Vibrio cholerae: Safety in numbers?

Vibrio cholerae is one of the most interesting micro-organisms inhabiting our oceans. When in water, V. cholerae can often be found in close association with zooplankton, particularly crustaceans because of the abundance of chitin present. Chitin is essential for Vibrio species.; it can be used as a sole nutrient source, furthermore, attachment to chitin causes up-regulation of many genes, and promotes HGT (horizontal gene transfer).  Grazing by protozoans has previously shown to have significant effects on the V. cholerae life-style by inducing biofilm formation, likely to be a protective mechanism. However, as V.cholerae would be found in large abundance in these biofilms, quorum sensing is likely to have a role. This study by Sun et al. (2015) aims to elucidate the role of quorum sensing (QS) and chitin metabolism on grazing resistance in V. cholerae biofilms, on both biotic and abiotic surfaces.

In order to demonstrate the importance of QS in chitin metabolism, and hence biofilm formation ΔhapR mutant V. cholerae were cultured, as were wild type V. cholerae. The gene, hapR is a ‘master response regulator’ of QS. To test for grazing resistance these were cultured on microtitre plates, with and without a carbon source and glucose, (abiotic), chitin flakes and the exoskeleton of Artemia (biotic) for 24 hours, reaching a final concentration of 10⁶ cells ml^-1 (determined using MTT staining), before being exposed to the grazer Rhynchomonas nasuta, a surface feeding flagellate at a concentration of 10⁴ cells ml^-1. To test whether any toxic products were produced by V. cholerae conferring the avoidance of predation, the cell-free supernatants of biofilms were collected. R. nasuta (10⁴ cells ml^-1) were exposed to this supernatant and enumerated microscopically, the ammonia concentration of the supernatant was also measured. The number of CFUs of V. cholerae present in glucose and chitin flakes was determined using a flow cell technique.

Biofilm production on abiotic surfaces was negligible, even when supplemented with a carbon source and glucose for both WT and ΔhapR, likewise no significant decrease in R. nasuta was observed. Abitoic surfaces supplemented with glucose, and chitin flakes did show an increase in the abundance in R. nasuta. However, biofilm formation on chitin flakes was x3053 that of the abiotic surfaces. Compared to the WT, a significant decrease in grazer mortality was noted on biofilms with the ΔhapR colonies present. Biofilms formed on Artemia caused inhibition of grazing by R. nasuta. This was attributed to the ammonium production during chitin metabolism by V. cholerae. Biofilms consisting of ΔhapR mutants showed a decreased grazing resistance compared with the WT (on chitin flakes), suggesting that QS is of some importance during this process. Analysis of the role of QS in chitin utilisation revealed that expression of several genes was significantly reduced in the QS negative ΔhapR mutant, including the entire chitin catabolic operon amongst others.

I think this study presents convincing evidence that QS plays a key role in chitin metabolism, biofilm formation and grazing protection. However, this study only tested the impact that one species of grazer has on biofilms composed of singular bacterial species. Therefore how representative this study is of the interactions that occur in the environment is questionable. Whilst a number of further projects can arise from this project, I think examining these interactions in the context of climate change and disease will be one of the most interesting, applicable and informative. Climate change is likely to augment many of the interactions that have been so well documented in the literature; what impact will these changes have on our biota, populations and ecosystems?

Jack 

Sun, S., Tay, X.M.T., Kjelleberg,S., Rice, S.A. and Mcdougald,D. (2015). Quorum sensing-regulated chitin metabolism provides grazing resistance to Vibrio cholerae biofilms. International Society for Microbial Ecology.  1751-7362/15, pp1-9.