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 Mycobacterium smegmatis, 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 M. smegmatis. 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 M. smegmatis 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.

The thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV oxidizes subatmospheric H2 with a high-affinity, membrane-associated [NiFe] hydrogenase

“…This is in agreement with other bacteria harboring Hyd-1h, e.g., Mycobacterium smegmatis and Pyrinomonas methylaliphatogenes [12,18]. This membrane association of Hyd-1h could be a determining factor in the high affinity for H 2 , as in R. eutropha H16 the low-affinity enzyme was purified from the cytoplasm [14,27] [41]. In addition, in the acidobacterium Pyrinomonas methylaliphatogenes the electron flow could be facilitated by hypothetical proteins in the Hyd-1h operon [18].…”

A widely distributed hydrogenase oxidises atmospheric H2during bacterial growth

“…4) . Several genes were commonly genomically associated with hucL genes in putative operons, including the hydrogenase small subunit ( hucS ), a Rieske-type iron-sulfur protein ( hucE ) [34], hypothetical proteins (including NHL-repeat proteins) [33], and various maturation factors (Fig. S4) .…”

“…This study also reports atmospheric H 2 oxidation for the first time in two globally dominant phyla, Proteobacteria and Gemmatimonadota, and uncovers A. ferrooxidans as the first H 2 -scavenging autotroph. Until recently, atmospheric H 2 oxidation was thought to be primarily mediated by heterotrophic Actinobacteriota [1, 10–12], but it is increasingly apparent that multiple aerobic lineages are responsible [4, 17–19, 22, 34]. Some six phyla have now been described that are capable of atmospheric H 2 oxidation and, given the group 2a [NiFe]-hydrogenase is encoded by at least eight other phyla, others will likely soon be described.…”

“…In M. smegmatis , this enzyme has a sufficiently high apparent affinity to oxidise H 2 even at sub-atmospheric levels [12, 23] and is maximally expressed during transitions between growth and persistence [23, 33]. In common with the group 1h hydrogenase also encoded by this bacterium, the group 2a hydrogenase requires potential electron-relaying iron-sulfur proteins for activity [34] and is obligately linked to the aerobic respiratory chain [23]. However, it remains unclear if atmospheric H 2 oxidation by the group 2a hydrogenase reflects a general feature of the enzyme or instead is a specific adaptation of the mycobacterial respiratory chain.…”

A Model of Aerobic and Anaerobic Metabolism of Hydrogen in the Extremophile Acidithiobacillus ferrooxidans

“…Given the overall low abundance (Figure 3), it would be anticipated that the membrane-bound respiratory Hyn only complements H 2 oxidation coupled to oxygen or ferric iron reduction. Transport of electrons from [NiFe] group 1-2 hydrogenases into the respiratory chain to quinones is assumed to be mediated by a protein carrying the [FeS] center (Islam et al, 2019). Therefore, the electrons derived from H 2 oxidation in the active [NiFe] center of an enzyme are further transported up to the Ips2 subunit, [FeS]-binding protein (4Fe-4S ferredoxin-type), which transfers them to quinones in the cytoplasmic membrane (Figure 4).…”