Stabilization of <i>Bacillus subtilis</i> Spx under cell wall stress requires the anti-adaptor protein YirB

<div><p>Spx is a global transcriptional regulator present in low-GC Gram-positive bacteria, including the model bacterium <i>Bacillus subtilis</i> and various human pathogens. In <i>B</i>. <i>subtilis</i>, activation of Spx occurs in response to disulfide stress. We recently reported, however, that induction of Spx also occurs in response to cell wall stress, and that the molecular events that result in its activation under both stress conditions are mechanistically different. Here, we demonstrate that, in addition to up-regulation of <i>spx</i> transcription through the alternative sigma factor σ^M, full and timely activation of Spx-regulated genes by cell wall stress requires Spx stabilization by the anti-adaptor protein YirB. YirB is itself transcriptionally induced under cell wall stress, but not disulfide stress, and this induction requires the CssRS two-component system, which responds to both secretion stress and cell wall antibiotics. The <i>yirB</i> gene is repressed by YuxN, a divergently transcribed TetR family repressor, and CssR~P acts as an anti-repressor. Collectively, our results identify a physiological role for the YirB anti-adaptor protein and show that induction of the Spx regulon under disulfide and cell wall stress occurs through largely independent pathways.</p></div

Model of regulation of Spx and <i>yirB</i> under cell wall stress.

<p>(A) Model of induction of the Spx regulon under cell wall stress. In exponentially growing cells (before induction), <i>spx</i> is constitutively expressed but Spx is actively degraded by the coordinated action of YjbH (i.e. the adaptor protein) and ClpXP (i.e. the protease). A fraction of Spx, however, remains stable at least in part due to the activity of YirB. This fraction of Spx, which is partially oxidized [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007531#pgen.1007531.ref004" target="_blank">4</a>], drives the expression of Spx-controlled genes. After treatment with cell wall antibiotics (i.e. early induction), σ^M activates the expression of <i>spx</i> through the P_M1 promoter, and CssR~P activates transcription of <i>yirB</i>. YirB then plays an important role in the stabilization of the newly synthesized Spx. Later in the process (i.e. late induction), induction of <i>spx</i> through P_M1 is maximal, which leads to an increase in the amount of newly synthesized Spx. At the same time, the expression of <i>yirB</i> and presumably its role in stabilization decreases. The accumulation of Spx leads to induction of the genes in its regulon. (B) Model for regulation of <i>yirB</i>. The protein YuxN (here presented as a dimer for simplicity) binds the YuxN boxes upstream and downstream the <i>yirB</i> promoter forming a DNA loop. Upon treatment with cell wall antibiotics, the CssR protein becomes phosphorylated by the CssS histidine kinase. CssR~P then binds the CssR BoxI, which overlaps the YuxN box, leading to derepression of the <i>yirB</i> promoter. The synthesized YirB protein binds YjbH and prevents Spx proteolysis.</p

A post-transcriptional event contributes to activation of Spx in response to cell wall stress.

<p>(A) The <i>spx</i> gene was placed under control of IPTG as the only source of <i>spx</i> for the cells. (B) WT cells were grown with a fixed concentration of inducer and treated or not with 2 μg ml^-1 ampicillin, 200 μg ml^-1 fosfomycin, or 1 μg ml^-1 vancomycin. Spx protein levels were monitored before and after 30 min of treatment using western blot. (C) The <i>spx</i> mRNA and <i>trxB</i> mRNA levels were simultaneously studied using northern blot. The blots are representative of at least two biological replicates. The “-” symbol indicates untreated cells.</p

The anti-adaptor YirB is required for Spx stabilization.

<p>(A) Accumulation of Spx in response to vancomycin treatment was determined in a time-course experiment in WT and <i>ΔyirB</i> cells using western blot. A representative blot is shown in the left panel. Relative quantification of Spx protein levels in both strains is plotted on the right panel. Data were normalized using the Spx levels of the WT strain before induction as reference. Error bars represent SEM of three biological replicates. One, two, and three asterisks indicate significant differences with P < 0.05, P < 0.01 and P < 0.001 respectively, as estimated using the T-test. The statistical analysis to compare Spx levels in both strains was independently performed for every time point. (B) Expression levels (i.e. β-galactosidase activity) of the Spx-controlled gene <i>trxB</i> in WT, <i>ΔyirB</i>, and complementation strain (<i>ΔyirB + amyE</i>::<i>yirB)</i> following treatment or not with 1 μg ml^-1 vancomycin. (C) Analysis of the activity of the <i>trxA</i> and <i>trxB</i> promoters in WT and <i>ΔyirB</i> during exponential and early stationary phase in LB medium (solid lines). The bacterial growth curves are also displayed on the figure (dashed lines) (D) Analysis of the activity of the <i>trxA</i> and <i>trxB</i> promoters in the strains <i>ΔyjbH</i> and <i>ΔyjbH ΔyirB</i>. (E)- (F) Effect of YirB on Spx stability during cell wall stress. The half-life of Spx was determined in exponentially growing cells treated or not with vancomycin. The percentage of remaining Spx was normalized with respect to time 0 min. (G) The concentration of YjbH-HA was determined by western blot in WT and Δ<i>yirB</i> cells. All experiments were performed in triplicate. Error bars indicate SEM.</p

Desarrollo y evaluación de un medio de cultivo alternativo para la multiplicación de Azospirillum brasilense C16 mediante diseños estadísticos secuenciados

For mass plant growth-promoting inoculant production, a high-yield culture medium is fundamental. Sequential application of statistical designs was used to optimize Azospirillum brasilense C16 biomass production. Six nutritional (glycerol, glutamate, mannitol, citric acid, yeast extract and K2HPO4 3H2O) and three mineral sources (MgSO4 7H2O, FeCl3 and NaCl) were evaluated using five statistical experiments Placket-Burman, factorial design, steepest ascent, response surface analysis, and mineral screening. The optimum medium composition (g L-1) was as follows: 28.33 glutamate, 2.92 yeast extract, 1.34 K2HPO4 3H2O, 0.5 MgSO4 7H2O and 0.02 FeCl3. After 24 hours of incubation, protein (32.04 μg) and dry biomass (1.51 g L-1) were 1.72 and 1.68 times higher than in conventional growth medium.  Para la producción masiva de inoculantes basados en bacterias promotoras de crecimiento vegetal (PGPR), es fundamental un medio de cultivo de alto rendimiento. La aplicación secuenciada de diseños estadísticos fue usada para optimizar la producción de biomasa de Azospirillum brasilense C16, seis fuentes nutricionales (glicerol, glutamato, manitol, acido cítrico, extracto de levadura y K2HPO4 3H2O) y tres fuentes minerales (MgSO4 7H2O, FeCl3 y NaCl) fueron evaluadas mediante cinco experimentos estadísticos - Placket-Burman, factorial fraccionado, diseño de paso ascendente, análisis de superficie de respuesta y screening mineral, para tal efecto. La composición optimizada del medio (g L-1) fue: 28,33 glutamato, 2,92 extracto de levadura, 1,34 K2HPO4 3H2O, 0,5 MgSO4 7H2O y 0,02 FeCl3, la cual luego de 24 h de incubación permitió producir una cantidad de proteína (32,04 μg) y biomasa seca (1,51 g L-1) del 1,72 y 1,68 veces más alta, respectivamente, en relación al medio de cultivo convencional.  

Vancomycin induces <i>yirB</i> through the CssRS two-component system.

<p>(A) The <i>yirB</i> transcript (~300 nt) is induced under cell wall stress, but not disulfide or ethanol stress. Total RNA was isolated from WT cells treated or not with 1 μg ml^-1 vancomycin, 500 μM diamide, and 5% ethanol, and <i>yirB</i> mRNA levels studied by northern blot. The “-” symbol indicates untreated cells. (B) Induction of <i>yirB</i> requires the two-component system CssRS. Transcriptional profile of <i>yirB</i> in WT, <i>ΔcssR</i>, and the complementation strain (<i>ΔcssR + amyE</i>::<i>cssR</i>) after treatment with 1 μg ml^-1 vancomycin as determined by northern blot. (C) Induction of <i>yirB</i> by CssR, under vancomycin-induced stress, requires the phosphorylation of the Asp52 residue. The expression of <i>yirB</i> was studied by northern blot in Δ<i>cssR</i> cells complemented ectopically with either <i>cssR</i> or <i>cssR</i>^D52A from the <i>cssR</i> native promoter. The presented blots are representative of three independent experiments.</p

CssR~P induces <i>yirB</i> expression by antagonizing YuxN repression.

<p>(A) Genetic context of the <i>yirB</i> 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 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007531#pgen.1007531.s004" target="_blank">S3 Fig</a>). (C) Promoter truncation analysis was used to determine the contribution of the upstream DNA sequences on <i>yirB</i> regulation. For this, the <i>yirB</i> gene along with the promoter truncations were integrated at the <i>amyE</i> site and used to complement a Δ<i>yirB</i> mutant. The positions were mapped with respect to the +1 site as shown in Fig 6B. The <i>yirB</i> mRNA levels were determined by northern blot. (D) The mRNA <i>yirB</i> 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 P_{<i>yirB(-538)</i>}<i>-yirB</i>. The three <i>yirB</i> promoter regions are identical except for the point mutations. The lower-case letters indicate the sites were the mutations were introduced (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007531#pgen.1007531.g006" target="_blank">Fig 6B</a>). (E) Analysis of the P_<i>yirB</i>-<i>lacZ</i> and P_<i>yuxN</i>-<i>lacZ</i> reporter fusions in cells lacking YuxN. (F) Activity of the <i>yirB</i> promoter in WT, as well as in the <i>ΔcssR</i>, <i>ΔyuxN</i>, and <i>ΔcssR ΔyuxN</i> knockout mutants. (G) The <i>yuxN</i> gene is upregulated in response to cell wall stress. (H) Analysis of the <i>yirB</i> promoter featuring truncations in the YuxN boxes. The different promoters were fused to the <i>lacZ</i> gene and its activity measured in WT, <i>ΔcssR</i>, and <i>ΔyuxN</i> 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.</p

Strains used in this study.

<p>Strains used in this study.</p

YirB also stabilizes Spx in cells with conditional expression of <i>spx</i>.

<p>(A) Graphical description of the experimental scenarios in Fig 3B and 3C. The regulation of <i>Spx</i> in wild-type cells was included for reference (right box). (B) Spx levels were monitored in WT and Δ<i>yirB</i> cells featuring conditional expression of <i>spx</i> (i.e. <i>spx</i> expression level was fixed using 60 μM IPTG, scenario i) using western blot. Cells were treated or not with 1 μg ml^-1 vancomycin. (C) Spx levels were monitored in WT and Δ<i>yirB</i> cells featuring conditional expression of <i>spx</i> (i.e. <i>spx</i> expression was fixed using 20 μM IPTG, scenario ii) using western blot. At the same time, expression of <i>spx</i> was upregulated by adding inducer to achieve 60 μM IPTG and cells were treated or not with 1 μg ml^-1 vancomycin. The blots presented are representative of three biological replicates, which produced similar results.</p