epr Is Transcribed from a D Promoter and Is Involved in Swarming of Bacillus subtilis

At the onset of stationary phase, Bacillus subtilis secretes at least seven extracellular proteases of which subtilisin (aprE) and a neutral metalloprotease E (nprE) are the most abundant (6). Of the remaining five minor extracellular proteases, Epr (epr) contributes 2 to 4% to this pool (18). The expression of these enzymes is stringently regulated, as best exemplified by the aprE gene (23). However, these proteases appear to be dispensable for growth (11, 19), and their relevance in the physiology of the cell remains to be determined. The transition state is also characterized by the acquisition of specific functions such as competence and motility (23). The expression of genes related to motility, chemotaxis, and flagellar assembly is governed by the alternate sigma factor D (14). D complexed to the core RNA polymerase (E ) recognizes and binds the bipartite promoter sequences 5 -CTAAA-3 ( 35) and 5 -CCGATAT-3 ( 10) (14). We present here evidence that epr is transcribed from a -dependent promoter and that this gene is involved in the swarming of B. subtilis. Although epr has been cloned and sequenced (4, 18), the identity of its promoter and the possible physiological role(s) of this gene have so far not been elucidated. To identify the promoter for epr, we examined the DNA sequence upstream of the ribosome-binding site (RBS) for a putative bipartite promoter sequence that may be related to known promoter sequences for the different sigma factors in B. subtilis. A -type promoter with a 35 (CTATT) and a 10 (CCGATAT) element was identified (Fig. 1) that showed matches of 3 of 5 and 7 of 7, respectively, with the consensus D promoter (14) and an optimal spacing of 17 bp between the two elements. To determine that indeed this promoter was utilized, primer extension analysis was employed to identify the epr transcription start site. Total RNA was isolated from B. subtilis 168/pIC56-4 and 1A716/pIC56-4 carrying the epr gene in a multicopy plasmid, pIC56-4 (Table 1). Since the levels of Epr are very low (4, 18), we were unable to detect epr mRNA from a single-copy gene and hence we have used a multicopy vector. A polyacrylamide gel electrophoresis-purified primer, KKR50 (5 -CGAG GATCCTGTACAACAAGTTTGCA-3 ) (Fig. 1), end labeled with [ -P]ATP (5,000 Ci/mM) and T4 polynucleotide kinase, was annealed with 10 g of RNA, and the primer was extended with Moloney murine leukemia virus reverse transcriptase (1, 17). The extended product(s) was analyzed on a 7 M urea–8% polyacrylamide denaturing gel. To obtain the template for the sequencing reactions, a DNA fragment of 360 bp that overlapped the putative start site was obtained from pIC56-4 by PCR amplification with the primers KKR76 (5 -CGAGATCT CTGCAGTTTTCCCGGCGAC-3 ) and KKR44 (5 CTGGAT CCAGGGGGCTGAAAAACAGAGTGAC-3 ) (Fig. 1) that carry PstI and BamHI restriction sites, respectively. The amplified product was cloned in M13mp19, and single-stranded circular DNA was isolated (17) and used as a template in the sequencing reactions with end-labeled KKR50 and Sequenase version 2.0 (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Figure 2 (lane 1) shows two extended products for RNA derived from 168/pIC56-4. The extended products correspond to a C (plus strand) and an A (plus strand) located six and seven bases downstream of the 10 position of the putative D promoter. This result demonstrates that epr transcription is governed by the D promoter. No extended products were obtained for RNA derived from 1A716/pIC56-4 (Fig. 2, lane 2), in which sigD is mutated, indicating that transcription of epr occurs only when D is present. The relative intensities of the two extended products were similar, suggesting that the two RNA transcripts are made in equal proportion. Alternatively, one of the products which terminates at A may have arisen as a result of premature termination during reverse transcription. To further confirm that the epr gene is transcribed by E , the promoter activity of epr was determined in both B. subtilis 168 and 1A716 containing the D promoter, fused to the -galactosidase gene of E. coli, in plasmid pRB381 (3). A 250-bp DNA segment (U7) containing the D promoter, the RBS, and the ATG was PCR amplified from pIC56-4 with the primers KKR77 (5 -CGAGATCTCTGCAGGCTCATCTTAAAAAC C-3 ) and KKR36 (5 -CTTAGGATCCATGATTCATCTCC3 ) (Fig. 1) that contain restriction enzyme sites for PstI and BamHI, respectively. The amplified product was digested with PstI-BamHI, cloned in pRB381 to give pRBU7, and transformed in B. subtilis 168 and 1A716 to give 168/pRBU7 and 1A716/pRBU7, respectively. The cells were grown at 37°C in Penassay broth to stationary phase (i.e., to an optical density at * Corresponding author. Mailing address: Gene Regulation Lab, Biotechnology Centre, Indian Institute of Technology, Powai, Mumbai 400076, India. Phone: (91) (22) 576-7776. Fax: (91) (22) 572-3480. E-mail: [email protected]. † Present address: Department of Physiology, College of Medicine, University of Tennessee Health Science Center, University of Tennessee, Memphis, TN 38163.