Molecular Microbiology (1991) 5(6). 1363-1373 AOONIS 0 9 5 0 3 8 2 X 9 1 0 0 1 5 1 L Sequential activation of dual promoters by different Sigma factors maintains spol/Jexpression during successive developmental stages of Bacillus subtilis D. Foulger* and J. Emngton Sir William Dunn School of Pathology. University of Oxford, South Parks Road, Oxford 0X1 3RE. UK. Summary The spoV^J gene of Baciiius subtiUs encodes a 36 kDa protein and is expressed only in the mother celt. spo VJ has an interesting pattern of regulation during sporulation because it is expressed from sequentially activated promoters. These promoters, designated P, and P2, are under the control of different sigma fac- tors, a^ and o**, which become active at separate times during sporulation. Removal of promoter P,, leaving promoter P^ active, resulted in about a 30- minute delay in the formation of heat-resistant spores and demonstrated that the expression of spoV^J from both promoters is essential for normal sporulation. A comparison is made between the sequences of the spoVJ promoters and the promoters of other genes dependent upon n^ and o**. ai, 1989; Foulger and Errington. 1989). A change in the active sigma factor would provide a means of sequentially turning on different sets of genes. It would also result in genes expressed during the earlier stages becoming inactive as the sigma factor required for their transcription is no longer produced (Losick and Pero. 1981). However, it might be predicted that some sporutation genes would be required during two or mere developmental stages. Such genes would need a mechanism for continued expression, despite changes in the active sigma factor. Here we report the identification of such a gene. spoVJ. controlled by sequentially activated dual promoters. Mutations in spoVJ block sporulation at a late stage. producing immature spores that are sensitive to organic solvents and heat but resistant to lysozyme (Hill, 1983). These observations suggest that the SpoVJ product only functions late in development. In order to elucidate the possible function of the SpoVJ product and the reason for the protracted expression of its gene, we have deter- mined its DNA sequence and examined the predicted product for homology to other proteins. Introduction Sporulation in Bacillus subtilis provides a relatively simple experimental system for use in the study of cellular devel- opment and differentiation. Sporulation involves two cell types, the mother cell and the prespore. and incorporates seven distinct morphological stages, and the coordinated expression of at least 50 spo genes (Piggot and Coote. 1976: Losick etai., 1986). Although many of these genes are involved in determining structural or functional proper- ties of the spore, others are known to be involved in regu- lating gene expression during sporulation (Mandelstam and Errington, 1987; Losick ef ai. 1989; Losick and Kroos, 1989). One class of regulatory genes encode Sigma factors, which bind to RNA polymerase thereby mediating promoter recognition. These clearly have a major role in control of gene expression during spore for- mation (Losick and Pero. 1981). although other types of regulatory control are also known to be involved (Kroos et Received 22 November, 1990: revised 11 February, 1991. "Forcorre- spondence. Tel. (0865) 275561: Fax (0865) 275556. Results DNA sequence of the spoVJ gene and its promoter We have previously shown, by integrational plasmid anal- ysis, that the transcriptional unit of spoVJ lies between faql and Smal restriction sites of the insert in plasmid pSGMUeO (Errington et ai.. 1989). One end of the open reading frame (ORF) was further defined using an addi- tional integrational piasmid, pSGMU262, containing the 0.75 kbp HaeW/Clal fragment of spo VJ (F\g. 1, nucieo- tides 586-1341). This plasmid generated a Spo* pheno- type when integrated into the chromosome of Spo* strain 168 by selection for chloramphenicol resistance (Cm"). Therefore pSGMU262 does not disrupt spoVJ and one end of the spoVJ transcriptional unit is upstream of the Cla\ site at nucleotide 1341 but, as shown by Errington et ai. (1989), downstream from the Sg/ll site. These results show that the downstream end of the transcriptional unit lies in the 95 bp region between these two restriction sites. The DNA between the Taq\ and Cla\ restriction sites was sequenced on both strands using Ml 3 and plasmid