Prolific S-layer shedding and associated proteins from the methanotroph <i>Methylomicrobium album</i> BG8

Some methanotrophs synthesize S-layers that overlay their outer membrane. Transmission electron microscopy imaging revealed that <i>Methylomicrobium album</i> BG8 constitutively sheds abundant S-layer units, a phenotype not found in seven other methanotrophs, even though <i>Methylotuvimicrobium buryatense</i> 5GB1 produces a similar structure. The release of S-layer units occurred regardless of carbon (methane or methanol) or nitrogen (ammonium or nitrate) source, with 50× trace metals, under copper deprivation, and at all growth phases. The released S-layer units were isolated from the culture medium of <i>M. album</i> BG8 by density gradient centrifugation for proteome analysis. The proteome revealed S-layer protein subunits, transporters for calcium uptake, including TolC and repeats-in-toxin (RTX) proteins, transporters for uptake of cobalamin and siderophores, cell wall biogenesis proteins, and proteins with Type I secretion system (T1SS) target domains. <i>M. album</i> BG8 adapted to grow at pH 4 lost its S-layer, and genome analysis revealed a frameshift mutation resulting in reduced expression of the S-layer unit gene, and an in-frame deletion mutation resulting in almost no expression of an S-layer-associated porin gene. Together, the results suggest that the biogenesis and secretion of <i>M. album</i> BG8 S-layer is mediated by its associated T1SS, the S-layer possesses metal acquisition functions, and low pH adaptation of <i>M. album</i> BG8 results in the loss of its S-layer, likely due to reduced or incomplete expression of S-layer units and the loss of an associated porin. The involvement of the T1SS and shedding phenotype of the S-layer in <i>M. album</i> BG8 could be applied toward selective secretion of proteins and other factors of bioindustrial interest.<h4>Importance</h4>The methanotrophic bacterium <i>Methylomicrobium album</i> 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 eight methanotrophic bacteria screened, only <i>M. album</i> 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. <i>M. album</i> BG8 adapted to grow at low pH lost the production of its S-layer due to mutations in the genes encoding S-layer units and an associated porin. Better understanding of <i>M. album</i> BG8 S-layer production and its shedding phenotype could be harnessed for exporting expressed proteins and bioproducts of industrial interest for the ease of collection and downstream processing.