Osmotic pressure can regulate matrix gene expression in Bacillus subtilis

Abstract

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non-specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non-specifically co-ordinate the behaviour of cells in communities composed of many different species.

Figure 1. Real time measurements of pellicle strength

(a) Pellicles were grown inside a cuvette cell on top of an Ares G2 strain controlled rheometer. The diameter of the outside well was 34 mm and that of the inside cylinder was 15 mm. The setup was held at a constant temperature of 30°. ( (b) Oscillatory torque, τ, was measured at a strain of 0.5% approximately every two hours for wild type bacteria in MSgg (red), MSgg supplemented with 20% by mass of 20 kDa PEG (blue), MSgg with 15% by mass of PEG with no bacteria (green) and pure MSgg inoculated with a matrix mutant that was not capable of synthesizing exopolysaccharide (black). The pellicle began to show rigid behavior after ~30 hours for both pure and PEG supplemented medium. Note, however, that the supplemented medium saturated faster and did not reach the level of robustness of the pure medium. (c) Oscillatory torque, τ, for wild type pellicles grown in MSgg medium supplemented with 20 kDa PEG at 0 - 25% mass fractions. D) 3610 was grown in a liquid biofilm medium for 3 days with/without polymer addition. Pictures were taken using a SPOT camera (Diagnostic Instruments, USA).

Figure 2. Osmotic pressure regulates matrix gene expression

(a) P_tapA activity in a wild type strain carrying P_tapA-lacZ (RL4582) was measured as a function of the mass fraction of 20 kDa PEG (blue) and 70 kDa dextran (black) added to the medium. (b) P_tapA activity in a wild type strain carrying P_tapA-lacZ (RL4582) was measured medium supplemented with various concentrations of different-sized PEG (right) and Dextran (left) polymers prepared at set osmotic pressures: dark blue diamonds, magenta squares and green triangles corresponded respectively to 5-8,1-3 and 0 atm additional osmotic pressure exerted by the polymers. (c) P_tapA-lacZ activity was measured in a sinR mutant (in which tapA is expressed constitutively) (RL4585). The curves represent 3 experiments, performed in duplicates, in which different polymers were used to increase the osmotic pressure; 70 kDa Dextran 600 Da PEG and 20 kDa PEG. Error bars are the standard deviation.

Figure 3. Polymer stimulates sporulation gene expression in a KinD-dependent manner

A wild type strain (IKG10; black columns) and its kinD mutant derivative (IKG603; white columns), both carrying a sporulation gene reporter P_spoIIA-lacZ, were grown with the indicated polymers. The columns represent 3 experiments, performed in duplicates., Error bars are the standard deviation.

Figure 4. Polymer inhibits matrix gene expression in a KinD-dependent manner

A wild type strain (RL4582; black columns) and its kinD mutant derivative (IKG602; white columns), both carrying a matrix gene reporter P_tapA-lacZ were grown with the indicated polymers. P_tapA activity was measured under conditions of low (LOW) and high (HIGH) osmotic pressure. The columns represent 3 experiments, performed in duplicates, in which different polymers described in figure 2B were used to increase the osmotic pressure. Error bars are the standard deviation.

Figure 5. The uncoupling of sporulation gene expression from increasing osmotic pressure is specifically dependent on KinD

Strain 3610 and its kinase mutant derivatives, kinA (IKG604), kinB (IKG605), kinC (IKG606), kinD (IKG603), all carrying P_spoIIA-lacZ (a sporulation promoter), were grown in biofilm-inducing medium. P_spoIIA activity is expressed in Miller units. The columns represent 3 experiments, performed in duplicates, in which different polymers described in figure 2B were used to increase the osmotic pressure. Error bars are the standard deviation.

Figure 6. Stimulatory effect of osmotic pressure on Spo0A-P-directed gene is specifically dependent on KinD

Strain 3610 carrying P_sdpA-lacZ (IKG609) and its derivatives with the kinase mutations ΔkinC (IKG611), ΔkinA ΔkinB (IKG612) and kinD (IKG610) were grown in LB (non-sporulation) medium in a shaking culture to early stationary phase. P_sdpA activity is expressed in Miller units. The curves represent 3 experiments, performed in triplicates. Error bars are the standard deviation.

Figure 7. Increasing osmotic pressure mimics the effect of matrix production on matrix and sporulation gene expression in a matrix mutant

Panels (A) and (B): The activities of P_tapA-lacZ in a wild type strain (RL4582) and its Δeps (IKG600) mutant derivative (A) and of P_spoIIA-lacZ in a wild type strain (IKG10) and its Δeps derivative (IKG601) (B) were determined without added polymer and with osmolarity increased (using the polymers described in Fig. 2B) to 2 atm (LOW) and 5-8 atm (High). The results shown are the average of using multiple polymers in triplicate. Panels (c) and (d): The activities of P_tapA-lacZ in a Δeps mutant lacking kinD (IKG607) (c) and of P_spoIIA-lacZ in an Δeps mutant lacking kinD (IKG608) (d) were determined without added polymer and at 2 atm (LOW) and 5-8 atm (High) as indicated above. The columns represent 3 experiments, performed in duplicates, in which different polymers described in figure 2B were used to increase the osmotic pressure. Error bars are the standard deviation.

Figure 8. Amino acid substitutions in a transmembrane segment of KinD impair osmosening

Panel A. Transmembrane segment II of KinD, which is similar to the transmembrane segment of EnvZ is highlighted in yellow. Panels B and C. The activities of P_spoIIA-lacZ (b) and P_tapA-lacZ (c) were measured at the indicated atms with derivatives of a mutant deleted for kinD (RL4552) and containing the indicated mutant and wild type alleles of kinD at sacA (IKG613). kinD-CACHE is mutant for the CACHE domain (Chen et al, 2012; (IKG614) and kinD-TMII contains the coding sequence for the transmembrane segment of KinB in place of the coding sequence for the second (II) transmembrane domain of KinD (IKG615). High osmolarity was as described for Fig. 7. The columns represent 3 experiments, performed in duplicates, in which different polymers described in figure 2B were used to increase the osmotic pressure. Error bars are the standard deviation.

Figure 9. Heterologous EPS inhibits matrix gene expression

An Δeps mutant containing P_tapA-lacZ (IKG600) was grown in medium supplemented with either 20 or 30% EPS extracted from B. subtilis 3610, Pseudomonas aeruginosa PA14 and E. coli W3100 pellicles. Percent EPS was calculated by dry weight in the total solution. The assay was performed in 96 well plates.