Characterization of the DNA insert in recombinant plasmid pCSBC14

Abstract

The recombinant plasmid pCSBC14, originally beleived to be a clone of protease gene, contains a 4.0 kb chromosomal DNA fragment of B.subtilis TISTR25 in pUC18. The pCSBC14 was digested with HindIII and EcoRI. Each of the 0.7, 0.9 and 1.6 kb fragment of pCSBC14 DNA insert was ligated to M13mp18 DNA vector and transformed into E.coli JM109. The 3 recombinant clones, comprising of 0.7, 0.9 and 1.6 kb insert fragments were named as mCSBC141, mCSBC142 and mCSBC143, respectively. The pCSBC14, mCSBC141, mCSBC142 and mCSBC143 were sequenced by dideoxynucleotide chain-termination method. The sequence of 2,308 bp in the 3 side of the insert in pCSBC14 was determined. The sequence revealed an open reading frame of 1,803 bp, capable of encoding a protein of 600 amino acids. This gene was compared with the GenBank deposited DNA sequences using BLADT. It showed 86% identity to L-glutamine D-fructose-6-phosphate amidotransferase (gcaA) gene of B.subtilis 168. The activity of gcaA protein in E. coli DH5alpha harboring pCSBC14 was assayed and confirmed. Therefore, the pCSBC14 was a clone of B. subtilis TISTR25 L-glutamine D-fructose-6-phosphate amidotransferase (gcaA) gene. By using Southern blot hybridization assay; it was confirmed that the protease genes for both neutral and alkaline protease exist in the genome of B. subtilis TISTR25.