ANALYSIS OF SPORE PHOTOPRODUCT LYASE OPERON (SPLAB) FUNCTION USING TARGETED DELETION-INSERTION MUTATIONS SPANNING THE BACILLUS-SUBTILIS OPERONS PTSHI AND SPLAB

Germinating Bacillus subtilis spores repair UV-induced DNA damage in p art using the enzyme spore photoproduct (SP) lyase. SP lyase is encode d by splB, the second cistron of the splAB operon. The sylAB operon is transcribed during sporulation from the P1 promoter, which partially over-laps 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 p hysical and functional boundaries of these contiguous operons, null mu tations were generated in the region by ill vitro site-directed mutage nesis, in which parts of the cloned ptsI-splAB region were res moved a nd replaced with an ermC antibiotic resistance cassette, then introduc ed by transformation into B. subtilis. A deletion-insertion spanning p tsI, splA, and splB abolished the ability of the resulting mutant to u tilize 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 compleme ntation system was developed using the deletion-insertion mutant lacki ng splAB which allows placement of alleles of the cloned splAB operon at the chromsomal amyE locus. The complementation system was used to e xplore the role of SP lyase in determining spore UV resistance.