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Journal of Bacteriology, February 2009, p. 1220-1229, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01269-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Expression of yeeK during Bacillus subtilis Sporulation and Localization of YeeK to the Inner Spore Coat using Fluorescence Microscopy{triangledown}

Hiromu Takamatsu,* Daisuke Imamura, Ritsuko Kuwana, and Kazuhito Watabe

Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan

Received 9 September 2008/ Accepted 25 November 2008

The yeeK gene of Bacillus subtilis is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in yabG mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the yeeK gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of yeeK did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in yabG mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.

* Corresponding author. Mailing address: Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan. Phone and fax: 81 (72) 866-3114. E-mail: takamatu{at}pharm.setsunan.ac.jp

{triangledown} Published ahead of print on 5 December 2008.

Journal of Bacteriology, February 2009, p. 1220-1229, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01269-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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