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.2007 Apr;189(8):2996-3005.
doi: 10.1128/JB.01819-06. Epub 2007 Feb 2.

The transcriptional regulators NorG and MgrA modulate resistance to both quinolones and beta-lactams in Staphylococcus aureus

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The transcriptional regulators NorG and MgrA modulate resistance to both quinolones and beta-lactams in Staphylococcus aureus

Que Chi Truong-Bolduc et al. J Bacteriol.2007 Apr.

Abstract

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four beta-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA.

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Figures

FIG. 1.
FIG. 1.
Nucleotide and amino acid sequences of the 1,321 bp ofS. aureus DNA containing thenorG gene from ISP794 (complete sequence shown). The −35 and −10 sequences of the putative promoter region and the putative helix-turn-helix (H-T-H) region are shown. The coding region ofnorG, preceded by a putative ribosome-binding site (RBS), is demarcated by the ATG start codon and the TAA stop codon.
FIG. 2.
FIG. 2.
(A) Gel mobility shift analyses of the interactions of the crude cell extracts (CE) from ISP794 and QT1 and the purified NorG protein with the biotinylated 150-bpnorA promoter fragment. Competing unlabeled herring sperm DNA (nonspecific) andnorA promoter DNA (specific) were used to determine the specificity of promoter binding. The amount of labeled DNA used was 2 ng per reaction. The amount of protein used was 50 ng per reaction for the NorG protein and 100 ng per reaction for the crude cell extracts. (B) Gel mobility shift analyses of the interactions of the crude cell extracts from ISP794 and the purified NorG protein with the biotinylated 150-bpnorB P1 and P2 promoter fragments. (C) Schematic representation of the positions ofnorB and the three adjacent ORFs on theS. aureus N315 published genome (11). The two putative promoters and the putativerho-dependent terminator are indicated.
FIG. 3.
FIG. 3.
(A) Gel mobility shift analyses of the interactions of the purified NorG and MgrA proteins with the biotinylated promoters ofmgrA,norG, andnorC. (B) Gel mobility shift analyses of the interactions of the crude cell extracts from ISP794 and QT1 and the purified NorG and MgrA proteins with the biotinylated 150-bpabcA promoter. (C) Schematic representation of the overlapping promoter regionabcA-pbpD as published by Domanski et al. (4). Primers designed to amplifyabcA DNA generated a fragment that included the inverted repeat located 8 bp downstream from theabcA transcriptional start (+1). This region was shown to be essential for the expression of both genes. The −35 and −10 consensus sequences of theabcA andpbpD promoters are underlined and/or in bold. The inverted repeat region is underlined and in bold. The asterisk indicates the transcription start site forabcA. The boxed DNA region indicates theabcA promoter that was used in the gel mobility shift binding assay. ThepbpD promoter region used for the gel mobility shift assay contains the inverted repeat as well as the −10 and −35 regions (underlined). The inverted repeat is located at a distance of 46 bp from the transcription start site of thepbpD gene (T).
FIG. 4.
FIG. 4.
(A) Northern blot analyses of RNAs isolated from the specifiedS. aureus strains. The same amount of RNA (10 μg) was loaded in each lane, and loading was verified by ethidium bromide staining of 16S rRNA before RNAs were transferred onto a nylon membrane and hybridized with specific probes (abcA ornorG). (B) RT-PCR using RNA extracted at exponential phase. Each reaction used 10 picograms of total RNA as the template and primers specific to an internal region ofnorB. We used 16S rRNA as an internal control for the RT-PCR assays as described previously (27). Photographs of ethidium bromide-stained gels were scanned and analyzed using the NIH Scion Image program as described previously (5).
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References

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