Here, acetate growth gave NVP-BEZ235 three-fold higher hdrA1 transcript levels versus methanol growth conditions. The participation of a soluble-type hdrABC enzyme in M. acetivorans metabolism is currently unknown but must now be considered. An orf following the hdrA1 gene is annotated as a polyferredoxin

(pfd), and this suggests a role for this protein in electron transfer to couple the soluble-type Hdr complex with an appropriate electron donor complex. SIS3 molecular weight In contrast, hdrA2 and hdrB2 transcript abundance was about two to twenty-fold lower under the corresponding conditions. This suggests a minor role for the second set of HdrABC-type genes (i.e., hdrA2B2C2) in methanogenesis. The hdrA1pfd and hdrC1B1genes for the soluble-type enzyme subunits are located at different chromosomal

loci, and are coordinately expressed since their mRNA abundance levels are alike (Figure 2C). Additionally, the PCR-based gene experiments also demonstrate that the hdrA1pfd and the hdrED1 genes are each expressed as operons (data not shown). Taken together, these data are consistent with a need for both a membrane-type and a soluble type Hdr enzyme for electron transfer/energy conservation under acetate and methanol cell growth conditions. This suggests that distinct electron transfer pathways are operating to service the alternative Hdr enzymes. The vht and frh gene clusters The M. acetivorans genome lacks an echABCDEF gene cluster encoding an Ech-type hydrogenase with described roles in hydrogen uptake

and ion 5-Fluoracil concentration translocation in M. mazei [3, 5]. Since M. acetivorans cells do not exhibit significant hydrogenase activity [8, 9], some other mechanism Proteasome inhibitor must provide a means for electron transfer from cellular donor(s) to Hdr. Interestingly, the M. acetivorans genome contains two sets of genes (designated vhtG1A1C1D1, and vhtG2A2C2) for F420-nonreducing hydrogenase-types (Figure 3A, 3B, Table 1). It also contains a set of frhADGB genes for a coenzyme F420-type hydrogenase (Figure 3A). Quantitative RT-PCR assays (Figure 3C) established that the vhtG1 and vhtC1genes were each expressed at four- to six-fold higher levels during methanol growth conditions, and this is within the range seen for the fpoL and fpoN genes needed for methyl group oxidation for methanol and acetate metabolism. In contrast, expression of the vhtG2 and vhtC2 genes was low under all conditions examined (Ca. about 17-20-fold lower than vhtG1 and vhtC1). Finally, the frhA and frhB gene expression levels were low relative to vhtG1 or fpoL (Figure 3C), and this suggests a minor role for the frhADGB and vhtG2A2C2 gene clusters in either methanol or acetate-dependent cell growth. Since vhtG1 transcript abundance was elevated and about half of that observed for the fpoL and fpoN genes that encode subunits of the F420 H2 dehydrogenase (Figure 3C), this implies a significant physiological role for the vhtG1A1C1D1 gene products during methanol growth.