Expression of the multidrug resistance transporter NorA from Staphylococcus aureus is modified by a two-component regulatory system - PubMed (original) (raw)

Expression of the multidrug resistance transporter NorA from Staphylococcus aureus is modified by a two-component regulatory system

B Fournier et al. J Bacteriol. 2000 Feb.

Abstract

To dissect genetically the regulation of NorA, a multidrug transporter of Staphylococcus aureus, we analyzed the differential expression of the norA promoter using a transcriptional fusion with a beta-lactamase reporter gene. Expression studies with an arlS mutant revealed that the norA promoter is ArlS dependent. The arlR-arlS locus was shown to code for a two-component regulatory system. The protein ArlR has strong similarity to response regulators, and ArlS has strong similarity to protein histidine kinases. We have also analyzed the 350-bp region upstream of the Shine-Dalgarno sequence of norA by gel mobility shift experiments. It was shown that only the 115-bp region upstream of the promoter was necessary for multiple binding of an 18-kDa protein. From transcriptional fusions, we have localized four different putative boxes of 6 bp, which appear to play a role in the binding of the 18-kDa protein and in the up-regulation of norA expression in the presence of the arlS mutation. Furthermore, the gel mobility shift of the 18-kDa protein was modified in the presence of the arlS mutation, and the arlS mutation altered the growth-phase regulation of NorA. These results indicate that expression of norA is modified by a two-component regulatory system.

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Figures

FIG. 1

Maps of the different DNA segments of the norA promoter examined in this study. The numbers indicate the nucleotide positions according to Yoshida et al. (38). (A) Schematic map of the norA promoter. The −35 and −10 consensus sequences are indicated by black boxes, and the Shine-Dalgarno site is marked SD. The repeated sequences, shown in panel D, are indicated by hatched boxes. (B) PCR fragments used in band shift experiments. (C) Schematic map showing the DNA cloned upstream of the β-lactamase gene in transcriptional fusions. (D) Sequence of the region upstream of the −35 consensus sequence. Repeated sequences are boxed, and the −35 and −10 sequences are underlined.

FIG. 2

Gel mobility shift analysis of the interaction of protein extracts from the wild-type strain ISP794 with different fragments of the norA promoter and the effect of unlabeled DNA. The radiolabeled fragment (arrow) was incubated with increasing amounts of protein extracts. The labeled fragments used in these experiments are L4-R1 (315 bp) (A), L2-R2 (153 bp) (B), L2-R3 (87 bp) (C), L1-R2 (60 bp) (D), and L5-R2 (39 bp) (E). The protein(s) binds to the tested fragment and retards its mobility (a different gel was used for each fragment). An unlabeled fragment of 350 bp amplified by PCR from a Klebsiella oxytoca promoter and an unlabeled fragment of the tested fragment serve as specificity control (NSPE DNA and SPE DNA, respectively). Protein and DNA concentrations and ratios of unlabeled fragments to labeled fragments used in this assay are indicated in the tables above the figures.

FIG. 3

Isolation of the protein from the wild-type strain ISP794 binding to different fragments of the norA promoter. Different fragments of DNA were immobilized on magnetic beads. Proteins binding to these fragments were then used for different analyses. (A) SDS-PAGE analysis of protein released from DNA affinity magnetic beads. Lane 1, standard proteins (in kilodaltons); lane 2, fragment L2-R2; lane 3, fragment L1-R2; lane 4, fragment L2-R3. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified extracts from strain ISP794. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of ISP794. Free DNA is indicated by an arrow.

FIG. 4

Gel mobility shift analysis of the interaction of the protein extracts from different strains with the complete norA promoter. The radiolabeled fragment L2-R2 (arrow) was incubated with increasing amounts of protein extracts. The protein(s) binds to the tested fragment and retards its mobility. Lanes: A, strain ISP794; B, arlS mutant BF15; C, arlS mutant BF15 containing pBF17.

FIG. 5

Isolation of the protein from the mutant BF15 binding to the fragment L2-R2. (A) SDS-PAGE analysis of protein released from affinity-purified extracts from different strains. Lane 1, standard proteins (in kilodaltons); lane 2, purified protein from ISP794; lane 3, purified protein from BF15. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified protein extracts from BF15 and fragment L2-R2. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of BF15. Free DNA is indicated by an arrow.

FIG. 6

Effect of the arlS mutation on NorA regulation during the growth. The parent strain MT23142 (circles) and the arlS mutant BF15 (squares) containing the plasmid pBF8-30 were grown at 37°C. The ratio of β-lactamase/OD600 was calculated as an estimate of specific activity. Open symbols indicate OD600, and solid symbols indicate the ratio of β-lactamase activity/OD600.

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Expression of the multidrug resistance transporter NorA from Staphylococcus aureus is modified by a two-component regulatory system - PubMed (original) (raw)