Sequencing of the rpoB gene in Legionella pneumophila and characterization of mutations associated with rifampin resistance in the Legionellaceae

Abstract

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis, was sequenced from 18 rifampin-resistant Legionella isolates representing four species (L. bozemanii, L. longbeachae, L. micdadei, and L. pneumophila), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae.

FIG. 1

Sequencing strategy for the rpoB gene in L. pneumophila. The thin bars represent the amplicons from the Vectorette PCR. The locations of sites for the restriction enzymes BamHI, ClaI, EcoRI, and HindIII are shown. The approximate positions upstream of the gene were found by Southern blotting with probe U. The C-terminal end of the rplL gene is also indicated.

FIG. 2

Partial DNA sequences of the rpoB genes of wild-type strains L. bozemanii K26, L. longbeachae K47 and K72, L. micdadei K74, and L. pneumophila K69 and K91. Conserved nucleotides are shaded. Numbering is according to that for the rpoB gene in L. pneumophila (GenBank accession number AF087812).

FIG. 3

Alignment of partial β-subunit sequences of the RNA polymerase genes of L. pneumophila (Lp; GenBank accession number AF087812), E. coli (Ec; GenBank accession number P00575), M. tuberculosis (Mt; GenBank accession number P47766), and H. pylori (Hp; GenBank accession number AE000625). Amino acid coordinates correspond to those in the sequence of L. pneumophila. The partial β-subunit sequence of L. pneumophila is identical to those of L. longbeachae (GenBank accession numbers AF101271 and AF113669), L. micdadei (GenBank accession number AF101272), and L. bozemanii (GenBank accession number AF101270), except that L. micdadei has the amino acid valine at position 517. The positions of the mutations found in these Legionella species are all indicated in the L. pneumophila sequence. Known amino acid substitutions associated with rifampin resistance are shaded.