Differential gene expression in a Bacteroides fragilis metronidazole-resistant mutant.
OBJECTIVE
The current work focused on molecular changes in a spontaneous Bacteroides fragilismutant selected by low concentrations of metronidazole as an adaptive response to the drug.


METHODS
A metronidazole-resistant strain derived from B. fragilis ATCC 25285 was selected by passage in the presence of drug using 0-4 mg/L gradient plates. Using a combination of proteomics for identification of differentially expressed proteins by two-dimensional electrophoresis and selected mutational analyses by single cross-over insertion and an allelic exchange, we have identified genes involved in the adaptive response to metronidazole.


RESULTS
There are significant changes in the protein profiles of resistant strains. These changes appeared to affect a wide range of metabolic proteins including lactate dehydrogenase (up-regulated) and flavodoxin (down-regulated), which may be involved in electron transfer reactions. Also, the enzymic activity of the pyruvate-ferredoxin oxidoreductase (PorA) complex was impaired. Mutant strains lacking the genes for flavodoxin and PorA were less susceptible to metronidazole than the sensitive parent, and a double flavodoxin/PorA mutant had even less susceptibility but none of the mutants were as resistant as the spontaneous metronidazole-resistant strain.


CONCLUSIONS
Overall, the data indicated that there were global changes in the regulation of the physiology of the metronidazole-resistant strain. In addition, flavodoxin was identified as an important contributor to metronidazole sensitivity in B. fragilis.
Diniz
Claudio Galuppo
CG
Department of Microbiology and Immunology, The Brody School of Medicine, East Carolina University, Greenville, NC, USA.
Farias
Luiz M
LM
Carvalho
Maria Auxiliadora R
MA
Rocha
Edson R
ER
Smith
C Jeffrey
CJ
D001441:Bacteroides fragilis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D015180:Electrophoresis, Gel, Two-Dimensional; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D008795:Metronidazole / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D004269:DNA, Bacterial; D008795:Metronidazole; D012313:RNA