Prolific release of S-layer units and associated proteins by the methanotrophic bacterium Methylomicrobium album BG8

Abstract Some methanotrophs synthesize S-layers that overlay their outer membrane. TEM imaging revealed that Methylomicrobium album BG8 constitutively sheds abundant S-layer units, a phenotype not found in 7 other methanotrophs, even though Methylotuvimicrobium buryatense 5GB1 produces a similar structure. Release of S-layer units occurred regardless of carbon (methane or methanol) or nitrogen (ammonium or nitrate) source, with 50X trace metals, under copper deprivation, and at all growth phases. The released S-layer units were isolated from the culture medium of M. album BG8 by density gradient centrifugation for proteome analysis. The proteome revealed the S-layer protein subunits, transporters for calcium uptake including TolC and Repeats-in-Toxin (RTX) proteins, transporters for uptake for cobalamin and siderophores, cell wall biogenesis proteins, and proteins with Type I secretion system (T1SS) target domains. M. album BG8 adapted to grow at pH 4 lost its S-layer and genome analysis revealed a frameshift mutation plus reduced expression of the S-layer unit gene plus the deletion and almost no expression of an S-layer-associated porin gene. Together, the results suggest that the biogenesis and secretion of M. album BG8 S-layer is mediated by its associated T1SS, the S-layer possesses metal acquisition functions, and low pH adaptation of M. album BG8 results in loss of S-layer, likely due to reduced, or incomplete, expression of S-layer units and loss of an associated porin. The involvement of the T1SS and shedding phenotype of the S-layer in M. album BG8 could be applied towards selective secretion of proteins and other factors of bioindustrial interest. Importance The methanotrophic bacterium M. album BG8 produces and sheds large quantities of S-layer units into the culture medium regardless of carbon or nitrogen source, metal availability or growth phase. Of the 8 methanotrophic bacteria screened, only M. album BG8 possessed this phenotype. Proteomics analysis of density gradient purified culture supernatant identified the S-layer protein units and proteins involved in metal uptake and S-layer biogenesis, some with secretion signals for the T1SS. M. album BG8 adapted to grow at low pH lost production of its S-layer due to mutations in the genes encoding S-layer units and an associated porin. Better understanding of M. album BG8 S-layer production and its shedding phenotype could be harnessed for exporting expressed proteins and bioproducts of industrial interest for ease of collection and downstream processing. Competing Interest Statement The authors have declared no competing interest. Footnotes -thoroughly revised text -addition of new experiments (pH adaptation, genome sequence comparison, RNAseq compartison) -new Figure, Table and Supp. Data