Biofilm Formation Drives Transfer of the Conjugative Element ICE Bs1 in Bacillus subtilis

Abstract

Horizontal gene transfer by integrative and conjugative elements (ICEs) is a very important mechanism for spreading antibiotic resistance in various bacterial species. In environmental and clinical settings, most bacteria form biofilms as a way to protect themselves against extracellular stress. However, much remains to be known about ICE transfer in biofilms. Using ICEBs1 from Bacillus subtilis, we show that the natural conjugation efficiency of this ICE is greatly affected by the ability of the donor and recipient to form a biofilm. ICEBs1 transfer considerably increases in biofilm, even at low donor/recipient ratios. Also, while there is a clear temporal correlation between biofilm formation and ICEBs1 transfer, biofilms do not alter the level of ICEBs1 excision in donor cells. Conjugative transfer appears to be favored by the biophysical context of biofilms. Indeed, extracellular matrix production, particularly from the recipient cells, is essential for biofilms to promote ICEBs1 transfer. Our study provides basic new knowledge on the high rate of conjugative transfer of ICEs in biofilms, a widely preponderant bacterial lifestyle in the environment, which could have a major impact on our understanding of horizontal gene transfer in natural and clinical environments.IMPORTANCE Transfer of mobile genetic elements from one bacterium to another is the principal cause of the spread of antibiotic resistance. However, the dissemination of these elements in environmental contexts is poorly understood. In clinical and environmental settings, bacteria are often found living in multicellular communities encased in a matrix, a structure known as a biofilm. In this study, we examined how forming a biofilm influences the transmission of an integrative and conjugative element (ICE). Using the model Gram-positive bacterium B. subtilis, we observed that biofilm formation highly favors ICE transfer. This increase in conjugative transfer is due to the production of extracellular matrix, which creates an ideal biophysical context. Our study provides important insights into the role of the biofilm structure in driving conjugative transfer, which is of major importance since biofilm is a widely preponderant bacterial lifestyle for clinically relevant bacterial strains.

Keywords: Bacillus subtilis; ICEBs1; biofilms; extracellular matrix; horizontal gene transfer.

FIG 1

Biofilm formation enhances ICEBs1 transfer. (A) Donor cells with a kanamycin resistance cassette inserted in ICEBs1 were mated with recipient cells bearing an intergenic chloramphenicol resistance cassette in a 1:1 ratio on non-biofilm-inducing solid and liquid media (LB and MSNc [white bars]) and biofilm-inducing solid and liquid (pellicle-inducing) media (LBGM and MSgg [gray bars]). Statistical analysis indicates a significant increase in ICEBs1 transfer when biofilm is formed (Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (B) Conjugation-deficient donor cells (nicK and cwlT) were mated with WT cells on MSgg to assess mating efficiency. Statistical analysis shows a significant decrease in mating efficiency with the nicK and cwlT donor cells (one-way ANOVA; **, P < 0.01). (C) Transformation-deficient cells (comK) were mated on MSNc and MSgg, and mating efficiency was compared to that of WT cells. Statistical analysis shows no significant difference in mating efficiencies between comK and WT cells (Student’s t test). (D) Single and double mutant ICEBs1 activation pathway donor cells were mated with WT recipient cells on MSgg. Statistical analysis shows a significant increase of ICEBs1 mating efficiency between recA and WT donor cells, but not with the rapI mutant (one-way ANOVA; ****, P < 0.0001). While the double mutant showed a decrease in mating efficiency, that difference was not significant. For all panels, mating efficiency was measured after 20 h for solid media and 28 h for liquid media at 30°C. The results shown are representative of at least three independent experiments, and error bars represent the standard error of the mean (SEM).

FIG 2

Lower donor/recipient ratio allows increased ICEBs1 transfer in biofilm. WT donor cells were diluted and mated with a fixed number of WT recipient cells on MSgg. Transfer efficiency was measured after 20 h at 30°C. Donor/recipient ratios of 1:10 and 1:100 show significantly more ICEBs1 transfer efficiency than the 1:1 ratio (one-way ANOVA; ****, P < 0.0001). The results shown are representative of at least three independent experiments, and error bars represent the SEM.

FIG 3

Biofilm formation and ICEBs1 conjugation activation are simultaneous. Recipient and donor cells harboring a P_tapA-yfp fluorescent marker in the amyE locus were mated on MSgg. Biofilms were then harvested after 8, 12, 16, 20, and 24 h. For each time point, cells were used to quantify biofilm expression by fluorescence-activated cell sorter (FACS) and to assess mating efficiency. The results shown are representative of at least three independent experiments, and error bars represent the SEM.

FIG 4

Matrix production is important for conjugation. (A) WT donor cells and ICEBs1⁰ attB-down recipient cells were mated on LB, LBGM, MSNc, and MSgg for 20 h at 30°C, and the donor attB site was amplified by qPCR. There was no significant difference in the ICEBs1 excision rate when there was biofilm formation (Student’s t test). (B) sinR donor and recipient cells were mated on non-inducing media, and mating efficiency was compared to that of WT cells mated on non-biofilm-inducing (LB and MSNc [white bars]) and biofilm-inducing solid media (LBGM and MSgg [gray bars]). Mating efficiency was measured after 20 h at 30°C. sinR mutants led to higher ICEBs1 transfer efficiency compared to WT cells on noninducing media. For all panels, the results shown are representative of at least three independent experiments, and error bars represent the SEM.

FIG 5

Both matrix components are important for ICEBs1 conjugation. (A) Donor and recipient cells deleted for the epsA–O (eps) and tasA operons were mated together or with WT cells on MSgg. The first genotype shown represents the donor genotype, while the second represent the recipient. Statistical analysis showed that absence of matrix and nonproduction from the recipient cells reduced significantly ICEBs1 transfer efficiency (one-way ANOVA; **, P < 0.01; ***, P < 0.001). (B) Donor and recipient cells mutated for either eps or tasA operon were mated on MSgg. Statistical analysis showed that absence of either exopolysaccharides or amyloid-like fibers in both donor and recipient decreases ICEBs1 transfer significantly. However, eps donors and tasA recipients can complement each other and restore the WT level of conjugation, while tasA donors and eps recipients are significantly different from WT pairs (one-way ANOVA; *, P < 0.05; **, P < 0.01). For both panels, mating efficiency was measured after 20 h at 30°C. The results shown are representative of at least three independent experiments, and error bars represent the SEM.