Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron

Abstract

Objectives: The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance.

Methods: The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays.

Results: A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose.

Conclusions: These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron.

Figure 1

Genomic arrangement of the rhamnose gene cluster in B. thetaiotaomicron VPI-5482 (accession number NC 004663) and the B. thetaiotaomicron Tn Met^R metronidazole-resistant mutant. (a) Position of the transposon insertion site. P_KIAPO and P_R indicate the two promoters that have been identified using sequencing and bioinformatic analysis.¹⁴ The dashed arrow shows the position of the Tn4400′ transposon insertion. (b) Schematic representation (not to scale) of the transposon insertion between rhaO and rhaR. IS4400L and IS4400R, insertion sequence elements; arrows indicate positions of primers used to confirm the insertion site (Table 2); tetQ, gene expressing tetracycline resistance in Bacteroides species; bla, gene expressing β-lactamase (ampicillin resistance) in E. coli; rhaO and rhaR, chromosomal genes of B. thetaiotaomicron VPI-5482.

Figure 2

Position of IS4400L in relation to the transcriptional start site of rhaR. Nucleotide sequence of IS4400L (italics) upstream of the B. thetaiotaomicron rhaR promoter region and nucleotide sequence; −7 and −33 promoter recognition sequences, the +1 transcriptional start site (bold underlined) and the ATG start codon of rhaR (bold, box) are shown. The putative −7 hexamers within the IS element are shown in bold italics and are underlined.³⁸