Quantitative analysis of the surficial and adhesion properties of the Gram-negative bacterial species Comamonas testosteroni modulated by c-di-GMP

Cyclic diguanylate monophosphate (c-di-GMP) is a ubiquitous intracellular secondary messenger which governs the transition from a bacterial cell’s planktonic state to biofilm formation by stimulating the production of a variety of exopolysaccharide material by the bacterial cell. A range of genes involved in c-di-GMP signaling in the Gram-negative species Comamonas testosteroni have been identified previously, yet the physical-chemical properties of the produced extracellular polymeric substances (EPS) and the bacterial adhesion characteristics regulated by c-di-GMP are not well understood. Here, we modulated the in vivo c-di-GMP levels of Comamonas testosteroni WDL7 through diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) gene editing. The strains and their adhesion properties were characterized by Fourier-transform infrared and two-dimensional correlation spectroscopy analysis (FTIR-2D CoS), contact angle and zeta potential measurements, atomic force microscopy (AFM) and extended-Derjaguin-Landau-Verwey-Overbeek (ExDLVO) analysis. Our results show that high c-di-GMP levels promoted the secretion of long-chain hydrophobic and electroneutral extracellular polysaccharides and proteins. The protein molecules on WDL7/pYedQ2 promoted the bacterial self-aggregation and adhesion onto negatively charged surfaces. In contrast, the reduction of intracellular c-di-GMP concentrations resulted in a nearly 80 % decrease in the adhesion of bacterial cells, although little change in the surface hydrophobicity or surface charge properties were observed for these cells relative to the wild type. These results indicate that the reduced adsorption of WDL7/YhjH that we observed may be caused by the flagellum-accelerated mobility at low c-di-GMP concentrations. Taken together, these results improve our mechanistic understanding of the effects of c-di-GMP in controlling bacterial physical-chemical properties and initial biofilm development.