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. 2016 Nov 23;3(11):160438.
doi: 10.1098/rsos.160438. eCollection 2016 Nov.

Deciphering a survival strategy during the interspecific competition between Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1

Brinta Chakraborty  1 , Anish Mallick  2 , Sumana Annagiri  1 , Supratim Sengupta  3 , Tapas K Sengupta  1
Affiliations

Deciphering a survival strategy during the interspecific competition between Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1

Brinta Chakraborty et al. R Soc Open Sci. .

Abstract

Interspecific competition in bacteria governs colony growth dynamics and pattern formation. Here, we demonstrate an interesting phenomenon of interspecific competition between Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1, where secretion of an inhibitor by Pseudomonas sp. is used as a strategy for survival. Although B. cereus grows faster than Pseudomonas sp., in the presence of Pseudomonas sp. the population of B. cereus reduces significantly, whereas Pseudomonas sp. do not show any marked alteration in their population growth. Appearance of a zone of inhibition between growing colonies of two species on nutrient agar prevents the expanding front of the MSM-S1 colony from accessing and depleting nutrients in the region occupied by MSM-M1, thereby aiding the survival of the slower growing MSM-M1 colonies. To support our experimental results, we present simulations, based on a chemotactic model of colony growth dynamics. We demonstrate that the chemical(s) secreted by Pseudomonas sp. is responsible for the observed inhibition of growth and spatial pattern of the B. cereus MSM-S1 colony. Our experimental results are in excellent agreement with the numerical results and confirm that secreted inhibitors enable Pseudomonas sp. to survive and coexist in the presence of faster growing B. cereus, in a common niche.

Keywords: Bacillus; Pseudomonas; interspecific interaction; mathematical model.

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Figures

Figure 1.
Figure 1.
Survivorship graphs of Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1 in nutrient-rich conditions. (a) Growth characteristics of Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1 in nutrient broth at 0.5 h intervals; (b) total CFUs of Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1 on 0.6% nutrient agar plate over 15 days (U = 36, d.f.1 = d.f.2 = 6, p = 0.002). Growth was measured in terms of OD of bacterial culture grown in liquid media and total CFUs were measured in the case of semi-solid agar media. All values are represented as means ± s.d. For both liquid media and nutrient agar plate n = 6. Mann–Whitney U-test was performed to determine statistical significance.
Figure 2.
Figure 2.
Comparative growth analysis of competing bacterial species. (a) Bacterial CFUs of Bacillus cereus MSM-S1 produced when grown alone versus Bacillus cereus MSM-S1 during interspecific competition, grown in the presence of Pseudomonas sp. MSM- M1 after 16 h in nutrient broth (U = 36, d.f.1,2 = 6, p = 0.002); CFUs of Pseudomonas sp. MSM-M1 produced when grown alone versus Pseudomonas sp. MSM-M1 during interspecific competition, grown in the presence of Bacillus cereus MSM-S1 after 16 h in nutrient broth. (b) Comparison of growth (total CFUs) of Bacillus cereus MSM S1 grown alone versus Bacillus cereus MSM-S1 grown in the presence of Pseudomonas sp. MSM M1 on 0.6% agar plates on day 1 (U = 20.5; d.f.1,2 = 6; p = 0.699), day 5 (U = 30; d.f.1,2 = 6; p = 0.065), day 7 (U = 36; d.f.1,2 = 6; p = 0.002) and day 15 (U = 36; d.f.1,2 = 6; p = 0.002). (c) Pseudomonas sp. MSM-M1 grown alone versus Pseudomonas sp. MSM-M1 grown in the presence of Bacillus cereus MSM-S1 on 0.6% agar plates on day 1 (U = 19; d.f.1,2 = 6; p = 0.937), day 5 (U = 19.5; d.f.1,2 = 6; p = 0.937), day 7 (U = 20.5; d.f.1,2 = 6; p = 0.699) and day 15 (U = 30; d.f.1,2 =6; p = 0.065). All results are shown as means ± s.d. Graphs (b,c) are represented in log10 scale. (d) The relative interaction between Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1 on nutrient agar plate across different days. Mann–Whitney U-test was performed to determine statistical significance.
Figure 3.
Figure 3.
CLSM studies of cellular orientation and morphology during interspecific interaction. (a) CLSM images of the non-interacting and the interacting edges of Bacillus cereus MSM-S1 (a(i)(ii)) and Pseudomonas sp. MSM-M1 (a(iii)(iv)) colonies grown for 7 days. (b) CLSM studies of non-interacting (b(i–iii)) and interacting edges of Bacillus cereus MSM-S1 (b(iv–vi)) colonies. All the figures represented here are stacked in z-axis.
Figure 4.
Figure 4.
FESEM studies of cellular level interactions between Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1. FESEM images of the non-interacting and the interacting edges of Bacillus cereus MSM-S1 (a,b) and Pseudomonas sp. MSM-M1 (c,d).
Figure 5.
Figure 5.
Studies on the role of inhibitor for the mechanism of interference. Agar pieces taken (in triplicates) from the regions close to growing colonies of MSM-M1 (I and IV) and MSM-S1 (III and VI) and from the inhibition zone between MSM-M1 and MSM-S1 (II and V) were placed over (a) a lawn of Bacillus cereus MSM-S1 and over (b) a lawn of Pseudomonas sp. MSM M1. (c) Induction of sporulation in Bacillus cereus (after 6 and 12 h) by agar pieces taken from control plate (i) and (iv), from the regions close to grown colonies of MSM-M1 (ii) and (v) and from the inhibition zone between MSM-M1 and MSM-S1 colonies (iii) and (vi).
Figure 6.
Figure 6.
Colony growth over time. Time lapse images of colony growth dynamics. Images were generated based on simulations. Parameters used: a1 = 0.8, a2 = 0.2, hc2 = 0.5, Γ1 = 0.01, Γ2 = 0.005, γ2 = 0.39, λ2 = 1, δ2 = 0.01, d1 = 0.001, d2 = 0.0001, dh2 = 0, c12 = 0.4, η1 = 0.4.
Figure 7.
Figure 7.
Minimum distance between colonies. Variation of minimum distance (a) and log(minimum distance) (b) between the two colonies with time. Parameters used are the same as in figure 6. Figure was generated based on simulations.
Figure 8.
Figure 8.
Temporal variation of cell population. Variation in cell number of the (a) red (S1) and (b) green (M1) colonies with time. Parameters used are the same as in figure 6. Figure was generated based on simulations.
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