Isolation of the Bacillus subtilis cdd downstream region and analysis of genetic structure around the cdd vicinity

A 310 bp EcoRI/HindIII fragment downstream of the Bacillus subtilis cdd gene was isolated from pSO52 which harbours the cdd gene and its vicinity, and was inserted into the pDIA5304. The hybrid vector pSO701 was integrated into the targeted locus of the B. subtilis chromosome and the cdd downstream region was rescued by furthermost BamHI walking toward the sigA locus. By sequencing and analyzing the intergenic region between the cdd and p23-dnaG-sigA operon from the selected clone pSO702, the era genes encoding glycyl tRNA synthetase alpha and beta chains with two unknown distal genes at both terminals were identified and mapped. The cdd was separated by 6,964 bp from the p23-dnaG-sigA operon. Combined data with an additional analysis of the cdd upstream from the sequenced 280 kb stretch in GenBank database (accession No. D84432) indicates that the revised gene order like hrcA-grpE-dnaK-dnaJ-phoH-dgk-cdd-era-tRNA( gly) synthetase alpha and beta-p23-dnaG-sigA-cccA with unidentified distal genes was established genetically on a counter clockwise orientation at 225 degrees of the linkage map.