Characterization of mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species

Abstract

Rickettsiae are gram-negative, obligately intracellular bacteria responsible for arthropod-borne spotted fevers and typhus. Experimental studies have delineated a cluster of naturally rifampin-resistant spotted fever group species. We sequenced the 4, 122- to 4,125-bp RNA polymerase beta-subunit-encoding gene (rpoB) from typhus and spotted fever group representatives and obtained partial sequences for all naturally rifampin-resistant species. A single point mutation resulting in a phenylalanine-to-leucine change at position 973 of the Rickettsia conorii rpoB sequence and present in all the rifampin-resistant species was absent in all the rifampin-susceptible species. rpoB-based phylogenetic relationships among these rickettsial species yielded topologies which were in accordance with previously published phylogenies.

FIG. 1

Distance matrix trees derived from rpoB data (A) and from data for the 16S rRNA-encoding gene (B). The evolutionary distances were determined by the method of Kimura (12). These values were used to construct a dendrogram by the neighbor-joining method. The numbers at nodes are the proportions of 100 bootstrap resamplings that support the topology shown. The scale bar shows 5% differences in nucleotide sequences. The GenBank accession numbers for the 16S rRNA gene sequences included are as follows: Escherichia coli, M24996; Salmonella enterica serovar Typhimurium, X80681; Buchnera aphidicola, Z19056; Haemophilus influenzae, M35019; Pseudomonas putida, X93997; Coxiella burnetii, M21291; Rickettsia massiliae, L36214; isolate Bar 29, L36102; Rickettsia conorii Moroccan, L36105; Rickettsia typhi, L36221; Rickettsia prowazekii, M21789; Mycoplasma genitalium, X77334; Thermotoga maritima, M21774; Borrelia burgdorferi, X85204; Staphylococcus aureus, X68417; Bacillus subtilis, AF058766; Mycobacterium smegmatis, M12872; and Mycobacterium tuberculosis, X52917. Accession numbers for the nonrickettsial rpoB sequences included are as follows: Escherichia coli, V00339; Salmonella enterica serovar Typhimurium, X13854; Buchnera aphidicola, Z11913; Haemophilus influenzae, U68759; Pseudomonas putida, X15849; Coxiella burnetii, U86688; Mycoplasma genitalium, U39717; Thermotoga maritima, X61562; Borrelia burgdorferi, X71024; Staphylococcus aureus, X64172; Bacillus subtilis, D83789; Mycobacterium smegmatis, U24494; and Mycobacterium tuberculosis, L27989. The GenBank accession numbers for the rickettsial rpoB sequences are given in Materials and Methods.