Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively AlkaliphilicBacillus pseudofirmusOF4 Lead to Characterization of an S-Layer Protein with a Role in Alkaliphily

Abstract

The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar 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 analysis 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 elevation 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 the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na ؉ -dependent transport of ␣-aminoisobutyric acid, or H ؉ -dependent synthesis of ATP. However, the capacity for Na ؉ -dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8.5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.Bacillus species have been a major component of the extremely alkaliphilic bacterial flora isolated both from highly selective environments such as alkaline lakes and from ostensibly unselective environments such as conventional soils (21,24,26). While many studies have focused on useful products of alkaliphilic bacilli (21), others have focused on the basis for alkaliphily itself (19,26,28). Among the questions that immediately arise are how can those membranous and protein structures that are exposed to the alkaline medium function, and how can cells growing above pH 10 maintain a cytoplasmic pH that is well below the external pH? With respect to the first question, recent structural studies of extracellular enzymes from extreme alkaliphiles and numerous deduced protein sequences of alkaliphile proteins have begun to indicate properties that may correlate with the ability to function at extremely high pH (26). The adaptations, moreover, appear to depend upon whether a high net charge is important to the function of the molecule or molecular segment. When that is the case, the...

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

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Hopanoids Play a Role in Membrane Integrity and pH Homeostasis in Rhodopseudomonas palustris TIE-1

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A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg 2+ uptake

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