Abstract
The organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans is able to grow with hydrogen as electron donor and with tetrachloroethene (PCE) as electron acceptor; PCE is reductively dechlorinated to cis-1,2-dichloroethene. Recently, a genomic survey revealed the presence of four gene clusters encoding NiFe hydrogenases in its genome, one of which is presumably periplasmic and membrane-bound (MBH), whereas the remaining three are cytoplasmic. To explore the role and regulation of the four hydrogenases, quantitative real-time PCR and biochemical studies were performed with S. multivorans cells grown under different growth conditions. The large subunit genes of the MBH and of a cytoplasmic group 4 hydrogenase, which is assumed to be membrane-associated, show high transcript levels under nearly all growth conditions tested, pointing toward a constitutive expression in S. multivorans. The gene transcripts encoding the large subunits of the other two hydrogenases were either not detected at all or only present at very low amounts. The presence of MBH under all growth conditions tested, even with oxygen as electron acceptor under microoxic conditions, indicates that MBH gene transcription is not regulated in contrast to other facultative hydrogen-oxidizing bacteria. The MBH showed quinone-reactivity and a characteristic UV/VIS spectrum implying a cytochrome b as membrane-integral subunit. Cell extracts of S. multivorans were subjected to native polyacrylamide gel electrophoresis (PAGE) and hydrogen oxidizing activity was tested by native staining. Only one band was detected at about 270 kDa in the particulate fraction of the extracts, indicating that there is only one hydrogen-oxidizing enzyme present in S. multivorans. An enrichment of this enzyme and SDS PAGE revealed a subunit composition corresponding to that of the MBH. From these findings we conclude that the MBH is the electron-donating enzyme system in the PCE respiratory chain. The roles for the other three hydrogenases remain unproven. The group 4 hydrogenase might be involved in hydrogen production upon fermentative growth.
Highlights
Molecular hydrogen (H2) is one of the primary electron donors for many anaerobic respiratory processes mediated by prokaryotes
To determine and compare the transcript levels of the hydrogenase genes under different growth conditions, S. multivorans was grown with the following substrate combinations: pyruvate plus fumarate, produced during tetrachloroethene (PCE), 5% O2, or without external electron acceptor, or H2 plus PCE or nitrate
For hydrogenase quantitative real-time PCR (qPCR), the following genes encoding the catalytic subunits of each hydrogenase gene cluster were chosen: hydB (SMUL_1424) of the MBH gene cluster, hupL (SMUL_1422) from the cytoplasmic uptake hydrogenase, echE (SMUL_1307) from the CooH-like hydrogenase and hyfG (SMUL_2388) from the Hyf hydrogenase
Summary
Molecular hydrogen (H2) is one of the primary electron donors for many anaerobic respiratory processes mediated by prokaryotes. Deduced from amino acid sequence similarity, HupSL was discussed to play a role in either the recycling of H2 produced cytoplasmically (e.g., during N2 fixation) or to deliver low-potential reducing equivalents for anabolic purposes comparable to Aquifex aeolicus hydrogenase III (Guiral et al, 2005) It could be involved in H2-dependent PCE respiration. In this study we wanted to (1) reveal the hydrogen uptake metabolism of S. multivorans during H2-driven PCE respiration and characterize the uptake hydrogenase involved in this process and (2) investigate the transcript profiles of hydrogenase catalytic subunit genes during different growth conditions, which might give information about the regulation of the four hydrogenases and their physiological role in S. multivorans
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