The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional

The role of quorum sensing in Pseudomonas aeruginosa biofilm formation is unclear. Some researchers have shown that quorum sensing is important for biofilm development, while others have indicated it has little or no role. In this study, the contribution of quorum sensing to biofilm development was found to depend upon the nutritional environment. Depending upon the carbon source, quorum-sensing mutant strains (lasIrhlI and lasRrhlR) either exhibited a pronounced defect early in biofilm formation or formed biofilms identical to the wild-type strain. Quorum sensing was then shown to exert its nutritionally conditional control of biofilm development through regulation of swarming motility. Examination of pilA and fliM mutant strains further supported the role of swarming motility in biofilm formation. These data led to a model proposing that the prevailing nutritional conditions dictate the contributions of quorum sensing and swarming motility at a key juncture early in biofilm development.     

Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms

In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express ‘cooperative traits'', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.     

Transition to quorum sensing in an Agrobacterium population: A stochastic model

Understanding of the intracellular molecular machinery that is responsible for the complex collective behavior of multicellular populations is an exigent problem of modern biology. Quorum sensing, which allows bacteria to activate genetic programs cooperatively, provides an instructive and tractable example illuminating the causal relationships between the molecular organization of gene networks and the complex phenotypes they control. In this work we—to our knowledge for the first time—present a detailed model of the population-wide transition to quorum sensing using the example of Agrobacterium tumefaciens. We construct a model describing the Ti plasmid quorum-sensing gene network and demonstrate that it behaves as an “on–off” gene expression switch that is robust to molecular noise and that activates the plasmid conjugation program in response to the increase in autoinducer concentration. This intracellular model is then incorporated into an agent-based stochastic population model that also describes bacterial motion, cell division, and chemical communication. Simulating the transition to quorum sensing in a liquid medium and biofilm, we explain the experimentally observed gradual manifestation of the quorum-sensing phenotype by showing that the transition of individual model cells into the “on” state is spread stochastically over a broad range of autoinducer concentrations. At the same time, the population-averaged values of critical autoinducer concentration and the threshold population density are shown to be robust to variability between individual cells, predictable and specific to particular growth conditions. Our modeling approach connects intracellular and population scales of the quorum-sensing phenomenon and provides plausible answers to the long-standing questions regarding the ecological and evolutionary significance of the phenomenon. Thus, we demonstrate that the transition to quorum sensing requires a much higher threshold cell density in liquid medium than in biofilm, and on this basis we hypothesize that in Agrobacterium quorum sensing serves as the detector of biofilm formation.     

Acyl-homoserine lactone-dependent eavesdropping promotes competition in a laboratory co-culture model

Many Proteobacteria use acyl-homoserine lactone (AHL)-mediated quorum sensing to activate the production of antibiotics at high cell density. Extracellular factors like antibiotics can be considered public goods shared by individuals within a group. Quorum-sensing control of antibiotic production may be important for protecting a niche or competing for limited resources in mixed bacterial communities. To begin to investigate the role of quorum sensing in interspecies competition, we developed a dual-species co-culture model using the soil saprophytes Burkholderia thailandensis (Bt) and Chromobacterium violaceum (Cv). These bacteria require quorum sensing to activate the production of antimicrobial factors that inhibit growth of the other species. We demonstrate that quorum-sensing-dependent antimicrobials can provide a competitive advantage to either Bt or Cv by inhibiting growth of the other species in co-culture. Although the quorum-sensing signals differ for each species, we show that the promiscuous signal receptor encoded by Cv can sense signals produced by Bt, and that this ability to eavesdrop on Bt can provide Cv an advantage in certain situations. We use an in silico approach to investigate the effect of eavesdropping in competition, and show conditions where early activation of antibiotic production resulting from eavesdropping can promote competitiveness. Our work supports the idea that quorum sensing is important for interspecies competition and that promiscuous signal receptors allow eavesdropping on competitors in mixed microbial habitats.     

Biofilm formation and sloughing in Serratia marcescens are controlled by quorum sensing and nutrient cues

We describe here a role for quorum sensing in the detachment, or sloughing, of Serratia marcescens filamentous biofilms, and we show that nutrient conditions affect the biofilm morphotype. Under reduced carbon or nitrogen conditions, S. marcescens formed a classical biofilm consisting of microcolonies. The filamentous biofilm could be converted to a microcolony-type biofilm by switching the medium after establishment of the biofilm. Similarly, when initially grown as a microcolony biofilm, S. marcescens could be converted back to a filamentous biofilm by increasing the nutrient composition. Under high-nutrient conditions, an N-acyl homoserine lactone quorum-sensing mutant formed biofilms that were indistinguishable from the wild-type biofilms. Similarly, other quorum-sensing-dependent behaviors, such as swarming motility, could be rendered quorum sensing independent by manipulating the growth medium. Quorum sensing was also found to be involved in the sloughing of the filamentous biofilm. The biofilm formed by the bacterium consistently sloughed from the substratum after approximately 75 to 80 h of development. The quorum-sensing mutant, when supplemented with exogenous signal, formed a wild-type filamentous biofilm and sloughed at the same time as the wild type, and this was independent of surfactant production. When we removed the signal from the quorum-sensing mutant prior to the time of sloughing, the biofilm did not undergo significant detachment. Together, the data suggest that biofilm formation by S. marcescens is a dynamic process that is controlled by both nutrient cues and the quorum-sensing system.     

细菌群体感应及其在食品变质中的作用

食品相关细菌引起的生物被膜形成和食品变质是食品工业中的重大问题。研究表明细菌群体感应(Quorum sensing,QS)与被膜形成、食品腐败变质密切相关。重点对细菌产生的各种QS信号分子及其在被膜形成的作用和被膜在食品工业中的重要性做了介绍。QS信号分子与食品变质密切相关,故对QS抑制剂作为新型食品防腐剂以延长储存期限及加强食品安全的前景进行了概述。     

Inhibition of Candida albicans Biofilm Formation by Farnesol, a Quorum-Sensing Molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 μM) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 μM farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

Quorum-Sensing Mutations Affect Attachment and Stability of Burkholderia cenocepacia Biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.     

The quorum-sensing system in a plant bacterium Mesorhizobium huakuii affects growth rate and symbiotic nodulation

Mesorhizobium huakuii is a free-living bacterium which is capable of establishing a specific symbiotic relationship with Astragalus sinicus, an important winter green manure widely used in Eastern Asia, allowing for nitrogen fixing during this process. Previous studies demonstrate that M.␣huakuii produces quorum-sensing molecules at high cell density and quorum sensing plays a role in biofilm formations. In this study, we isolated and characterized two quorum-sensing deficient mutants in M. huakuii. Analysis of the flanking region of transposon insertions indicated that autoinducer synthase related genes are not homologous to acyl homoserine lactone (AHL) synthase genes that are shared among many Gram-negative bacteria, but related to peptide synthesis, indicating that M. huakuii quorum-sensing signals are distinct from AHLs. Compared with the wild-type strains, these quorum-sensing deficient mutants promoted their growth rate and were defective in nodule formation on host plants, indicative of a critical role of quorum sensing in M.␣huakuii during the host–bacterium symbiotic interaction.Yijing Gao and Zengtao Zhong contributed equally to this work.     

Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C₆-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C₉-CPA), had a strong inhibitory effect on prodigiosin production. C₉-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C₉-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C₆-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C₉-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Inhibition of quorum sensing in a computational biofilm simulation

Bacteria communicate through small diffusible molecules in a process known as quorum sensing. Quorum-sensing inhibitors are compounds which interfere with this, providing a potential treatment for infections associated with bacterial biofilms. We present an individual-based computational model for a developing biofilm. Cells are aggregated into particles for computational efficiency, but the quorum-sensing mechanism is modelled as a stochastic process on the level of individual cells. Simulations are used to investigate different treatment regimens. The response to the addition of inhibitor is found to depend significantly on the form of the positive feedback in the quorum-sensing model; in cases where the model exhibits bistability, the time at which treatment is initiated proves to be critical for the effective prevention of quorum sensing and hence potentially of virulence.     

Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-acylhomoserine lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C(6)-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C(9)-CPA), had a strong inhibitory effect on prodigiosin production. C(9)-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C(9)-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C(6)-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C(9)-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Quorum-sensing mutations affect attachment and stability of Burkholderia cenocepacia biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.     

The Natural Antimicrobial Carvacrol Inhibits Quorum Sensing in Chromobacterium violaceum and Reduces Bacterial Biofilm Formation at Sub-Lethal Concentrations

The formation of biofilm by bacteria confers resistance to biocides and presents problems in medical and veterinary clinical settings. Here we report the effect of carvacrol, one of the major antimicrobial components of oregano oil, on the formation of biofilms and its activity on existing biofilms. Assays were carried out in polystyrene microplates to observe (a) the effect of 0–0.8 mM carvacrol on the formation of biofilms by selected bacterial pathogens over 24 h and (b) the effect of 0–8 mM carvacrol on the stability of pre-formed biofilms. Carvacrol was able to inhibit the formation of biofilms of Chromobacterium violaceum ATCC 12472, Salmonella enterica subsp. Typhimurium DT104, and Staphylococcus aureus 0074, while it showed no effect on formation of Pseudomonas aeruginosa (field isolate) biofilms. This inhibitory effect of carvacrol was observed at sub-lethal concentrations (<0.5 mM) where no effect was seen on total bacterial numbers, indicating that carvacrol''s bactericidal effect was not causing the observed inhibition of biofilm formation. In contrast, carvacrol had (up to 8 mM) very little or no activity against existing biofilms of the bacteria described, showing that formation of the biofilm also confers protection against this compound. Since quorum sensing is an essential part of biofilm formation, the effect of carvacrol on quorum sensing of C. violaceum was also studied. Sub-MIC concentrations of carvacrol reduced expression of cviI (a gene coding for the N-acyl-L-homoserine lactone synthase), production of violacein (pigmentation) and chitinase activity (both regulated by quorum sensing) at concentrations coinciding with carvacrol''s inhibiting effect on biofilm formation. These results indicate that carvacrol''s activity in inhibition of biofilm formation may be related to the disruption of quorum sensing.     

The Influence of Fluid Flow on Modeling Quorum Sensing in Bacterial Biofilms

In this paper, we study quorum sensing in Pseudomonas aeruginosa biofilms. Quorum sensing is a process where bacteria monitor their population density through the release of extra-cellular signalling molecules. The presence of these molecules affects gene modulation leading to changes in behaviour such as the release of virulence factors. Here, we use numerical methods to approximate a 2-D model of quorum sensing. It is observed that the shape of the biofilm can have a profound effect on the onset of quorum sensing. This has serious repercussions for experimental observations since biofilms of the same biomass but different shapes can produce quite different results.     

Heterologous overexpression of quorum-sensing regulators to study cell-density-dependent phenotypes in a symbiotic plant bacterium <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis>

Quorum-sensing is widespread among many prokaryotic lineages. In order to investigate quorum regulation in the plant bacterium Mesorhizobium huakuii which produces an N-acyl homoserine lactone (AHL) quorum signal, the Agrobacterium quorum-sensing regulator TraR was heterologously expressed in this bacterium. The resulting strains showed reduced AHL production in the supernatant compared to wild-type, but similar intracellular levels of AHLs were detected, suggesting that M. huakuii AHLs can be bound to intracellular TraR proteins and thus become unavailable for its own quorum systems. M. huakuii overexpressing TraR formed thinner biofilms than the wild-type, suggesting a role played by quorum-sensing in biofilm formation.Hui Wang and Zengtao Zhong contributed equally to this work.     

Is there a role for quorum sensing signals in bacterial biofilms?

Bacteria form multicellular biofilm communities on most surfaces. Genetic analysis of biofilm formation has led to the proposal that extracellular signals and quorum-sensing regulatory systems are essential for differentiated biofilms. Although such a model fits the concept of density-driven cell-cell communication and appear to describe biofilm development in several bacterial species and conditions, biofilm formation is multifactorial and complex. Hydrodynamics, nutrient load and intracellular carbon flux have major impacts, presumably by altering the expression of cellular traits essential for bacterial adaptation during the different stages of biofilm formation. Hence, differentiated biofilms may also be the net result of many independent interactions, rather than being determined by a particular global quorum sensing system.