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Journal of Bacteriology, November 2000, p. 5969-5981, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Raymond Gilmour,1,dagger Paul Messner,2 Arthur A. Guffanti,1 Rebecca Kent,1 Andrea Scheberl,2 Nancy Kendrick,3 and Terry Ann Krulwich1,*

Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York1; Zentrum für Ultrastrukturforschung und Ludwig-Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria2; and Kendrick Laboratories Inc., Madison, Wisconsin3

Received 1 June 2000/Accepted 9 August 2000

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 alpha -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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Box 1020, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-7280. Fax: (212) 996-7214. E-mail: terry.krulwich{at}mssm.edu.

dagger Present address: Eli Lilly and Company, Indianapolis, IN 46285.

Journal of Bacteriology, November 2000, p. 5969-5981, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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