Abstract
BackgroundBiofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation.ResultsIn this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain.ConclusionsThis suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.
Highlights
Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide
We have previously shown that biofilms of P. aeruginosa disperse upon carbon source starvation and that starvation results in a rapid reduction in c-di-GMP [4]
We investigated biofilms of P. aeruginosa after starvation-induced dispersal and show that the nondispersing biomass has a high proportion of morphotypic variants and that these variants have single nucleotide polymorphism (SNP) in genes associated with biofilm formation, in particular, genes associated with the production or degradation of c-di-GMP
Summary
Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Bacteria typically disperse from biofilms in response to environmental cues, such as nutrient starvation, oxygen limitation and some signal molecules, e.g. nitric oxide (NO) [2,3,4]. While it is clear that these cues or pathways can be manipulated for biofilm control, e.g. by exposing biofilms to exogenously added NO donors, exposure to dispersing conditions rarely results in complete biofilm dispersal [8, 9]. A monospecies biofilm growing on a single carbon substrate will develop zones of differing oxygen or nutrient availability, e.g. oxygenic, limited oxygenic and anoxic zones, due to incomplete diffusion of oxygen from the medium or as a consequence of rapid utilisation by the bacteria within the biofilm [12,13,14]. Dispersal as a consequence of these environmental cues reflects cellular physiology and it is likely that incomplete dispersal is a reflection of physiological heterogeneity across the biofilm [16]
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