ORGANIZATION OF THE BACILLUS-SUBTILIS-168 CHROMOSOME BETWEEN KDG AND THE ATTACHMENT SITE OF THE SP-BETA PROPHAGE - USE OF LONG ACCURATE PCRAND YEAST ARTIFICIAL CHROMOSOMES FOR SEQUENCING

Within the Bacillus subtilis genome sequencing project, the region bet ween lysA and ilvA was assigned to our laboratory. In this report we p resent the sequence of the last 36 Wb of this region, between the kdg operon and the attachment site of the SP beta prophage. A two-step str ategy was used for the sequencing. In the first step, total chromosoma l DNA was cloned in phage M13-based vectors and the clones carrying in serts from the target region were identified by hybridization with a c ognate yeast artificial chromosome (YAC) from our collection. Sequenci ng of the clones allowed us to establish a number of contigs. In the s econd step the contigs were mapped by Long Accurate (LA) PCR and the r emaining gaps closed by sequencing of the PCR products. The level of s equence inaccuracy due to LA PCR errors appeared to be about 1 in 10 0 00 which does not affect significantly the final sequence quality. Thi s two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence conta ins 38 coding sequences (CDSs), 19 of which encode unknown proteins. S even genetic loci already mapped in this region, xpt, metB, ilvA, ilvD , thyB, dfrA and degR were identified. Eleven CDSs were found to displ ay significant similarities to known proteins from the data banks, sug gesting possible functions for some of the novel genes: cspD may encod e a cold shock protein; bcsA, the first bacterial homologue of chalcon e synthase; exol, a 5' to 3' exonuclease, similar to that of DNA polym erase I of Escherichia coli; and bsaA, a stress-response-associated pr otein. The protein encoded by yplP has homology with the transcription al NifA-like regulators. The arrangement of the genes relative to poss ible promoters and terminators suggests 19 potential transcription uni ts.