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Mechanisms of transcription modulation by low concentrations of rifampicin in Salmonella typhimurium

University of British Columbia

Screening of a S. typhimurium promoter-luxCDABE fusion library revealed that the antibiotic rifampicin modulated transcription of a subset of the fusions. This thesis examines promoter sequences of three down-modulated (fliA, flgK, invF) and three up-modulated (spvA, traS, STM3595) promoters in an effort to elucidate the mechanisms by which sub-minimal inhibitory concentrations of rifampicin modulate transcription. Reverse transcriptase polymerase chain reaction was used as a second method to examine expression in response to rifampicin and the results were consistent with reporter data. Transcription modulation was abolished in a strain carrying a rifampicin resistance mutation in the gene for the beta subunit of RNA polymerase suggesting that rifampicin effects are mediated by its binding to RNA polymerase. Transcription modulation of the selected genes was observed after cells were treated with several antimicrobials; the effects on transcription differed from those of rifampicin. DNA deletion studies of two promoters for genes involved in flagellar biosynthesis, PfliA and PflgK, showed that ~50 bp fragments containing the δ²⁸-dependent promoters were sufficient to observe rifampicin mediated down-modulation in culture. For PinvF, a promoter specific activator binding site was required for down-modulation. In vitro transcription from PinvF and PflgK was hypersensitive to rifampicin when compared to a control. The results indicate that the selectivity of rifampicin mediated down-modulation reflects variation in the direct interaction of rifampicin with different promoter:RNA polymerase complexes. In contrast to the down-modulated promoters, factors others than rifampicin and RNA polymerase holoenzyme may be involved in up-modulation. Transcription up-modulation from the promoters for STM3595 and traS could not be shown in vitro. In vivo, time course assays of rifampicin induced transcription up-modulation showed a lag period between rifampicin addition and induction. Use of spent media containing rifampicin was not able to shorten the lag phase, suggesting that an intracellular rather than a secreted factor was involved. DNA deletion studies suggested that nucleotides downstream of the transcription start site were involved with up-modulation. A potential intrinsic terminator was found in the untranslated region of the STM3595 transcript, suggesting that transcription attenuation may be involved in rifampicin mediated up-modulation.

Text

2011-05-25

Attribution-NoDerivs 3.0 Unported

http://hdl.handle.net/2429/34803

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nd/3.0/