Diverse bacteriohemerythrin genes of <i>Methylomonas denitrificans</i> FJG1 provide insight into the survival and activity of methanotrophs in low oxygen ecosystems

Proteobacterial methanotrophs are dependent on the oxidation of methane for ATP production and assimilation of carbon into biomass. Interestingly, some types of gammaproteobacterial methanotrophs thrive in oxygen-depleted zones of lakes and other aquatic ecosystems despite their reliance on oxygen to support methane oxidation. The model gammaproteobacterial methanotroph, <i>Methylomonas denitrificans</i> FJG1, oxidizes methane coupled to nitrate reduction under hypoxia and highly upregulates its expression of bacteriohemerythrin (Bhr). Bhr is a homolog of eukaryotic hemerythrin, which is a protein associated with oxygen binding. Ten <i>bhr</i> homologs were identified in the genome of <i>M. denitrificans</i> FJG1, requiring phylogenetic and gene expression analyses to pinpoint which homolog is likely responsible for delivering oxygen to support methane oxidation under low oxygen conditions. This study examined the prevalence and phylogeny of the 10 <i>bhr</i> homologs from <i>M. denitrificans</i> FJG1 in the genomes of other methanotrophs and across the Bacteria. The homolog denoted "<i>bhr-</i>00" was specific to methanotroph genomes, was highly expressed in <i>M. denitrificans</i> FJG1 under hypoxia, and its predicted structure was nearly identical to a purified oxygen-scavenging hemerythrin protein from <i>Methylococcus capsulatus</i> Bath. Other <i>bhr</i> homologs upregulated from denitrifying cultures of <i>M. denitrificans</i> FJG1 included those with gene neighborhoods related to oxygen sensing, denitrification, and chemotaxis. Together, this study uncovered potential multifunctional roles of bacteriohemerythrin genes of <i>M. denitrificans</i> FJG1 under low oxygen conditions and identified the <i>bhr</i> homolog that most likely enables and supports oxygen delivery to methane monooxygenase enzymes in anoxic ecosystems.IMPORTANCEAerobic gammaproteobacterial methanotrophs can survive and grow in anoxic lakes, but mechanisms that provide them with oxygen to support methane oxidation remain uncharacterized. <i>Methylomonas denitrificans</i> FJG1 encodes 10 copies of bacteriohemerthyrin (<i>bhr</i>), of which seven are expressed at the mRNA level under low oxygen conditions. Comparing the 10 <i>bhr</i> homologs from <i>M. denitrificans</i> FJG1 with those from other methanotrophs and bacterial genomes shows that two are specific to methanotrophs. Gene neighborhoods surrounding conserved <i>bhr</i> genes in methanotrophs suggest a range of potential functions, including oxygen respiration, oxygen sensing, chemotaxis, and nitrate reduction. The results from this study illuminate a previously undescribed diversity of structures and potential functions of <i>bhr</i> homologs in <i>M. denitrificans</i> FJG1 and related methanotrophic bacteria. The results pinpoint a methanotroph-specific homolog, <i>bhr</i>-00, that is likely responsible for oxygen binding and delivery to methane monooxygenase enzymes to promote methane oxidation in low oxygen ecosystems.