"New intermediates, pathways, enzymes and genes in the microbial metabolism of organosulfonates"

Abstract

Taurine (2-aminoethanesulfonate), isethionate (2-hydroxyethanesulfonate) and sulfoacetate are natural C2 sulfonates which exist in the environment and are to our current knowledge solely degraded by microorganisms which are able to cleave the chemically stable C-sulfonate bond.Anaerobic and aerobic degradation of taurine is well investigated in diverse (marine and terrestrial) bacteria.The aerobic dissmilation of taurine proceeds via the central intermediate sulfoacetaldehyde (SAA) and subsequent desulfonation by sulfoacetaldehyde acetyltransferase (Xsc) to acetylphosphate and sulfite. Acetylphosphate is then metabolized (by two different possible pathways, e.g. by Pta, phosphate acetyltransferase) to acetyl-CoA and thereby channeled into central metabolism. Sulfite is, for the purpose of detoxification, oxidized to sulfate by sulfite dehydrogenase.It is a generally accepted hypothesis that the degradative pathways of isethionate and sulfoacetate converge at SAA with other (C2) sulfonates.In this study, this hypothesis was confirmed in the bacterium Cupriavidus necator H16: the organism was able to utilize taurine, isethionate and sulfoacetate as a sole source of carbon and energy for growth, and it excreted stoichiometric amounts of sulfate into the growth medium. Inducible enzyme activities were measured during growth with each of the three sulfonates. Additionally, transcription experiments (RT-PCR, reverse transcription PCR) with the appropriate genes showed inducible transcription of each of the genes in mRNA extracted from sulfoacetate-grown cells. Furthermore, a transport protein (TauE) was presumed to represent a sulfite exporter wich is responsible for the translocation of sulfite into the periplasm, its site of oxidation by SorAB.In addition, the previously uncharacterized initial steps of isethionate and sulfoacetate, leading to SAA, were investigated: The microbial dissimilation of isethionate is presumed to occur via inducible isethionate dehydrogenase (IseJ) in marine and terrestrial bacteria.A gene cluster was found which presumably encodes the isethionate degradative genes. The gene products include a putative transcriptional regulator (IseR), isethionate dehydrogenase (IseJ) as well as a transport system (IseU in terrestrial bacteria, IseKLM in marine bacteria).The microbial degradation of sulfoacetate in C. necator H16 proceeds via an ATP-dependent activation of sulfoacetate to the novel CoA-ester sulfoacetyl-CoA. This intermediate was identified by MALDI-TOF mass spectrometry. The enzyme catalyzing this reaction was sulfoacetate-CoA ligase (SauT) which could be measured in a discontinuous enzyme assay at the HPLC. In the next step, sulfoacetyl-CoA was converted to SAA by sulfoacetaldehyde dehydrogenase (SauS). Both SauT and SauS were inducibly active during growth with sulfoacetate. SauS was purified to homogeneity, characterized and assigned to the coding gene (H16_A2747) by PMF (peptide mass fingerprinting).The gene sauS was part of a cluster consisting of four genes encoding regulation (SauR), activation (SauT), reduction (SauS) and transport (SauU). The catabolic genes sauSTU were inducibly transcribed during growth with sulfoacetate, as confirmed by RT-PCR. In addition, the involvement of the genes in sulfoacetate degradation was proven by site-directed mutagenesis.By comparative genomics, 25 microorganisms, both marine and terrestrial, were found to contain sulfoacetate gene clusters. Thereby, a different putative activation enzyme (heteromeric sulfoacetate-CoA ligase SauPQ), different types of regulators (SauI, SauV) and an alternative transport system (TTT, tripartite tricarboxylate transporter, SauFGH) for sulfoacatate were discovered. Some of these variants of the newly discovered sulfoacetate degradation pathway were investigated in e.g. Roseovarius nubinhibens ISM and Oligotropha carboxidovorans OM5 by means of growth experiments, enzyme activity tests and RT-PCR.