Protozoan grazing is an important mortality factor of bacteria in aquatic habitats. Vibrio cholerae, the etiological agent of cholera and natural inhabitant of brackish and seawater, relies on biofilm formation to defend against predation. Here, we investigated the ‘reverse grazer effect’ of V. cholerae biofilms induced by predation by the ciliate, Tetrahymena pyriformis.

V. cholerae was exposed to T. pyriformis in artificial seawater and biofilm biomass was determined by confocal laser scanning microscopy and crystal violet staining. Co-cultures were also performed using filter inserts to physically separate V. cholerae and T. pyriformis to determine if physical contact was required for the increase in biofilm biomass. The nutrients in the cell-free supernatants of T. pyriformis were quantified by biochemical assays and LC-MS, and addback experiments were performed to confirm their effects on V. cholerae biofilm formation. RNA-Seq and RT-PCR were used to identity V. cholerae genes involved in supernatant-induced biofilm formation.

When V. cholerae was co-incubated with T. pyriformis, the bacterium was eliminated in the planktonic phase, but microcolony numbers and surface coverage of the biofilm increased. The stimulation of biofilm formation was also observed when T. pyriformis was physically separated from the bacterial biofilm by filter inserts. The supernatants were analysed and it was determined that phosphate concentration (0.59 mM) and the presence of 4 amino acids (arginine 3.3 μM, asparagine 1.2 μM, glutamine 3.4 μM and serine 2.7 μM) in the T. pyriformis supernatant were important for stimulation of the V. cholerae biofilms, while carbon and nitrogen were not critical. RNA-Seq revealed that 112 transcripts were up-regulated and 371 transcripts were down-regulated in response to the cell-free supernatant. RT-PCR confirmed that the 4 amino acids induced V. cholerae biofilm formation through csrA. This data shows that V. cholerae has the ability to sense and respond to the presence of a protozoan predator by forming grazing resistant biofilms.