CsrA maximizes expression of the AcrAB multidrug resistance transporter

Abstract

Carbon Storage Regulator A (CsrA) is an RNA binding protein that acts as a global regulator of diverse genes. Using a combination of genetics and biochemistry we show that CsrA binds directly to the 5' end of the transcript encoding AcrAB. Deletion of csrA or mutagenesis of the CsrA binding sites reduced production of both AcrA and AcrB. Nucleotide substitutions at the 5' UTR of acrA mRNA that could potentially weaken the inhibitory RNA secondary structure, allow for more efficient translation of the AcrAB proteins. Given the role of AcrAB-TolC in multi-drug efflux we suggest that CsrA is a potential drug target.

Figure 1.

Transcriptional regulation of acrAB expression in Escherichia coli and Salmonella enterica. In both E. coli and S. enterica the acrAB regulon (•) can be activated by the transcriptional activators, MarA (•), SoxS (•) and Rob (•). In S. enterica ramRA (•) is the master regulator of the acrAB regulon. MarA, SoxS, Rob and RamA activate the acrAB regulon by directly binding to a degenerate nucleotide sequence upstream of the acrAB locus known as the mar/sox/rambox. In E. coli and S. enterica acrR is the local repressor of acrAB, in S. enterica ramR acts as the local repressor of ramA.

Figure 2.

Output from FACS following transformation of pMW82-pramA into Salmonella enterica serovar Typhimurium transposon library post 2 h recovery incubation at 37°C. Cell populations expressing greater GFP than the negative control were gated into populations P4, P5, P6 and P7 before sorting.

Figure 3.

Effect of csrA inactivation on protein levels of AcrA, AcrB and RamA3XFLAG. (A) Expression of AcrA in SL1344 (WT), ΔacrA, ramR::aph and csrA::aph mutants. (B) Expression of AcrB in SL1344 (WT), ΔacrB, ramR::aph and csrA::aph mutants. (C) Expression of RamA3XFLAG in SL1344 (WT), ΔramA, ramR::aph and csrA::aph mutants. Internal control used in all the experiments was the β subunit of RNA polymerase. The RNA polymerase western blot confirms that there is an equal amount of protein in each well. Band intensity was determined using GeneSys, this information was used to assign a relative concentration of AcrA, AcrB and RamA3XFLAG to each respective band, expressed as a percentage of wild-type. For negative controls, the respective deleted gene mutant i.e. ΔacrA, ΔacrB and ΔramA mutants, were used.

Figure 4.

DNA sequence of the ramA and acrAB promoter regions used in this study. (A) DNA sequence of the Salmonella enterica serovar Typhimurium (SL1344) 288 bp ramRA intergenic region. The ramA promoter −10 and −35 elements, and transcription start site (TSS) are in bold type. The ribosomal binding site is underlined and the putative CsrA binding site is in bold and shaded gray. (B) DNA sequence of the S. Typhimurium (SL1344) 141 bp acrAB promoter region. The acrAB promoter −10 and −35 elements, and predicted TSS are in bold type. The ribosomal binding site is underlined and the putative CsrA binding sites are in bold and shaded gray. The deleted sequence containing putative RNase E cleavage sites is indicated with dotted lines.

Figure 5.

RNA electrophoretic mobility shift analysis to evaluate ramA and acrAB RNA interactions with CsrA. (A) In vitro transcribed ramA RNA (0.5 nM) was incubated with the various concentrations of CsrA indicated at the bottom of each lane. The positions of bound and free RNA are shown at the left of each panel. (B) In vitro transcribed acrAB RNA (0.5 nM) was incubated with the various concentrations of CsrA indicated at the bottom of each lane. The positions of bound and free RNA are shown at the left of each panel. (C) Effect of mutating putative CsrA binding sites on CsrA-acrAB RNA interactions. RNA electrophoretic mobility shift assays were performed using mutant acrAB RNA (0.5 nM). The concentration of CsrA was indicated at the bottom of each lane. The positions of bound and free RNA are shown. (D) Binding curve for the reaction shown in B.

Figure 6.

Absence of CsrA and CsrA binding sites on acrB and acrA-gfp mRNA stability. (A) Analysis of acrB mRNA stability in Salmonella enterica serovar Typhimurium wild-type and csrA mutant strain. (B) Analysis of acrA-gfp mRNA stability in S. Typhimurium containing pAcrA-TF-WT and pAcrA-TF-SDM translational GFP fusions.

Figure 7.

Effect of CsrA on acrA-GFP translation. Coupled transcription-translation reactions were performed with a PURExpress kit using pT7-acrA-GFP and pT7-acrA-GFP Mut CsrA BS translational fusions in the presence and absence of purified CsrA-His protein (320 nM). Fluorescence was measured at excitation and emission wavelengths of 492 and 520 nm, respectively using a FLUOstar Optima. Each experiment was performed three times.

Figure 8.

Primary sequence and secondary RNA structures of WT and mutant acrAB leader regions. Primary DNA sequence of wildtype 5′ UTR of acrAB and mutated 5′ UTR of acrAB in which the nucleotides making up the CsrA binding sites have been substituted (gray shaded box). Ribosome binding site (dashed rectangle box). (A) The RNA structures of the acrAB upstream region in the two plasmids of pAcrA-GFP-WT (B) and pAcrA-GFP-SDM (C) were predicted by RNAfold and their folding free energies were −12.30 kcal/mol and −6.0 kcal/mol, respectively. (Substituted nucleotides in gray shaded box)

Figure 9.

AcrA-GFP expression from the wildtype and mutated acrAB leader region. GFP expression from the wildtype (pAcrA-GFP-WT) and mutated (pAcrA-GFP-SDM) translational fusions in SL1344 and SL1344 csrA::aph. GFP levels were calculated from three biological replicates; error bars indicate the standard deviation.

Figure 10.

Induction of AcrB in SL1344 and SL1344 csrA::aph in the presence of 2 mM indole. GFP levels were calculated from three biological replicates; error bars indicate the standard deviation.

Figure 11.

AcrB expression levels in SL1344, MDR mutants of SL1344 and respective csrA mutants. Expression of AcrB in SL1344 (WT), csrA::aph, ΔacrB, ramR::aph, ΔramR/ csrA::aph, MDR mutant and MDR mutant/csrA::aph. Internal control used in the experiment was the β subunit of RNA polymerase. The RNA polymerase Western blot confirms that there is an equal amount of protein in each well. Band intensity was determined using GeneSys, this information was used to assign a relative concentration of AcrB to each band, expressed in percentage of wild-type. A ΔacrB mutant was used as a negative control.