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PreprintCited by 8Microbial metabolism and enzyme functionMethane Hydrates and Related PhenomenaMetal-Catalyzed Oxygenation Mechanisms

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

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Weiblen, Cerrise, Kits, K. Dimitri, Kleiner, Manuel, Sauvageau, Dominic, Stein, Lisa Y.·2024·mBio
DOI 10.1101/2024.01.07.574550PMC PMC12607906

Preprint — published as 10.1128/mbio.01533-25 · Paper page

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Abstract

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, Methylomonas denitrificans 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 bhr homologs were identified in the genome of M. denitrificans FJG1, requiring phylogenetic and gene expression analyses to pinpoint which homolog is likely responsible for delivering oxygen to support methane oxidation to under low oxygen conditions. This study examined the prevalence and phylogeny of the 10 bhr homologs from M. denitrificans FJG1 in the genomes of other methanotrophs and across the Bacteria. The homolog denoted “ bhr- 00” was specific to methanotroph genomes, was highly expressed in M. denitrificans FJG1 under hypoxia, and its predicted structure was nearly identical to a purified oxygen-scavenging hemerythrin protein from Methylococcus capsulatus Bath. Other bhr homologs upregulated from denitrifying cultures of M. denitrificans FJG1 included those with gene neighborhoods related to oxygen sensing, denitrification and chemotaxis. Together, this study uncovered potential multifunctional roles of bacteriohemerythrin genes of M. denitrificans FJG1 under low oxygen conditions and identified the bhr homolog that most likely enables and supports oxygen delivery to methane monooxygenase enzymes in anoxic ecosystems. Importance: Aerobic gammaproteobacterial methanotrophs can survive and grow in anoxic lakes, but mechanisms that provide them with oxygen to support methane oxidation remain uncharacterized. Methylomonas denitrificans FJG1 encodes 10 copies of bacteriohemerthyrin ( bhr ), of which 7 are expressed at the mRNA level under low oxygen conditions. Comparing the 10 bhr homologs from M. denitrificans FJG1 with those from other methanotrophs and bacterial genomes shows that two are specific to methanotrophs. Gene neighbourhoods surrounding conserved bhr 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 bhr homologs in M. denitrificans FJG1 and related methanotrophic bacteria. The results pinpoint a methanotroph-specific homolog, bhr -00, that is likely responsible for oxygen binding and delivery to methane monooxygenase enzymes to promote methane oxidation in low oxygen ecosystems.

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Figures

Fig 1 Unrooted phylogram of microbial genes related to the 10 bhr genes from M. denitrificans FJG1. Homologs were identified from the GenBank database using BLASTn (Geneious Prime 2019 v1.2.3). Sequences from M. denitrificans FJG1 are highlighted in pink, and genes from other proteobacterial methanotrophs are highlighted in blue. Non-methanotrophs are colored gray. Each color block is labeled with the number of organisms represented. Figure S1 is a version of the taxon names. Data Sheet S2 lists the accession numbers and provides the distance matrix for the included taxa.
Fig 1 Unrooted phylogram of microbial genes related to the 10 bhr genes from M. denitrificans FJG1. Homologs were identified from the GenBank database using BLASTn (Geneious Prime 2019 v1.2.3). Sequences from M. denitrificans FJG1 are highlighted in pink, and genes from other proteobacterial methanotrophs are highlighted in blue. Non-methanotrophs are colored gray. Each color block is labeled with the number of organisms represented. Figure S1 is a version of the taxon names. Data Sheet S2 lists the accession numbers and provides the distance matrix for the included taxa.CC BY · PubMed Central
Fig 2 Cladogram created with full-length CDSs matching the bhr-66 gene of M. denitrificans FJG1 (rooted with eukaryotic outgroup Themiste pyroides (KY007473–KY007479; Geneious Prime 2019 v1.2.3). Red: Gammaproteobacteria. Blue: Betaproteobacteria. Black: Actinobacteria. Gray: Deltaproteobacteria. Genes from methanotrophic organisms are highlighted with a yellow bar.
Fig 2 Cladogram created with full-length CDSs matching the bhr-66 gene of M. denitrificans FJG1 (rooted with eukaryotic outgroup Themiste pyroides (KY007473–KY007479; Geneious Prime 2019 v1.2.3). Red: Gammaproteobacteria. Blue: Betaproteobacteria. Black: Actinobacteria. Gray: Deltaproteobacteria. Genes from methanotrophic organisms are highlighted with a yellow bar.CC BY · PubMed Central
Fig 3 Cladograms of phylogenetic pairings between bhr-00 and bhr-66 with 16S rRNA genes in methanotrophs and of bhr-00 with bhr-99 genes (Geneious Prime 2019 v1.2.3). Yellow highlights indicate multiple branch differences, green highlights indicate one branch difference, and blue highlights indicate consistent branch topology.
Fig 3 Cladograms of phylogenetic pairings between bhr-00 and bhr-66 with 16S rRNA genes in methanotrophs and of bhr-00 with bhr-99 genes (Geneious Prime 2019 v1.2.3). Yellow highlights indicate multiple branch differences, green highlights indicate one branch difference, and blue highlights indicate consistent branch topology.CC BY · PubMed Central
Fig 4 Gene neighborhoods and color-coded gene annotations surrounding bhr-00, bhr-66, and bhr-99 (Geneious Prime 2019 v1.2.3). Key genes were color-coded to discriminate functional categories, as shown above each gene neighborhood.
Fig 4 Gene neighborhoods and color-coded gene annotations surrounding bhr-00, bhr-66, and bhr-99 (Geneious Prime 2019 v1.2.3). Key genes were color-coded to discriminate functional categories, as shown above each gene neighborhood.CC BY · PubMed Central
Fig 5 Gene neighborhoods of bhr-00, -11, -66, and -77/-88 with gene expression and protein abundance data. Gene neighborhoods are shown on the left in their operon structures and are color-coded by functional group as per the legend. Up arrows indicate genes encoded on the forward DNA strand, and down arrows indicate genes encoded on the reverse DNA strand. Columnar data displayed to the right represents mRNA and protein expression abundance for M. denitrificans FJG1 cells transitioning from oxic (24 h) through hypoxic (48 h) to anoxic (72 h) conditions in rows from left to right as noted in the column headings. mRNA expression (TPM) in NMS: low (yellow) to high (red) and AMS: low (yellow) to high (green). Protein expression (NSAF) for NMS: low (pink) to high (purple). Rows are aligned with the center point of corresponding gene icons in the neighborhoods to the left. CDS lengths are proportional, and gaps are preserved. (Neighborhood genes colorized in Geneious Prime 2019 v1.2.3. Data columns prepared in LibreOffice Calc. Components organized and legend added in GIMP 2.10.
Fig 5 Gene neighborhoods of bhr-00, -11, -66, and -77/-88 with gene expression and protein abundance data. Gene neighborhoods are shown on the left in their operon structures and are color-coded by functional group as per the legend. Up arrows indicate genes encoded on the forward DNA strand, and down arrows indicate genes encoded on the reverse DNA strand. Columnar data displayed to the right represents mRNA and protein expression abundance for M. denitrificans FJG1 cells transitioning from oxic (24 h) through hypoxic (48 h) to anoxic (72 h) conditions in rows from left to right as noted in the column headings. mRNA expression (TPM) in NMS: low (yellow) to high (red) and AMS: low (yellow) to high (green). Protein expression (NSAF) for NMS: low (pink) to high (purple). Rows are aligned with the center point of corresponding gene icons in the neighborhoods to the left. CDS lengths are proportional, and gaps are preserved. (Neighborhood genes colorized in Geneious Prime 2019 v1.2.3. Data columns prepared in LibreOffice Calc. Components organized and legend added in GIMP 2.10.CC BY · PubMed Central
Fig 6 AlphaFold2 predicted protein structures visualized in Mol* Viewer. (A) Bhr protein structures predicted from M. denitrificans FJG1 genes from the AlphaFold2 database. Br-55 was not found using AlphaFold2 and was instead created using Robetta, as described in Materials and Methods. (B) Structural comparison of predicted AlphaFold2 structure for Bhr-00 (ID: A0A140E3I1) with experimentally determined Bhr-Bath structure (11), PDB ID: 4XPX. Diiron cofactor is shown in orange. Animated superposition in .mp4 format is available as Movie S1.
Fig 6 AlphaFold2 predicted protein structures visualized in Mol* Viewer. (A) Bhr protein structures predicted from M. denitrificans FJG1 genes from the AlphaFold2 database. Br-55 was not found using AlphaFold2 and was instead created using Robetta, as described in Materials and Methods. (B) Structural comparison of predicted AlphaFold2 structure for Bhr-00 (ID: A0A140E3I1) with experimentally determined Bhr-Bath structure (11), PDB ID: 4XPX. Diiron cofactor is shown in orange. Animated superposition in .mp4 format is available as Movie S1.CC BY · PubMed Central

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Metadata

Authors
Weiblen, C., Kits, K. D., Kleiner, M., Sauvageau, D., & Stein, L. Y.
Affiliations
  • University of Alberta (CA)
  • North Carolina State University (US)
Corresponding author
Stein, Lisa Y.
ORCID
Venue
mBio
Publisher
openRxiv
Published
2024-01-07
Volume
16
Issue
11
Pages
e0153325-e0153325
ISSN
2150-7511 (ISSN-L)
DOI
10.1101/2024.01.07.574550
PMC
PMC12607906
OpenAlex
W4414510811
Cited by
8
References
2
Citations by year
2026: 4 · 2025: 3 · 2024: 1
FWCI
2.10
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • University of Alberta
  • Mitacs
  • Canada First Research Excellence Fund
  • Natural Sciences and Engineering Research Council of Canada
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial metabolism and enzyme function
  • Physical Sciences › Environmental Science › Environmental Chemistry › Methane Hydrates and Related Phenomena
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
Keywords
methanotrophic bacteria, methane oxidation, hypoxia, oxygen delivery, methane monooxygenase, denitrification, gene expression, oxygen sensing, anoxic lakes, Gammaproteobacteria, Methylococcus capsulatus, phylogenetic analysis
MeSH terms
Bacterial Proteins, Hemerythrin, Methane, Oxidation-Reduction, Oxygen, Phylogeny, Ecosystem, Methylomonas

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CoChat. (2026). Citation check: Diverse bacteriohemerythrin genes of Methylomonas denitrificans FJG1 provide insight into the survival and activity of methanotrophs in low oxygen ecosystems. https://app.cochat.ai/papers/10.1101/2024.01.07.574550
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Weiblen, C., Kits, K. D., Kleiner, M., Sauvageau, D., & Stein, L. Y. (2024). Diverse bacteriohemerythrin genes of Methylomonas denitrificans FJG1 provide insight into the survival and activity of methanotrophs in low oxygen ecosystems. mBio. https://doi.org/10.1101/2024.01.07.574550