KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/ reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to -lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression.
Two ampD homologues, ampD I and ampD II , of Stenotrophomonas maltophilia have been cloned and analyzed. Comparative genomic analysis revealed that the genomic context of the ampD II genes is quite different, whereas that of the ampD I genes is more conserved in S. maltophilia strains. The ampD system of S. maltophilia is distinct from that of the Enterobacteriaceae and Pseudomonas aeruginosa in three respects. (i) AmpD I of S. maltophilia is not encoded in an ampDE operon, in contrast to what happens in the Enterobacteriaceae and P. aeruginosa. (ii) The AmpD systems of the Enterobacteriaceae and P. aeruginosa are generally involved in the regulation of ampR-linked ampC gene expression, while AmpD I of S. maltophilia is responsible for the regulation of two intrinsic -lactamase genes, of which the L2 gene, but not the L1 gene, is linked to ampR. (iii) S. maltophilia exhibits a one-step L1 and L2 gene derepression model involving ampD I , distinct from the two-or three-step derepression of the Enterobacteriaceae and P. aeruginosa. Moreover, the ampD I and ampD II genes are constitutively expressed and not regulated by the inducer and AmpR protein, and the expression of ampD II is weaker than that of ampD I . Finally, AmpD II is not associated with the derepression of -lactamases, and its role in S. maltophilia remains unclear.
Isogenic L1 and L2 gene knockout mutants of Stenotrophomonas maltophilia KJ (KJ⌬L1 and KJ⌬L2, respectively) were constructed by xylE gene replacement. Induction kinetics of the L1 and L2 genes were evaluated by testing catechol 2,3-dioxygenase activity in the mutants. The results suggested that the induction of the L1 and L2 genes was differentially regulated.
BackgroundFusaric acid (5-butylpicolinic acid), a mycotoxin, is noxious to some microorganisms. Stenotrophomonas maltophilia displays an intrinsic resistance to fusaric acid. This study aims to elucidate the mechanism responsible for the intrinsic fusaric acid resistance in S. maltophilia.MethodologyA putative fusaric acid resistance-involved regulon fuaR-fuaABC was identified by the survey of the whole genome sequence of S. maltophilia K279a. The fuaABC operon was verified by reverse transcriptase-PCR. The contribution of the fuaABC operon to the antimicrobial resistance was evaluated by comparing the antimicrobials susceptibility between the wild-type strain and fuaABC knock-out mutant. The regulatory role of fuaR in the expression of the fuaABC operon was assessed by promoter transcription fusion assay.ResultsThe fuaABC operon was inducibly expressed by fusaric acid and the inducibility was fuaR dependent. FuaR functioned as a repressor of the fuaABC operon in absence of a fusaric acid inducer and as an activator in its presence. Overexpression of the fuaABC operon contributed to the fusaric acid resistance.SignificanceA novel tripartite fusaric acid efflux pump, FuaABC, was identified in this study. Distinct from the formally classification, the FuaABC may constitute a new type of subfamily of the tripartite efflux pump.
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