Isolation of Tn917 insertional mutants of Bacillus subtilis that are resistant to the protonophore carbonyl cyanide m-chlorophenylhydrazone

Tn917 transposition libraries prepared from Bacillus subtilis were screened for mutants that had insertions in the chromosome resulting in resistance to the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Five such strains were characterized. Three of these were found to have distinct insertion sites that resulted in changes in the fatty acid composition of the membrane lipids. The lipid changes were qualitatively similar to changes observed earlier in CCCP-resistant strains of B. subtilis that had been isolated after chemical mutagenesis. However, the extent of the changes was more modest, correlating with a lower level of protonophore-resistance. One of these three mutants was disrupted in a gene homologous to the Escherichia coli rho gene, as reported earlier (Quirk et al. (1993) J. Bacteriol. 175, 647-654), one was disrupted in a new member of the two-component signalling systems, and the third was disrupted in a new gene of unknown function that apparently forms an operon with transporter genes. The other two CCCP-resistant mutants were disrupted in genes that are likely to encode membrane transporters; the disruption of these genes may have reduced the transmembrane ion leaks during growth, thus conferring modest protonophore-resistance. In one of these strains, the disrupted gene is part of an apparent operon that is a homologue of iron uptake operons from other prokaryotes.