Testing the Hypothesis of Quorum Sensing in Vibrio fischeri : Luminescence, Motility, and Biofilm

Abstract

The individual behaviour of prokaryotic organisms such as bacteria often gives rise to complexity that is commonly associated with multicellular behaviour. The transition from unicellular to multicellular behaviour occurs in response to chemical signals, called autoinducers, which bacteria generate and receive internally within a given population. These autoinducers control the gene expression necessary for the emergence of group-behaviour-phenotype. This phenomenon is called quorum sensing (QS). An example of the quorum sensing control of gene regulation has been the luminescence (lux) operon in Vibrio fischeri. The luxI and ainS quorum signalling systems work in conjunction to regulate luminescence in V. fischeri. LuxI and AinS are acyl-synthases that catalyse the production of the autoinducers C6-HSL and C8-HSL respectively. These autoinducers bind to LuxR, a transcriptional activator of the lux operon, which activates expression of the lux genes causing an increase in luminescence. It was shown that quorum signalling also affects motility and biofilm formation in bacteria. However, the evidence with respect to these phenotypes is conflicting and inconclusive, the reason being the state of quorum is ambiguously defined. It is not properly known whether the observed collective behaviour is purely a result of physical crowding of bacteria, or that both chemical signalling and crowding contribute to this phenomenon. This work attempts to address these issues by studying luminescence, motility, and biofilm, a diverse set of behaviours, yet closely linked to each other in V. fischeri-squid symbiosis.

We studied the luminescence response of V. fischeri to both endogenous and externally added signals at per-cell and population level. Experiments with ES114, a wild-type strain of V. fischeri, and ainS mutant showed that (i) luminescence per cell does not mutually correlate with the cell-density, indicating that bacteria do not show greater response to the signal at higher densities; (ii) the activity of the lux signalling circuit shows a strong dependence on the growth stage, (iii) the cells do not show enhanced growth, i.e., they do not derive fitness benefits at higher densities in response to the signal. We anticipated that the culture with a higher cell-density should exhibit greater per-cell-luminescence. However, we found that the luminescence curve of the culture with lower density crosses that of the cultures with higher densities during the exponential phase. Kinetic modelling of the luxI mRNA expression showed that the expression profile qualitatively agrees with the luminescence trend observed in the cultures, supporting the observation that growth-phase plays a major role in regulating the luminescence gene expression.

We also studied the effect of autoinducers on motility of V. fischeri. V. fischeri uses flagella to move into the inner crypts of the light organ of the squid. The bacterium secretes autoinducers, encounters secretions of the light organ, and slows down during the final stage of colonization process. Studies have shown that flagellar elaboration is repressed as a consequence of ainS signalling. However, those studies were soft-agar migration assays and carried out with the mutant strain of ainS. We measured real-time planktonic motility of ES114 and the signalling mutant strains of V. fischeri in response to autoinducers added exogenously at different concentrations. We found that the autoinducers do not affect the motility of the strains. We also showed that reduction in motility is purely a consequence of physical crowding of bacteria, and chemical signalling may not be involved in the process. It was shown that reduction in motility leads to biofilm formation. Motile bacteria must lose flagella in order to form biofilm, and signalling controls biofilm formation in many species. Our study on motility showed that reduction in motility occurs because of physical crowding in V. fischeri. Hence, we explored the possibility that physical crowding might lead to formation of biofilm rather than signalling in this species. We quantified exopolysaccharide production by crystal violet assay, which revealed that planktonic cells produce exopolysaccharides, in addition to biofilm cells. The study revealed that V. fischeri cells always produce exopolysaccharides irrespective of their physiological state. We examined the effect of signalling on biofilm in ES114 and the mutant strains using gene-expression analysis. We quantified the expression of various genes involved in biofilm formation and found that both ES114 and the mutants expressed rscS and sypP indicating that exopolysaccharide production is not under the control of autoinducers. Therefore, we hypothesized that biofilm formation in V. fischeri may be a result of physical agglomeration of cells. Our observations indicate that the state of quorum is inadequately defined and there is no direct measure of the underlying process. Multicellular behaviour in V. fischeri is regulated by a complex interplay of cell-density, signalling, and other factors such as the growth phase of the culture, indicating that the state of quorum employs different mechanisms to regulate various phenotypes. Our study reveals that QS is an intricate process, and the accepted mechanisms for QS are incomplete at best.