Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

Abstract

The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe-and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H 2 as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H 2 was also present, growth in lactateand sulfate-containing media lacking H 2 was less efficient. The hyd mutant produced, transiently, significant amounts of H 2 under these conditions, which were eventually all used for sulfate reduction. Sulfate-reducing bacteria of the genus Desulfovibrio contain the genes for several hydrogenases, including the hynBA and hysBA genes for the NiFe-and NiFeSe-hydrogenases, the hydAB genes for Fe-only hydrogenase, and the hndABCD genes for NADP-reducing hydrogenase. The first three enzymes are translocated by the twin-arginine translocation (tat) system and are thus either periplasmic or membrane bound with the active site facing the periplasm. Only the NADPreducing hydrogenase has been shown elsewhere to be cytoplasmic (11). Desulfovibrio vulgaris strain Hildenborough has the Fe-only hydrogenase as well as the NiFe-and NiFeSehydrogenases. The former is a soluble periplasmic enzyme, whereas the latter two are membrane bound. Searching of the database for the D. vulgaris genome at http://www.tigr.org indicated that D. vulgaris has an HndD homolog but lacks the hndA, hndB, and hndC genes. D. vulgaris is thus unlikely to have a cytoplasmic, NADP ϩ -reducing hydrogenase. The hydAB genes encode the 46-kDa ␣ and the 10-kDa ␤ subunit of Fe-only hydrogenase from D. vulgaris (25). The structures of Fe-only hydrogenase have been determined recently both for CpI, the cytoplasmic enzyme from Clostridium pasteurianum (16), and for the periplasmic enzyme from Desulfovibrio desulfuricans (14). The sequences of the ␣ and ␤ subunits of the periplasmic enzyme in D. desulfuricans and D. vulgaris form a contiguous, single polypeptide of 60 kDa in CpI. The splitting of the sequence into two polypeptides in Desulfovibrio spp. is for export: the ␤ subunit has a long twinarginine-type signal sequence for this purpose (24). The function of CpI in the fermentative metabolism of C. pasteurianum is to reoxidize reduced ferredoxin, using protons as the electron acceptor to produce H 2 (2). A similar function has been proposed elsewhere for Fe-only hydrogenase in lactate metabolism by D. vulgaris (23). Reduction of the Fe-only hydrogenase content by expression of hydAB antisense RNA reduced the growth rate and growth yield of D. vulgaris in lactate-and sulfate-containing medium. Observation of a reduced H 2 burst in the initial stages of growth on this medium also pointed to a decreased H 2 production act...

A New Function of the Desulfovibrio vulgaris Hildenborough [Fe] Hydrogenase in the Protection against Oxidative Stress

“…To specify the importance of this enzyme, the effect of oxygen was also studied on a mutant strain, D. vulgaris HYD100, in which the gene encoding for this hydrogenase had been deleted (27). Changes in total hydrogenase activity and cytochrome content of HYD100 cells, induced by the presence of oxygen, were followed.…”

“…This hydrogenase activity rise was related to an increase in the [Fe] hydrogenase is a key enzyme for the dissimilatory sulfate reduction. Its physiological function has been shown to be hydrogen uptake when both hydrogen and lactate are electron donors for sulfate reduction (27) and hydrogen production with pyruvate and lactate as electron donors in the absence of sulfate (37). In vitro experiments have shown that hydrogenases are oxygen-sensitive enzymes (38,39).…”

“…Evidence is given for a peculiar function of periplasmic [Fe] hydrogenase in the protection against oxidative stress. Hildenborough (26), D. vulgaris SOD100 (SOD100) (23), and D. vulgaris HYD100 (HYD100) (27) were cultured in medium C and on solid medium E (26) at 32°C in an anaerobic chamber (COY) filled with a 5% H 2 /5% CO 2 / 90% N 2 mixed gas atmosphere. Growth medium was supplemented with kanamycin (50 g/ml) and chloramphenicol (10 g/ml) when appropriate.…”

Genome sequence of the dissimilatory metal ion–reducing bacterium Shewanella oneidensis

“…Hydrogenases are ubiquitous in prokaryotes and serve as intermediaries in numerous pathways involved in hydrogen production and/or consumption. Hydrogenases are classified into two metalloenzyme families based on the metal content of the active site: the Ni-containing hydrogenases (i.e., 19,20 .…”

Molecular Biology of Microbial Hydrogenases

“…This type of enzyme is found in anaerobic prokaryotes, such as clostridia and sulfate reducers (reviewed by Adams, 1990;Atta andMeyer, 2000 and) and in eukaryotes (Akhmanova et al, 1998;Horner et al, 2000Horner et al, , 2002Florin et al, 2001, Wünschiers et al, 2001b, Happe and Kaminsky, 2002Voncken et al, 2002;Forestier et al, 2003 (Pohorelic et al, 2002). Recently, components of an [FeFe]-H 2 ase have been found associated with formate dehydrogenases from Eubacterium acidaminophilum (Graentzdoerffer et al, 2003).…”