Germinating Bacillus subtilis spores repair UV-induced DNA damage in part using the enzyme spore photoproduct (SP) lyase. SP lyase is encoded by splB, the second cistron of the splAB operon. The splAB operon is transcribed during sporulation from the P1 promoter, which partially overlaps the transcriptional terminator of the upstream ptsHI operon, which in turn encodes the Hpr protein and Enzyme I components of the PEP:sugar phosphotransferase (PTS) system. In order to determine the physical and functional boundaries of these contiguous operons, null mutations were generated in the region by in vitro site-directed mutagenesis, in which parts of the cloned ptsI-splAB region were removed and replaced with an ermC antibiotic resistance cassette, then introduced by transformation into B. subtilis. A deletion-insertion spanning ptsI, splA, and splB abolished the ability of the resulting mutant to utilize the PTS sugar glucose. Deletions removing either splB alone or both splA and splB did not affect glucose utilization, thus indicating that splAB gene products are not involved in PTS function. A complementation system was developed using the deletion-insertion mutant lacking splAB which allows placement of alleles of the cloned splAB operon at the chromosomal amyE locus. The complementation system was used to explore the role of SP lyase in determining spore UV resistance.