organisms, and their capacity for survival in soil remains poorly understood [39].

1.3.3 Escherichia coli in tropical Fresh Water Environment:

High concentrations of E. coli have been found in numerous tropical locations in the absence of known fecal sources [47]. For example, the waters of Puerto Rico are highly contaminated with human waste, yet at sampling sites upstream of known sewage drainage points there are large numbers of fecal coliforms, similar to the numbers at sites downstream [47]. Likewise, the number of viable E. coli cells in the upper one-third of the Mameyes River in Puerto Rico (in a cloud rain forest) shows a strong positive correlation with water temperature and nutrient concentrations [48]. The total bacterial count found at this unspoiled tropical location exceeds by twofold the total count reported for the polluted, temperate Anacostia River in Washington, D.C. High respiration rates and extended survival suggest that tropical waters harbor natural populations of E. coli [48]. However, studies performed with a pure culture of an E. coli strain that was not isolated from the Mameyes River showed that the size of the population decreased by 90% after 12 days, raising the possibility that a natural isolate in the region may be genetically adapted for increased survival in this environment [47]. In spite of this, the survival time and the percentage of physiologically active cells (55 to 90%) suggest that E. coli is able to grow in a tropical freshwater environment [49]. Water sources in uninhabited rain forests provide elevated concentrations of nutrients for thriving microbial communities in the absence of human pollution [50]. Epiphyte species living in trees 15 m above the ground, such as bromeliads, form large cups with their leaves, which collect rainwater and runoff, thus serving as microcosms that are rich in nutrients. For example, bromeliad water, which can contain up to 50 times the typical concentration of nitrites and nitrates, was shown to contain an average of 1.5x106 coliforms per 100 ml, 51.6% of which were undergoing respiration and 49.1% of which were engaged in protein synthesis. E. coli comprises 72% of the total bacterial species living in bromeliad water microcosms. The presence of active E. coli populations in bromeliad water at different forest elevations throughout the year suggests that these populations did not originate from the small population of Puerto Rican birds or tree-climbing mammals [50]. Natural E. coli populations are not limited to tropical freshwater. The soil of the North Fork of the New River in Ft. Lauderdale, Fla., is a major source of E. coli [51]. This subtropical riverbank environment is characterized by warm and humid conditions with cyclic periods of wet and dry weather, which are conducive to E. coli growth [52].

1.3.4 T4 Bacteriophage in Natural Environment:

Bacteriophages are very common in all natural environments and are directly related to the numbers of bacteria present. They are thus very common in soil and have shaped the evolution of bacteria. It is thought that predatory T4 phages could control the numbers of bacteria and facilitate gene transfer between bacteria by transduction [53]. In addition, estimates of T4 phage abundance in aquatic habitats suggest their numbers are 10 times greater than those of bacteria [54]. Extrapolating this estimate to the biosphere at large would make phages the most abundant organisms on earth [54].

T4 phages also impact the movement of nutrients and energy within ecosystems primarily by lysing bacteria. T4 phage can also impact abiotic factors via the encoding of exotoxins (a subset of which are capable of solubilizing the biological tissues of living animals) [55]. Phage ecosystem ecologists are primarily concerned with the phage impact on the global carbon cycle, especially within the context of a phenomenon known as the microbial loop. The interaction of phage with bacteria is the primary concern of phage community ecologists. Phage, however, are capable of interacting with species other than bacteria, e.g., such as phage-encoded exotoxin interaction with animals [56].

Bacteriophages can also show biogeography because studies have shown that some phage have a global distribution while others may be endemic to particular environments [57]. Generally phages are pretty stable if the environment is not hostile. They are broken down in UV light, and can be damaged by abrasion, or exposure to chemicals, but researchers have been known to keep phages in their fridges for over 40 years with no reduction in titre [58].

Hankin ME in 1896 reported that something in the waters of the Ganges and Jumna rivers in India had marked antibacterial action against the bacteria responsible for the disease, cholera. Testing these waters showed that this inhibition of the bacterial growth remained even if the water was passed through a very fine porcelain filter. That meant that whatever was responsible for killing off the Vibrio cholera cultures was smaller than any known bacterium, none of which could pass through the tiny pores present in that dense ceramic filter. Unfortunately this observation was not followed up further [59].

1.3.5 Interaction between Phages and Hosts in their Physical Environment:

The environments created by the animal-host physiology, and by the microbial activity in different densely populated niches appears to profoundly influence the mode of interaction between bacterial and phage populations. The intestinal coliphage ecology is one of the best studied examples of this complex interaction and the impact of the phages on host populations, mechanisms of phage-host mutual regulation and adaptation have been shown to vary considerably in different animal species [60]. Furthermore, recent work confirmed that in a natural situation, healthy horses frequently do excrete coliphages. This work also showed that coliphage populations exhibit significant temporal variation, with up to 4 orders of magnitude difference in phage abundance during 15 days of monitoring [61]. In terms of coliphages in other animals, low fecal coliphage prevalence has been reported in dogs [62].

The role that phages play in the ecology of bacteria also differs between specific ecosystems within different body sites in the same animal species. The failure to obtain phage isolates from the vaginal and oral cavity in humans suggests that the phage impact in these systems is less than their impact in other environments, such as colonic ecosystems of the same species [63].

1.4 ESCHERICHIA COLI AS A MODEL ORGANISM:

Escherichia coli is a Gram-negative, non sporulating and facultative anaerobic rod. It is about 2.0 micrometers (μm) in length and its diameter is 0.25-1.0 μm (Fig-1.4) [64]. Those strains which have flagella are motile. Structurally flagella have peritrichous arrangement [65]. 37 °C (98.6 °F) is the optimal temperature for multiplication of E. coli but few of the laboratory strains can grow up to 49 °C (120 °F) of temperature [66]. Multiplication can be driven by utilizing a large number of redox pairs involving “reduction” of substrates like oxygen, fumarate, trimethylamine N-oxide and dimethyl sulfoxide plus “oxidation” of substances like formic acid, pyruvic acid, amino acid and hydrogen [67].

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Figure-1.4: After gram staining E. coli were found as pinkish rods giving gram negative staining reaction under oil immersion (100X) lens. Most organisms appeared singly but some were also found in diplobacilli and streptobacilli

1.4.1 Life cycle of Escherichia coli :

In life cycle of Escherichia coli there is division of one cell into two daughter cells. This process is known as binary fission. Under circumstances when no mutation occurs, the daughter cells are identical genetically to the parent cell. Thus there is "local doubling" of the E. coli population (Fig-1.4.1).

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Figure-1.4.1: Escherichia coli reproduce through the process of Binary Fission, an asexual process in which the cell grows in size, replicates its DNA, and then separates the cytoplasm and DNA into two new cells. E. coli usually takes about 20 minutes to duplicate

However, it is not necessary that both daughter cells survive but E. coli population undergoes exponential growth if the numbers of surviving daughter cells exceed unity on an average [68].

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Figure-1.2.1: Life cycle of Escherichia coli

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Figure-1.4.1: Life cycle of Escherichia coli

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Figure-1.2.1: Life cycle of Escherichia coli

1.4.2 Escherichia coli Transmission:

Escherichia coli is found commonly in the distal part of intestine in warm-blooded organisms (endotherms) [69]. They constitute about 0.1% of normal flora of gut [70]. Major route of transmission for E. coli is oro-fecal after which bacterial pathogenic strains cause disease. E. coli cells can only survive outside the body for a limited period of time so they can be considered as ideal indicator organisms in order to test samples from environment for fecal contamination [71]. However, research work carried out in this regard has showed that environmental samples may have E. coli strains that can survive for relatively long period of time even outside the host [39].

1.4.3 Escherichia coli Genera History:

A German pediatrician named odor Escherichia in 1885 discovered E. coli in the feces of healthy individuals and named it Bacterium coli because of the fact it is found in the colon. Early prokaryotic classification placed Bacterium coli in a genera based on their motility and shape. Afterwards Ernst Haeckel's bacterial classification placed bacteria in the Monera kingdom [72]. In 1895 Migula reclassified bacteria in the genus Escherichia which was named so after its discoverer [73]. This genus belongs to the bacterial group formally called "coliforms" which are member of the "the enterics” known as Enterobacteriaceae family [74].

1.4.4 Escherichia coli Phenotypic Diversity:

Escherichia coli includes a vast population of bacteria that demonstrate a very high degree of both phenotypic and genetic diversity. Taxonomic reclassification is required after looking at the genomic sequencing of a great number of isolates of the E. coli and also related bacteria [75]. E. coli is still one of the most varied bacterial specie and only about 20% of the genome seems to be common in all strains [76].

As a matter of fact, the members of Shigella genus (S. flexneri, S. dysenteriae , , S. sonnei and S. boydii) must be classified as strains of E. coli from the evolutionary point of view. This phenomenon is termed as taxa in disguise. In the same way, other E . coli strains (e.g. the K- 12 strain commonly used in recombinant DNA work) are very different, thus they warrant reclassification [77].

A specie subgroup, which has distinctive characteristics thus distinguishing it from other, is called a strain. One can find these minute characteristic differences only at the molecular level which are responsible for changes in the lifecycle or physiology of the bacterium as for example, a strain may attain the ability to use a unique carbon source, take upon particular ecological niche, resist antimicrobial agents or gain pathogenic capacity. However, strains of E. coli are usually host-specific thus making it easy to determine the source of fecal contamination in samples obtained from environment. A good example is that if researchers know that which E. coli strains are present in a sample of water it will allow them to hypothesize that whether the contamination has its origin from a human or some other mammal and even from a bird [71].

On the bases of evolutionary relatedness, there is a common subdivision system of E. coli known as serotype. Serotype is based on antigens of surface (i.e. O antigen which is a part of lipopolysaccharide layer, K antigen, H:flagellin and capsule for example O157:H7) [78]. However it is common to quote only the serogroup that is the O-antigen. Presently up to 190 serogroups are known till date. The common strain of laboratory is non type able because it has a mutation which prevents the formation of an O-antigen [79].

New strains of E. coli evolved like all life forms i.e. through the natural biological processes of horizontal gene transfer, gene duplication and mutation. About 18% laboratory strain MG1655 genome was acquired horizontally from Salmonella divergence [80]. All E. coli strains are derived from either E. coli B or E. coli K-12 strains. In microbiology, few strains developed traits which can be harmful to a animal host [81]. O157:H7 is a more virulent strain which causes serious illness and even death in immunocompromised persons, the elderly people and the very young [82].

1.4.5 Escherichia coli Strains:

Many of the E. coli strains have been characterised and isolated. Most of the strains of E. coli which are commonly used in research work are derived from Clifton's K-12 strain (λ+ F+; O16) and to a lesser extent from Bacillus coli strain (B strain; O7) [83].

1.4.6 DNA Sequence of Escherichia coli:

About 60 genomic sequences of Shigella and Escherichia species are available which have complete genomic sequences. A significant amount of diversity is seen when these sequences are compared. Over 20% of each genome represents those sequences which are present in each isolates and about 80% of genome can differ among the isolates [76]. An individual genome consists about 4,000 to 5,500 genes, however there are more than 16,000 gene sequences in E. coli strains (the pan-genome). This fact reveals that a large variety of component genes exist which have been interpreted and about two-thirds of pan-genome of the E. coli originated in other species by horizontal gene transfer process [84].

1.4.7 Escherichia coli Proteins:

Many E. coli proteins have been isolated and studied. A study purified 4,339 proteins from cultures of strain K-12 and found interacting partners for 2,667 proteins, many of which had unknown functions at that time [85]. But another study found 5,993 interactions between proteins of the same E. coli strain though this data showed little overlap with that of the above data [86].

1.4.8 Escherichia coli as a Model Organism:

In a laboratory setting, the E. coli can be grown inexpensively and easily. E. coli has been widely studied for about 60 years. It is the most extensively investigated prokaryotic model organism and considered to be very important species in biotechnology and microbiology [87].

1.4.9 Nonpathogenic Escherichia coli Advantages:

Nonpathogenic strains of Escherichia coli serve as probiotic agents in the field of medicine especially to treat various diseases of gastrointestinal tract [88], which include inflammatory bowel disease [89].

1.4.10 Role of Escherichia coli in Research Field:

E. coli holds an important position in industrial microbiology and modern biological engineering because of its easy manipulation and also long history of its laboratory cultures [90]. The research work of Herbert Boyer and Stanley Norman Cohen regarding use of restriction enzymes and plasmids in order to create recombinant DNA by E. coli became the base of biotechnology [91].

E. coli is considered to be a very flexible host for the heterologous proteins production [92]. Recombinant protein production involves various protein expressions in E. coli. Plasmids have been used to introduce genes into the microbes by researchers which have lead to high level of protein expression. Such proteins can be produced by the fermentation process in the industries at mass level. A very useful and important application of recombinant DNA technology was production of human insulin by E. coli manipulation [93].

Many folded forms of proteins have been successful in expressing them in E. coli which was previously thought to be difficult and even impossible. A good example is that proteins which have multiple disulphide bonds may have the ability to be produced in periplasmic space or in cytoplasm of those mutants having sufficiently oxidizing agent in order to allow the formation of disulphide-bonds [94]. On the other hand proteins that need post-translational modification glycosylation for function or stability use the system of N-linked glycosylation which is found in Campylobacter jejuni engineered into E. coli for their expression [95].

E. coli cells in modified form have been used in the development of vaccine, bioremediation, biofuels production [96], and formation of immobilized enzymes [97].

In microbiology studies, E. coli is widely used as a model organism . Unlike wild type strains, cultivated strains (e.g. E. coli K12) used in the laboratories are well-adapted to the lab environment and cannot survive in intestine. Many laboratory strains have lost their ability of biofilms formation. By this way wild type strains are protected from antibodies and also other chemical attacks, however, this requires expenditure of large number of material resources and energy [98].

E. coli was used as a model bacterium in order to describe bacterial conjugation [99], and it is still the primary model to study this process. E. coli has helped in understanding phage genetics [100].

1.5 T4 BACTERIOPHAGE AS A MODEL ORGANISM:

The Escherichia coli are infected by a bacteriophage named T4 bacteriophage. Length of its DNA genome is about 169 kbp which is double stranded. DNA genome is held in an icosahedral head which is also called capsid. As compared to other bacteriophages T4 is a relatively big bacteriophage, which is about 200 nm long and 90 nm wide (most of the bacteriophages range in length from 25 to 200 nm). Its tail fibers permit attachment to host cell while the tail of T4 bacteriophage is hollow so that it can transmit its nucleic acid to the host cell thus infecting it during attachment (Fig-1.5). T4 bacteriophage does not undergo lysogenic lifecycle but has the ability to undergo a lytic lifecycle [101].

Abbildung in dieser Leseprobe nicht enthalten

Figure-1.5: Schematic of T4 bacteriophage

1.5.1 Infection and Lifecycle of T4 Bacteriophage:

Long tail fibers (LTF) of T4 bacteriophage recognize surface receptors of E . coli thereby initiating the infective process. LTFs send a recognition signal to the base plate. The short tail fibers (STF) are then unraveled thus binding irreversibly to the E. coli cell surface. The conformation of base plate changes which results in contraction of tail sheath causing GP5 present at the end of the tail tube to puncture the outer cellular membrane. The periplasmic peptidoglycan layer is degraded by the activated lysozyme domain of GP5. When remaining part of the membrane is also degraded then DNA from the phage’s head enters the E. coli by traveling through the tail tube (Fig-1.5.1) [102].

Abbildung in dieser Leseprobe nicht enthalten

Figure-1.5.1: Injection process of T4 bacteriophage DNA into a bacterial cell. The T4 phage initiates an E. coli infection by binding OmpC porin proteins and Lipopolysaccharide (LPS) on the surface of E. coli cells with its long tail fibers (LTF)

T4 bacteriophage takes about 30 minutes to complete lytic lifecycle i.e. from entering a bacterium to its destruction (at 37 °C) and composed of:

- Adsorption and penetration (starting immediately)
- Arrest of host gene expression (starting immediately)
- Enzyme synthesis (starting after 5 min)
- DNA replication (starting after 10 min)
- Formation of new virus particles (starting after 12 min)

The host cell bursts open and releases the newly built viruses after the completion of lytic lifecycle thus destroying the host cell. The burst size of T4 bacteriophage is about 100-150 viral particles per infected host. T4 bacteriophage infects a host cell by their information afterwards blowing up the host cell thus propagating their progeny and increasing themselves [103].

1.5.2 T4 Bacteriophage Unique Feature:

There are some unique features in T4 bacteriophage which are as follows: [103].

- Eukaryote-like introns
- High speed DNA copying mechanism, with only 1 error in 300 copies
- Special DNA repair mechanisms
- It infects E. coli O157:H7
- genome terminally redundant

First of all genome is replicated and form several units and then there is end-to-end recombination of these genomic units which results in the formation of a concatemer. The concatemer is cut into same length at unspecific points during packaging. Thus several genomes are formed which correspond to Circular permutations of the original genome [104].

1.5.3 Bacteriophage Classification:

Phages are classified by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid. T4 phages belong to order caudovirales and family Myoviridae. Nineteen families are currently recognised that infect bacteria and archaea. Of these, only two families have RNA genomes and only five families are enveloped. Of the viral families with DNA genomes, only two have single-stranded genomes. Eight of the viral families with DNA genomes have circular genomes, while nine have linear genomes. Nine families infect bacteria only, nine infect archaea only, and one (Tectiviridae) infects both bacteria and archaea [105].

1.5.4 Lysis from within" and "Lysis from without:

T4 bacteriophage signifies two totally different types of lysis, which are "Lysis from without” and "Lysis from within". Lysis from without occurs almost instantaneously by T4 bacteriophage adsorption at a threshold which is almost equal to the adsorption capability of that bacterium. None of the T4 bacteriophage is liberated in this case, on the contrary, the adsorbed T4 bacteriophages are lost. While lysis from within is the usual lysis that is observed when the latent periods of lytic T4 bacteriophage end. The cell wall of the bacterium is attacked by T4 bacteriophage in such a way which permits swelling of the cell and its deformation into a spherical body. Lysis from within is initiated by adsorption of single or few T4 bacteriophage particles. In favorable conditions there is multiplication of one T4 bacteriophage particle within the bacterium up to a threshold value during the latent period. Threshold value is equal to the adsorption capacity and when this threshold value reaches only then there is abrupt destruction of protoplasmic membrane which results in liberation of the T4 bacteriophage particle and permits a rapid release of the cell contents with no deformation of the cell wall [106].

1.5.5 T4 Bacteriophage as Model System:

E. coli is infected by a virus i.e. bacteriophage T4 which has played key roles in few of the most important advances in the field of molecular biology including the recognition of the chemical nature of genes, explaining the mechanism of DNA replication, discovering genes code for proteins and even deciphering how the genetic code is read [107].

In the development of modern genetics and molecular biology, T4 bacteriophages have been considered important model systems since the 1940s. Many investigators have taken benefit of T4 phage’s practical degree of complexity and its capability to gain complete genetic and physiological information with quite easy experiments. T4 bacteriophage was considered useful in the formulations of many basic biological concepts. These comprise of clear-cut recognition of nucleic acids as the genetic material; defining gene by fine-structure through recombinational, mutational and functional analyses; demonstrating the triplet nature genetic code; mRNA discovery; the significance of recombination in DNA replication; mechanism of light-independent and light-dependent DNA repair; modification and restriction of DNA; translational bypassing; prokaryote’s self splicing introns and many others [108]. The benefit in view of T4 bacteriophage as a model system is T4 phage’s complete inhibition of host gene expression thus allowing investigators to distinguish between phage and host macromolecular syntheses [109].

1.5.6 T4 Bacteriophage Therapy:

The reason of attraction towards T4 bacteriophage as therapeutic agent is that they are lethal and highly specific for targeted bacteria while on the other hand safe for humans. Moreover, T4 bacteriophages can develop rapidly to battle the antibiotic-resistant pathogenic bacteria emergence [110].

The Eli Lilly Company (Indianapolis, Ind.) in 1940s formed T4 bacteriophage products against Escherichia coli for human use [111]. In a most recent paper from Brussow and colleagues [60], systematical experiments were conducted with E. coli phages. T4 bacteriophages were tested against both non-pathogenic and pathogenic (EPEC and EHEC) strains of E. coli [112]. T4 coli phages which showed the complementary and widest infection range among the strains were chosen for the experiments.

1.5.7 T4 Bacteriophage Lysins as Antimicrobials:

Many of recent studies have revealed that rather than intact phage the T4 bacteriophage endolysins has been potentially used in therapeutics [113]. T4 bacteriophage endolysins are enzymes which hydrolyze the four major bonds in peptidoglycan component of cell wall thus spoiling the cell wall’s integrity. Most of the phage lysins studied till date is in fact modular in structure that composed of two clearly divided functional domains which are N-terminal catalytic domain and C-terminal cell-wall binding domain. The C-terminal directs the enzyme to its target while the catalytic domain can consist of one or more than one of the following types of peptidoglycan hydrolases: muramidases (lysozyme), endopeptidases, glucosamidases and N-acetylmuramoyl- L-alanine amidases. The majority of the lysins are amidases [114].

1.5.8 T4 Bacteriophage Display:

Display technology of T4 bacteriophage is a powerful molecular tool that had a key impact on the discovery of drugs, immunology, pharmacology and plant science. Display technology is a technique by which foreign proteins, peptides or fragments of antibody are present at the T4 bacteriophage particles surface. As a result of transcriptional fusion, the heterologous protein or peptide is cloned with one of the coat protein genes into phagemid genome or a T4 bacteriophage. Thus T4 bacteriophages become vehicles for expression that along with carrying the nucleotide sequence within them encode the expressed proteins thereby permitting the gene sequence to be retrieved and also have the ability to replicate [115].

1.5.9 Function of T4 Bacteriophage in Vaccines:

An exciting and novel use of T4 bacteriophages is to deliver vaccines either as delivery vehicles for DNA vaccines or as a form in which immunogenic peptides are attached to the modified coat proteins of T4 bacteriophage. Display of T4 bacteriophage is helpful for the recognition of immunogenic mimotopes or epitopes on displayed peptides in turn which can become the base of peptide vaccines [116].

1.5.10 T4 Bacteriophage for Detection of Pathogens:

E. coli can be detected by T4 bacteriophage. Small outer capsid (SOC) protein of T4 bacteriophage was used in order to present green fluorescent protein (GFP) which is an easily detectable protein marker present on the capsid of phage. The T4e(-) phage was used to detect E. coli, which does not produce the lysozyme responsible for the lysis of host cell. The intensity of green fluorescence was increased by the propagation of T4e(-)/GFP in the host cells thus it makes the differentiation of E. coli cells from other cells very effective and simple. This method allows the rapid and conclusive quantitation of E. coli cells in an hour [117].

1.6 GOAL OF OUR STUDY :

It’s reported that phage-bacterium interactions depends on physical-chemical factor (Concentration, pH and Temperature) and depend on host physical state and culture condition [118], so we checked the effect of above condition on T4 phage- E. coli interaction.

There are two reports that quorum sensing effects the infection and phage-bacterium interactions. One reported said that bacterial cell-cell signaling provides clues about the density of potential host cells, phages could benefit from that signals and start attachment to the host cell and release progeny phages specifically when new host cells are abundant. Equally, as the risk of phage infection increases with increased bacterial density, bacteria could be expected to use cell-cell signaling to tie the regulation of their anti-phage defense mechanisms to the cell density of their population [119].

Other report said that quorum sensing can be used as a mean of regulating phage-bacterium interactions. Phage's predation pressure is of considerable importance, as phages exceed bacterial cell numbers by 10-fold in many natural environments [120]. As a consequence, bacteria have worked out an extensive range of antiphage mechanisms, which includes ways of bricking the early attachment of phages, phage genome's degradation or abortive infection by host suicide, and thereby arresting the spread of phage progeny in the population to some extent [121]. Phages need a bacterial host to expand and proliferate, they are supposed to be more in number and diverse in heavily populated mixed species than in environments which are sparsely populated. For this reason, the danger of suffering phage attack is brightened at high microbial cell population levels. Thus the key factor in designing the evolutionary advancement between the phage and host is the bacterial phage resistance mechanism [122]. Bacteria make use of quorum sensing in order to regulate and govern their antiphage activities, they can enhance their defense mechanisms thus avoiding infection during their growth under high-risk conditions, besides saving the metabolic accountability and maintaining a steadily elevated antiphage strategy.

Another goal of our research was to clarify the role of bacterial quorum sensing, in shaping the interactions between bacteria and the bacteriophage that prey on them. I presented evidence for this hypothesis by using model system of Escherichia coli and T4 bacteriophage.

CHAPTER No: 2 THE EFFECT OF DIFFERENT pfu, TEMPERATURE AND pH ON T4 BACTERIOPHAGE INFECTION ACTIVITY AND PRODUCTION:

The bacteriophage rate of interaction and adsorption to its host directly depended on physical-chemical factors such as pH and temperature [118]. Bacteriophages are the most numerous form of life on earth; ten times more numerous than bacteria [123]. They can be found in all environments where bacteria grow [124]. Phages are detected in ground and surface water, soil, food (e.g., sauerkraut, wine), sewage, and sludge [125]. They have also been isolated from humans and animals, for example from feces, urine, saliva, spit, rumen, and serum [126]. Phages are able to penetrate different organs and tissues, including the central nervous system, and are a part of intestinal flora together with their bacterial hosts [127]. They are responsible for 10–80% of total bacterial mortality in aquatic ecosystems and are important factors limiting bacterial populations [56].

Various external physical and chemical factors can inactivate a bacteriophage through damage of its structural elements (head, tail, and envelope), lipid loss, and/or DNA structural changes [128].

2.1 MATERIALS AND METHODS:

2.1.1 Bacteria and Bacteriophage:

For conducting the study, the E. coli BL21 strains and T4 bacteriophage were used. The E. coli BL21 and T4 bacteriophage were obtained from the American Type Culture Collection (ATCC). All bacterial stock cultures prepared/obtained were stored at - 80 0C in Luria-Bertani broth (Oxiod) containing 50% (v/v) glycerol. The frozen cultures were plated onto LB agar (Oxiod) on the need basis. For looking at the effect of different temperature, pH and dilution regimes, first an overnight culture of E. coli BL21 was prepared by inoculating LB broth with a single isolated E. coli BL21 colony from an LB plate and incubating it in a 37°C until the OD600 reached 1. Bacteria and phages were propagated in LB broth.

2.1.2 Culture Media:

LB medium consisted of tryptone (10g), yeast extract (5g) and sodium chloride (10g) per 1,000 (pH 7) [129].

2.1.3 Semi Solid Media:

For phage-plaque formation semi solid medium containing 1.5 and 0.5% agar was used for the upper layer, respectively [130].

2.1.4 Plaque Count Assay:

The purpose of the plaque assay is to grow T4 phage particles within a lawn of bacteria. We can visualize the clearing of bacterial growth on the agar media, demonstrating the effect of T4 phage on E. coli [131]. The progress of infection and production were recorded by plaque count assays to check the infection activity of T4 bacteriophage against Escherichia coli.

2.1.5 Plating of T4 Bacteriophage:

For dilution, temperature and pH experiments 0.2 ml of E. coli (1 OD at 600nm) was taken and added 0.1 ml of T4 bacteriophage. The effects of different parameters were checked by double agar overlay method similar to that of Adams [132]. Experiment was repeated three times to obtained well defined results.

2.1.6 Dilution of T4 Bacteriophage Stock:

For different pfu experiment T4 bacteriophage stock was serially diluted in sterile broth. Aseptically aliquot 900 μl LB broth into one micro centrifuge tube for each dilution that will be made. Label the tubes with the dilution number. Remove 100 μl of the “neat” (undiluted) phage sample and add it to the first dilution tube. Vortex for five seconds to mix. Remove 100 μl from the now-mixed 10-1 tube and add to the 10-2 tube. Vortex for five seconds. Repeat this process until the required numbers of dilutions have been made.

2.1.7 Media pH:

While for the different pH of media we used HCl and NaOH and for temperature experiment plates were incubated in different temperature incubator.

2.1.8 Sterilization of Instruments and Media:

Autoclaves are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards (e.g., heat, steam and pressure) associated with autoclaving, extra care must be taken to ensure their safe use. Sterilization time should not exceed 15-20 minutes at 120°C and 15 psi.

2.1.9 Statistical Analysis:

Statistical analysis included t test for the comparison of change in outcome variables with methods described by sigma stat. The analysis was carried out with Graph Pad Prism 5 software.

Abbildung in dieser Leseprobe nicht enthalten

Figure-3.5A: Schematic E.coli cell growth curve

2.2 RESULT:

2.2.1 Effect of Different pfu of T4 Bacteriophage on its Host Interaction:

Bacteriophage numbers are very important for the phage infection. High concentration of phage is required for infection of its host [132]. It was found that in mixtures containing less than 106 phage “particles" per c.c., no “killing “of E. coli could be detected. However, when the amount of phage was increased tenfold (to 107"particles “per c.c.) it was able to “kill “95 per cent. of the E. coli in the culture; this was true over a range of E. coli concentration from 103 to 107 per c.c.; with thicker E. coli suspensions it was no longer true. E. coli in high concentration required comparatively more concentrated phage for comparable “killing." Undiluted phage containing 5 x 109 “particles” per c.c. sufficed to “kill)" 99 per cent of E. coli in a suspension containing 5 x 108 per c.c. The absolute number of phage “particles” per c.c. seemed thus over a wide range to be more important than the ratio of phage to E. coli [132] . It has been found that lysis of the bacterial host is the final event in the infection cycle of a lytic bacteriophage [133]. The lysis result showed that T4 bacteriophage did lysis from 10-1 to 10-7 pfu while from 10-8 to 10-10 pfu T4 bacteriophage had no infection activity. It means that at highest dilutions, T4 bacteriophage failed to interact to its host as shown in Fig -2.2.1.

Abbildung in dieser Leseprobe nicht enthalten

Figure -2.2.1: Effect of Different pfu of T4 Bacteriophage on its host interaction

Abbildung in dieser Leseprobe nicht enthalten

Figure-3.1: Effect of dilution on T4 phage lysis activity

2.2.2 Effect of Temperature on T4 Bacteriophage and its Host Interaction:

Temperature is a crucial factor for bacteriophage survivability [134]. It plays a fundamental role in attachment, penetration, multiplication, and the length of the latent period (in the case of lysogenic phages). At lower than optimal temperatures, fewer phages genetic materials penetrate into bacterial host cells; therefore, fewer of them can be involved in the multiplication phase. Higher temperatures can prolong the length of the latent stage [135].

At room temperature phage interact bacteria very rapidly but there is a very remarkable temperature dependence of the yield of virus [136]. The temperature sensitivity of the capacity to support phage growth was found more sensitive to temperature than the formation of bacterial colonies [137].

Present study also intended to look at the relationship that exists between T4 bacteriophage and its host at different temperature. For conducting this experiments predator/prey system was used, thereby investigating the potential of environmental factors that effect the evolution of predator and prey populations and communities. In this regard review of literature revealed that a number of studies have been conducted to look at the effect of temperature on the formation of bacteriophage. Among these Luria [138] has commented briefly on the failure of phage production at temperatures at which bacteria are able to grow and divide. Similarly Pollard and Woodyatt [139] found that phage growth was more sensitive to temperature than the formation of bacterial colonies. While looking at the results of these studies, bacteriophage T4, which is a viral predator, was used against its bacterial prey i.e. Escherichia coli to investigate the effects of the thermal environment on the infection activity.

While conducting the study on the T4 bacteriophage system, it was observed that the yield of T4 bacteriophage is highly dependent upon temperature. Study results indicated that low temperature regimes such as 4°C did not permit T4 bacteriophage to develop and perform lysis on E. coli. At temperature regimes 15°C, 25°C and 30°C the T4 bacteriophage did good interaction with its host but the infection activity was little bit delayed manner. Similarly thermophilic temperature of 41°C also permitted the T4 bacteriophage to develop and perform infection on the host bacteria. While, temperature regimes of 45°C, 55°C and 70°C proved as limiting factor and caused the actual inactivation of the bacteriophage. The most important result of the study was the finding that the ideal temperature for T4 bacteriophage to perform bacteriolytic activity against E. coli is 37°C. Results of the study are compiled in Fig- 2.2.2.

Abbildung in dieser Leseprobe nicht enthalten

Figure-2.2.2: Effect of temperature on T4 bacteriophage lysis activity

2.2.3 Effect of pH on T4 Bacteriophage and its Host Interaction:

Study results showed that besides the temperature regimes, the pH of water and media is also a factor that indirectly influences virus survival through affecting virus adsorption to other surfaces and particles. Virus adsorption, at pH levels of natural water, involves diffuse double layer which includes charged surface of virion. Due to this characteristic of charged surface and so called hydrophilic property, viruses have a great surface potential which is very similar to colloidal particles present in aqueous media. In such a situation, the cation concentration as well as medium’s pH affects virus adsorption thus helping in calculating the thickness of diffused layer. This ultimately paves the way for inhibiting and promoting Van der Waals forces of attraction [140].

The effect of pH on the infection of T4 bacteriophage against E. coli was also observed in this study and results revealed that T4 bacteriophage showed a stable growth/development/infection in the pH ranging from 4 to 10. Results compiled in this regard are shown in Fig -2.2.3.

Abbildung in dieser Leseprobe nicht enthalten

Figure-2.2.3: Effect of pH on T4 bacteriophage lysis activity

2.3 DISCUSSION:

Molecular mechanisms of phage infection have been studied quite extensively for the bacteriophage control of bacterial virulence in animals and human [141]. It has been found/concluded that lysis of the bacterial host is the final event in the infection cycle of a lytic bacteriophage [133]. Present study was an effort for looking at effect of dilution, temperature and pH against infection of T4 bacteriophage. Results obtained during the study showed that T4 bacteriophage infection is highly depended on concentration, temperature and pH.

The result showed that T4 bacteriophage did infection from 10-1 to 10-7 pfu while from 10-8 to 10-10 pfu T4 bacteriophage had no infection activity. It means that at highest dilutions, T4 bacteriophage fails to do lysis. Lysis can be produced by phage pfu up to a certain point and it’s reported that at high dilution phage give incomplete lysis [142].

Temperature is one of the most important environmental factor that strongly affects many aspects of the biological systems. One of the important characteristic of the temperature, as environmental factor, is its fluctuation over a wide range of spatial and temporal scales that makes possible as well as limits existence of life in different niches. Influence of temperature upon the biological system is very vivid and it has been observed that evolution of phenotypic traits, species distributions, and extinctions in many cases can be traced to changes in temperature regimes [143]. Present study results are in confirmation with the above findings as during the experiment it was observed that yield of T4 bacteriophage was highly temperature dependent. The T4 bacteriophage was unable to develop and perform lysis on E. coli BL21 at 4°C, while on 15°C, 25°C and 30°C, the activity was carried out but in a little bit delayed manner. These findings of the study are in line with those of D. Herelle [144], who observed that lysis by phage was delayed if the incubation temperature was below 37°C. Similarly, this study showed that at thermophilic temperature 41°C, T4 phage developed and performed infection on his host bacteria and support the results of Pollard and Woodyatt [139], who reported that bacteriophage developed at 41.2°C. Study results regarding the inactivation of T4 phage at temperature regimes 45°C, 55°C and 70°C are in confirmation with those observed by Yates et al., [145] who reported that increase in temperature decreases virus survival and production. In the same way, findings by Pollard and Woodyatt [139], that indicate an increase in phage yield till 39°C corroborates the present study results which revealed that 37°C was ideal temperature for bacteriolytic activity of T4 bacteriophage against E. coli BL21.

Present study findings regarding exertion of indirect influence on the survival of virus by pH of water media through affecting the extent of virus adsorption to other particles and surfaces confirm the results obtained by the Gerba [140]. Similarly the findings of the present study regarding the stability shown by T4 bacteriophage at different pH regimes ranging from 4 to 10 confirm Pollard and Connolly [146], results which indicated that virus exhibited stability at wide range of pH regimes.

CHAPTER No: 3 VARIOUS CULTURE MEDIA EFFECT ON T4 BACTERIOPHAGE INFECTION ACTIVITY AND PRODUCTION:

The bacteriophage rate of interaction and adsorption to its host directly depended on nutrition level and culture condition [118]. Studies about growth and development of bacteriophage played important role in the molecular biological history [108]. The information, particularly with the model species T4 phage was accumulated during the 1940s and this placed the base of the evolving field [147]. Ellis & Delbruck [148], performed classical one-step growth experiment which defined the latent period, rise time and burst size, and the eclipse period. Eclipse period was discovered by the effective procedure planned to disrupt infected bacteria before their lysis to occur spontaneously and the mature phages were not damaged [149].

There is a highly specific binding of phage to one of the cell envelope layers of bacteria which initiates phage-host interaction. Fibers of the phage tail attach to specific receptors present in the bacterial envelope [150]. This process is called as adsorption and depends on various factors such as nutrition level in the environment [151].

Lysis of the bacterial host is the last event in lytic bacteriophage infection cycle [133], but standardization of media is very important factor for phage infection and its production. The ultimate solution to this issue would be to have a synthetic medium which is chemically defined but preserving the sensitivity [152]. The culture media composition is also very important [153] . Apart from the nature of the culture medium, plaque formation also depends on the medium from which the culture was obtained [154]. Infection in liquid media is even more dependent on the cultures history than is solid media dependence of plaque formation [154].

3.1 MATERIALS AND METHODS:

3.1.1 Bacteria and Bacteriohage:

In order to conduct the study, the E. coli BL21 strain was used as the primary host for infection of the bacteriophage named E. coli bacteriophage. An overnight culture of E. coli BL 21 was prepared by inoculating in different broth with a single isolated E. coli BL21 colony from a plate and incubating it in a 37°C.

3.1.2 Culture Media:

3.1.2.1 Luria-Bertani Media:

Luria-Bertani (LB) medium consisted of 10 g tryptone, 5 g yeast extract and 10 g sodium chloride per 1,000 ml of ddH2O ; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 20 min at 120°C.

3.1.2.2 Luria-Bertani Pulse 0.8% Glucose Media:

Luria-Bertani (LBG) pulse 0.8% glucose medium consisted of 10 g tryptone, 5 g yeast extract, 10 g sodium chloride and 0.8% glucose per 1,000 ml of water ; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 20 min at 120°C.

3.1.2.3 Minimal Media:

Minimal Media (MM) contains M9 minimal salts solution (5X concentrate) (64g sodium phosphate, penta-hydrate -- Na2HPO4-7H2O ,15g potassium phosphate (dibasic) -- KH2PO4, 2.5g table salt – NaCl and 5.0g ammonium chloride -- NH4Cl per 1,000 ml of distilled water) 1M solution of magnesium sulfate (MgSO4), 20% solution wt/wt of glucose and 1M solution of calcium chloride (CaCl2) ; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 20 min at 120°C.

3.1.2.4 Nutrient Media:

Nutrient medium (NM) consisted of peptone 5 g, sodium chloride 5 g, beef extract 1.5 g, yeast extract 1.5 g per 1,000 ml of water; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 20 min at 120°C.

3.1.3 Semi Solid Media:

For phage-plaque formation semi solid medium containing 1.5 and 0.5% agar was used for the upper layer, respectively [130].

3.1.4 Plaque Count Assay:

The purpose of the plaque assay is to grow T4 phage particles within a lawn of bacteria. We can visualize the clearing of bacterial growth on the agar media, demonstrating the effect of T4 phage on E. coli [131]. The progress of infection and production were recorded by plaque count assays to check the infection activity of T4 bacteriophage against Escherichia coli.

3.1.5 Plating of T4 Bacteriophage:

For different media experiment LB, LBG, MM and NM were used. 0.2 ml of bacterial culture was taken in 4 ml tube and then 0.1 ml of T4 bacteriop hage was added into suspension. The suspension was then added to the soft agar (different media) and poured onto base plate. Agar tube was rolled between palms to mix for 2 or 3 seconds, and quickly poured onto agar surface of warm base plate. In order to disperse soft agar over the surface of the base plate agar they were gently moved in the pattern of figure eight. Soft agar was allowed to harden and then Incubated at 37°C [132]. Experiment was repeated three times to obtained well defined results.

3.1.6 Sterilization of Instruments and Media:

Autoclaves are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards (e.g., heat, steam and pressure) associated with autoclaving, extra care must be taken to ensure their safe use. Sterilization time should not exceed 15-20 minutes at 120°C and 15 psi.

3.1.7 Statistical Analysis:

Statistical analysis included t test for the comparison of change in outcome variables with methods described by sigma stat. The analysis was carried out with Graph Pad Prism 5 software.

Abbildung in dieser Leseprobe nicht enthalten

Figure-3.5A: Schematic E.coli cell growth curve

3.2 RESULT :

An ideal medium would be one on which maximal bacterial growth occurs and on which the phage has optimal activity. Some factors favoring growth of the bacteria may have a tendency to inhibit phage action and some factors favoring phage activity may tend to limit growth of the bacterial host. A balance may be reached however in which both activities are maintained at a high level [155].

E . coli cells grow faster and larger cells are obtained in richer media, because a higher proportion of their mass is included in the protein-synthesizing system (PSS) [156].

In present study we observed that the maximum growth and infection of T4 phage was on LB and nutrient media. T4 phage production and infection was also good in LB plus glucose media but a little less than LB and nutrient media while in minimal media rate of growth and lysis activity was lowest as compared to other mentioned medium as showed in Fig-3.2.

Abbildung in dieser Leseprobe nicht enthalten

Figure-3.2: Studies about growth and development of bacteriophage played important role in the molecular biological history. In study we checked the effect of LB, LBG, NA and MM on T4 bacteriophage infection and production. The maximum infection of T4 bacteriophage was observed at LB and NA media. p<0.0004

3.3 DISCUSSION:

Most of the previous studies on bacteriophage development have been performed under optimal conditions for the host cell but these conditions may not be optimal for the phage. In nature E. coli faces unfavorable growth conditions such as those prevailing in the human gut. The rate of phage production is proportional to the amount per cell of the PSS at the time of infection and the increased rate of phage production results in larger burst sizes in the bigger cells.

This study characterizes the effects of well-defined physiological conditions on T4 bacteriophage growth and also its interactions with the bacterial host. In the present study we observed that the maximum growth and infection of T4 bacteriophage was on LB and nutrient media. T4 bacteriophage production and infection was also good in LB plus glucose media but a little less than LB and nutrient media while in minimal media rate of growth and lysis activity was lowest as compared to other mentioned medium as Sula and Sulova [152], reported that media can effect phage infection activity and production.

C HAPTER No: 4 EFFECT OF DIFFERENT PHASE ESCHERICHIA COLI ON T4 BACTERIOPHAGE INFECTION ACTIVITY AND PRODUCTION:

The bacteriophage rate of interaction and adsorption to its host directly depended on host physiological state [118]. Escherichia coli is the most extensively studied bacterium in microbiology. However, the majority of E. coli studies are performed using fresh or log phase cultures and may not accurately represent the characteristics of such cells in nature [157]. Studies have shown that at different phase of cell the general biosynthetic processes, metabolism and protein production are not same [158]. These dramatic physiological differences lead to an interest in performing studies on different phase E. coli, as well the relationship between E. coli and T4 bacteriophage.

T4 bacteriophage is a commonly used and highly studied virus that infects E. coli [159]. However, bacteriophage T4 phage is able to adjust its development to the physiological state of the host cell [151]. This adjustment depends not only on the ability of the bacterial cell to produce progeny phage particles and lysis proteins but also on other factors and processes [160]. In particular, the development of bacteriophage T4 is generally longer than the time necessary to form a sufficient number of progeny phages. Interestingly, under conditions of a very slow bacterial growth, T4 phage development may even be stopped [160]. It also stops at the early phases of development if the bacterial host is not growing at all [161]. When host bacteria are starved, T4 phage does not form progeny virions. Instead, its genome can be maintained in the infected host cell until the host’s growth resumes, and then a small burst of progeny phage is produced and the cell is killed by lysis [161].

However, as the physiology of E. coli continuously changes as the culture ages, there is a possibility that infective center production may increase or decrease at different time points so we studied the effect of different phase E. coli effect of T4 phage infection and production.

4.1 MATERIALS AND METHODS:

4.1.1 Bacteria and Bacteriohage:

In order to conduct the study, the E. coli BL21 strain was used as the primary host for lysis activity of the bacteriophage named E. coli bacteriophage (ATCC11303-B4). An overnight culture of E. coli BL 21 was prepared by inoculating LB broth with a single isolated E. coli B21 colony from an LB plate and incubating it in a 37°C. The different phase E. coli was taken according to their phase stages in experiment (Fig-4.1.1)

Abbildung in dieser Leseprobe nicht enthalten

Figure-4.1.1: Growth curve of E. coli

4.1.2 Luria-Bertani Media:

Luria-Bertani (LB) medium consisted of 10 g tryptone, 5 g yeast extract and 10 g sodium chloride per 1,000 ml of ddH2O ; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 20 min at 120°C.

4.1.3 Semi Solid Media:

For phage-plaque formation semi solid medium containing 1.5 and 0.5% agar was used for the upper layer, respectively [130].

4.1.4 Plaque Count Assay:

The purpose of the plaque assay is to grow T4 phage particles within a lawn of bacteria. We can visualize the clearing of bacterial growth on the agar media, demonstrating the effect of T4 phage on E. coli [131]. The progress of lysis and production were recorded by plaque count assays to check the infection activity of T4 bacteriophage against Escherichia coli.

4.1.5 Plating of T4 Bacteriophage:

For different phase of E. coli experiment 0.2 ml of bacterial culture ( different phase of E. coli) was taken in 4 ml tube and then 0.1 ml of T4 bacteriop hage was added into suspension. The suspension was then added to the soft agar and poured onto base plate. Agar tube was rolled between palms to mix for 2 or 3 seconds, and quickly poured onto agar surface of warm base plate. In order to disperse soft agar over the surface of the base plate agar they were gently moved in the pattern of figure eight. Soft agar was allowed to harden and then Incubated at 37°C [132]. This experiment was repeated three times to obtained well defined results.

4.1.6 Sterilization of Instruments and Media:

Autoclaves are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards (e.g., heat, steam and pressure) associated with autoclaving, extra care must be taken to ensure their safe use. Sterilization time should not exceed 15-20 minutes at 120°C and 15 psi.

4.1.7 Statistical analysis:

Statistical analysis included t test for the comparison of change in outcome variables with methods described by sigma stat. The analysis was carried out with Graph Pad Prism 5 software.

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Figure-3.5A: Schematic E.coli cell growth curve

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Figure-3.5A: E. coli Growth Curve

4.2 RESULT:

Bacteriophage studies played a key role in setting the foundations of molecular biology [147]. As a result, phage ranks among the best-characterized organisms at the molecular level. Mathematical models or computer (in silico) simulations can add value to this wealth of phage information by showing how the molecular components and interactions, when taken together, can define developmental processes. In recent years simulations have shown how the physicochemical interactions that govern gene regulation in lambda phage correlate with its lysis-lysogeny decision [162], how the coupling of RNA and replicase production enables rapid takeover of the host during phage Qβ infections [163], and how the intracellular development of phage T7 depends on the organization of its genome [164].

The relative simplicity of phage developmental processes, compared with those of microbes or higher organisms, is balanced in part by the complexity of the resources that they need for growth. Phages require at least nucleic acid precursors, protein precursors, and translation machinery from their hosts. Consequently, phage infection processes depend not only on the physicochemical characteristics of their genome-encoded functions but also on the intracellular resources of their hosts, which depend further on the physiological state of their hosts. Studies spanning 60 years have demonstrated this dependence [151]. Different stages of phage growth, including the attachment of the phage particle to its host, the penetration of phage DNA into its host, and the synthesis of the phage components, have been found to be sensitive to the physiological state of the host, which has been modulated by its growth medium, temperature, oxygen tension, and pretreatment using chemical agents [151].

Production of phages with lowered affinity to bacterial cells, or phages which can bind only to growing bacterial cells seems to be an effective strategy for phages which are constantly endangered by adsorption to cells which are starving or are suspected to be excreted outside of the relatively safe environment of the mammalian gut. The ability of phage T4 to produce a fraction of virions unable to infect starved cells is linked to the functions of genes rI and rIII, as well as rIIA. This may represent the adaptation of phage T4 in order to persist in unfavorable environmental conditions [165]. In our experiments we observed that the maximum growth and lysis of T4 phage was in stationary phase. T4 phage production and lysis was also good in log phase but in lag phase and death phase production and infection activity was less as compared to other mentioned phase as shown in Fig-4.2.

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Figure-4.2: Different phase E. coli effect on T4 bacteriophage infection activity and production

4.3 DISCUSSION:

Earlier work on the bacterium-bacteriophage reaction has stressed the importance of bacteria growth as a condition factor for phage production. Our result showed that E. coli growth to be primer condition factor for T4 phage production and in the present study we observed that the maximum growth and infection activity of T4 bacteriophage was in stationary phase. T4 bacteriophage production and infection activity was also good in log phase but in lag phase and death phase production and infection activity was less as compared to other mentioned phase as Golec et al., [165] reported that bacteriophage is able to adjust its development to the growth parameters of the host cell.

CHAPTER No: 5 LYSIS INHIBITION EFFECT T4 BACTERIOPHAGE PLAQUE BURST SIZE:

There is still a gap in our understanding of the lysis mechanism of T4 bacteriophage despite the level to which T4 bacteriophage is otherwise understood physiologically, genetically and molecularly. On the other hand, the lysis mechanism of phages such as 4X174, MS2 and Lambda has been well understood and characterized genetically molecularly. The reason for which there had been lack of efforts made toward the understanding of T4 phage lysis mechanism over the past 20 years could be that this is a result of its complexity and especially a consequence of the existence of a phenomenon known as lysis inhibition (LIN) [166].

Basically lysis inhibition is a continuation of the latent period of T4 phage infected cell and an amplification of the T4 phage burst size. Lysis inhibition occurs by the adsorption of a second T4 bacteriophage and this lysis inhibition induction requires at least six genes functions which are rI, rIL4, rnIB, rIII, rIV, and rV [167]. So cells, which are infected by T4 phage may lyse at the end of a normal latent period even if lysis inhibition is not induced. This occurs in a way that is most probably similar to that of phage lambda infected cells [166]. However this lysis occurs at the end of a considerably longer lysis inhibition latent period.

T4 phage particles have multiple complications during lytic cycle [166]. In the induction of the lysis inhibition state there is secondary T4 phage adsorption which causes collapse of lysis inhibition. There is loss of plaque-forming ability (secondary trauma) due to multiple T4 phage secondary adsorptions. This association between adsorption and lysis inhibition suggests a mechanism of extracellular induction of lysis inhibition collapse which could be related to lysis from without or secondary trauma. Surprisingly, lysis from without and secondary trauma are distinguishable genetically i.e different T4 phage genes code for the bulk of resistance to each phenomenon, gene imm for resistance to secondary trauma and gene sp for resistance to lysis from without trauma [168].

The study of this inhibition was considered to be the most promising point of attack as lysis inhibition seems to be the cause of the difference in plaque morphology. Our study deals with experiments which are designed to give additional information about the effect of lysis inhibition on T4 phage plaque size.

5.1 MATERIALS AND METHODS:

5.1.1 Bacteria and Bacteriophage:

In order to conduct the study, the E. coli BL21 strain was used as the primary host for lysis activity of the bacteriophage named E. coli bacteriophage. An overnight culture of E. coli BL 21 was prepared by inoculating LB broth with a single isolated E. coli B21 colony from an LB plate and incubating it in a 37°C.

5.1.2 Luria-Bertani Media:

Luria-Bertani (LB) medium consisted of 10 g tryptone, 5 g yeast extract and 10 g sodium chloride per 1,000 ml of ddH2O ; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 20 min at 120°C.

5.1.3 Semi Solid Media:

For phage-plaque formation semi solid medium containing 1.5 and 0.5% agar was used for the upper layer, respectively [130].

5.1.4 Plaque Count Assay:

The purpose of the plaque assay is to grow T4 phage particles within a lawn of bacteria. We can visualize the clearing of bacterial growth on the agar media, demonstrating the effect of T4 phage on E. coli [131]. The progress of lysis and production were recorded by plaque count assays to check the lysis activity of T4 bacteriophage against Escherichia coli.

5.1.5 Plating of T4 Bacteriophage:

For lysis inhibition experiment 0.2 ml of bacterial culture was taken in 4 ml tube and then 0.1 ml of T4 bacteriop hage was added into suspension. The suspension was then added to the soft agar and poured onto base plate. Agar tube was rolled between palms to mix for 2 or 3 seconds, and quickly poured onto agar surface of warm base plate. In order to disperse soft agar over the surface of the base plate agar they were gently moved in the pattern of figure eight. Soft agar was allowed to harden and then Incubated at 37°C [132]. This experiment was repeated three times to obtained well defined results.

We have noticed that particles of T4 phage which get adsorbed secondarily are enough to effect inhibition. We can accomplish that primary and secondary infections must be separated by more than one minute, however, the primary infection could be followed by secondary infection by as little as 4 or 5 minutes.

5.1.6 Sterilization of Instruments and Media:

Autoclaves are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards (e.g., heat, steam and pressure) associated with autoclaving, extra care must be taken to ensure their safe use. Sterilization time should not exceed 15-20 minutes at 120°C and 15 psi.

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Figure-3.5A: E. coli Growth Curve

5.2 RESULT:

The results described the following picture of the actual events occurring under conditions of lysis inhibition. Bacteriophages T4 infect bacteria that reproduce the phage during the latent period, at the end of which lysis begins, first in a few bacteria, but increasing rapidly. Immediately after liberation of the first new phage particles they become reabsorbed on those bacteria which have not lysed. The reactions involved in and following this secondary adsorption of T4 phage delay the lysis of the secondarily infected bacteria.

LIN should be observed all of the time. However, this is not what happens during T4 infection. LIN can only be observed when superinfection takes place; therefore, LIN is the result of superinfection. How superinfection causes LIN at the molecular level is not clear, but it occurs when there is a high concentration of free phage and a low concentration of uninfected cells. At this point, T4 phage has two choices: either undergoes lysis, which produces more free phages, but free phage concentration does not increase after lysis occurs; or undergo lysis inhibition, which does not produce free phages, but accumulates phages intracellularly. From the standpoint of the phage, it is better to increase the number of progeny linearly within the cell rather than releasing progeny in a host-poor environment. Thus, high free phage concentration and low uninfected cell concentration provide a quorum sensing mechanism so that T4 phage can decide whether it is the optimal time to lyse the host.

LIN takes place only when the superinfecting phage is intact; i. e. T4 ghosts do not induce LIN. Thus, the signal for LIN is either the T4 phage genomic DNA or the internal proteins that are carried in the head of superinfecting phages but missing from ghosts. During the primary infection, the phage DNA is injected into the cytoplasm and genetic markers from the primary phage are inherited in the progeny. However, as shown by Anderson et al. [169], DNA of the superinfecting phage ended up in the periplasm of the host, and thus cannot participate in the genetic make-up of the progeny. Gene endA of E. coli encodes periplasmic endonuclease I [170] and degradades DNA that ends up in the periplasm from the injection of superinfecting phage [171]. The fact that genetic markers of superinfecting phage are not inherited in the progeny is called superinfection exclusion. This superinfecting phage DNA can be visualized by electron microscopy coupled with 32P labeling and radiography when the host is endA [169]. The degradation of the DNA injected from the superinfecting phage by EndA is called superinfection breakdown. The length of LIN in endA+or endA-strains is not known, but both strains allow genetic exclusion from the superinfecting phage. It is thought that the presence of Imm protein from the primary infection or expressed from a plasmid alters the E. coli envelope so that the DNA of the superinfecting phage is diverted to the periplasmic [172].

One further point that deserves mention is the fact that there is a decided increase in the number of phage particles liberated per bacterium after lysis inhibition has been effected as shown in Fig-5.2C. . Fig-5.2A showed that the step size is higher when T4 infected bacteria are inhibited than when they are not inhibited. The burst size of plaque is 12mm in secondary infected bacteria as compared to control (6mm) one as showed in Fig-5.2 B.

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Figure-5.2 A: Plaque after lysis inhibition

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Figure-5.2 B: Normal Plaque size

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Figure-5.2 C: Effect of lysis inhibition on burst size. Comparison with control plate

5.3 DICUSSION:

Burst size is extensively affected by cell lysis time as well as delay in cell lysis; this delay occurs due to superinfection and in turn yields much more phages. Lysis time is related to lysozyme synthesis i.e. its synthesis time, rate and effective concentration. These all parameters depend on cell dimensions (volume and surface area). Superinfection maximizes lysis inhibition which in turn increases burst size. Present study findings showed that lysis inhibition increased the plaque size of phage and support the results of Hadas et al. [151], who reported that lysis inhibition delay the lysis time and increased the plaque size of bacteriophage.

C HAPTER No: 6 QUORUM SENSING MOLECULES ACYL-HOMOSERINE LACTONES AND INDOLE EFFECT ON T4 BACTERIOPHAGE INFECTION AND PRODUCTION:

It’s well known that in infection cycle of a lytic bacteriophage, lysis of the bacterial host makes the last event [141], and there are number of different agents which effect the lytic activity of bacteriophage like mitomycin, antibiotics, UV light and some chemicals [173]. There are certain chemical signals in the ecosystem that effect the lytic cycle of phage [174]. It is concluded therefore, that in terrestrial ecosystems some mechanisms do exist. These chemical signaling molecules also known as autoinducers [175], are synthesized and used by bacteria thus they play an important role in quorum sensing. Bacterial population use quorum sensing to coordinate their specific behaviors which are dependent on bacterial local density. Quorum sensing can take place within as well as between different species of bacteria, and thereby serve as a communication network. Common classes of molecules used as signals in quorum sensing are acyl homoserine lactones (AHL) in gram-negative bacteria, oligopeptides in gram-positive bacteria and a family of autoinducers i.e. autoinducer-2 (AI-2) in both gram-positive and negative bacteria [2].

However, quorum sensing has been a burning topic in gram-negative Escherichia coli research for very long period of time. In the recent last 8 years, many researchers have elaborated that E. coli use many quorum-sensing systems [31], and indole is one of the most important inter-species quorum sensing molecule [176]. Indole acts as a signaling molecule and activates the transcription of astD, tnaAB and gabT genes. Among these genes activation of the tnaAB operon induces indole production, while the other two genes i.e. astD and gabT are involved in the degradation of amino acids to pyruvate or succinate. Indole signaling pathway has its role in adaptation of bacteria in a poor nutrient environment, where amino acids degradation is a very important source of energy [177]. But it is worth to note that E. coli do not produce AHL molecules which are commonly found in other gram-negative bacteria and are important signaling molecules. However, E. coli has a receptor which detects AHLs formed by other bacteria thereby changing their expression of genes according to the ubiquity of other "quorate" communities of gram-negative bacteria [178].

This study was inspired by the hypothesis that quorum sensing can be used as a mean of regulating phage-bacterium interactions. Phage's predation pressure is of considerable importance, as phages exceed bacterial cell numbers by 10-fold in many natural environments [120]. As a consequence, bacteria have worked out an extensive range of antiphage mechanisms, which includes ways of bricking the early attachment of phages, phage genome's degradation or abortive infection by host suicide, and thereby arresting the spread of phage progeny in the population to some extent [110]. Phages need a bacterial host to expand and proliferate, they are supposed to be more in number and diverse in heavily populated mixed species than in environments which are sparsely populated. For this reason, the danger of suffering phage attack is brightened at high microbial cell population levels. Thus the key factor in designing the evolutionary advancement between the phage and host is the bacterial phage resistance mechanism [179]. We suppose that if bacteria make use of quorum sensing in order to regulate and govern their antiphage activities, they can enhance their defense mechanisms thus avoiding infection during their growth under high-risk conditions, besides saving the metabolic accountability and maintaining a steadily elevated antiphage strategy. There is another report that quorum sensing effects the infection and phage-bacterium interactions. Reported said that bacterial cell-cell signaling provides clues about the density of potential host cells, phages could benefit from that signals and start attachment to the host cell and release progeny phages specifically when new host cells are abundant. Equally, as the risk of phage infection increases with increased bacterial density, bacteria could be expected to use cell-cell signaling to tie the regulation of their anti-phage defense mechanisms to the cell density of their population [119].

As a first step to test that quorum sensing is used in order to regulate phage-bacterium interactions. We investigated the effect of AHL and Indole on the infection activity of T4 phage against E. coli BL21 (DE3) pLysS. This is the first ever report on the infection activity of T4 phage in the narrated above condition.

6.1 MATERIALS AND METHODS:

6.1.1 Bacteria and Bacteriophage:

In order to conduct the study, the E. coli BL21 (DE3) pLysS strain was used as the primary host for lysis activity of the bacteriophage named E. coli bacteriophage (ATCC11303-B4). The E. coli BL21 (DE3) pLysS was obtained from the American Type Culture Collection (ATCC). All bacterial and phage stock cultures prepared/obtained were stored at -80ºC in Luria-Bertani broth (Oxiod) containing 50% (v/v) glycerol. Phage titer was determined as plaque-forming units (pfu/ml) using the double layer agar plate method.

6.1.2 Luria-Bertani Media:

LB medium containing 10 g tryptone, 5 g yeast extract and 10 g sodium chloride per 1,000 ml of water (pH 7).

6.1.3 Semi Solid Media:

For phage-plaque formation semi solid medium containing 1.5 and 0.5% agar was used for the upper layer, respectively [130].

6.1.4 Chemicals:

N -Butyryl-DL-Homoserine lactone C8H13NO3 (>97%, HPLC) and Indole C8H7N (>99%) were obtained from Sigma–Aldrich (USA). C8H13NO3 (0.1%) concentration working solutions (1,000 mg L−1) of the analytes were prepared in acetonitrile (ACN). The solutions were kept at −20°C and stored for four weeks, while to obtain indole working solution; it was diluted in water to receive a final concentration of 0.5%.

N -Acyl homoserine lactones (AHLs or N-AHLs) are a class of signaling molecules involved in bacterial quorum sensing. N-Butyryl-DL-homoserine lactone is a member of N-acyl-homoserine lactone family (Fig-6.1.4A).

Abbildung in dieser Leseprobe nicht enthalten

Figure-6.1.4A: General chemical structure of an N -acyl homoserine lactone

Indole is an aromatic heterocyclic organic compound. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. Indole is a common component of fragrances and the precursor to many pharmaceuticals. Compounds that contain an indole ring are called indoles. The amino acid tryptophan is an indole derivative and the precursor of the neurotransmitter serotonin (Fig-6.1.4B).

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Figure-6.1.4B: General chemical structure of an indole

6.1.5 Plaque Count Assays:

The progress of lysis and production were recorded by plaque count assays to check the effect of AHL and indole on the lysis activity of T4 bacteriophage (4x109 pfu/ml) against E. coli BL 21(DE3) pLysS (1 OD at 600nm). The effect of AHL and indole on diluted bacteriophage was also checked, for dilution test T4 bacteriophage was diluted in LB broth through a serial dilution. In order to avoid indole production by E. coli, 12 hours incubated E.coli cells in LB media were centrifuged (13000rpm/15min). By centrifugation all the indole produced during incubation in media was removed. The E. coli cells were taken and suspended in fresh LB media broth for experimental use.

6.1.6 Plating of T4 Bacteriophage:

For AHL and indole experiment 20 µl of AHL/indole, was added to 0.2 ml of bacterial culture ( E. coli cells from fresh LB media) and then allowed to adsorb to the E. coli for 3 minutes followed by addition of 0.1 ml of T4 bacteriop hage to suspension. The suspension was then added to the soft agar and poured onto base plate. Agar tube was rolled between palms to mix for 2 or 3 seconds, and quickly poured onto agar surface of warm base plate. In order to disperse soft agar over the surface of the base plate agar they were gently moved in the pattern of figure eight. Soft agar was allowed to harden and then Incubated at 37°C.

6.1.7 Plaque size Determination:

Pictures of plates with T4 bacteriophage plaques were taken using the gel documentation system and diameters of plaques were measured manually.

6.1.8 Sterilization of Instruments and Media:

Autoclaves are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards (e.g., heat, steam and pressure) associated with autoclaving, extra care must be taken to ensure their safe use. Sterilization time should not exceed 15-20 minutes at 120°C and 15 psi.

6.1.9 Statistical Analysis:

Statistical analysis included t test for the comparison of change in outcome variables in response to AHL and Indole with methods described by sigma stat. The analysis was carried out with Graph Pad Prism 5 software.

6.2 RESULT :

6.2.1 Quorum Sensing Molecules AHL Effect on T4 Bacteriophage Infection and Production:

As bacteriophages are obligate parasites, they accomplish one or another gene functions of their host thus exploiting host's gene function for the sake of their own growth [180]. Host's machinery that is RNA and DNA is used by viruses for protein synthesis and as a source of generating energy [181]. Despite this voracious behavior of phages, bacteria have stably coexisted with them for a long period of time and this existence is due to mechanism of quorum sensing [182]. It is also worth to note that prophages can follow the lytic pathway if they are induced so, thus killing the host cell in order to release phage progeny. These prophages have evolved in a way which incorporates sensory inputs into the genetic switches that rules this developmental decision. The prophages are provided with information on the metabolic state of host through induction signals and level of stress of the host cell [183].

In our study, we addressed and tested the relationship of quorum sensing molecules on infection of T4 phage and its production experimentally by using a model predator/prey system. We used bacteriophage T4, a viral predator, and its bacterial prey i.e. E. coli BL21 (DE3) pLysS.

The enterobacteria including E. coli cannot produce AHLs because they do not have LuxI-type AHL synthase, so can only detect and recognize AHLs which are emitted by other bacteria [184]. Present study finding showed that AHL increased the infection activity and production by 115.2% of T4 phage as shown in Fig-6.2.1.

Abbildung in dieser Leseprobe nicht enthalten

Figure-6.2.1: E. coli has a receptor which detects AHLs and due to AHL E. coli changes their genes expression according environment so therefore we checked the effect of AHL on the infection activity of T4 phage and result showed that AHL increased the lysis activity of T4 bacteriophage. Reported differences were evaluated using t test. p<0.0003

6.2.2 Quorum Sensing Molecule Indole Effect on T4 Bacteriophage Infection and Production:

In nature, bacteria normally occur in polymicrobial communities. Interactions between community members typically involve several mechanisms, including responses to antimicrobial compounds, nutritional interactions, and signaling [185]. Chemical signaling is widespread in bacteria, and in E. coli it involves several compounds, including N-acyl derivatives of homoserine lactone (AHLs) and indole [3]. A common diagnostic marker for E. coli identification is indole [186], which is formed by tryptophanase enzyme from tryptophan [187]. Indole can also act as an extracellular signaling molecule [177]. In this study, we have demonstrated that quorum sensing molecule indole can reduce the infection activity and production up to 41.9% of T4 phage as shown in Fig-6.2.2.

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Figure-3.8: Indole signaling pathway has its role in adaptation of bacteria in a poor nutrient environment, where amino acids degradation is a very important source of energy while in our experiment we checked the effect of indole on the lysis activity of T4 phage and result showed that indole decreased the lysis activity of T4 bacteriophage. Reported differences were evaluated using t test. p<0.0005

Abbildung in dieser Leseprobe nicht enthalten

Figure-6.2.2: Indole signaling pathway has its role in adaptation of bacteria in a poor nutrient environment, where amino acids degradation is a very important source of energy while in our experiment we checked the effect of indole on the lysis activity of T4 phage and result showed that indole decreased the lysis activity of T4 bacteriophage. Reported differences were evaluated using t test. (P<0.005)

6.2.3 The AHL and Indole Effect on Diluted T4 Bacteriophage Production and Lysis Activity:

The effect of quorum sensing molecules AHL and indole on diluted T4 phage was also checked and the results showed that AHL increased the lysis activity by 55.8% in diluted T4 phage while indole decreased the lysis activity by 27.8% in diluted T4 phage as shown in Fig-6.2.3.

Abbildung in dieser Leseprobe nicht enthalten

Figure-6.2.3: AHL and indole effect the lysis activity of diluted T4 bacteriophage. The results showed that AHL increased the lysis activity in diluted T4 phage while indole decreased the lysis activity in diluted T4 phage as shown in Fig. (P<0.004)

6.2.4 Quorum Sensing Molecules AHL and Indole Effect on the Plaque Size of T4 Bacteriophage:

The effect of AHL and indole on plaque size was checked and result showed that AHL increased the plaque size by 100% as compared to the control and indole reduced the plaque size as compared to the control as shown in Fig-6.2.4. The plaque size of AHL was 10mm and indole was 5mm while the size of control plaque was 6mm after 16 hours.

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Figure-6.2.4: T4 bacteriophage and E. coli grown in LB medium. AHL and indole effect plaques size of T4 bacteriophage. AHL increased the plaque size and indole reduced the plaque size as compared to the control as shown in Fig.

6.3 DISCUSSION:

Many bacterial species use a intercellular communication mechanism known as quorum sensing [188] , which itself is mediated by tiny diffusible molecules known as autoinducers which are synthesized within the cell and then released into the surrounding environment [189] .

Quorum sensing plays an important role in both symbiotic and pathogenic bacteria-host interactions [190] . In addition, bacteria have evolved to use quorum sensing as a way of regulating their interaction with their prey that is virus which is the most numerous biological entities on earth [191] .

The present study was an effort to look at the effect of quorum sensing molecules which are acyl-homoserine lactones and indole on production and infection activity of T4 bacteriophage. Results obtained during the study showed that acyl-homoserine lactones (AHL), which are essential signaling molecules of quorum sensing in many gram-negative bacteria, can increase T4 phage production and infection against Escherichia coli BL21 (DE3) pLysS as Ghosh et al., [192], reported that acyl-homoserine lactones can induce virus production and interaction with its host.

In addition, our study showed that indole which can also act as a signaling molecule and is a common diagnostic marker for the identification of Escherichia coli reduced the infection activity and production of T4 bacteriophage as Mahyar [193], reported that indole can reduced virus attachment to host.

Our study also showed that AHL increased the lysis activity by 54.4% in diluted bacteriophage while indole decreased the lysis activity by 27. 4% in diluted bacteriophage. The effect of AHL and indole on plaque size was checked and result showed that AHL increased the plaque size by 100% as compared to the control and indole reduced the plaque size as compared to the control. The plaque size of AHL was 10mm and indole was 5mm while the size of control plaque was 6mm after 16 hours.

CHAPTER No: 7 SEPERATION AND PURIFICATION OF TWO NEW QUORUM SENSING MOLECULES W1 AND W2:

Numerous species of bacteria employ a mechanism of intercellular communication known as quorum sensing. This signaling process allows the cells comprising a bacterial colony to coordinate their gene expression in a cell-density dependent manner. Quorum sensing is mediated by small diffusible molecules termed autoinducers that are synthesized intracellularly (throughout the growth of the bacteria) and released into the surrounding milieu. As the number of cells in a bacterial colony increases, so does the extracellular concentration of the autoinducer. Once a threshold concentration is reached (at which point the population is considered to be “quorate”), productive binding of the autoinducer to cognate receptors within the bacterial cells occurs, triggering a signal transduction cascade that results in population-wide changes in gene expression. Thus, quorum sensing enables the cells within a bacterial colony to act cooperatively, facilitating population-dependent adaptive behavior [189] .

Quorum sensing plays an important role in both symbiotic and pathogenic bacteria-host interactions [190] . In addition, bacteria have evolved to use quorum sensing as a way of regulating their interaction with their prey that is virus which is the most numerous biological entities on earth [191] .

7.1 MATERIALS AND METHODS:

7.1.1 Bacteria and Bacteriophage:

In order to conduct the study, the E. coli BL21 strain was used as the primary host for lysis activity of the bacteriophage named E. coli bacteriophage. An overnight culture of E. coli BL 21 was prepared by inoculating LB broth with a single isolated E. coli B21 colony from an LB plate and incubating it in a 37°C.

7.1.2 Minimal Media :

Minimal Media contains M9 minimal salts solution (5X concentrate) (64g sodium phosphate, penta-hydrate -- Na2HPO4-7H2O ,15g potassium phosphate (dibasic) -- KH2PO4, 2.5g table salt – NaCl and 5.0g ammonium chloride -- NH4Cl per 1,000 ml of distilled water) 1M solution of magnesium sulfate (MgSO4), 20% solution wt/wt of glucose and 1M solution of calcium chloride (CaCl2) ; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 20 min at 120°C.

7.1.3 Semi Solid Media:

For phage-plaque formation semi solid medium containing 1.5 and 0.5% agar was used for the upper layer, respectively [130].

7.1.4 Plaque Count Assay:

The purpose of the plaque assay is to grow T4 phage particles within a lawn of bacteria. We can visualize the clearing of bacterial growth on the agar media, demonstrating the effect of T4 phage on E. coli [131]. The progress of lysis and production were recorded by plaque count assays to check the lysis activity of T4 bacteriophage against Escherichia coli.

7.1.5 Plating of T4 Bacteriophage:

For lysis activity experiment 0.2 ml of bacterial culture was taken in 4 ml tube and then 0.1 ml of T4 bacteriop hage was added into suspension. The suspension was then added to the soft agar and poured onto base plate. Agar tube was rolled between palms to mix for 2 or 3 seconds, and quickly poured onto agar surface of warm base plate. In order to disperse soft agar over the surface of the base plate agar they were gently moved in the pattern of figure eight. Soft agar was allowed to harden and then Incubated at 37°C [132]. This experiment was repeated three times to obtained well defined results.

7.1.6 0.1M Tris-Base Buffer:

121.14 x 0.1 x 0.1=1.2114g/100ml

121.14 x 0.1 x 0.5=6.05g/500ml

7.1.7 0.1 M Hcl Buffer:

37% Hcl=12M

3ml Hcl+357ml dd water (360ml 0.1M Hcl)

7.1.8 0.05M Tris-Hcl Buffer (100ml):

Abbildung in dieser Leseprobe nicht enthalten

Autoclave or filter with 0.2um membrane filter and then

7.1.9 0.05M Tris-Hcl with 2M Nacl Buffer (100ml):

Abbildung in dieser Leseprobe nicht enthalten

Autoclave or filter with 0.2um membrane filter and then

7.1.10 20% Ethanol/ Alcohol:

20 ml ethanol/Alcohol + 80 ml dd water. Autoclave or filter with 0.2um membrane filter and then ultrasonication for 30 minutes.

7.1.11 Acetonitrile:

Acetonitrile was supplied from Sigma Aldrich for our research work

7.1.12 Cutt-off membrane (MWCO 9 and 5 KDa):

Designed for fast, non denaturing concentration of biological samples by membrane ultrafiltration. Up to 30-fold concentration of the sample can be achieved with recovery of the target molecule typically exceeding 95%.

7.1.13 HiPrep QFF Column and its Preparation:

HiPrep QFF is strong anion exchange prepacked with Q Sepharose Fast Flow. Ready to used for fast, preparative separation of protein and bimolecular using ion exchange (IEX) chromatography.

Wash away storage solutions and preservatives before using any IEX medium. Use prepacked columns to ensure the best performance and reproducible results. The volume required for the packed bed is determined by the amount of sample to be purified and the binding capacity of the medium. Pack a column that will have approximately 5-fold excess of the binding capacity required with a bed height up to 20 cm.

Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove contaminants. It is recommended to reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire length of the column. The number of column volumes and time required for each cleaning step may vary according to the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be taken when changing a filter as this may affect the column packing and interfere with performance.

The following procedure should be satisfactory to remove common contaminants:

- Wash with at least 2 column volumes (CV) of 2 M NaCl.
- Wash with at least 4 CV of 1 M NaOH (same flow as step 1).
- Wash with at least 2 CV of 2 M NaCl (same flow as step 1).
- Rinse with at least 2 CV of distilled water (same flow as step 1) until the UV-baseline and eluent pH is stable.
- Wash with at least 4 CV of start buffer or storage buffer (same flow as step 1) until pH and conductivity values have reached the required values.

7.1.14 Dialysis Tube:

We used Spectrum Ready-to-Use Devices with ultra-pure Biotech Membrane. Which provide excellent sample recovery without membrane impurities. We used Float-A-Lyzer G2 10 ml (MWCO 0.5 KDa) for rapid dynamic dialysis.

7.1.15 Zorbax semi-preparative C18 (9.4 × 250 mm) column:

The HPLC column was obtained from Agilent Technologies, Palo Alto, CA, USA. This column has following qualities.

- For rapid preparative separations and fast re-equilibration in the 15-50 mg range.
- Finger tight connections supply high pressure seal up to 7,000 psi
- End-fittings are reusable - only the column barrel itself is replaced
- For combinatorial chemistry, medicinal chemistry, or other high-throughput applications
- Complementary scale up from rapid analytical separations
- For fast gradient or fast isocratic separations

7.1.16 Sterilization of Instruments and Media:

Autoclaves are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards (e.g., heat, steam and pressure) associated with autoclaving, extra care must be taken to ensure their safe use. Sterilization time should not exceed 15-20 minutes at 120°C and 15 psi.

7.1.17 Protocol for Purification of W1 and W2 Quorum Sensing Molecules:

30 ml of MM in flask was taken and E. coli were inoculated in it. The culture was incubated at 37 oC/150rpm for 12 hrs (OD 1.8) and then 4ml (109 pfu/ml) T4 bacteriophage was added in flask, after 6 to 8 hrs complete lysis of E. coli was happen. While after complete lysis the solution was ultra filter by using two different molecular weight cut-off (MWCO 9 and 5 kDa) membranes (Yadong Hitech Co. Ltd, Shanghai, China) at 3000g and temperature was kept at 4 °C. The elution which we got after filtration was checked for phage contamination through spot test.

7.1.18 Spot Test:

For spot test we added 0.2 ml (200 μl) of the E. coli BL21 culture into a sterile test tube. Then heat the top agar in a microwave, interrupting and shaking periodically, until it bubbles and no visible solid chunks remain. The top agar was used immediately because if it cools it will begin to re-solidify too early. We used a serological pipette to add 3 ml top agar to tube containing E. coli BL 21. Mix quickly by pipetting up and down. Then Pour/pipet the inoculated agar onto LB agar labeled plate. Immediately but gently swirl the plate in a figure 8 pattern to spread the agar across the surface of the plate. Allow the plates to cool and harden (15-30 minutes). Using a micropipettor, transfer 5, 10 and 20 μl of elution sample onto plate in the appropriate place. Try not to physically touch your pipette tip to the agar, as this will interfere with even lawn growth. Also avoid making bubbles, as these will scatter the sample across plate. As a negative control, spot 5 μl of water in an appropriate place. Allow the liquid from the spots to absorb into the agar, and then invert. Incubate at 37 0C overnight (16-24 hours, or until a visible bacterial lawn grows). The next day, check result. If spot test was positive then do filtrations step again to remove phage from the elution and if was negative then we go for dialysis to remove salt from elution.

7.1.19 Dialysis Process :

The elution solution was passed through semi permeable membrane, such as a cellulose membrane with pores. Molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis tube or bag (MWCO 0.5 KDa), whereas smaller molecules and ions traverse the pores of such a membrane and emerge in the dialysate outside the bag or tube. This technique is useful for removing a salt or other small molecule. For dialyzation process 0.05 M Tris-base (pH 7.5) buffer. The total volume of dialysis buffer should be about 10 -20 times the volume of sample is a good number, but can use 5 times depending on the total volume. We perform 2 to 4 buffer changes. The first buffer change can take place 4 and second 6 hours after starting. Let the third buffer change dialyze overnight. After dialysis process we used elution for AKTA FPLC.

7.1.20 Ion-Exchange Chromatography:

Elution was loaded onto an anion exchange column (2.6 × 50 cm) with a QFF GE-Healthcare equilibrated with Tirs-Hcl buffer (0.05 M, pH 7.5). The column was washed with the same buffer and eluted with a linear gradient of NaCl 2 M at a flow rate of 1 ml/min and monitored at 215 and 280 nm. 3 mL of W1 and W2 fraction were obtain from FPLC and then both fraction were dialyzed to remove all salt from them. After dialysis W1 and W2 fraction were used for HPLC.

7.1.21 High-Performance Liquid Chromatography (HPLC):

The fraction obtained from chromatography was further separated by high performance liquid chromatography (HPLC) on a Zorbax semi-preparative C18 (9.4 × 250 mm) column (Agilent Technologies, Palo Alto, CA, USA), using a linear gradient of acetonitrile containing 0.1% TFA (5–30%, in 30 min) at a flow rate of 1.0 mL/min. The W1 and W2 molecules isolated from HPLC were used for TOF MS/MS.

7.2 RESULT :

7.2.1 Fast Protein Liquid Chromatography (FPLC):

After Ultra filtration and dialysis, Ion-Exchange Chromatography was performed. QFF GE-Healthcare was one of the strong anion exchangers and it was widely utilized for separating peptides. In order to separate quorum sensing molecules ion exchange chromatography was performed and, result showed two peaks, named W1 and W2 (Fig-7.2.1 ).

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.1: W1 and W2 peaks separated by FPLC

7.2.2 High-Performance Liquid Chromatography (HPLC):

The fraction W1 and W2 obtained from chromatography were further separated by high performance liquid chromatography (HPLC) on a Zorbax semi-preparative C18 (9.4 × 250 mm) column. HPLC also separated two peaks W1 and W2 as shown in Fig-7.2.2.

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.2: W1 and W2 peaks separated by HPLC

7.2.3 Time-of-Flight Mass Spectrometry (TOF MS):

For the identification of molecular mass of W1 and W2 TOF MS was performed. The result showed that the molecular mass of W1 is 13056 Da as shown in Fig-7.2.3A and the molecular mass of W2 is 2069 Da as shown in Fig-7.2.3B.

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.3A: W1 molecular mass after TOF MS

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.3B: W1 molecular mass after TOF MS

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.3B: W2 molecular mass after TOF MS

7.2.4 Quorum Sensing Molecules W1 and W2 Effect on T4 Bacteriophage Infection and Production:

We checked the effect of new quorum sensing molecules W1 and W2 on T4 bacteriophage infection activity and production. Result showed that W1 and W2 effect the infection property of T4 bacteriophage and increased its production. For this trail W1/W2 was (200ul, 150ul, 100ul and 50ul) added to 0.2 ml of bacterial culture and then allowed to adsorb to the E. coli for 3 minutes followed by addition of 0.1 ml of T4 bacteriop hage to suspension. The suspension was then added to the soft agar and poured onto base plate. Soft agar was allowed to harden and then Incubated at 37°C. In this study, we have demonstrated that W1 and W2 can increased the lysis activity and production of T4 phage. We used the different concentration of W1 and W2 and results showed that activity reduced when we reduces the concentration of W1 and W2 as shown in Fig-7.2.4.

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.4: Different concentration of W1 and W2 quorum sensing effect on T4 bacteriophage infection and production activity

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.4: Different concentration of W1 and W2 effect of T4 bacteriophage infection activity and production

7.2.5 To check the Effect of Different Condition on W1 and W2 Quorum Sensing Molecules Efficiency:

The effects of -20 oC and liquid nitrogen on W1 and W2 molecules were checked. The W1 and W2 were kept in -20 oC and liquid nitrogen till 1hr after this the lysis activity of both were checked. The results showed that after treating with -20 oC and liquid nitrogen W1 and W2 still has activity as shown in Fig-7.2.5.

The W1 and W2 were treated with equal amount of chloroform for 10 minutes and then lysis activity was checked. The results showed that after treating with chloroform, W1 and W2 still has activity as shown in Fig-7.2.5. While after boiling the lysis activity of W1 and W2 lost as shown in Fig-7.2.5.

Abbildung in dieser Leseprobe nicht enthalten

Figure-7.2.5: Different condition effect on W1 and W2 quorum sensing molecules efficiency

7.3 DISCUSSION:

Quorum sensing is a regulatory mechanism which enables bacteria to make collective decisions with respect to the expression of a specific set of genes. These may include genes involved in biofilm formation and virulence. They play a key role in orchestrating the expression of exoproteases, siderophores, exotoxins and several secondary metabolites, and participate in the development of biofilms.

Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N- acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out new quorum sensing molecules.

In this study, we identified two new quorum sensing molecules due to interaction of T4 phage with E. coli. These molecules have effect on the infection activity and production of T4 bacteriophage. W1 and W2 can increase the lysis activity and production of T4 bacteriophage. In our study we also found that these two molecules are stable when treated with -20 oC, liquid nitrogen and chloroform because their activity still remain but after boiling the activity lost. Our result showed that the molecular mass of W1 is 13056 Da and the molecular mass of W2 is 2069 Da.

CHAPTER No: 8 SUMMERY AND OUTLOOK

Escherichia coli is a member of the coliform group of bacteria which grow and stay alive in open environments such as soils and water. Escherichia coli is debatably the most finely understood and extensively studied free-living organism on our planet. Escherichia coli is an ever-present constituent of all human beings and form a fraction of the normal flora of gut. Escherichia coli is well acknowledged by the shortened name of E. coli.

E. coli is the first choice for researchers to investigate numerous basic biological processes which are essential for life and is the most extensively used organism in molecular genetics. The reason of widespread use of E. coli for study purpose is the ease of its maintenance and breeding in a laboratory environment plus its meticulous experimental advantages. As compared to other living organisms more is known about E. coli because of its simple nutritional requirements, rapid growth rate and most important it’s well established genetics. Rate of cell division of E. coli is average of once in every 30 minutes thus enabling quick environmental adaptation. This fast division rate of E. coli has helped in evolutionary experiments which are conducted in the laboratories.

Bacteriophages are very common in all natural environments and are directly related to the numbers of bacteria present. They are thus very common in soil and have shaped the evolution of bacteria. In order to study many of the essential processes of life, T4 bacteriophage has become a "model organism" due to many reasons. It’s easy to maintain and breed T4 bacteriophage in a laboratory. It is presently the most vastly used organism in molecular genetics due to its rapid adsorption, penetration, DNA replication, formation of new particles, enzyme synthesis and completed genomic sequence.

T4 bacteriophage play key role in molecular biology field in order to identify the chemical nature of the gene, discovering genes code for proteins, deciphering how the genetic code is read and elucidating the mechanism of DNA replication.

The reason of attraction towards T4 bacteriophage as therapeutic agent is that they are lethal and highly specific for targeted bacteria while on the other hand safe for humans. T4 bacteriophages are considered as natural antimicrobial agents to fight against Escherichia coli infections in animals and humans. Moreover research work on Escherichia coli and its T4 bacteriophages played an important part in the revolution of molecular biology. Endolysin or lysins of T4 bacteriophage are used as antimicrobial agents and T4 bacteriophage is used as a particle to deliver vaccine. T4 bacteriophage also play major role in detecting the pathogen.

Molecular mechanisms of T4 phage infection have been studied quite extensively for the bacteriophage control of bacterial virulence in animals and human. It has been found/concluded that lysis of the bacterial host is the final event in the infection cycle of a lytic bacteriophage. The result showed that T4 bacteriophage did infection activity from 10-1 to 10-7 pfu while from 10-8 to 10-10 pfu T4 bacteriophage had no lysis activity. It means that at highest dilutions, T4 bacteriophage fails to do lysis. Lysis can be produced by phage pfu up to a certain point.

Temperature is one of the most important environmental factor that strongly affects many aspects of the biological systems. One of the important characteristic of the temperature, as environmental factor, is its fluctuation over a wide range of spatial and temporal scales that makes possible as well as limits existence of life in different niches. Influence of temperature upon the biological system is very vivid and it has been observed that evolution of phenotypic traits, species distributions, and extinctions in many cases can be traced to changes in temperature regimes. Present study results are in confirmation with the above findings as during the experiment it was observed that yield of T4 bacteriophage was highly temperature dependent. The T4 bacteriophage was unable to develop and perform infection on E. coli BL21 at 4°C, while on 15°C, 25°C and 30°C, the activity was carried out but in a little bit delayed manner. Similarly, this study showed that at thermophilic temperature 41°C, T4 phage developed and performed infection on his host bacteria. During study it was also observed that at temperature regimes of 45°C, 55°C and 70°C, the T4 bacteriophage was completely inactive. Present study results indicated that the ideal temperature for bacteriolytic activity of T4 bacteriophage against E. coli BL21 was 37°C.

During research the pH of media was also found affecting the virus survival indirectly by influencing the extent of virus adsorption to other particles and surfaces. Present study results also indicated that T4 bacteriophage was stable in the pH range from 4 to 10.

Most of the previous studies on bacteriophage development have been performed under optimal conditions for the host cell but these conditions may not be optimal for the phage. In nature, E. coli faces unfavorable growth conditions such as those prevailing in the human gut. The rate of phage production is proportional to the amount per cell of the PSS at the time of infection and the increased rate of phage production resulted in larger burst sizes in the bigger cells.

This study characterizes the effects of well defined physiological conditions on T4 phage growth and also its interactions with the bacterial host. In the present study we observed that the maximum growth and infection activity of T4 phage is on LB and nutrient media. T4 phage production and infection was also good in LB plus glucose media but a little less than LB and nutrient media while in MM media rate of growth and infection activity in lowest as compared to other mentioned medium.

Earlier work on the bacterium-bacteriophage reaction has stressed the importance of bacteria growth as a condition factor for phage production. Our result showed that bacterial growth to be primer condition factor for phage production and in the present study we observed that the maximum growth and infection of T4 phage was in stationary phase. T4 phage production and lysis was also good in log phase but in lag phase and death phase production and infection activity was less as compared to other mentioned phase as.

The cellular content of PSS dictate burst size which is a measure of rate of phage synthesis. Burst size is extensively affected by cell lysis time as well as delay in cell lysis; this delay occurs due to superinfection and in turn yields much more phages. Lysis time is related to lysozyme synthesis i.e. its synthesis time, rate and effective concentration. These all parameters depend on cell dimensions (volume and surface area). Superinfection maximizes lysis inhibition which in turn increases burst size.

Present study findings showed that lysis inhibition increased the plaque size of T4 phage and we know that lysis inhibition delay the lysis time and increased the plaque size of bacteriophage.

Many bacterial species use a intercellular communication mechanism known as quorum sensing, which itself is mediated by tiny diffusible molecules known as autoinducers which are synthesized within the cell and then released into the surrounding environment.

Quorum sensing plays an important role in both symbiotic and pathogenic bacteria-host interactions. In addition, bacteria have evolved to use quorum sensing as a way of regulating their interaction with their prey that is virus which is the most numerous biological entities on earth.

AHLs are the most common class of autoinducer used by Gram-negative bacteria but E. coli do not produce AHL molecule commonly found in other Gram-negative bacteria. The present study findings showed that AHL increased the infection activity and production of T4 bacteriophage. We know that acyl-homoserine lactones can induce virus production.

Indole production is a common diagnostic marker for the identification of Escherichia coli. Indole is formed from tryptophan by the tryptophanase enzyme and indole can act as an extracellular signaling molecule. In this study, we have found that indole can reduce the infection activity and production of T4 phage. We found that indole can reduce virus attachment to host.

Our study also showed that AHL increased the infection activity by 54.4% in diluted bacteriophage while indole decreased the lysis activity by 27. 4% in diluted bacteriophage. The effect of AHL and indole on plaque size was checked and result showed that AHL increased the plaque size by 100% as compared to the control and indole reduced the plaque size as compared to the control. The plaque size of AHL was 10mm and indole was 5mm while the size of control plaque was 6mm after 16 hours.

In this study, we identified two new quorum sensing molecules due to interaction of T4 phage with E. coli. These molecules have effect on the lysis activity of T4 phage. W1 and W2 can increase the lysis activity and production of T4 phage. In our study we also found that these two molecules are stable when treated with -20 oC, liquid nitrogen and chloroform because their activity still remain but after boiling the activity lost. Our result showed that the molecular mass of W1 is 13056 Da and the molecular mass of W2 is 2069 Da.

ACKNOWLEDGEMENT

I am thankful to almighty “ALLAH” who gave me courage and strength to undertake this research and academic qualification.

The author has special debt of gratitude to Mr. Dr Wei Yunlin, Faculty of Life Science and Technology, Kunming University of Science and Technology Yunnan, P.R China, for his enthusiastic interest, priceless assistance and cordial help in conduction and completion of research work and voluminous thesis write up. Thank you Prof Wei Yunlin for giving me the opportunity to attend a variety of conferences and being the first referee of my thesis.

I would like to express my particular gratitude to Xiuling Ji, my working supervisor. She showed me different ways to come up a research problem and the need to be persistent to accomplish any goal. Her insights have strengthened this study significantly. Her broad discussions around my work and interesting explorations in operations have been very helpful for this study. Throughout my thesis writing period, she provided encouragement, good teaching, sound advice, good company, and lots of good ideas.

The author is very much grateful to, Lianbing Lin, Qi Zhang, Wang Nan, Qin Kun Hao and Pan Bo for their cooperation without which it would have not been possible to complete the task.

I also thank all of my past and present lab mates, classmates and chinese friends for their company in study period.

My specially thanks to University of Balochistan for the financial support which gave me the chance to pursuit my PhD study in China.

In the end I express my heart full and deepest affection to my parent’s, wife, brother, sisters, friends and others who always prayed for my success.

Taj Muhammad Kamran ( 太极 )

Kunming University of Science and Technology Yunnan China

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[185]. Russell AB, Hood RD, Bui NK, LeRoux M, Vollmer W and Mougous JD. 2011. Type VI secretion delivers bacteriolytic effectors to target cells. Nature, 475: 343-347.

[186]. Srinivasan S, Aslan A, Xagoraraki I, Alocilja E and Rose JB. 2011. Escherichia coli, enterococci, and Bacteroides thetaiotaomicron q PCR signals through wastewater and septage treatment. Water Research, 45 (8): 2561-2572.

[187]. Pandey R, Swamy KV and Khetmalas MB. 2013. Indole a novel signaling molecule and its applications. Indian Journal of Biotechnology, 12(3): 297-310.

[188]. Fuqua C, MR Parsek and Greenberg EP . 2001. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet, 35: 439-68.

[189]. Galloway WR, Hodgkinson JT, Bowden SD, Welch M and Spring DR. 2011. Quorum sensing in gram-negative bacteria: small-molecule modulation of AHL and AI-2 quorum sensing pathways. Chem Rev, 111: 28-67.

[190]. Boyer M and Wisniewski-Dye F. 2009. Cell-cell signalling in bacteria: not simply a matter of quorum. FEMS Microbiol Ecol, 70(1): 1-19.

[191]. Hoyland-Kroghsbo NM, Maerkedahl RB and Svenningsen SL. 2013. A quorum-sensing-induced bacteriophage defense mechanism. mBio, 4 (1): 00362-12.

[192]. Ghosh D, Roy K, Williamson KE, Srinivasiah S, Wommack KE and Radosevich M. 2009. Acyl homoserine lactones can induce virus production in lysogenic bacteria: an alternative paradigm for prophage induction. Applied and Environmental Microbiology, 75 (22): 7142-52.

[193]. Mahyar N, Ghasem G, Parviz A, Abdolreza T and Shahab S. 2012. Linear and non linear quantitative structure activity relationship models on indole substitution patterns as inhibitors of HIV-1 attachment. Indian J Bioch Bioph, 49: 202-210.

PAPERS DETAILS

Published:

1. Muhammad Kamran Taj, Wei Yunlin, Imran Taj, Taj Muhammad Hassani and Zohra Samreen. 2013. Lysis Inhibition Effect T4 Bacteriophage Burst Size. International Journal of Applied and Natural Sciences (IJANS). 2(4): 91-94. Impact Factor (JCC): 2.4758.
2. Muhammad Kamran Taj, Xiuling Ji, Imran Taj, Taj Muhammad Hassani, Zohra Samreen and Wei Yunlin. 2013. Different Phase Escherichia coli Effect on T4 Bacteriophage Lysis and Production. i nternational Journal of Applied and Natural Sciences (IJANS). 2(5): 73-76. Impact Factor (JCC): 2.4758.
3. Muhammad Kamran Taj, Wei Yunlin, Imran Taj, Taj Muhammad Hassani, Zohra Samreen and Ji Xiu Ling. 2013 . Various Culture Media Effect on T4 Phage Lysis and Production. International Journal of Innovation and Applied Studies. 4(3): 543-546. Impact Factor (GIF): 0.4.
4. Muhammad Kamran Taj, Zohra Samreen, Imran Taj, Taj Muhammad Hassani, Ji Xiu Ling and Wei Yunlin. 2014. T4 Bacteriophage as a Model Organism. International Journal of Research in Applied, Natural and Social Sciences (IMPACT: IJRANSS). 2(2): 19-24. Impact Factor (JCC): 1.0174.
5. Muhammad Kamran Taj, Wei Yunlin, Zohra Samreen, Imran Taj, Taj Muhammad Hassani and Ji Xiu Ling. 2014. Quorum Sensing and its Different Signals System In Bacteria. International Journal of Research in Applied, Natural and Social Sciences (IMPACT: IJRANSS). 2(2): 117-124. Impact Factor (JCC): 1.0174.
6. Muhammad Kamran Taj, Zohra Samreen, Ji Xiu Ling, Imran Taj, Taj Muhammad Hassani and Wei Yunlin. 2014. Escherichia coli as a Model Organism. International Journal of Engineering Research and Science & Technology. 3(2): 1-8.

Accepted:

1. Muhammad Kamran Taj, Lin Lian Bing, Zhang Qi, Ji Xiu Ling, Imran Taj, Taj Muhammad Hassani, Zohra Samreen, Aslam Mangle and Wei Yunlin. 2014. Quorum Sensing Molecules Acyl-Homoserine Lactones and Indole Effect on T4 Bacteriophage Production and Lysis Activity (Pakistan Veterinary Journal Manuscript No: 13-224). Impact Factor (SCI): 1.365.

Under Review:

1. Muhammad Kamran Taj, Ji Xiu Ling, Lin Lian Bing, Zhang Qi, Imran Taj, Taj Muhammad Hassani, Zohra Samreen and Wei Yunlin. Effect of Dilution, Temperature and pH on the Lysis Activity of T4 Bacteriophage. (The Journal of Animal and Plant Sciences Paper ID 13-0612). Impact Factor (SCI) 0.638.
2. Muhammad Kamran Taj and Wei Yunlin. Quorum Sensing Effect the Lysis Mechanism of T4 Bacteriophage. International Journal of Innovation and Applied Studies. Impact Factor (GIF): 0.4.
3. Muhammad Kamran Taj, Xiuling Ji, Lianbing Lin, Qi Zhang, Zohra Samreen and Yunlin Wei. Diversity and Phylogenetic Analysis of Pseudomonas Strains Isolated from Mingyong Glacier China. (Pakistan Veterinary Journal Manuscript No: PVJ-13-463). Impact Factor (SCI): 1.365.

ANNEXURE-1 INSTRUMENTS

Abbildung in dieser Leseprobe nicht enthalten

ANNEXURE-2 COLUMNS AND DIALYSIS TUBLE/BAG

AKTA FPLC column:

Principle of Ion Exchange Chromatography:

Ion exchange (IEX) chromatography can separate molecules or groups of molecules that have only slight differences in charge. Separation is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. Typically, conditions are selected to ensure that the molecules of interest bind to the medium as they are loaded onto the column. Conditions are then altered so that the bound substances are eluted differentially. Elution is most often performed by a continuous gradient or a stepwise increase in ionic strength, most commonly using NaCl. Proteins are built up of many different amino acids containing weak acidic and basic groups (i.e. ionizable groups) that can be titrated. Hence, the net surface charge of a protein is highly pH dependent and will change gradually as the pH of the environment changes. Each protein has its own unique net charge versus pH relationship which can be visualized as a titration curve. This curve reflects how the overall net charge of the protein changes according to the pH of the surroundings. IEX can be repeated at different pH values to separate several proteins which have distinctly different charge properties.

Choice of ion exchanger:

Begin with a strong exchanger (Q, S, SP) to enable development work to be performed over a broad pH range. Use a strong anion exchanger (Q) to bind the protein(s) of interest if their isoelectric point is below pH 7.0 or unknown. Use a strong exchanger in those cases where maximum resolution occurs at an extreme pH and the proteins of interest are stable at that pH. Consider using a weak exchanger (DEAE, ANX, CM) if the selectivity of the strong ion exchanger is unsatisfactory, but remember that the ion exchange capacity of a weak ion exchanger varies with pH. Multimodal ligands (MMC, adhere) provide ionic interaction, hydrogen bonding and hydrophobic interaction. MMC behaves like a weak cation exchanger, but allows binding at high conductivity. Adhere behaves as a strong anion exchanger.

Chromatography media selection:

Select the ion exchange medium according to the objective of the purification step and the condition of the starting material. Other factors such as sample stability, scale, speed, binding capacity and equipment available may also influence the final choice.

Sample preparation:

Correct sample preparation is essential in order to achieve optimal separation and avoid deterioration in column performance. Samples must be clear and free from particulate matter. To remove particulate matter, filter (see Buffer Preparation for filter sizes) or centrifuge (10 000 g for 15 min). Desalt samples and transfer into the chosen start buffer using HiTrap™ Desalting 5 ml (volumes up to 1.5 ml) or HiPrep™ 26/10 Desalting (volumes up to 15 ml). Very small sample volumes in high salt concentration, with no major contaminants, can be diluted with start buffer to lower the salt concentration to a level that does not interfere with binding to the medium.

Column preparation:

Wash away storage solutions and preservatives beforeusing any IEX medium. Use prepacked columns to ensure the best performance and reproducible results. The volume required for the packed bed is determined by the amount of sample to be purified and the binding capacity of the medium. Pack a column that will have approximately 5-fold excess of the binding capacity required with a bed height up to 20 cm.

Column cleaning:

Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove contaminants. It is recommended to reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire length of the column. The number of column volumes and time required for each cleaning step may vary according to the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be taken when changing a filter as this may affect the column packing and interfere with performance.

Removal of common contaminants:

The following procedure should be satisfactory to remove common contaminants:

1. Wash with at least 2 column volumes (CV) of 2 M NaCl.
2. Wash with at least 4 CV of 1 M NaOH (same flow as step 1).
3. Wash with at least 2 CV of 2 M NaCl (same flow as step 1).
4. Rinse with at least 2 CV of distilled water (same flow as step 1) until the UV-baseline and eluent pH are stable.
5. Wash with at least 4 CV of start buffer or storage buffer (same flow as step 1) until pH and conductivity values have reached the required values.

Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems:

- Protein:

0.5 M sodium hydroxide/ 15 minutes

- Nucleic acids:

1 M sodium hydroxide/15 minutes

High pressure liquid chromatography (HPLC) column:

C18 columns from Thermo Electron Corporation provide a versatile C18 phase for a wide range of application areas. C18 columns combine a C18 phase with a hydrophillic end-capping to offer a unique material for reversed phase chromatography, offering alternative selectivity, up to twice the retention for polar compounds, and no phase collapse under 100% aqueous conditions. These columns maintain selectivity with reduced concentrations of buffers and additives, making them ideal for use with LC/MS. In this Technical Guide we review the C18 packing, which has been tailored to go beyond the limitations of traditional C18 packing materials.

- Exhibits different retention & selectivity than conventional C18
- Excellent peak shapes for basic, acidic and neutral compounds
- Retains polar molecules twice as strongly as conventional C18
- Compatible with 100% aqueous mobile phase
- Excellent results with low buffer concentrations
- Stable for LC/MS application

Chromatographic Characterization:

Packings that offer additional modes of interaction give rise to quite different retention behavior and selectivity. In general, analytes with the greatest polar functionality will typically show the greatest changes in selectivity and retention. The difference in behavior between the C18 column and the BetaBasic™ 18 column (a highly base deactivated and densely bonded C18 column that has a very similar percent carbon value). where analyte interactions are based purely on hydrophobic (or dispersive) interactions, the C18 column is slightly less retentive than the BetaBasic 18 column.The C18 packing was designed for the reversed phase separation of polar molecules. Despite its relatively high concentration of C18 groups, it also has hydrophilic sites that help to provide increased retention of highly polar water soluble compounds. The C18 column offers the nearly twice the retention for several polar, basic compounds when compared to a BetaBasic 18 column. The retention of basic compounds and also polar acidic compounds on the C18 column are both significantly increased compared to the BetaBasic 18 column. This illustrates clearly how useful the C18 column can be when increased retention of polar compounds or alternative selectivity is required.

Increased Retention of Polar Compounds:

Polar compounds often elute near or at the unretained marker when run on typical C18 HPLC columns. C18 columns provide additional analyte-ligand interactions to reversed phase hydrophobic interactions, leading to increased retention of analytes with polar functionality. C18 columns maintain retention of neutral compounds while offering increased retention for both acidic and basic compounds.

Applications:

- Highly polar compounds
- Nucleosides and Nucleotides
- Organic acids
- Vitamins
- Peptides
- Catecholamines

Chromatographic Interactions:

Dispersive interactions are the primary interactions generally associated with retention on traditional alkyl C18 type packings. Secondary interactions associated with residual silanols have been significantly reduced by end-capping, improvements in silica quality and increased density of the derivatized ligand. Silanol interactions that previously gave rise to broad tailing peaks for basic analytes have therefore, to some extent, been eliminated. These secondary interactions are also responsible in part for the retention of compounds with polar functionality, either by hydrogen bonding interactions or via ion exchange interactions.The progressive elimination of the secondary silanol interactions has resulted in columns that give good peak shape for basic compounds but reduced retention of polar compounds in general. C18 columns provide an excellent combination of traditional reversed phase interactions and polar interactions to retain more polar analytes.

Highly Aqueous Mobile Phases:

The inclusion of polar functionality to the stationary phase also increases the wetting characteristics of the packing in highly aqueous mobile phases. The C18 column can be run in 100% aqueous mobile phase conditions and shows no tendency towards phase collapse (Figures 3 and 4). Phase collapse is often seen with C18 packings unless a small amount of organic solvent (1-5%) is added to the mobile phase. As a result of phase collapse, the retention and selectivity of the phase is lost and the column must be regenerated using a pure organic solvent wash. The C18 packing is immune to this folding due its unique polar functionality.

Spectra/Por® Float-A-Lyzer® G2 Tube for Dialysis:

Spectrum introduces the Spectra/Por® Float-A-Lyzer® G2, the next generation in Ready-to-Use laboratory dialysis devices featuring proprietary Ultra-pure Biotech Cellulose Ester (CE) Membrane.

- 95 - 98% Sample Recovery
- Highest Membrane Purity
- Superior Handling & Leak Prevention
- Volume Specific Dilution Control
- Complete & Ready-to-Use
- Perfect for dialyzing electro-eluted proteins or chromatography fractions
- Sample Recovery, Purity and Dilution Control:

Spectrum's proprietary Biotech Grade CE is a low protein-binding synthetic membrane available in 9 precise MWCO's with no heavy metal and sulfide contaminants. The cylindrical tubing geometry prevents sample dilution (associated with cassette-type devices) and provides open access for total volume retrieval by pipette. Only the Float-A-Lyzer G2 assures a 95-98% sample recovery while maintaining 99% sample purity and <1% sample dilution.

Designed for Ease-of-Use and Leak Prevention:

The leak-proof screw-on cap with sealing o-ring provides easy access with included pipette for loading, in-process testing and sample retrieval, without the risk of needle punctures. The included floatation ring improves sample buoyancy and vertical orientation during dialysis. The sleek design allows multiple samples to be dialyzed in the same reservoir.

No additional kits, buoys or accessories need to purchased separately. The Float-A-Lyzer G2 is packaged dry and ready-to-go with all components included.

- Rinse with water
- Load sample with Pipette
- And dialyze

Features:

Abbildung in dieser Leseprobe nicht enthalten

Dimensions:

Abbildung in dieser Leseprobe nicht enthalten

ANNEXURE-3 FPLC AND HPLC PROCEDURE

Fast Performance Liquid Chromatography operating procedure (FPLC):

Experiment procedure:

Pre-run checklist:

1. Check that the main waste bottle is empty (lower refrigerator chamber).
2. Check that the pump waste bottle is empty (placed at the left side of the FPLC, with two waste tubes).
3. Check that the fraction collector base unit status light is green colored and that the carousel is positioned securely in it’s place.
4. Check that there are enough clean tubes in fraction rack, with the “A” row filled (the system’s default choice). PC monitor checklist:

Preparing the system for a run:

Note: In order to simultaneous execute several commands at the control panel, press “insert” and at the end of the command listing press “execute”.

1. Switch on PC and initiate the “Unicorn” software package (see desktop icon); four windows will open.

a. Login info ⇒ User: default

2. In the Unicorn software, prompt the system control window; Go to Manual in the menu items (or press Ctrl+M).

3. PC monitor checklist:

a. Choose Flowpath:

- Check that “injection valve” is set to load
- Column position ⇒ check that it is set at “Position1bypass”
- Buffer ValveA1 ⇒ Check that the pump inlet is set to A11
- Pump inlet ⇒ A1

b. Choose “Pump” and check the following before continuing:

- Flow rate ⇒ should be set to the appropriate flow rate of the column (consult column’s accompanying leaflet for the recommended flow rate).
- Gradient ⇒ Check that inlet %B is set to “0”.
- Alarm & monitor ⇒ set alarm pressure to 0.25MPa.

4. Wash system’s A1 pump:

a. Go to “pump”⇒”PumpWashExplorer”, Choose “Inlet A11”. In this automatic program, the pump is washed for 4 minutes with MQ pumped from inlet A11.

5. If pump B1 is to be used, wash pump B1 with MQ (wash and transfer tubing B11 to MQ bottle).

6. Wash B11 tubing with it’s appropriate buffer (if it’s MonoQ it is usually high salt buffer).

7. (Optional: if lysate or large volume of protein solution is to be used wash inlet A18 tubing):

a. Verify that the system is at standby (check to see that flow rate is “0.00”).
b. Rinse A18 tubing end thoroughly with MQ and place in 50ml flacon with 30ml of freshly filled MQ.
c. Change flow path⇒”BufferValaveA1” ⇒ “A18”
d. Wash tubing with 20ml of MQ at flow rate ⇒ 10ml/min
e. Set flow rate ⇒ 0ml/min
f. Fill falcon tube with 30ml of protein’s buffer
g. Wash tubing with 20ml of protein’s buffer at flow rate ⇒ 10ml/min
h. Set flow rate ⇒ 0ml/min
i. Port A18 is ready for injection.

8. Wash inlet A12 tubing with MQ:

a. Verify that the system is at standby (check to see that flow rate is “0.00”).
b. Rinse A12 tubing end thoroughly with MQ and place in MQ bottle
c. Change flow path⇒”BufferValaveA1” ⇒ “A12”
d. Set flow rate ⇒ 10ml/min
e. Wash in 20ml of MQ

9. (optional) Preparation of loop for small volume injection (up to 5ml):

a. Connect loop to injection valve via ports 2 and 6
b. Wash loop with MQ - Fill 10ml syringe with MQ, connect syringe to the injection port (screw-type) and pump the MQ while monitoring the outlet tubing drizzle (if it is not constant there might be air bubbles in the system; continue pumping till drizzle is contant)
c. Repeat wash with protein’s buffer (according to the column used).
d. Injection valve is ready for run.
10. Connect column:

a. Remove plastic capping from the column ends and mount column on column arms.

i. If it is a small column, it is possible to connect the column directly to the pump valve#3 at any available position.

b. Connect column’s lower part to pump valve#3 (upper left module) via tubing on position# 8

c. Connect the column’s upper port to pump valve#2 via position#8 in a drop-to-drop style (this step ensures that no air bubbles will enter the column):

i. Go to Flow path ⇒ “column position” ⇒ “A18”
ii. Check Flow path ⇒ “BufferValaveA1” ⇒ Choose appropriate tubing
iii. Go to Flow rate ⇒ 0.5ml/min1 (make sure the system is at “Run” mode)

d. The inlet tube edge should start to drizzle – fill the tubing socket with liquid and only then screw in the connecter while pressing down.

e. Monitor the curve on screen and see that the curves stabilize after several minutes.

b. Set flow rate ⇒ 0ml/min

11. Wash column with MQ:

a. Verify that the system is at standby (check to see that flow rate is “0.00”).
b. Check A12 tubing is placed in MQ bottle
c. Set flow rate ⇒ 0.5ml/min
d. Wash column in one column volume
e. Set flow rate ⇒ 0ml/min

12. Perform pump wash with appropriate buffer:

a. Go to “pump”⇒”PumpWashExplorer”, Choose the appropriate buffer inlet (usually A12).

13. Wash column with protein’s buffer2:

a. Verify that the system is at standby (check to see that flow rate is “0.00”).
b. Transfer A12 tubing to buffer’s bottle
c. Set flow rate ⇒ 0.5ml/min
d. Wash column with two column volume
e. Column is ready for sample run.

High pressure liquid chromatography (HPLC):

Section 1 - Personal Protective Equipment:

1. Lab Gown/lab coat
2. Proper enclosed footwear
3. Gloves when handling solvents
4. Safety glasses

Section 2 – Potential Hazards + Safety precautions:

1. Solvents used as mobile phases (eg. methanol, acetonitrile, hexane) are TOXIC. Wear personal protective equipment (lab coat, enclosed shoes, gloves, safety glasses/goggles). Do not allow skin to come into contact with HPLC solvents and do not breathe the fumes of these solvents.
2. Solvents used as mobile phases (eg. methanol, acetonitrile, hexane) are FLAMMABLE. Ensure solvent reservoirs and waste container are gas-tight, and do not allow vapours of solvents to enter the lab. HPLC waste vessels should be located in a fume hood, or should exhaust into a fume hood.
3. Liquids under pressure could cause eye injury if a sudden leak occurs. Electrical faults could lead to electrocution or fire. Ensure the HPLC is routinely serviced / inspected by a qualified individual at regular intervals. If any electrical or mechanical faults are suspected, stop using the machine, and get it serviced immediately.
4. Samples being analysed may be TOXIC (eg. pollutants, metabolic inhibitors etc) or/and BIOHAZARDS (derived from pathogenic microbes, clinical samples, recombinant organisms). Treat both HPLC solvents and the analysis samples with care, and be aware of the hazards associated with them. (eg. toxic/biohazard/flammable).
5. Read and understand the risk assessments and SOPs for flammables and toxics before starting – the most commonly used HLPC solvents (methanol and acetonitrile) fall into both these categories.

Section 3 – Procedure:

1. Know the location of spill kits, eyewashes, safety showers, fire extinguishers and fire blankets before starting work.
2. Do not attempt to use the equipment till you have read/completed risk assessment sheets Part A and Part B and read this SOP.
3. Do not operate the HPLC machine till you have been given training on the equipment and the software that controls the machine.
4. Do not turn the HPLC pump till all mobile phase containers to be used are filled with appropriate mobile phases.
5. Ensure that your mobile phase is compatible with the HPLC column.
6. Ensure that your mobile phase is compatible with the previously used mobile phase, or wash the column with eg. water first. If you run 100% methanol or 100% acetonitrile into a column containing a buffer with a salt dissolved in it, this can precipitate the salt and damage the column.
7. Ensure that the pump lines have been purged of air bubbles and check for leaks of mobile phase in the HPLC fluid lines.
8. Ensure that there is no fluctuation in the pressure and that the pressure is well below the maximum pressure for the HPLC system.
9. Ensure that the waste solvent flows properly into the waste solvent bottle/drum.
10. Waste solvent bottle/drum should be fitted with a filter that allows air to escape, but not solvent vapours, eg. granular activated carbon in a plastic column.
11. Do not let the mobile phase bottles run dry.
12. Keep hands clear of the injection system – the needle could cause injury.
13. If doing manual injections, be aware of the hazards of using sharps (see Sharps risk assessment / SOP) .
14. Wash HPLC column for at least half an hour before and after the HPLC analysis run with 50% methanol (or other appropriate mobile phase for cleaning the column).

ANNEXURE-4 STERILIZATION OF INSTRUMENTS

1) Autoclave all glassware before washing, at 120°C, 15psi for 20min.
2) The outside surface of glassware can be cleaned with Vim and inside with only Teepol and scrubbed with a clean brush , which is only meant for brushing glassware. Wash in tap water and leave in 5% Teepol solution overnight (can be boiled)
3) Next morning wash in tap water at least 20 times and leave in 10% HC1 overnight.
4) Next day wash in tap water and rinse in 2 changes of demineralised distilled water at least 10 times in each, leave in third bucket of demineralised distilled water overnight.
5) Take out next day, dry it in hot air oven, pack and sterilize.

Packing and sterilization

Pipettes: Wrap the pipette with brown paper and sterilize in a hot air oven at 160°C for two hours.

Petridishes : Wrap the Petri dishes in brown paper and tie with

Twine and sterilize by hot air oven at 160°C for two hours.

Culture & media storage bottles, measuring cylinders and beakers :

Cover mouth with aluminum foil, over which brown paper is tied with twine at the neck and sterilize in a hot oven at 160°C. Plastic measuring cylinders and beakers are autoclaved at 120°C, psi for 30min.

Plastic centrifuge tubes, Eppendorf tubes, screw cap vials & tubes:

Arrange neatly the following items in a plastic or glass beaker and cover the mouth with aluminum foil and over that wrap brown paper tied with twine at the neck.

Sterilize by autoclaving at 120°C, 15 psi for 30min.

Coverslips: Coverslips are put into a Petri dish , which is covered with brown paper and tied with twine. Sterilize in a hot air oven at 160°C for two hours.

Filter Apparatus: Wrap filter apparatus first with aluminum foil and then with brown paper, tie it with twine tightly. Sterilize by autoclaving at 120° C, 15 psi for 30min.

Solutions: Plug the mouth of the container with cotton, wrap it with brown paper and tie it with twine. Sterilize by autoclaving at 121° C , 15 psi for 20 min.

Autoclaves are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards (e.g., heat, steam and pressure) associated with autoclaving, extra care must be taken to ensure their safe use. Sterilization time should not exceed 15-20 minutes at 121°C (250°F) and 15 psi.

ANNEXURE-5 RECIPES OF MEDIA

Luria Broth (LB) and Luria Agar (LA) Media:

History:

LB is a widely used bacterial culture medium today but it has its origins in the field ofbacteriophage genetics. Guiseppi Bertani created the LB recipe while trying to optimize plaque formation on a Shigella indicator strain (Bertani, 1952). According to Bertani, LB has been variously misconstrued to stand for "Luria Broth", "Luria-Bertani" medium, and "Lennox Broth"; however, the acronym originally stood for "Lysogeny Broth" (Bertani, 2004). The agar form of the medium should be designated LA but it is often referred to as LB. Although originally developed for bacteriophage studies and Shigella growth, LB subsequently became the medium of choice for growth of Escherichia coli and other related enteric species.

Purposes:

LB medium is a rich medium that is commonly used to culture members of the Enterobacteriaceae as well as for coliphage plaque assays. LB and related media (SOC, Terrific Broth, 2xYT, etc) are used extensively in recombinant DNA work and other molecular biology procedures. Often an antibiotic is added to the sterilized medium to select for cells that contain a specific genetic element such as a plasmid, a transposon, or a gene disruption via an antibiotic resistance cassette. X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) may be added to sterile medium when using the blue-white screen for plasmids bearing the alpha fragment of the beta-galactosidase gene (a-complementation analysis). IPTG (isopropyl-beta-D-thiogalactopyranoside) is sometimes added to induce expression of genes controlled by the lac promoter. In addition to (or perhaps because of) its prominent position in the molecular biology field, LB has also been used as a general-purpose bacterial culture medium for a variety of facultative organisms. In the undergraduate microbiology teaching labs, LB is sometimes used as the growth surface when attempting to analyze bacterial colony morphology (See LB agar medium images).

RECIPE:

Many, slightly different recipes for LB exist. LB (broth and agar formulation) is also commercially available in a premixed form.

Standard Recipe for 1 Liter of LB:

(Sambrook and Russell, 2001 Gerhardt, et al. 1994).

Tryptone 10 g

Yeast Extract 5 g

NaCl 10 g

Dissolve components in 1 liter of distilled or deionized water.

For LB agar* add agar to a final concentration of 1.5%.

Heat the mixture to boiling to dissolve agar and sterilize by autoclaving at 15 psi, from 121-124°C for 15 minutes.

* Strictly speaking, LB agar should be called LA.

LB Variation:

Variations of the LB recipe can be found in the literature and lab manuals. One variation of LB ("Lennox Broth"), calls for the use of 5 grams per liter of NaCl (Lennox, 1955; Gerhardt, et al. 1994). In some research studies, a diluted version of LB (0.1% LB) has been used to culture environmental microbe isolates (Lee, 2000). To isolate marine microbes such as Vibrio spp., some researchers have used 30 grams per liter of NaCl (Nandi, et al., 2000). Glucose is sometimes added to a final concentration of 1 to 2 grams per liter of medium. Many recipes call for the use of a concentrated NaOH solution (~1-5 M) to adjust the pH to 7.0 prior to autoclaving. To maximize growth, especially when a carbon source such as glucose is added, phosphate buffer or Tris-HCl buffer may be added to maintain the pH. If the medium is to be used for bacteriophage growth, a sterile stock solution of CaCl2 is often added to a final concentration of 2.5 x10-3 M after autoclaving. Top agar (0.75 % agar plus the other LB components) is routinely used for plating of bacteriophage. The use of top agar facilitates diffusion of phage particles.

PROTOCOLS:

Streak Plate for Elucidation of Colony Morphologies:

Streak a plate of LB agar with a pure or mixed bacterial culture. Incubate inverted plate at organism's ideal temperature for 24 to 48 hours. Observe colony morphology. For more information on colony morphology, see Description of Colonial Morphology of Microorganisms.

Plating of Bacteriophage:

See Plaque Assay entry.

Safety:

The ASM advocates that students must successfully demonstrate the ability to explain and practice safe laboratory techniques. For more information, read the laboratory safety section of the ASM Curriculum Recommendations: Introductory Course in Microbiology and the Guidelines for Biosafety in Teaching Laboratories.

Comments and Tips:

This section is to evolve as feedback on the protocol is discussed at ASMCUE. Please contact the project manager for further information.

Nutrient Media

Microorganisms can only be determined precisely subsequent to being cultured in a pure culture medium. The precondition for this is optimal nutrient media. Correct selection of the nutrient medium but also its quality is determined to a large extent by the quality of the culture. The quality of a particular medium is largely determined by its batch constancy which is the basis for reproducible results.

Composition of nutrient media:

The most important components of the nutrient medium are carbohydrates as a source of carbon, peptones as a source of nitrogen, minerals as enzyme activators and for maintaining osmotic pressure and growth factors (essential amino acids, vitamins etc.). Solid media are prepared by adding 1.5 to 2 % of agar-agar, an extract from algae of the species gelidium, or 10 to 15 % gelatine.

As each microorganism has its own optimum pH range, buffer systems, mostly phosphate buffers, are normally added. This has the effect of buffering acids resulting from the degradation of carbohydrates.

Types of nutrient medium:

Nutrient media can be differentiated according to the components involved, their consistency and their function.

Components:

One can differentiate between media containing natural biological components and those that are categorised as either semi- or fully synthetic. As biological materials are always subject to a certain degree of fluctuation and which are undesired in the case of certain determinations (e.g. sensitivity testing), media have been developed consisting either partially or wholly of precisely defined chemical substances.

Consistency:

One can differentiate between solid media usually containing 1.5 to 2 % agar, semi-solid media containing less than 1 % (e.g. for mobility testing) and nutrient broths.

Manufacture of nutrient media:

Dissolving:

Almost all known nutrient media are available in a premixed and tested form and supplied by commercial companies. Ready-to-use solutions, concentrates or preprepared mixtures of powders are available. Dry media are “dehydrated“ products and may be strongly hygroscopic. They are dissolved in demineralised or distilled water at a pH of 7. Dehydrated products are those from which water has been removed during the manufacturing process. Dry nutrient media must be closed in their original packages and stored air-tight. In preparing such media, the instructions provided by the manufacturer should be followed precisely. The required quantity of dry substance should be suspended in the prescribed volume of water and allowed to stand for approximately 15 minutes so that the agar/gelatine mixture can swell. To complete the solution, the suspension should be stirred using a magnetic stirrer, warmed and subsequently sterilised. This is normally done by autoclaving for 15 minutes at 121 °C. Heat-sensitive media should be sterilised using steam. Ready-to-use nutrient media are suitable for use in small laboratories; they are available commercially and can be called up from the supplier at regular intervals as and when required. The expiry date should be kept in mind when storing.

Minimal Media

Minimal Media contains the essentials for bacterial species to grow. The media is often used to define if a particular microbial species is a heterotroph, namely an organism that does not have any nutritional requirements beyond core sources of carbon (sugars) and nitrogen (to synthesize amino acids and nucleic acid bases). Auxotrophs or organisms with nutritional requirements will not be able to grow on minimal media.

Important components are:

- A source of carbon, commonly the sugar glucose (C6H12O6).
- A source of nitrogen, needed to synthesize amino acids for proteins and bases for nucleic acids. Ammonium chloride is commonly used (NH4Cl), particularly given that the majority of bacterial species cannot fix nitrogen (convert atmospheric N2 into biologically usable ammonia or amine groups).
- Ions/electrolytes - like us, bacterial cells need core nutrients such as sodium, chloride, potassium, calcium, phosphorus and magnesium. These components are provided in the "M9 salts" solution

Reagents/Materials:

1. Erlenmeyer flask of volume at 2X for amount of media to be made (i.e. 1L flask to make 500mL)

2. M9 minimal salts solution (5X concentrate)

- To 800mL of distilled/deionized water add:
- 64g sodium phosphate, penta-hydrate -- Na2HPO4-7H2O
- 15g potassium phosphate (dibasic) -- KH2PO4
- 2.5g table salt -- NaCl
- 5.0g ammonium chloride -- NH4Cl
- Stir until dissolved.
- Adjust volume to 1000mL (1L).
- Sterilze by autoclaving or filter sterilization if autoclave is not available.

3. Distilled water

4. 1M solution of magnesium sulfate (MgSO4).

5. 20% solution wt/wt of glucose (20g gulcose in 80g of water). Add powdered glucose (also called "dextrose") to water solution slowly while mixing, else the glucose will clump and not easily dissolve. Filter sterilize when finished - do not autoclave as the heat will carmelize the sugar.

6. 1M solution of calcium chloride (CaCl2)

7. To make solid media, include agar base and sterile Petrie plates.

Method

- To prepare 1L of media, add:
- 200mL 5X M9 salts solution
- to ~800mL of distilled water
- Add 15g of agar media if agar plates are to be poured.

After autoclaving, swirl to mix evenly and "temper" at room temperature until you can place your hand on the flask for 2 second (an adult should do this!), then add the following:

- 2mL of 1M MgSO4 solution
- 0.1mL of 1M CaCl2 solution
- 20mL of 20% glucose

Swirl to mix evenly.

Liquid media should be distributed to sterile bottles or tubes with a pipette and pipettor. Small bottles can hold 250mL of media for use when needed, for instance, or aliquots of 5mL or 10mL can be transferred to sterile, capped test tubes.

If plates are to be poured, 25mL of media should be added per Petrie dish and allowed to solidify at room temperature. Poured/solid plates can be stacked and replaced in the original plastic sleeve in which they came. Label the sleeve with the type of media and date poured.