Sulfitdehydrogenasen in organotrophen Bakterien : Enzyme, Gene und deren Regulation

Sulfite is the product of desulfonation in organotrophic bacteria. Due to its high reactivity with biomolecules (DNA, proteins) it has to be detoxified by oxidation to sulfate catalyzed by sulfite-oxidizing enzymes (SOEs). In prokaryotes the responsible enzymes are sulfite dehydrogenases (SDHs) which are molybdopterin cofactor (Moco)-binding enzymes belonging to the DMSO reductase family. SDHs are highly diverse in their overall structures and up to date two bacterial SDHs have been characterized: SorA and/or SorT. About 50% of the organisms which are known to degrade organosulfonates excrete the sulfonate moiety as sulfate but do not share any sequence similarities to SorA or SorT. Therefore, the focus of this study was filling up the gap of knowledge about sulfite dehydrogenases in organotrophic bacteria.Physiological studies with taurine and cysteate as sole carbon and energy source in Ruegeria pomeroyi DSS-3 revealed that taurine is completely utilized concomitant with the excretion of sulfate. However, negligible sulfite-oxidizing activity was measured. In contrast, cysteate was not quantitatively utilized and sulfite was excreted. The highest SDH activity was found in these cell extracts using a previously described enzyme assay. The activity was purified and it turned out that a homotrimer, consisting of three monomers of 11 kDa, is responsible for this enzyme activity. This 11 kDa protein is a soluble protein located in the periplasm which is encoded by a gene (SPO3124 for R. pomeroyi DSS-3) within a three-gene operon with genes encoding for an ECF26-type RNA polymerase σ-factor (SPO3125) and an anti σ-factor (SPO3126). Reverse transcription (RT)-PCR experiments and regulator studies using lacZ-fusion plasmids revealed that the SPO3124-26 gene operon is induced by sulfite. The discovered 11 kDa protein was interpreted to represent a periplasmic sensor protein for an ECF-type signal transduction through the cytoplasmatic membrane, e. g. for sensing of sulfite stress or of other environmental stress, and that the apparent SDH activity measurable for this protein is a reflection of its ‘sensory domain’.Bioinformatic analyses revealed that two-thirds of the organisms with an unknown SDH possess a three-gene cluster: soeABC, which is in close neighbourhood of genes involved in desulfonation. A hypothesis for the function of SoeABC was developed in which SoeA, annotated as a Moco containing oxidoreductase, oxidizes sulfite while transferring the electrons to SoeB, a [4Fe-4S]-cluster binding protein which itself transfers the electrons to SoeC, a transmembrane protein, for which it would be conceivable that the electrons are shuttled to the respiratory chain. First hints for the involvement of SoeABC in sulfite oxidation were obtained by orbitrap analyses and gradient SDS-PAGE indicating that SoeA is inducibly expressed in taurine-grown cell extracts of R. pomeroyi DSS-3 (SPO3559), R. nubinhibens ISM (ISM_10700) and Roseovarius sp. strain 217 (ROS217_11926). Further, Clark-type electrode experiments revealed that the enzyme is located in the membrane supporting the hypothesis that SoeABC is attached to the membrane. An soeA knockout mutant of R. pomeroyi DSS-3 was created by insertional mutation. The mutant was not able to grow with taurine or isethionate within the first two days of growth. However, revertants occurred within six days of growth. It was shown that the soeABC gene cluster is co-transcribed and also co-transcribed with the upstream genes xsc and pta. These findings led to the assumption that TauR is induced by sulfoacetaldehyde (SAA), which is an intermediate in taurine- and in isethionate-degradation. To test this hypothesis, SAA was supplied intracellularly to cysteate-grown R. pomeroyi DSS-3 cells. These cells were now able to quantitatively utilize cysteate while sulfate was excreted; only traces of sulfite were detected. Phylogenetic analyses of SoeA (R. pomeroyi DSS-3) within the DMSO reductase family revealed a close relationship with PsrA which is required for sulfide oxidation. This protein shares 24% sequence identity to the PsrLC complex which was previously described as a novel sulfite dehydrogenase in some green sulfur bacteria.In this study, an 11 kDa protein induced by sulfite stress was discovered. In the absence of an active sulfite-oxidizing enzyme, the 11 kDa protein presumably acts as sensor protein in R. pomeroyi DSS-3. However in this bacterium as well as in members of the order of Rhodobacterales, a novel type of sulfite dehydrogenase was discovered, which was termed SoeABC