Kin Discrimination Modifies Strain Distribution, Spatial Segregation, and Incorporation of Extracellular Matrix Polysaccharide Mutants of Bacillus subtilis Strains into Mixed Floating Biofilms

Abstract

Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivated Bacillus subtilis strains as mixtures of isogenic, kin, and nonkin strain combinations in the biofilm-promoting minimal medium under static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation, and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtilis. IMPORTANCE Biofilm communities have beneficial and harmful effects on human societies in natural, medical, and industrial environments. Bacillus subtilis is a biotechnologically important bacterium that serves as a model for studying biofilms. Recent studies have shown that this species engages in kin discriminatory behavior during swarming, which may have implications for community assembly, thus being of fundamental importance. Effects of kin discrimination on fitness, genotype segregation, and success of extracellular matrix (ECM) polysaccharide (EpsA-O) mutant invasion into biofilms are not well understood. We provide evidence that kin discrimination depends on the antagonism of the dominant strain against nonkin by using environmental strains with determined kin types and integrated fluorescent reporters. Moreover, this antagonism has important implications for genotype segregation and for when the bacteria are mixed with ECM producers. The work advances the understanding of kin-discrimination-dependent bacterial sociality in biofilms and its role in the assembly of multicellular groups.

Keywords: Bacillus subtilis; biofilms; incorporation of extracellular matrix polysaccharide mutant; kin discrimination.

FIG 1

Relative cell frequency of two strains in mixed pellicles is influenced by nonkin interactions. The relative cell frequencies in pellicles of two isogenic (the same strain labeled with different fluorescent markers), two kin, and three nonkin strain combinations are shown. The first strain in the combination is always represented by the darker bar with the diagonal pattern. Two strains differentially labeled with different antibiotic resistance markers were mixed as revitalized spores in given combinations (designated numbers of PS strains). They were grown in 2 mL of MSgg for 24 h at 37°C, and the relative frequency of each strain was assessed in the formed pellicles after sonication by CFU counts. The proportions of the two strains in the pellicles were significantly different in kin and isogenic strain combinations than in nonkin strain combinations (Student t test; two-tail, P < 0.05). Mean values of relative frequency of the first strain in each strain combination (±SD) are shown as darker bars with a diagonal pattern. Experiments were performed in at least three biological replicates (n ≥ 3).

FIG 2

Fluorescent microscopy imaging of isogenic, kin, and nonkin strains in floating biofilms. Pellicles were formed by mixing of two isogenic, kin, and nonkin B. subtilis PS strains that constitutively express fluorescent reporters. Biofilms were examined by confocal laser scanning microscopy (CLSM) (bar, 200 μm) after 16 h of biofilm growth. This time point was chosen to ensure imaging before sporulation started. Images were pseudocolored yellow (for YFP-tagged B. subtilis strains PS-216, PS-218, PS-18, and PS-68) and magenta (for mKate2-tagged strains B. subtilis PS-216, PS-218, and PS-196). Representative images of at least three biological replicates are shown.

FIG 3

Multiscale spatial segregation analysis (MSSA) of the floating biofilm’s image stacks obtained by confocal laser scanning fluorescence microscopy (CLSM). (A) Plots of calculated segregation levels ($\widehat{S_d}$) in mixed strain biofilms are shown for the kin pair (PS-68 YFP + PS-216 mKate2) strains and nonkin pair (PS-218 YFP + PS-216 mKate2) strains as a function of the dimension of field of view. Examined field of view, which represents what is seen under the microscope at different zoom levels, is a square with the dimensions d by d. $\widehat{S_d}$ was calculated for simulated maximal and minimal segregations. $\widehat{S_d}$ is 1 if only one strain is present in the field of view and 0 if both strains are present in the expected ratios (ratio of the two strains in all images of the stack) in the field of view, graphically represented as the average field of view. (B) Microscopy images (middle slice of CLSM stack) representing the field of view with d of 12 μm, 120 μm, and 1,200 μm. Top and bottom rows show corresponding simulated images; middle row represents original images as taken by the microscope. For the simulation of maximal segregation, it was assumed that one strain completely excluded the other strain.

FIG 4

Effect of epsA–O mutation on floating biofilm formation in different B. subtilis strains. (A) Indicated strains were inoculated in exponential growth phase as monocultures in 12-well plates in 2 mL MSgg medium and incubated at 37°C. Images of representative biofilms in the wells (diameter, 20.5 mm) after 20 h of growth are shown. (B) Close-up images of pellicles of the indicated strains obtained by stereomicroscope; bar, 5 mm.

FIG 5

Incorporation of isogenic (or kin) ΔepsA–O mutant strain into the pellicle when mixed with the wild-type (WT) EpsA-O producer and exclusion of nonkin strains. Two strains differentially labeled with different antibiotic resistance markers were mixed in exponential growth phase in given combinations (numbers represent specific PS strain; Δeps is an indication of the mutant). They were grown in 2 mL of MSgg for 24 h at 37°C. Log₁₀ values of the final mean ratios between ΔepsA–O mutants and WTs (±SD) determined by CFU counts in the formed pellicles after sonication are shown. Experiments were performed in at least four biological replicates (n ≥ 4), except for the kin combination PS-18 ΔepsA–O mixed with PS-216 (n = 2).