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Journal of Bacteriology, August 2008, p. 5635-5641, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00670-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Localization of the Germination Protein GerD to the Inner Membrane in Bacillus subtilis Spores

Patricia L. Pelczar and Peter Setlow^*

Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3305

Received 13 May 2008/ Accepted 6 June 2008

GerD of Bacillus subtilis is a protein essential for normal spore germination with either L-alanine or a mixture of L-asparagine, D-glucose, D-fructose, and potassium ions. GerD's amino acid sequence suggests that it may be a lipoprotein, indicating a likely location in a membrane. Location in the spore's outer membrane seems unlikely, since removal of this membrane does not result in a gerD spore germination phenotype, suggesting that GerD is likely in the spore's inner membrane. In order to localize GerD within spores, FLAG-tagged GerD constructs were made, found to be functional in spore germination, and detected in immunoblots of spore extracts as not only monomers but also dimers and trimers. Upon fractionation of spore extracts, GerD-FLAG was found in the inner membrane fraction from dormant spores and was present at 2,000 molecules/spore. GerD-FLAG in the inner membrane fraction was solubilized by Triton X-100, suggesting that GerD is a lipoprotein, and the protein was also solubilized by 0.5 M NaCl. GerD-FLAG was not processed proteolytically in a B. subtilis strain lacking gerF (lgt), which encodes prelipoprotein diacylglycerol transferase (Lgt), indicating that when GerD does not have a diacylglycerol moiety, signal sequence processing does not occur. However, unprocessed GerD-FLAG still gave bands corresponding to monomers and dimers of slightly higher molecular weight than that of GerD-FLAG from a strain with Lgt, further suggesting that GerD is a lipoprotein. Upon spore germination, much GerD became soluble and then appeared to be degraded as the germinated spores outgrew and initiated vegetative growth. All of these results suggest that GerD is a lipoprotein associated with the dormant spore's inner membrane that may be released in some fashion from this membrane upon spore germination.

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* Corresponding author. Mailing address: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3305. Phone: (860) 679-2607. Fax: (860) 679-3408. E-mail: setlow{at}nso2.uchc.edu

Published ahead of print on 13 June 2008.

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Journal of Bacteriology, August 2008, p. 5635-5641, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00670-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Mongkolthanaruk, W., Robinson, C., Moir, A. (2009). Localization of the GerD spore germination protein in the Bacillus subtilis spore. Microbiology 155: 1146-1151 [Abstract] [Full Text]  
• Ghosh, S., Setlow, P. (2009). Isolation and Characterization of Superdormant Spores of Bacillus Species. J. Bacteriol. 191: 1787-1797 [Abstract] [Full Text]