Differences in hydrogenase gene expression between Methanosarcina acetivorans and Methanosarcina barkeri

Abstract

Methanosarcina acetivorans C2A encodes three putative hydrogenases, including one cofactor F(420)-linked (frh) and two methanophenazine-linked (vht) enzymes. Comparison of the amino acid sequences of these putative hydrogenases to those of Methanosarcina barkeri and Methanosarcina mazei shows that each predicted subunit contains all the known residues essential for hydrogenase function. The DNA sequences upstream of the genes in M. acetivorans were aligned with those in other Methanosarcina species to identify conserved transcription and translation signals. The M. acetivorans vht promoter region is well conserved among the sequenced Methanosarcina species, while the second vht-type homolog (here called vhx) and frh promoters have only limited similarity. To experimentally determine whether these promoters are functional in vivo, we constructed and characterized both M. acetivorans and M. barkeri strains carrying reporter gene fusions to each of the M. acetivorans and M. barkeri hydrogenase promoters. Generally, the M. acetivorans gene fusions are not expressed in either organism, suggesting that cis-acting mutations inactivated the M. acetivorans promoters. The M. barkeri hydrogenase gene fusions, on the other hand, are expressed in both organisms, indicating that M. acetivorans possesses the machinery to express hydrogenases, although it does not express its own hydrogenases. These data are consistent with specific inactivation of the M. acetivorans hydrogenase promoters and highlight the importance of testing hypotheses generated by using genomic data.

FIG. 1.

Alignments of Methanosarcina genomic regions encoding hydrogenases. The black arrows represent hydrogenase and hydrogenase-related genes. All other genes are represented by gray arrows.

FIG. 2.

Maximum parsimony phylogenetic trees for Methanosarcina Vht and Vhx subunits. Mm, M. mazei; Mb, M. barkeri; Ma, M. acetivorans; RC-I, rice cluster I methanogen; Af, Archaeoglobus fulgidus. Bootstrap scores greater than 50 are indicated at the nodes. (A) VhtA/VhxA subunits; (B) VhtC/VhxC subunits; (C) VhtG/VhxG subunits.

FIG. 3.

Hydrogenase promoter activity in M. barkeri. β-Glucuronidase activity (UidA activity) was measured for M. barkeri strains carrying single-copy reporter gene fusions to each M. barkeri and M. acetivorans hydrogenase promoter. The growth substrates used were methanol (black bars), methanol plus H₂ (open bars), H₂-CO₂ (gray bars), and acetate (striped bars).

FIG. 4.

Hydrogenase promoter activity in M. acetivorans. β-Glucuronidase activity (UidA activity) was measured for M. acetivorans strains carrying single-copy reporter gene fusions to each M. barkeri and M. acetivorans hydrogenase promoter. The growth substrates used were methanol (black bars), methanol plus H₂ (open bars), and acetate (striped bars).