Deciphering the influence of NaCl on social behaviour of Bacillus subtilis

Abstract

Various environmental signals, such as temperature, pH, nutrient levels, salt content and the presence of other microorganisms, can influence biofilm's development and dynamics. However, the innate mechanisms that govern at the molecular and cellular levels remain elusive. Here, we report the impact of physiologically relevant concentrations of NaCl on biofilm formation and the associated differences in an undomesticated natural isolate of Bacillus subtilis. NaCl exposure and its uptake by bacterial cells induced substantial changes in the architecture of pellicle biofilm and an upsurge in the expansion of biofilm colonies on agar surfaces. We have observed the upregulation of genes involved in motility and the downregulation of genes involved in the biosynthesis of extracellular matrix components through the transcription factor sigD, suggesting the possible underlying mechanisms. To further support these observations, we have used ΔsigD and ΔsrfAC null mutants, which showed compromised NaCl-induced effects. Our results indicate that NaCl induces a lifestyle shift in B. subtilis from a sessile biofilm state to an independent unicellular motile state. Overall, we present evidence that NaCl can reprogramme gene expression and alter cellular morphology and the state of cells to adapt to motility, which facilitates the expansion of bacterial colonies.

Keywords: Bacillus subtilis; NaCl; biofilm; flagella; surface motility; surfactin.

Figure 1.

Pellicle formation of Bacillus subtilis and quantification in static liquid media. (a) Top view of pellicles of B. subtilis IITKSM1 (wild-type) under different NaCl concentrations [(i)W _NaCl = 0% (ii) W _NaCl = 0.5% (iii) W _NaCl = 1% (iv) W _NaCl = 1.5% (v) W _NaCl = 2%] in rich liquid culture media at ca. 48 h of incubation at 30°C (24 well plate, well diameter—15.5mm). All images are contrast-enhanced for better visibility. (b). Dry weight of pellicles (W _pellicle) as a function of W _NaCl (n = 9, triplicates with three independent experiments). The data plotted is the mean obtained from three independent experiments. (c) Optical density was measured for wild-type planktonic cells (A ₆₀₀) and crystal violet-stained surface-adhered biofilm (A ₅₉₅) as a function of W _NaCl (n = 9) after 48 h. (d) Secreted carbohydrate concentration was estimated using the phenol-sulphuric acid method for varying NaCl concentrations (n = 9, triplicates with three independent experiments) after 48 h. (e). Intracellular sodium concentration in the absence and presence of 2 wt.% NaCl as measured by ICP-MS (triplicates). Standard error (s.e.) = [stdev/sqrt (count)]. W _NaCl represents the amount of NaCl in the media by weight percentage.

Figure 2.

Mutually exclusive lifestyle between biofilm and planktonic state of B. subtilis IITKSM1 under the influence of NaCl. (a) Changes in the gene expression level of motility and biofilm-inducing genes in the presence of NaCl (2 wt.%) compared to cells grown without NaCl (n = 9, triplicates with three independent experiments). (b) TEM images showing cells grown without NaCl. (c) Cells grown with 2 wt.% NaCl (right) in liquid media. The dark blue arrows (b) indicate cell chaining, and the dark blue arrows (c) point towards the flagella. Standard error (s.e.) = [stdev/sqrt (count)].

Figure 3.

Pellicle formation of ΔsigD strain of B. subtills and quantification in static liquid media. (a) Top view of pellicles of ΔsigD B. subtilis IITKSM1 under different NaCl concentrations [(i)W _NaCl = 0% (ii) W _NaCl = 0.5% (iii) W _NaCl = 1% (iv) W _NaCl = 1.5% (v) W _NaCl = 2%] in rich liquid culture media after ca. 48 h of incubation at 30°C (24 well plates, well diameter—15.5 mm). All images are contrast-enhanced for better visibility. (b) Dry weight of pellicles (W _pellicle) as a function of W _NaCl (n = 9) after 48 h. The data plotted is the mean obtained from triplicates with three independent experiments. (c) Optical density was measured for wild-type planktonic cells (A ₆₀₀) and crystal violet-stained surface adhered biofilm (A ₅₉₅) as a function of W _NaCl (n = 9) after 48 h. (d) TEM images showing ΔsigD cells grown without NaCl. (e) ΔsigD cells grown with 2 wt.% NaCl (right) in liquid media. Standard error (s.e.) = [stdev/sqrt (count)]. W _NaCl represents the amount of NaCl in the media by weight percentage.

Figure 4.

Biofilm colony expansion of B. subtilis on agar surface. (a) Representative time-lapse images showing the lateral expansion of wild type, Δ sigD and Δ srfAC cells in the absence and the presence of 2 wt.% NaCl (Plate diameter: 86 mm) (b) Diameter [mm] of the expanding biofilms (n = 9, triplicates with three independent experiments) on 1.2% rich agar media. (c) Profilometric images of wild-type biofilm colony centre grown without NaCl and with 2 wt.% NaCl, which clearly shows differences in height features n = 3. (d) Surface wettability of the colony centre grown without NaCl and with 2 wt.% NaCl (1.2% agar) is captured via the contact angles of water on the respective surfaces. Contact angles for 0% NaCl: 139.6° and 2% NaCl: 76.5°. Standard error (s.e.) = [stdev/sqrt (count)].

Figure 5.

Optical micrographs and analysis of B. subtilis motility on agar with and without NaCl. Time series of optical micrographs every 2 min capturing the lateral expansion of biofilms at the cellular level in (a) the absence of NaCl, (b) in the presence of 1 wt.% NaCl and (c) 2 wt.% NaCl. Microscopy experiments were performed ca. 8 h after the inoculation of culture on the agar plates. (d) The probability that a cell has displaced at least 3.5 times larger than the length of the bacteria, i.e. P (Δr²|_max) = ∑_i P_i(Δr²|_max), where √Δr²|_max > 3.5. Inset of (d) Representative trajectories (black and red correspond to the trajectories in the absence and presence of NaCl, respectively) of bacteria in both cases. (e) Mean square displacement of motile cells in the presence of 2 wt.% NaCl (red circles), 1 wt.% NaCl (grey circles), and in the absence (black squares) of NaCl. Distribution of exponents characterizing the dynamics of bacteria, in the presence and absence of NaCl, (f) for the initial regime (Δt < 3 s) and (g) the later regime (Δt > 3 s).