Two-dimensional gel electrophoresis analyses of pH-dependent protein expression in facultatively alkaliphilic Bacillus pseudofirmus OF4 lead to characterization of an S-layer protein with a role in alkaliphily

The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 gr own at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis a nalyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominen ce of some apparent breakdown products of SlpA in gels from pH 10.5-grown c ells led to discovery of the alkaliphile S-layer, the largest and major Slp A forms were present in large amounts in gels from pH 7.5-grown cells as we ll. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was simi lar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic ana lysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na+-replete or suboptimal Na + concentrations. The extent of the adaptation deficit increased with pH el evation and suboptimal Na+. By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7.5 under Na+-replete and suboptimal Na+ conditions, respectively. Logarithmically growing cells of t he two strains exhibited no significant differences in growth rate, cytopla smic pH regulation, starch utilization, motility, Na+-dependent transport o f alpha-aminoisobutyric acid, or H+-dependent synthesis of ATP. However, th e capacity for Na+-dependent pH homeostasis was diminished in RG21 upon a s udden upward shift of external pH from 8.5 to 10.5. The energy cost of reta ining the SlpA layer at near-neutral pH is apparently adverse, but the cons titutive presence of SlpA enhances the capacity of the extremophile to adju st to high pH.