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Journal of Bacteriology, September 2005, p. 6443-6453, Vol. 187, No. 18
0021-9193/05/$08.00+0     doi:10.1128/JB.187.18.6443-6453.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Gene Encoding a Holin-Like Protein Involved in Spore Morphogenesis and Spore Germination in Bacillus subtilis

Gonçalo Real,¹ Sérgio M. Pinto,¹ Ghislain Schyns,^2, Teresa Costa,¹ Adriano O. Henriques,^1,2 and Charles P. Moran Jr.^2*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras, Portugal,¹ Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322²

Received 23 March 2005/ Accepted 25 May 2005

We report here studies of expression and functional analysis of a Bacillus subtilis gene, ywcE, which codes for a product with features of a holin. Primer extension analysis of ywcE transcription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a ^A-type promoter bearing the TG dinucleotide motif of "extended" –10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from the ywcE promoter by purified ^A-containing RNA polymerase (E^A), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by E^A during growth was subjected to repression. Studies with a lacZ fusion revealed that the transition-state regulator AbrB repressed the transcription of ywcE during growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise to ywcE transcription. We found ywcE to be required for proper spore morphogenesis. Spores of the ywcE mutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid. ywcE was also found to be important for spore germination.

Present address: DSM Nutritional Products, Bau 203/25a, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.