Microfluidic study of the chemotactic response of Escherichia coli to amino acids, signaling molecules and secondary metabolites.

Quorum sensing and chemotaxis both affect bacterial behavior on the population level. Chemotaxis shapes the spatial distribution of cells, while quorum sensing realizes a cell-density dependent gene regulation. An interesting question is if these mechanisms interact on some level: Does quorum sensing, a density dependent process, affect cell density itself via chemotaxis? Since quorum sensing often spans across species, such a feedback mechanism may also exist between multiple species. We constructed a microfluidic platform to study these questions. A flow-free, stable linear chemical gradient is formed in our device within a few minutes that makes it suitable for sensitive testing of chemoeffectors: we showed that the amino acid lysine is a weak chemoattractant for Escherichia coli, while arginine is neutral. We studied the effect of quorum sensing signal molecules of Pseudomonas aeruginosa on E. coli chemotaxis. Our results show that N-(3-oxododecanoyl)-homoserine lactone (oxo-C12-HSL) and N-(butryl)-homoserine lactone (C4-HSL) are attractants. Furthermore, we tested the chemoeffector potential of pyocyanin and pyoverdine, secondary metabolites under a quorum sensing control. Pyocyanin is proved to be a weak attractant while pyoverdine are repellent. We demonstrated the usability of the device in co-culturing experiments, where we showed that various factors released by P. aeruginosa affect the dynamic spatial rearrangement of a neighboring E. coli population, while surface adhesion of the cells is also modulated.

Modeling bacterial quorum sensing in open and closed environments: potential discrepancies between agar plate and culture flask experiments.

Quorum sensing (QS) is a process of bacterial communication and cooperation mediated by the release of jointly exploited signals and "public goods" into the environment. There are conflicting reports on the behavior of mutants deficient in the release of these materials. Namely, mutants that appear perfectly viable and capable of outgrowing wild type cells in a closed model system such as a culture flask, may not be viable or invasive on open surfaces such as agar plates. Here we show via agent-based computational simulations that this apparent discrepancy is due to the difference between open and closed systems. We suggest that the experimental difference is due to the fact that wild type cells can easily saturate a well-mixed culture flask with signals and public goods so QS will be not necessary after a certain time point. As a consequence, QS-deficient mutants can continue to grow even after the wild type population has vanished. This phenomenon is not likely to occur in open environments including open surfaces and agar plate models. In other words, even if QS is required for survival, QS deficient mutants may grow faster initially in short term laboratory experiments or computer simulations, while only WT cells appear stable over longer time scales, especially when adaptation to changing environments is important.

Stability of multispecies bacterial communities: signaling networks may stabilize microbiomes.

Multispecies bacterial communities can be remarkably stable and resilient even though they consist of cells and species that compete for environmental resources. In silico models suggest that common signals released into the environment may help selected bacterial species cluster at common locations and that sharing of public goods (i.e. molecules produced and released for mutual benefit) can stabilize this coexistence. In contrast, unilateral eavesdropping on signals produced by a potentially invading species may protect a community by keeping invaders away from limited resources. Shared bacterial signals, such as those found in quorum sensing systems, may thus play a key role in fine tuning competition and cooperation within multi-bacterial communities. We suggest that in addition to metabolic complementarity, signaling dynamics may be important in further understanding complex bacterial communities such as the human, animal as well as plant microbiomes.

Effect of antimicrobial peptide-amide: indolicidin on biological membranes.

Indolicidin, a cationic antimicrobial tridecapeptide amide, is rich in proline and tryptophan residues. Its biological activity is intensively studied, but the details how indolicidin interacts with membranes are not fully understood yet. We report here an in situ atomic force microscopic study describing the effect of indolicidin on an artificial supported planar bilayer membrane of dipalmitoyl phosphatidylcholine (DPPC) and on purple membrane of Halobacterium salinarum. Concentration dependent interaction of the peptide and membranes was found in case of DPPC resulting the destruction of the membrane. Purple membrane was much more resistant against indolicidin, probably due to its high protein content. Indolicidin preferred the border of membrane disks, where the lipids are more accessible. These data suggest that the atomic force microscope is a powerful tool in the study of indolicidin-membrane interaction.

Co-swarming and local collapse: quorum sensing conveys resilience to bacterial communities by localizing cheater mutants in Pseudomonas aeruginosa.

BACKGROUND: Members of swarming bacterial consortia compete for nutrients but also use a co-operation mechanism called quorum sensing (QS) that relies on chemical signals as well as other secreted products ("public goods") necessary for swarming. Deleting various genes of this machinery leads to cheater mutants impaired in various aspects of swarming cooperation. METHODOLOGY/PRINCIPAL FINDINGS: Pairwise consortia made of Pseudomonas aeruginosa, its QS mutants as well as B. cepacia cells show that a interspecies consortium can "combine the skills" of its participants so that the strains can cross together barriers that they could not cross alone. In contrast, deleterious mutants are excluded from consortia either by competition or by local population collapse. According to modeling, both scenarios are the consequence of the QS signalling mechanism itself. CONCLUSION/SIGNIFICANCE: The results indirectly explain why it is an advantage for bacteria to maintain QS systems that can cross-talk among different species, and conversely, why certain QS mutants which can be abundant in isolated niches, cannot spread and hence remain localized.

Locality versus globality in bacterial signalling: can local communication stabilize bacterial communities?

BACKGROUND: Microbial consortia are a major form of life; however their stability conditions are poorly understood and are often explained in terms of species-specific defence mechanisms (secretion of extracellular matrix, antimicrobial compounds, siderophores, etc.). Here we propose a hypothesis that the primarily local nature of intercellular signalling can be a general mechanism underlying the stability of many forms of microbial communities. PRESENTATION OF THE HYPOTHESIS: We propose that a large microbial community can be pictured as a theatre of spontaneously emerging, partially overlapping, locally recruited microcommunities whose members interact primarily among themselves, via secreted (signalling) molecules or cell-cell contacts. We hypothesize that stability in an open environment relies on a predominantly local steady state of intercellular communication which ensures that i) deleterious mutants or strains can be excluded by a localized collapse, while ii) microcommunities harbouring useful traits can persist and/or spread even in the absence of specific protection mechanisms. TESTING THE HYPOTHESIS: Some elements of this model can be tested experimentally by analyzing the behaviour of synthetic consortia composed of strains having well-defined communication systems and devoid of specific defence mechanisms. Supporting evidence can be obtained by in silico simulations. IMPLICATIONS OF THE HYPOTHESIS: The hypothesis provides a framework for a systematic comparison of bacterial community behavior in open and closed environments. The model predicts that local signalling may enable multispecies communities to colonize open, structured environments. On the other hand, a confined niche or a host may be more likely to be colonized by a bacterial mono-species community, and local communication here provides a control against spontaneously arising cheaters, provided that survival depends on cooperation.

A simple model for the early events of quorum sensing in Pseudomonas aeruginosa: modeling bacterial swarming as the movement of an "activation zone".

BACKGROUND: Quorum sensing (QS) is a form of gene regulation based on cell-density that depends on inter-cellular communication. While there are a variety of models for bacterial colony morphology, there is little work linking QS genes to movement in an open system. RESULTS: The onset of swarming in environmental P. aeruginosa PUPa3 was described with a simplified computational model in which cells in random motion communicate via a diffusible signal (representing N-acyl homoserine lactones, AHL) as well as diffusible, secreted factors (enzymes, biosurfactans, i.e. "public goods") that regulate the intensity of movement and metabolism in a threshold-dependent manner. As a result, an "activation zone" emerges in which nutrients and other public goods are present in sufficient quantities, and swarming is the spontaneous displacement of this high cell-density zone towards nutrients and/or exogenous signals. The model correctly predicts the behaviour of genomic knockout mutants in which the QS genes responsible either for the synthesis (lasI, rhlI) or the sensing (lasR, rhlR) of AHL signals were inactivated. For wild type cells the model predicts sustained colony growth that can however be collapsed by the overconsumption of nutrients. CONCLUSION: While in more complex models include self-orienting abilities that allow cells to follow concentration gradients of nutrients and chemotactic agents, in this model, displacement towards nutrients or environmental signals is an emergent property of the community that results from the action of a few, well-defined QS genes and their products. Still the model qualitatively describes the salient properties of QS bacteria, i.e. the density-dependent onset of swarming as well as the response to exogenous signals or cues.

Studying the structural properties of polyalanine and polyglutamine peptides.

Poly-(Ala) and poly-(Gln) peptides have important biological effects, and can cause various human illnesses and neurodegenerative diseases. Conformational analysis of these homo-oligopeptides (HOPs) was carried out by simulated annealing in order to identify their structural properties regarding secondary structures and intramolecular H-bonding patterns. Poly-(Ala) and poly-(Gln) peptides composed of 7, 10, 14 or 20 amino acids were modelled in both charged and terminally blocked forms. In the case of conformers derived from simulated annealing calculations, the presence of various secondary structural elements (different types of beta-turns, alpha-helix, 3(10)-helix, poly-proline II helix, parallel and antiparallel beta-strands) was investigated. Moreover, the intramolecular H-bonding patterns formed either between the backbone atoms for both HOPs or between the backbone and side-chain atoms for the poly-(Gln) peptides were examined. Our results showed that different secondary structural elements (type I and type III beta-turns, alpha-helix, 3(10)-helix, antiparallel beta-strand) could be observed in both poly-(Ala) and poly-(Gln) peptides and, according to their presence, characteristic H-bonding patterns formed mainly by i<--i+3 and i<--i+4 H-bonds could be found.