Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants

Abstract

Background Biofilms 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. Results In 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. Conclusions This 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.

Complex Ecotype Dynamics Evolve in Response to Fluctuating Resources

“…In response to starvation conditions, microbes often respond by dispersing and initiating detachment from biofilm structures ( 36 – 38 ). However, recent investigation of heterogeneity in biofilms has suggested that carbon starvation can select for dispersal-insensitive mutants ( 39 ). To determine if repeated conditioning to longer starvation conditions affects the evolution of E. coli biofilms, we quantified biofilm density in 96-well plates after 24 h of static growth.…”

Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas

“…The molecular mechanisms by which different nutrients influence biofilm formation or stability are being explored from various perspectives, from signal transduction through chemoreceptors [22] to population dynamics and evolutionary aspects [23]. In a recent study using P. fluorescens Pf01 as a model organism, the impact of a wide array of nutrients on biofilm formation was analysed, using glycerol as a carbon source to define the effect of compounds that may not be strictly "nutrients" for this microorganism but still act as environmental signals [24].…”

Dispersal of a dominant competitor can drive multispecies coexistence in biofilms

“…Less drastically, the growing edge of a biofilm often loses population members continuously, though in lower numbers relative to sloughing events, in an erosion-like process 99 . The environmental cues that drive dispersal are diverse and vary between different species, but quorum sensing regulation and starvation response are commonly involved 72,73,[100][101][102][103] . How biofilms' often high variance in structure over time and space impacts microbial ecosystem species richness remains an important direction for future work using both in vitro systems accessible to live time lapse microscopy, as well as more naturalistic systems and settings [104][105][106] .…”