Molecular Cloning of a Gene Affecting the Autolysin Level and Flagellation in Bacillus subtilis

Abstract

Summary: A 2.8 kb PstI fragment of Bacillus subtilis 168W DNA has been cloned into Escherichia coli HB101 and B. subtilis AG5 using pAC3 as a shuttle plasmid. The new plasmid (pBRG1), of 10.2 kb, complemented flaD mutations which show reduced production of autolysin(s), filamentation and non-motility (deficiency of flagella). Deletion experiments showed that the suppressive gene is located between the HindIII and XbaI sites (1.0 kb apart) in pBRG1. The integration of a plasmid having chloramphenicol resistance closely linked to the flaD gene into the B. subtilis AC703 chromosome and its genetic analysis indicated that the cloned fragment contained the flaD gene itself. A high-copy-number plasmid carrying the cloned gene did not lead to an increase in autolysin production above the wild-type level, but it changed the colony morphology from smooth to rough. Among several autolysin-deficient mutations, lyt-151 was suppressed only by the high-copy-number plasmid carrying the cloned gene.