Molecular cloning of a chitinase gene from Bacillus circulans C-2

The 2-10 kb DNA fragments of the PstI partially digested total DNA of Bacillus circulans C-2 were cloned into the PstI site of vector pUC19 and the resulting hybrid DNA molecules were then transformed into Escherichia coli. One chitinase gene-containing clone (named pCHT1) was selected from about 8000 recombinants on chitin overlay plates. Analysis of pCHT1 cut with 12 restriction enzymes showed that the inserted fragment in this clone was about 3.0 kb in size and contained one site for each of the three restriction enzymes: KpnI, SacI, and SspI. Cells harboring the recombinants plasmid (pCHT1-R) in which the insert was in an inverted orientation also displayed chitinase activity, indicating that the cloned fragment from B. circulans C-2 contained an intact chitinase gene and its own promoter could be recognized by the Escherichia coli transcriptional system. Southern hybridization analysis proved that the inserted fragment of pCHT1 was really from the genome of B. circulans C-2, and there was only one copy existing in the genome. This fragment could not hybridize with the total DNAs from the other seven chitinase-producing bacteria.