Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Fri, 22 Apr 2016 07:23:41 Identification of sporulation genes by genome-wide analysis of the s E regulon of Bacillus subtilis Andrea Feucht, Louise Evans and Jeff Errington Correspondence Jeff Errington jeff.errington@path.oxford.ac.uk Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK Received 9 April 2003 Revised 16 July 2003 Accepted 17 July 2003 Differentiation in the spore-forming bacterium Bacillus subtilis is governed by the sequential activation of five sporulation-specific transcription factors. The early mother-cell-specific transcription factor, s E , directs the transcription of many genes that contribute to the formation of mature, dormant spores. In this study, DNA microarrays were used to identify genes belonging to the s E regulon. In total, 171 genes were found to be under the control of s E . Of these, 101 genes had not previously been described as being s E dependent. Disruption of some of the previously unknown genes ( ydcC, yhaL, yhbH, yjaV and yqfD) resulted in a defect in sporulation. INTRODUCTION Starvation induces the Gram-positive bacterium Bacillus subtilis to initiate a simple, two-cell developmental process that results in the formation of dormant spores. Early in sporulation, the developing cell divides asymmetrically to produce a smaller compartment, the prespore, which becomes the spore, and a larger compartment, the mother cell, which participates in the maturation of the spore and finally lyses to release it. Differential gene expression in the two cells is governed by the tightly regulated synthesis and activation of a series of sporulation-specific transcription factors. These sigma factors redirect RNA polymerase to specific new sets of promoters. The first s-factor, s F , becomes active only in the prespore and is required also for the activation of the first mother-cell-specific s-factor, s E . Then s F and s E are later replaced by s G and s K respectively. Intercompartmental signal transduction pathways link the activation of s E and s K in the mother cell to the action of s F and s G in the prespore, respectively, thereby ensuring the correct sequence of gene expression in the two compart- ments (reviewed by Kroos & Yu, 2000; Piggot & Losick, 2002). The early mother-cell-specific s-factor, s E , is encoded by the sigE (spoIIGB) gene and is expressed upon the initiation of sporulation from a promoter that is recognized by RNA polymerase containing the housekeeping s-factor s A , in conjunction with SpoOA, the key regulator for entry into sporulation (Kenney & Moran, 1987; Satola et al., 1991). s E - dependent gene expression is, however, not observed until 2 h after the initiation of sporulation as the protein is synthesized in an inactive form, which must undergo proteolytic cleavage to become active (LaBell et al., 1987). The protease responsible for the cleavage is probably SpoIIGA (Jonas et al., 1988; Stragier et al., 1988), encoded by the gene spoIIGA upstream of, and co-transcribed with, sigE. Cleavage of pro-s E occurs only in the mother cell and requires the SpoIIR protein, which is expressed in the prespore from a s F -dependent promoter, thereby coupling the activation of s E to s F activity (Karow et al., 1995; Londono-Vallejo & Stragier, 1995). As the first s-factor to become active in the mother cell, s E is responsible for the expression of the genes encoding the late mother cell s- factor, s K , and SpoIIID, a transcription factor required for fine-tuning the regulation of many s E -dependent genes (reviewed by Errington, 1993). Other s E -dependent genes are involved in the formation of the spore coat and cortex and some are necessary for proper germination (reviewed by Piggot & Losick, 2002). At least one s E -dependent gene is involved in the regulation of transcription of sigG, encoding the late prespore-specific s-factor s G , because mutations in the spoIIG operon block transcription at the sigG promoter (Partridge & Errington, 1993). DNA microarrays are increasingly being used for transcrip- tional profiling in various organisms. This technique enables an overview of which genes are being expressed under particular conditions and can produce vast quantities of informative data. Recently, DNA microarrays have been used to identify genes of Bacillus subtilis dependent on s B (Price et al., 2001), s H (Britton et al., 2002), SpoOA and s F (Fawcett et al., 2000) and CodY (Molle et al., 2003). Here we used DNA microarrays to compare the transcrip- tional profile of two different s E mutants to the profile found in wild-type cells. In addition to the identification of previously known s E -dependent genes, we were able to assign 124 additional genes, of which 88 are organized in operons, to the s E regulon. Furthermore, disruption of some of the previously unknown genes resulted in a defect in sporulation. 0002-6413 G 2003 SGM Printed in Great Britain 3023 Microbiology (2003), 149, 3023–3034 DOI 10.1099/mic.0.26413-0