Microbal Degradation of Tauropine. An investigation

Table of Contents

Introduction
Opines
The degradation pathway of tauropine in marine invertebrates is well known
The metabolism of tauropine in microorganisms is not yet clarified

Results and Discussion
Dissimilation pathway of tauropine
Detection of the metabolites of tauropine degradation
Investigation of enzymes involved in the tauropine dissimilation pathway
Identification of the tauropine dehydrogenase in one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis
Synthesis of tauropine4,15
Isolation and identification of potential tauropine-degrading strains
Screening for antibacterial and antifungal activity of Ralstonia solanacearum
Summary and Outlook

Methods
Synthesis, purification, identification, and quantification of tauropine
Isolation and cultivation of tauropine-degrading strains from soil
16S rDNA analysis of the model organisms
Screening for antibacterial and antifungal activity
Growth curves
Preparation of cell-free extract
Enzymatic activity tests for clarification of the tauropine degradation pathway
Identification of the tauropine dehydrogenase in one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Appendix
Nucleotide sequences
NMR spectra of tauropine

Introduction

Opines

Tauropine, which is a C5-amino sulfonate (Figure 1), belongs to the group of opines. Opines are molecules, that are typically formed in a reductive condensation reaction of an α-keto acid with an L-amino acid.

The first opine to be isolated was D-octopine, which was found in sepia in 19271. D-octopine probably serves as functional analog of lactate for fast energy supply in cephalopods2.

Opines in the past have mainly been known to play a role in marine animals, such as in slugs, shells, and worms. Under anoxic circumstances, these phylogenically lower invertebrates would exceedingly use an opine dehydrogenase system than the lactate dehydrogenase in anaerobic glycolysis3. As this reaction is essential for adjustment of redox levels in cells, opines occur as end products in the anaerobic metabolism.

The degradation pathway of tauropine in marine invertebrates is well known

The product of the reaction of pyruvate and taurine has been called tauropine. Equivalent names for tauropine are D-rhodoic acid or N -(D-1-Carboxyethyl)-taurine.

For tauropine, not only its significance for the anaerobic metabolism in various marine invertebrate phyla3 has been clarified. Scientists also succeeded several years ago in isolating the underlying enzyme, called tauropine dehydrogenase.

The tauropine dehydrogenase is responsible for the reductive condensation of taurine (a C2 sulfonate) and pyruvate to tauropine. Thereby NAD+ is involved as electron acceptor. The tauropine dehydrogenase also catalyzes the backward reaction.

This enzyme occurs widely among different animal and plant phyla. For example, it could be isolated from the adductor muscles of Haliotis lamellosa4, Arabella iricolor5, Asterina pectinifera6, Halichondria japonica7, and even from a red alga, Rhodoglossum japonicum8.

The metabolism of tauropine in microorganisms is not yet clarified

Tauropine, besides other opines, has also been reported in the context of bacteria. In fact, it was found in plants, which were infected by agrobacteria with a virulent Ti plasmid9. The resulting genetic modification leads to tumor formation, and the plant is triggered to produce opines. As plants cannot use opines themselves, the opines serve as nutrition for the agrobacteria10 and other opine-degrading bacterial strains11.

But so far, compared to marine animal phyla, the intermediate steps in the degradation of tauropine in microorganisms are widely unknown. Preliminary investigation in marine bacteria like Ruegeria pomeroyi DSS-3 and Roseovarius nubinhibens ISM has shown, that they can use tauropine as source of carbon and nitrogen.

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Figure 1: Potential degradation pathway of tauropine. Inner rectangle refers to the cytosol; the area outside the rectangles refers to the periplasm. Abbreviations: cyt cox = cytochrome c oxidized; cyt cred = cytochrome c reduced; Pi = pyrophosphate.

Sulfate thereby occurs as end product.

It is possible, that the tauropine degradation in bacteria is analogous to that in invertebrates. This would mean, that a dehydrogenase is involved. If in microorganisms tauropine can be degraded into pyruvate and taurine by a tauropine dehydrogenase, it is also possible, that taurine is further metabolized in processes, which are already quite well understood12-14. Those processes could include the taurine dehydrogenase and desulfonation by sulfoacetaldehyde acetyltransferase (Figure 1) .

To conclude, initially one interesting aspect is the involvement and the relevance of one sole enzyme in the microbial tauropine degradation pathway: the tauropine dehydrogenase.

Therefore three main questions were studied. The first was to verify the action of a tauropine dehydrogenase in microorganisms. The second step was to further characterize this enzyme by its molecular weight and its localization within bacterial cells. In addition, the degradation pathway downstream of the potential tauropine dehydrogenase should be clarified.

Therefore, in this study, the metabolism of tauropine in four different model organisms was investigated. As model organisms a Ralstonia strain from fresh water was used and in addition three terrestrial bacterial strains were isolated.

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Figure 4: 1D SDS-PAGE of different cultures and fractions. Lane 1-8 and 10: cell lysates, lane 9: marker. Abbreviations: CF = cell-free fraction; MF = membrane fraction; Tp = tauropine; Tau = taurine; Ac- = acetate; act = activity of tauropine dehydrogenase. Roman numbers indicate replicates from different days. White numbers indicate the molecular

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Figure 5: Agarose gel of the isolated 16S rDNA of the different bacteria strains after PCR reaction, stained with ethidium bromide. The size of the marker bands is written in white numbers and indicates number of base pairs. The captions of each lane are written directly above (strain “KD” was not further investigated in this research).

Results and Discussion

Dissimilation pathway of tauropine

Based on previous findings in different phyla3 and the well known degradation pathway of taurine in bacteria14, a potential dissimilation pathway of tauropine was set up, as depicted in Figure 1. For investigations of this pathway, a previously selected strain Ralstonia solanacearum was used as a model organism. From previous experiments it is proven, that this strain is tauropine-degrading.

To prove this pathway, different experiments were conducted to validate the presence and action of the tauropine dehydrogenase. Additionally, the presence of the enzymes downstream of the tauropine dehydrogenase, namely the taurine dehydrogenase, sulfoacetaldehyde acetyltransferase, and sulfite dehydrogenase (not shown in Figure 1) as well as the end products (sulfate ions and ammonium ions) were investigated. Besides verifying the degradation pathway, new tauropine degrading terrestrial microorganisms were isolated and identified.

Detection of the metabolites of tauropine degradation

Ralstonia solanacearum was cultured in liquid medium, containing tauropine. Figure 2 shows the growth curve. The first proof of the previously shown pathway (Figure 1) is the concentration increase of the metabolites sulfate and ammonium and the decrease of tauropine. Figure 3 shows the concentration levels of the substrate tauropine, and the metabolites sulfate and ammonium as function of the optical density, which correlates with the time for growth. Although a one-to-one conversion of tauropine to sulfate was assumed, the amount of degraded tauropine is higher than the resulting sulfate ions. The detection method used was not a high resolution method, leading to this small deviation. The level of sulfate is higher than the ammonium level, because nitrogen is partially incorporated into the bacteria.

These findings support the proposed dissimilation pathway.

Investigation of enzymes involved in the tauropine dissimilation pathway

For detailed investigation of the degradation pathway, the individual enzymes were tested in photometric assays. The wavelength was fixed in each assay and was dependent on the conditions used (for details see section “Methods”). The general approach is the administration of the enzyme (dissolved in a buffer), the respective substrates, a chromophore as a marker, and an electron acceptor. The solution changes its color intensity upon the reaction. Only tests with linear incline or regression of the absorption were considered as positive.

For these assays, three different bacteria cultures were grown using tauropine, taurine, and acetate as carbon source. From these cultures a cell-free, a soluble, and a membrane fraction were prepared, respectively. Roman numerals (Table 1) indicate replicates from different days. For validation of enzyme activity, substrate linearity and in single cases the backwards reaction were investigated.

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Figure 2: Growth curve of a Ralstonia solanacearum culture over a period of 50 h. OD = optical density. Transition from lag- to log-phase not specifically shown

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Figure 3: Concentrations the substrate tauropine and the metabolites sulfate and ammonium in relation to the optical density. Values for optical density are equal to Figure 2.

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Figure 6: Environmental isolates grown on LB plates. A: root stock isolate; B: forest soil isolate; C: compost isolate

Investigation of the tauropine dehydrogenase

The correct parameters of the assays, regarding the conditions and the fraction used, had to be established first. As it was not known yet, whether the tauropine dehydrogenase is membrane-associated or soluble, the three different fractions described above were investigated separately. The assay itself was modified using several buffers (CAPS, TRIS, KP, MOPS, MES) with pH values between 9.4 and 5.0. The electron acceptor was varied as well, using natural ones (cytochrome c, NAD+, FMN), and artificial ones (2,6-Dichlorophenolindophenol, abbreviated DCPIP). The most promising conditions appeared to be MES/NaOH with pH=5 as buffer and DCPIP as an electron acceptor.

Table 1 shows the results of all enzyme assays regarding the tauropine dehydrogenase. It was induced only in the cultures grown with tauropine as carbon source. When taurine or acetate were used as carbon sources, no enzymatic activity could be determined, except in the soluble fraction II. This might be considered as false positive result, because of a high endogenous activity. In general, the enzyme activities are always low, which might be partially owed to the low amount of protein within the fractions. The measured activities have a high standard deviation as well, which leads to an imprecise determination of the activity of the potential tauropine dehydrogenase. It was not possible to determine, whether the enzyme is membrane-bound or not, because every fraction shows enzymatic activity. But the data strongly supports the assumption of the presence of a tauropine dehydrogenase.

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Table 1: Overview of tauropine dehydrogenase assays. Roman numerals indicate replicates from different days.

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