Expression of the multi-drug efflux genes acrAB of Escherichia coli

Danby, Simon G. (2005) Expression of the multi-drug efflux genes acrAB of Escherichia coli. Doctoral thesis, University of Central Lancashire.

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The action of AcrAB-ToIC, a multidrug efflux system of Escherichia co/i, is largely responsible for the increased intrinsic resistance of this organism to many antimicrobial agents. The level of mRNA specific to the acrAB efflux genes is elevated by six-fold during exponential phase of growth in LB broth. In MOPS minimal medium supplemented with 0.4 % (w/v) glucose, levels of acrA mRNA remain higher for a prolonged period of time concurrent with the delayed onset of stationary phase,
suggesting that transcription of this gene is linked with active growth. A case for transcriptional activation of this gene by RNA polymerase associated with the "housekeeping" sigma factor, a 7o, is presented.
Investigation of acrAB transcription in E. co/i SD1648 using the acrAB::lacZ reporter system encoded on a plasmid (pNN602) appears to contradict these fmdings, suggesting that acrAB transcription is induced upon entry into stationary phase. This paradox is attributed to fundamental differences between the two methods of investigating transcription, and suggests that acrAB expression is subject to post-transcriptional regulation or that the plasmid based reporter system fails to convey an important aspect of control. Notably acrA niRNA levels suggest transient induction of acrA transcription upon transition into stationary phase of growth; however this is secondary to the observed exponential phase peak in transcript levels.
In addition to the general stress conditions already found to induce acrAB transcription including 0.4 % (v/v) ethanol and 0.5 M sodium chloride, bile salts (cholate and deoxycholate), ampicillin, and growth on certain carbon sources, were found to induce acrAB transcription.
A family of isogenic mutant strains based on the lacZ derivative of E. co/i MG1655 (SD 1648) harbouring specific gene disruptions was constructed in order to investigate the effect of key regulatory factors on transcription of the acrAB genes. CRP-cAMP, FIS, H-NS, Rob, AcrR, OmpRJEnvZ, CsrAB, and SdiA were all found to have significant effects on acrAB transcription.
A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, using the Roche LightCyclerTM Instrument, was developed for the purpose of this investigation of acrA gene transcription. The assay was used specifically to quantify acrA mRNA levels; however the method can readily be applied to the study of other E. coli genes. The method was uniquely applied to the investigation of acrAB transcription throughout the growth cycle of E. co/i during batch culture in LB broth, in order to ascertain the patterns of expression for this gene.
Following this, the RT-PCR assay was used as a novel approach to determining the start of transcription for the acrAB genes. Where transcription is initiated from more than one promoter, this method could be applied further to determine the relative strengths of each promoter, in vivo, under different conditions. In the case of the acrAB genes a promoter was located between 224 and 383 bases upstream from the acrA start of translation. This suggests that at least two promoters exist upstream of the acrAB genes, the implications of which are discussed.

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