Stabilization of Bacillus subtilis Spx under cell wall stress requires the anti-adaptor protein YirB
Fig 6
CssR~P induces yirB expression by antagonizing YuxN repression.
(A) Genetic context of the yirB gene. (B) The transcription start site was mapped using 5’ RACE, and the identification of the putative -10 and -35 boxes was performed manually. Two putative CssR boxes were located upstream the -35 box, which exhibited similarity with the consensus CssR binding sequences (S3 Fig). (C) Promoter truncation analysis was used to determine the contribution of the upstream DNA sequences on yirB regulation. For this, the yirB gene along with the promoter truncations were integrated at the amyE site and used to complement a ΔyirB mutant. The positions were mapped with respect to the +1 site as shown in Fig 6B. The yirB mRNA levels were determined by northern blot. (D) The mRNA yirB levels were studied by northern blot in cells harboring the mutant CssR BoxI* (5’-TGACttTtTAGatAtt-3’) or CssR BoxII* (5’-aGaaATAAAATTAAaC-3’), and compared with PyirB(-538)-yirB. The three yirB promoter regions are identical except for the point mutations. The lower-case letters indicate the sites were the mutations were introduced (see Fig 6B). (E) Analysis of the PyirB-lacZ and PyuxN-lacZ reporter fusions in cells lacking YuxN. (F) Activity of the yirB promoter in WT, as well as in the ΔcssR, ΔyuxN, and ΔcssR ΔyuxN knockout mutants. (G) The yuxN gene is upregulated in response to cell wall stress. (H) Analysis of the yirB promoter featuring truncations in the YuxN boxes. The different promoters were fused to the lacZ gene and its activity measured in WT, ΔcssR, and ΔyuxN cells before and after 20 min of treatment with 1 μg ml-1 vancomycin. Error bars represent SEM of at least three independent replicates. One, two, and three asterisks indicate significant differences with P < 0.05, P < 0.01 and P < 0.001 respectively, as estimated using one-way ANOVA and the Tukey’s HSD test for Fig 6E, and the T-test for Fig 6F–6H. NS indicates no significant differences.