Isolation of rifampin-resistant mutants of Listeria monocytogenes and their characterization by rpoB gene sequencing, temperature sensitivity for growth, and interaction with an epithelial cell line

The sequence of the rpoB gene from Listeria monocytogenes was determined. R ifampin-resistant (Rif(r)) mutants arising from L. monocytogenes cultures e xposed to rifampin were isolated, and by partial sequencing of their rpoB g enes, seven different point mutations affecting five different amino acids (473Asp-->Asn or Gly, 479Gly-->Asp, 483His-->Tyr or Leu, 528Ile-->Phe, and 530Ser-->Tyr), which led to MICs of 0.5 to 100 mu g/ml for the organisms, w ere determined. These mutants showed various deficiencies for growth at 42 degrees C, with only one being comparable to the wild-type strain. The inte raction of these Rif(r) mutants with human Caco-2 cells was examined by usi ng an immunofluorescence technique. Three mutants failed to interact, while three showed a reduced interaction compared to that of the wild type. It i s believed that these pleiotropic phenotypes have arisen as a result of mut ations within the DNA-dependent RNA polymerase holoenzyme.