Single mutations in BraRS confer high resistance against nisin A in Staphylococcus aureus

Abstract

Nisin A is a lantibiotic produced by Lactococcus lactis that is widely used as a food preservative. In Staphylococcus aureus, the BraRS two-component system (TCS) senses nisin A and regulates the expression of the ABC transporter VraDE, which is responsible for nisin A resistance. In this study, we exposed S. aureus to a sub-minimum inhibition concentration of nisin A and obtained three spontaneous mutants that were highly resistant to this lantibiotic, designated as SAN (S. aureus nisin resistant) 1, SAN8, and SAN87. In the wild-type S. aureus strain, VraDE expression was induced by nisin A. In contrast, SAN8 and SAN87 showed constitutively high VraDE expression, even in the absence of nisin A, while SAN1 showed higher BraRS expression, which resulted in high VraDE expression in the presence of nisin A. We identified a single mutation in the promoter region of braXRS in SAN1, whereas SAN8 and SAN87 had single mutations in braR and braS, respectively. Interestingly, even the unphosphorylated form of the mutant BraR protein induced VraDE expression. These results indicate that conformational changes in BraS or BraR resulting from the point mutations may result in the constitutive expression of VraDE, allowing S. aureus to adapt to high concentrations of nisin A.

Keywords: ABC transporter; mutation; nisin A; resistance; two-component system.

Figure 1

Expression of vraD in the MW2 and nisin A‐resistant strains. Protein and mRNA expression levels of vraD were evaluated by immunoblotting and quantitative PCR, as described in the Section 2. (a) Quantitative analysis of vraD expression in the MW2, SAN1, SAN8, and SAN87 strains. *p < 0.05, as determined by Dunnett's post hoc tests compared to untreated MW2. (b) Immunoblotting analysis of VraD expression in the MW2, SAN1, SAN8, and SAN87 strains

Figure 2

Mutation sites in the braXRS region in isolated mutants. The mutation sites in the braXRS region in the isolated mutants are indicated by white arrows. The nucleotide sequence upstream of braXRS in the MW2 strain is shown, with the −35 and −10 regions indicated in the box. The transcription initiation start sites are labeled with an asterisk, and the ATG translation initiation codons are indicated in bold

Figure 3

Expression of vraD in wild‐type MW2 and in the mutants. The protein and mRNA expression levels of vraD were evaluated by quantitative PCR (a) and immunoblotting (b), as described in the Section 2. The wild‐type strain MW2 and the SAN1, SAN8, and SAN87 mutants, as well as their braRS‐inactivated and braRS‐complemented strains, were investigated. *p < 0.05, as determined by Dunnett's post hoc tests compared to untreated MW2

Figure 4

Expression of braR and the braXRS promoter activity in the MW2 and SAN1 strains. The expression of braR mRNA in the MW2 and SAN1 strains was evaluated by quantitative PCR (a) as described in the Section 2. The promoter activity of braXRS was evaluated using a β‐galactosidase reporter system (b), as described in the Section 2. *p < 0.05, as determined by t test

Figure 5

Activity of the vraDE promoter in the mutants. The vraDE promoter activity was evaluated using a β‐galactosidase reporter system as described in the Section 2. The plasmid for the reporter assay was constructed by fusing the vraDE promoter region with the gene encoding β‐galactosidase. Next, the plasmid was transduced into various strains, and β‐galactosidase activity was evaluated. *p < 0.05, as determined by Dunnett's post hoc tests compared to untreated MW2

Figure 6

Effect of the mutated braR gene on the expression of vraD. The expression of vraD in the MW2, SAN8, and braRS‐inactivated strains and in the MW2 or SAN8 braRS‐inactivated strains complemented with braRS (MW2 or SAN8) (a) or braR (MW2 or SAN8) (b) was investigated by quantitative PCR as described in the Section 2. *p < 0.05, as determined by Dunnett's post hoc tests compared to untreated MW2

Figure 7

BraR electrophoretic mobility shift assay (EMSA). (a) The nucleotide sequence of the vraDE promoter region and the DNA fragments used in this study. DNA fragments with or without the BraR‐binding site were used. Gray shadow, palindromic sequence: squares, −35, −10 box; *, vraD transcriptional start site; bold, vraD translation initiation codon. (b) EMSA of BraR using two DNA fragments. Fragments were labeled with DIG and incubated with recombinant unphosphorylated (rBraR, left) or phosphorylated BraR protein (rBraR‐phos, right) as described in the Section 2. After electrophoresis, DNA bands were detected as described in the Section 2

Figure 8

The vraDE promoter activity in the braS‐inactivated mutants. The vraDE promoter activity was evaluated using a β‐galactosidase reporter system as described in the Section 2. The plasmid for the reporter assay was constructed by fusing the wild‐type vraD promoter region or the vraD promoter region with the BraR‐binding site deleted with the gene encoding β‐galactosidase. Next, the plasmid was transduced into various strains, and β‐galactosidase activity was evaluated. *p < 0.05, as determined by Dunnett's post hoc tests compared to untreated MW2

Figure 9

Protein alignments of BraR with other proteins and amino acid sequence of BraS. Protein alignment of BraR with other response regulators exhibiting homology with BraR (a). The active site aspartate residue (arrow), the two switch residues (dashed arrows), and the dimer interface regions (shown in the box and the dashed box) are shown. The triangle represents the mutation site in the mutant. Protein sequence of BraS (b). The dashed underline, double underline, and underline represent the region for the membrane‐spanning region, histidine kinase domain, and ATPase domain region, respectively. The active site histidine residue (the arrow), the mutation site in the SAN87 strain (black triangle), and the mutation sites reported previously (white triangle) are shown