Cloning of the aroI+ gene regions of Bacillus subtilis chromosomal DNAs by B. subtilis temperate phage .RHO.11 and Escherichia coli vector systems, and a comparison of physical maps of the gene regions.

Abstract

The gene order around amyE+ and aroI+ of the B. subtilis chromosomal DNA is lint-tmrA-amyR-amyE-tmrB-aroI (J. Bacteriol., 136: 818-821, 1978). After EcoRI-digestion of the DNA, tmrB and aroI+ were includedin one DNA fragment but amyE+ was not. The tmrB aroI+ DNA fragment generated by EcoRI-digestion of the chromosomal DNA from B. subtilis B8 was cloned in a temperate B. subtilis phage ρ11 genome. The molecular size of the EcoRI-insert containing tmrB and aroI+ in the constructed transducing phage genome was 5.14kb. The fragment was recloned in E. coli vector systems, λgtWES and pBR322. The recombinant plasmid was designated as pTUE1.The tmrB aroI+ DNA fragment generated by EcoRI-digestion of the chromosomal DNA from B. subtilis T2N26, an ultrahyper α-amylase producing strain, was directly cloned in another vector system, charon 4A, using [32P]-labeled pTUE1 as a probe. The molecular size of the tmrB aroI+ fragment inserted in the recombinant phage (λ144A) genome was 11.7kb. Southern hybridization analysis using the two tmrB aroI+ DNA fragments showed that the molecular size of the EcoRI fragments from the B8 and T2N26 chromosomal DNA were 13.5kb and 11.7kb, respectively. The physical map of the aroI+ gene regions was differerent between the B8 T2N26 chromosomal DNA. The physical maps of the EcoRI-fragments containing the tmrB aroI+ genes of pTUE1 and the B8 chromosomal DNA were quite different but they hybridized. The small molecular size of the DNA fragment from pTUE1 seemed to be caused by deletion(s) which might have occurred at the construction and stabilization of the tmrB aroI+ genes in the ρ11 genome.