Prolific S-layer shedding and associated proteins from the methanotroph Methylomicrobium album BG8

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.