A Model of the Quorum Sensing System in Vibrio fischeri Using P Systems

Quorum sensing is a cell density dependent gene regulation system that allows an entire population of bacterial cells to communicate in order to regulate the expression of certain or specific genes in a coordinated way depending on the size of the population. In this paper we present a model of the Quorum Sensing System in Vibrio fischeri using a variant of membrane systems called P systems. In this framework each bacterium and the environment are represented by membranes and the rules are applied according to an extension of Gillespie’s Algorithm called Multicompartmental Gillespie’s Algorithm. This algorithm runs on more than one compartment and takes into account the disturbance produced when chemical substances diffuse from one compartment or region to another. Our approach allows us to examine the individual behaviour of each bacterium as an agent as well as the emergent behaviour of the colony as a whole and the processes of swarming and recruitment. Our simulations show that at low cell densities bacteria remain dark while at high cell densities some bacteria start to produce light and a recruitment process takes place that makes the whole colony of bacteria to emit light. Our computational modelling of quorum sensing could provide insights that may enable researchers to develop new applications where multiple agents need to robustly and efficiently coordinate their collective behaviour based only on a very limited information of the local environment

Implications of Rewiring Bacterial Quorum Sensing

Bacteria employ quorum sensing, a form of cell-cell communication, to sense changes in population density and regulate gene expression accordingly. This work investigated the rewiring of one quorum-sensing module, the lux circuit from the marine bacterium Vibrio fischeri. Steady-state experiments demonstrate that rewiring the network architecture of this module can yield graded, threshold, and bistable gene expression as predicted by a mathematical model. The experiments also show that the native lux operon is most consistent with a threshold, as opposed to a bistable, response. Each of the rewired networks yielded functional population sensors at biologically relevant conditions, suggesting that this operon is particularly robust. These findings (i) permit prediction of the behaviors of quorum-sensing operons in bacterial pathogens and (ii) facilitate forward engineering of synthetic gene circuits

Host-selected mutations converging on a global regulator drive an adaptive leap towards symbiosis in bacteria

Host immune and physical barriers protect against pathogens but also impede the establishment of essential symbiotic partnerships. To reveal mechanisms by which beneficial organisms adapt to circumvent host defenses, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri by colonization and growth within the light organs of the squid Euprymna scolopes. Serial squid passaging of bacteria produced eight distinct mutations in the binK sensor kinase gene, which conferred an exceptional selective advantage that could be demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion that were mediated by cell-associated matrices including symbiotic polysaccharide (Syp) and cellulose. binK variation also altered quorum sensing, raising the threshold for luminescence induction. Preexisting coordinated regulation of symbiosis traits by BinK presented an efficient solution where altered BinK function was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations

Directed evolution of Vibrio fischeri LuxR for improved response to butanoyl-homoserine lactone

LuxR is the 3-oxohexanoyl-homoserine lactone (3OC6HSL) dependent transcriptional activator of the prototypical acyl-homoserine lactone (AHL) quorum sensing system of Vibrio fischeri. Wild-type LuxR exhibits no response to butanoyl-HSL (C4HSL) in quantitative bioassays at concentrations of up to 1 µM; a previously described LuxR variant (LuxR-G2E) exhibits a broadened response to diverse AHLs, including pentanoyl-HSL (C5HSL), but not to C4HSL. Here, two rounds of directed evolution of LuxR-G2E generated variants of LuxR that responded to C4HSL at concentrations as low as 10 nM. One variant, LuxR-G4E, had only one change, I45F, relative to the parent LuxR-G2E, which itself differs from wild-type at three residues. Dissection of the four mutations within LuxR-G4E demonstrated that at least three of these changes were simultaneously required to achieve any measurable C4HSL response. The four changes improved both sensitivity and specificity towards C4HSL relative to any of the other 14 possible combinations of those residues. These data confirm that LuxR is evolutionarily pliable and suggest that LuxR is not intrinsically asymmetric in its response to quorum sensing signals with different acyl-side chain lengths

Modeling the Quorum Sensing Signaling Regulatory Network in \u3cem\u3eVibrio fischeri\u3c/em\u3e

Quorum sensing is a mechanism by which bacteria can sense the levels of signaling molecules and respond by controlling the expression of target genes. The marine bacterium, Vibrio fischeri, has been extensively studied as a model for the quorum sensing mechanism in Gram-negative bacteria. In order to systematically investigate the quorum sensing regulatory network in V. fischeri, a conceptual model was first established based on the existing knowledge. Next, molecular microbiology and bioinformatics techniques were employed to both qualitatively and quantitatively characterize the system. These techniques included the quantification of the 3-oxo-C6-HSL concentrations in the cell culture supernatant using a bioluminescent bioreporter strain of E. coli, the measurements of the messenger RNA levels of quorum sensing genes (luxI, luxR, ainS and litR) using the reverse transcription-polymerase chain reaction (RT-PCR), as well as the sequence analysis of the promoter regions of quorum sensing related genes. A mathematical model composed of ordinary differential equations was created to characterize the regulatory process. The simulated annealing method was used to minimize the weighted discrepancy between the modeling output and the experimental data with correlations ranging from 0.85 to 0.99. This study, mathematically modeled the comprehensive quorum sensing regulatory system, which encompasses 3-oxo-C6-HSL, lux operon (luxR and luxICDABEG), C8-HSL, ainS, ainR, luxO, and litR, and can benefit the understanding of dozens of similar quorum sensing regulatory systems

Modelling Vibrio fischeri’s behaviour Using P Systems

Quorum sensing is a cell density dependent gene regulation system that allows an entire population of bacterial cells to communicate in order to regulate the expression of certain or specific genes in a coordinated way depending on the size of the population. In this paper we present a model of the Quorum Sensing System in Vibrio fischeri using a variant of membrane systems called P systems. In this framework each bacterium and the environment are represented by membranes and the rules are applied according to probabilities computed using mass action law. This approach allows us to examine the individual behaviour of each bacterium as an agent as well as the behaviour of the colony as a whole and the processes of swarming and recruitment. Our simulations show that at low cell densities the bacteria remain dark while at high cell densities some bacteria start to produce light and a recruitment process takes place that makes the whole colony of bacteria to emit light. The above mentioned behaviour of our in silico bacteria maps well experiments and in vitro observations.Ministerio de Ciencia y Tecnología TIC2002-04220-C03-0

GacA regulation of luminescence in the symbiotic bacterium Vibrio fischeri

The symbiosis of Euprymna scolopes and Vibrio fischeri depends on the ability of the bacterium to colonize the squid and produce light. GacA is an activator of symbiosis phenotypes including luminescence though it is not understood how GacA functions. My hypothesis, based on other bacterial models, is that GacA activates expression of CsrBs, functional RNAs, which sequester and repress a translational repressor, CsrA. I approached this question with mutagenesis and complementation techniques. We found mutation of CsrA or addition of a CsrB in trans could complement the GacA mutant for symbiosis and luminescence phenotypes. The inability of other luminescence regulators including a constitutively expressed lux operon to complement GacADelta indicated that luminescence was regulated post-transcriptionally. A putative CsrA binding site found in the sequence of the lux mRNA increased light production when deleted. These data demonstrate that GacA regulates luminescence post-transcriptionally via CsrB/CsrA antagonism, supporting my hypothesis

Spontaneous phenotypic suppression of GacA-defective Vibrio fischeri is achieved via mutation of csrA and ihfA

Background: Symbiosis defective GacA-mutant derivatives of Vibrio fischeri are growth impaired thereby creating a selective advantage for growth-enhanced spontaneous suppressors. Suppressors were isolated and characterized for effects of the mutations on gacA-mutant defects of growth, siderophore activity and luminescence. The mutations were identified by targeted and whole genome sequencing. Results: Most mutations that restored multiple phenotypes were non-null mutations that mapped to conserved domains in or altered expression of CsrA, a post-transcriptional regulator that mediates GacA effects in a number of bacterial species. These represent an array of unique mutations compared to those that have been described previously. Different substitutions at the same amino acid residue were identified allowing comparisons of effects such as at the R6 residue, which conferred relative differences in luminescence and siderophore levels. The screen revealed residues not previously identified as critical for function including a single native alanine. Most csrA mutations enhanced luminescence more than siderophore activity, which was especially evident for mutations predicted to reduce the amount of CsrA. Although CsrA mutations compensate for many known GacA mutant defects, not all CsrA suppressors restore symbiotic colonization. Phenotypes of a suppressor allele of ihfA that encodes one subunit of the integration host factor (IHF) heteroduplex indicated the protein represses siderophore and activates luminescence in a GacA-independent manner. Conclusions: In addition to its established role in regulation of central metabolism, the CsrA regulator represses luminescence and siderophore as an intermediate of the GacA regulatory hierachy. Siderophore regulation was less sensitive to stoichiometry of CsrA consistent with higher affinity for the targets of this trait. The lack of CsrA null-mutant recovery implied these mutations do not enhance fitness of gacA mutants and alluded to this gene being conditionally essential. This study also suggests a role for IHF in the GacA-CsrB-CsrA regulatory cascade by potentially assisting with the binding of repressors of siderohphore and activators of luminescence. As many phosphorelay proteins reduce fitness when mutated, the documented instability used in this screen also highlights a potentially universal and underappreciated problem that, if not identified and strategically avoided, could introduce confounding variability during experimental study of these regulatory pathways

Genetic and Ecological Characterization of Indigoidine Production by Phaeobacter sp. strain Y4I

The Roseobacter clade is a widely distributed, abundant, and biogeochemically active lineage of marine alpha-proteobacteria. Members of the Roseobacter lineage are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage in colonization. In this work, Phaeobacter sp. strain Y4I was found to produce the water soluble, blue pigment indigoidine via a nonribosomal peptide synthase-based biosynthetic pathway encoded by a novel series of genetically linked genes, termed igiBCDFE. Comparison of wildtype, non-pigmented, and hyper-pigmented Y4I insertional mutants demonstrated a perfect correlation between indigoidine production and the inhibition of Vibrio fischeri on agar plates, revealing a previously unrecognized bioactivity of this molecule. Competitive co-cultures of V. fischeri and Y4I showed that the production of indigoidine by Y4I significantly inhibits surface colonization of V. fischeri. Subsequent experiments identified a role for quorum sensing in the production of this secondary metabolite. Y4I has two independent quorum sensing systems, termed pgaIR and phaIR. Transposon insertions in each of the phaIR genes resulted in defects in indigoidine production. A transposon insertion in pgaR confers a null indigoidine phenotype. All of these quorum sensing mutants are unable to inhibit the growth of V. fischeri in competition experiments. These strains also have altered biofilm and motility phenotypes suggesting a role for the quorum sensing systems in regulation of these activities. Identification of the N-acyl homoserine lactone signaling molecules that are produced by Y4I was achieved using a combination of (AHL) bioreporters and mass spectrometry analyses. The two dominant AHLs were found to be N-octanoyl homoserine lactone (C8-HSL) and a putative monounsaturated N-3-hydroxydodecanoyl homoserine lactone (3OHC12:1-HSL) when the strain is grown on a complex medium. Evidence is provided that AHL production is not wholly cell-density dependent in this strain. Finally, a comprehensive analysis of the luxRI-type quorum sensing systems in sequenced roseobacter genomes provide evidence that these genetic systems are closely related among lineage members and likely share a common ancestor