c-di-GMP distribution across single and mixed species biofilms
Harikrishnan A. S. Nair
Date of Issue2016-11-09
Bacteria in nature preferentially grow as a surface associated, matrix encapsulated structures, called biofilms. Biofilms play a major role in society, such as degrading waste and in the recycling of used water. At the same time, biofilms are responsible for many chronic, infectious diseases, biocorrosion of engineered structures and membrane fouling of water treatment plants. Bis-(3 ́-5 ́)-cyclic dimeric guanosine monophosphate (c-di-GMP) is an intracellular signaling molecule that plays a central role in the biofilm life cycle. While the molecular mechanisms of the c-di-GMP signaling pathway have been well studied, the differential distribution of c-di-GMP across biofilms, which commonly display physiological heterogeneity, is less well understood. This is partially due to the limitation of current methodologies for the quantification of c-di-GMP, which are based on chemical extraction. Chemical methodologies also fail to take the physiological heterogeneity of biofilm into consideration and thus represent an average c-di-GMP concentration across the entire biofilm. To address these issues, a ratiometric, image-based quantification method was developed here based on expression of the green fluorescence protein under the control of the c-di-GMP responsive cdrA promoter (Rybtke., et al. 2012). The approach has been successfully applied to biofilms at different developmental stages and as well as during dispersal. Using this dynamic, real-time monitor, a transient state of increased c-di-GMP (up to 2 h) before the expected decrease in c-di-GMP was observed for biofilms under starvation conditions. The observation has been validated by comparison with chemical measurements of c-di-GMP with increased temporal resolution. Transcriptomic analysis of starved biofilms and planktonic cultures indicated that 50 out of 52 signal transduction genes were induced upon starvation, including three diguanylate cyclase and three phosphodiesterase genes. Additionally, it was observed that c-di-GMP was localized to the outer boundary of mature colonies (diameter >50 μm) rather than showing a uniform distribution in smaller colonies (diameter <50 μm). After continuous starvation for up to 3 days, some of the biofilm remained in the flow cell and that biomass displayed high reporter fluorescence, suggesting those cells contained high concentrations of c-di-GMP. Isolation and genetic characterization of these cells lead to the conclusion that these colonies that failed to disperse upon carbon starvation were mutants. Ratiometric imaging was applied to a model mixed species biofilm consisting of Pseudomonas aeruginosa PAO1, Pseudomonas protegens Pf5 and Klebsiella pneumoniae KP1 to understand the distribution of c-di-GMP during mixed species biofilm growth. The c-di-GMP localization patterns of P. aeruginosa in the mixed species community were generally similar to that of monospecies biofilm although the total c-di-GMP level of P. aeruginosa was much less than that of the monospecies biofilm. Interestingly, the total c-di-GMP measured for the mixed species biofilm using LC-MS was in fact higher than that of any of the mono species biofilms. Starvation of the mixed species biofilm leads to dispersal and a transient increase of c-di-GMP similar to the monospecies biofilm. While some of the mixed species biofilm did not disperse, no morphotypic variants were detected, which contrasted with the monospecies biofilms. In conclusion, a method for the visualization and semi-quantification of c-di-GMP in situ, in real time was developed and validated. Using this method, c-di-GMP was found to be localized at the boundary of mature colonies. Under the improved temporal and spatial revolution, a transient increase in c-di-GMP was observed during starvation-induced dispersal and the reporter system was equally applicable to investigate c-di-GMP distributions in a mixed species biofilm system.