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PreprintCited by 1Microbial metabolism and enzyme functionBacterial Genetics and BiotechnologyMethane Hydrates and Related Phenomena

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Microbial Hauberks: Composition and Function of Surface Layer Proteins in Gammaproteobacterial Methanotrophs

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Hamilton, Richard, Gebbie, William, Bowman, Chynna, Mantanona, Alex, Kalyuzhnaya, Marina G.·2024·Applied and Environmental Microbiology
DOI 10.1101/2024.07.09.602694PMC PMC11784148

Preprint — published as 10.1128/aem.01364-24 · Paper page

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Abstract

Many species of proteobacterial methane-consuming bacteria (methanotrophs) form a hauberk-like envelope represented by a surface (S-) layer protein matrix. While several proteins were predicted to be associated with the cell surface, the composition and function of the hauberk matrix remained elusive. Here we report the identification of the genes encoding the hauberk-forming protein in two gamma-proteobacterial (Type I) methanotrophs, Methylotuvimicrobium buryatense 5GB1 (EQU24_15540) and Methylotuvimicrobium alcaliphilum 20Z R (MEALZ_0971 and MEALZ_0972). The proteins share 40% AA identity with each other and are distantly related to the RsaA proteins from Caulobacter crescentus (20% AA identity). Deletion of these genes resulted in loss of the characteristic hauberk pattern on the cell surface. A set of transcriptional fusions between the MEALZ_0971 and a superfolder green fluorescent protein (sfGFP) further confirmed its surface localization. The functional roles of the hauberk and cell-surface associated proteins, including MEALZ_0971, MEALZ_0972, EQU24_15540, and a copper-induced CorA protein, were further investigated via gene expression studies and phenotypic tests. The hauberk core protein shows constitutive expression across 18 growth conditions. The M. alcaliphilum 20Z R Δ0971 showed reduced growth at high salinity, high methanol and metal-limited conditions, suggesting a role in cell-envelope stability and metal scavenging. Overall, understanding the genetics, composition and cellular functions of the S-layers contributes to our knowledge of methanotroph adaptation to environmental perturbations and opens a promising prospect for (nano)biotechnology applications.

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Figures

Fig 1 Heatmap of transcriptomic and proteomic expression levels in the putative outer membrane protein in M. alcaliphilum 20ZR and M. buryatense 5GB1genes compared to key metabolic enzymes across different growth conditions.
Fig 1 Heatmap of transcriptomic and proteomic expression levels in the putative outer membrane protein in M. alcaliphilum 20ZR and M. buryatense 5GB1genes compared to key metabolic enzymes across different growth conditions.CC BY · PubMed Central
Fig 2 Comparison of identified gene loci in the genome and protein homology in M. alcaliphilum 20ZR and M. buryatense 5 GB1 to known SLP protein (RsaA) in C. crescentus and identified homolog in M. album BG8. The homology of RsaA to each predicted SLP protein is highlighted in blue. RsaD and RsaE proteins for T1SS were compared for similarity, highlighted in yellow and green, respectively. C. crescentus is lacking the putative type 1 secretion outer membrane protein (OMP),which was compared for similarity in 20ZR, 5GB1, and BG8, highlighted in red. Lastly MEALZ_0972 highlighted in purple was solely identified in M. alcaliphilum 20ZR and was in the same operon as the predicted S-layer protein.
Fig 2 Comparison of identified gene loci in the genome and protein homology in M. alcaliphilum 20ZR and M. buryatense 5 GB1 to known SLP protein (RsaA) in C. crescentus and identified homolog in M. album BG8. The homology of RsaA to each predicted SLP protein is highlighted in blue. RsaD and RsaE proteins for T1SS were compared for similarity, highlighted in yellow and green, respectively. C. crescentus is lacking the putative type 1 secretion outer membrane protein (OMP),which was compared for similarity in 20ZR, 5GB1, and BG8, highlighted in red. Lastly MEALZ_0972 highlighted in purple was solely identified in M. alcaliphilum 20ZR and was in the same operon as the predicted S-layer protein.CC BY · PubMed Central
Fig 3 AlphaFold predictions of the putative SLP protein from M. alcaliphilum 20ZR (protein MEALZ_0971) in comparison to the RsaA protein. A–B. RsaA structure, adapted from Bharat, T.A., et al. 2017. A: RsaA oriented in hexamer structure (PDB Code:5N97). B: RsaA monomer. Blue to red colors indicate the order of amino acid residues of the RsaA protein from N-terminal to C-terminal regions, respectively; The dark blue region represents the LPS-binding domain of the RSA protein (PDB Code:6Z7P). C–D. AlphaFold prediction of the MEALZ_0971, a putative SLP protein structure (C) and the structure of the MEALZ_0971-sfGFP protein fusion. The gold regions represent the SLP protein region homologous to RsaA. The predicted transmembrane region of the MEALZ_0971 protein is marked in blue. The putative periplasmic region of the protein is marked in pink. The light green represents the sfGFP structure. E. Confidence scores from high (blue) to low (orange) for the AlphaFold prediction of the MEALZ_0971 protein structure. F. Confidence scores from high (blue) to low (orange) for the AlphaFold prediction of the MEALZ_0971–sfGFP fusion structure. G–H. Predicted structure of the MEALZ_0971 (G) and MEALZ_0971–sfGFP fusion proteins. N-terminal and C-terminal residues are marked in red and purple, respectively.
Fig 3 AlphaFold predictions of the putative SLP protein from M. alcaliphilum 20ZR (protein MEALZ_0971) in comparison to the RsaA protein. A–B. RsaA structure, adapted from Bharat, T.A., et al. 2017. A: RsaA oriented in hexamer structure (PDB Code:5N97). B: RsaA monomer. Blue to red colors indicate the order of amino acid residues of the RsaA protein from N-terminal to C-terminal regions, respectively; The dark blue region represents the LPS-binding domain of the RSA protein (PDB Code:6Z7P). C–D. AlphaFold prediction of the MEALZ_0971, a putative SLP protein structure (C) and the structure of the MEALZ_0971-sfGFP protein fusion. The gold regions represent the SLP protein region homologous to RsaA. The predicted transmembrane region of the MEALZ_0971 protein is marked in blue. The putative periplasmic region of the protein is marked in pink. The light green represents the sfGFP structure. E. Confidence scores from high (blue) to low (orange) for the AlphaFold prediction of the MEALZ_0971 protein structure. F. Confidence scores from high (blue) to low (orange) for the AlphaFold prediction of the MEALZ_0971–sfGFP fusion structure. G–H. Predicted structure of the MEALZ_0971 (G) and MEALZ_0971–sfGFP fusion proteins. N-terminal and C-terminal residues are marked in red and purple, respectively.CC BY · PubMed Central
Fig 4 Analysis of phenotypic changes of the surface of the cells with proposed surface layer protein mutants using both scanning electron microscopy (SEM) and fluorescent microscopy. A: SEM of the SLP on 20ZR WT cells, 20ZR with MEALZ_0971 knocked out, and 20ZR with MEALZ_0972 knocked out. B: Quantification using SEM micrographs of the surface of M. alcaliphilum 20ZR, M. alcaliphilum 20ZR∆0971, and M. alcaliphilum 20ZR∆0972. Surface represented as displaying Hauberk crystallized SLP, bumpy unorganized SLP, or smooth cell surface. C: Fluorescence microscopy of sfGFP integrated into the SLP at the C-terminal, N-terminal, and at the NA5,546 position from the start codon.
Fig 4 Analysis of phenotypic changes of the surface of the cells with proposed surface layer protein mutants using both scanning electron microscopy (SEM) and fluorescent microscopy. A: SEM of the SLP on 20ZR WT cells, 20ZR with MEALZ_0971 knocked out, and 20ZR with MEALZ_0972 knocked out. B: Quantification using SEM micrographs of the surface of M. alcaliphilum 20ZR, M. alcaliphilum 20ZR∆0971, and M. alcaliphilum 20ZR∆0972. Surface represented as displaying Hauberk crystallized SLP, bumpy unorganized SLP, or smooth cell surface. C: Fluorescence microscopy of sfGFP integrated into the SLP at the C-terminal, N-terminal, and at the NA5,546 position from the start codon.CC BY · PubMed Central
Fig 5 Growth rate of M. alcaliphilum 20ZR and ∆MEALZ_0971 and ∆MEALZ_0972 mutants. A. Growth rates at different salinity 0.09–1.02 M (0.5%–6% NaCl wt/vol). B: Growth curve of 20ZR WT, 20ZR ∆0971, and 20ZR ∆0972 grown with 6% NaCl. C: Growth rates at different methanol supplementation (0.2%–2% vol/vol). D: Growth curve of 20ZR WT, 20ZR ∆0971, and 20ZR ∆0972 grown in 2% MeOH. E: Growth rates with trace element additions (T/E). F: Growth curve of 20ZR WT, 20ZR ∆0971, and 20ZR ∆0972 grown without T/E. Unpaired t-test was used for statistical analysis of the growth rates of mutant strains compared to 20ZR.
Fig 5 Growth rate of M. alcaliphilum 20ZR and ∆MEALZ_0971 and ∆MEALZ_0972 mutants. A. Growth rates at different salinity 0.09–1.02 M (0.5%–6% NaCl wt/vol). B: Growth curve of 20ZR WT, 20ZR ∆0971, and 20ZR ∆0972 grown with 6% NaCl. C: Growth rates at different methanol supplementation (0.2%–2% vol/vol). D: Growth curve of 20ZR WT, 20ZR ∆0971, and 20ZR ∆0972 grown in 2% MeOH. E: Growth rates with trace element additions (T/E). F: Growth curve of 20ZR WT, 20ZR ∆0971, and 20ZR ∆0972 grown without T/E. Unpaired t-test was used for statistical analysis of the growth rates of mutant strains compared to 20ZR.CC BY · PubMed Central

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Citations

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Metadata

Authors
Hamilton, R., Gebbie, W., Bowman, C., Mantanona, A., & Kalyuzhnaya, M. G.
Affiliations
San Diego State University (US)
Corresponding author
Kalyuzhnaya, Marina G.
ORCID
Venue
Applied and Environmental Microbiology
Publisher
openRxiv
Published
2024-07-09
Volume
91
Issue
1
Pages
e0136424-e0136424
ISSN
0099-2240 (ISSN-L)
DOI
10.1101/2024.07.09.602694
PMC
PMC11784148
OpenAlex
W4405944986
Cited by
1
References
1
Citations by year
2026: 1
FWCI
0.14
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Applied and Environmental Microbiology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Defense
  • Defense Advanced Research Projects Agency
  • Advanced Research Projects Agency (DE-AR000350)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial metabolism and enzyme function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Physical Sciences › Environmental Science › Environmental Chemistry › Methane Hydrates and Related Phenomena
Keywords
surface layer protein, methanotrophic bacteria, Gammaproteobacteria, cell envelope, gene deletion, gene expression, metal scavenging, salt tolerance, methanol tolerance
MeSH terms
Bacterial Proteins, Membrane Glycoproteins, Methane, Methylococcaceae, Gammaproteobacteria

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