Ionizing radiation assays. Stationary phase cultures of B. subtilis wild type, ykoV ykoU or ykoV ykoU double mutants, and B. subtilis recA mutant cells were treated with the indicated doses of x-rays using a Faxitron

Construction of YkoU and YkoV mutant B. subtilis strains. Strains BFS1845 and BFS1846 were constructed using the plasmid pMUTIN4 (S1). PCR-amplified fragments of an internal region of YkoU or YkoV were cloned into the Eco RI–Bam HI sites of pMUTIN4. A 32–base pair (bp) PCR fragment of the YkoU gene, synthesized using the primers SM44 (5 &&**$$77&&$$$7**$&7**$&& DQG 60 CGCGGATCCGACATATCCTCACAGAC-3 ZDV FORQHG LQWR S087,1 WR PDNH WKH plasmid pDU1845. A 253-bp fragment of the YkoV gene, synthesized with the primers SM46 (5 &&**$$77&77&$**$$$77*$7&&* DQG 60 CGCGGATCCGATTGATCAGGTACACC-3 ZDV FORQHG LQWR S087,1 WR PDNH pDU1846. The plasmids, pDU1845 and pDU1846, were propagated in E. coli and were then transformed into the B. subtilis chromosome generating strains BFS1845 and BFS1846. Strains were verified by PCR. In these strains the gene of interest is inactivated and a transcriptional lacZ fusion has been generated in each case. B. subtilis strain SL7360 [recA::neo; (S2)] was generously provided by P. J. Piggot (Temple University School of Medicine, Philadelphia). The parent strain of SL7360 is B. subtilis BR151 (trpC2 lys-3 metB10). In order to make all the strains isogenic, chromosomal DNA from SL7360 was transformed into B. subtilis 168 to generate SL7360-168. Chromosomal DNA from SL7360 was also transformed into BFS1845 and BFS1846 to create the strains ES33 and ES34, respectively. Sequence analysis indicates that YkoV and YkoU are probably in the same operon. It should be noted that integration of the pMUTIN4 plasmid into YkoV generated a strain (ykoV) in which YkoV is inactivated and the downstream gene, YkoU, is under the control of the IPTG-inducible PSPAC promoter. When this strain is grown in the absence of IPTG neither YkoV or YkoU are expressed and this represents the “double-mutant” (ykoU ykoV). When the strain is grown in the presence of IPTG ykoU is expressed under the control of the PSPAC promoter. This is also the case for the recA ykoV strain.