Insusceptibility of members of the class Mollicutes to rifampin: studies of the Spiroplasma citri RNA polymerase beta-subunit gene

In order to study the mechanism of insusceptibility of Spiroplasma citri to rifampin, we have cloned and sequenced its rpoB gene, which encodes the beta subunit of RNA polymerase. By comparison of the deduced amino acid sequence with sequences of beta subunits from susceptible and resistant bacteria, it was possible to identify several differences in the so-called Rif region (encompassing rpoB codons 500 to 575 in the Escherichia coli sequence). We constructed a chimeric rpoB gene made of the E. coli rpoB gene in which the Rif region was replaced by the equivalent region from S. citri. E. coli cells harboring this chimeric gene were resistant to rifampin. Subsequent experiments involving site-directed mutagenesis demonstrated that a single amino acid substitution (asparagine at position 526) was able to provide high-level rifampin resistance in E. coli.