Putative Iron-Sulfur Proteins Are Required for Hydrogen Consumption and Enhance Survival of Mycobacteria

Abstract

Aerobic soil bacteria persist by scavenging molecular hydrogen (H₂) from the atmosphere. This key process is the primary sink in the biogeochemical hydrogen cycle and supports the productivity of oligotrophic ecosystems. In <i>Mycobacterium smegmatis</i>, atmospheric H₂ oxidation is catalyzed by two phylogenetically distinct [NiFe]-hydrogenases, Huc (group 2a) and Hhy (group 1h). However, it is currently unresolved how these enzymes transfer electrons derived from H₂ oxidation into the aerobic respiratory chain. In this work, we used genetic approaches to confirm that two putative iron-sulfur cluster proteins encoded on the hydrogenase structural operons, HucE and HhyE, are required for H₂ consumption in <i>M. smegmatis</i>. Sequence analysis show that these proteins, while homologous, fall into distinct phylogenetic clades and have distinct metal-binding motifs. H₂ oxidation was reduced when the genes encoding these proteins were deleted individually and was eliminated when they were deleted in combination. In turn, the growth yield and long-term survival of these deletion strains was modestly but significantly reduced compared to the parent strain. In both biochemical and phenotypic assays, the mutant strains lacking the putative iron-sulfur proteins phenocopied those of hydrogenase structural subunit mutants. We hypothesize that these proteins mediate electron transfer between the catalytic subunits of the hydrogenases and the menaquinone pool of the <i>M. smegmatis</i> respiratory chain; however, other roles (e.g., in maturation) are also plausible and further work is required to resolve their role. The conserved nature of these proteins within most Hhy- or Huc-encoding organisms suggests that these proteins are important determinants of atmospheric H₂ oxidation.