Acyl-homoserine Lactone Quorum Research Articles

Role of acyl-homoserine lactone quorum-sensing system in oral biofilm formation: A review

Background: The function of Acyl homoserine Lactone (AHL) as a communication system in oral biofilm formation by oral microorganisms is essential. A complete understanding of the role of AHL in oral microflora leads to new strategies for controlling biofilms and promoting oral health. The relationship between AHL and oral biofilm formation is not fully understood. Closing this knowledge gap leads to a better understanding of how oral AHL influences the microbiota and enables the development of more effective oral healthcare strategies. Objective: To summarise the connection between AHL and oral biofilm formation, including the mechanisms involved and the impact of Quorum sensing (QS) on oral and dental health. Methods: The authors performed a comprehensive literature review utilising Google Scholar and PubMed to investigate the association between the AHL and the development of biofilms by oral microflora. Result: The research included a comprehensive overview of the existing knowledge regarding the interplay between AHL signalling and the formation of oral biofilms. Conclusion: The AHL quorum sensing system plays a fundamental function in developing and organising oral biofilms, which contribute to oral and dental diseases such as gum disease and dental caries.

A Mesorhizobium japonicum quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal.

Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti) synthesizes and responds to N-[(2E, 4E)-2,4-dodecadienoyl] homoserine lactone (2E, 4E-C12:2-HSL). We show that the 2E, 4E-C12:2-HSL QS circuit involves one of four luxR-luxI-type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2E, 4E-C12:2-HSL. The 2E, 4E-C12:2-HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2E, 4E-C12:2-HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S-adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits.

Deciphering the role of granular activated carbon (GAC) in anammox: Effects on microbial succession and communication

Anaerobic ammonium oxidation (anammox) offered an energy-efficient option for nitrogen removal from wastewater. Granular activated carbon (GAC) addition has been reported that improved biomass immobilization, but the role of GAC in anammox reactors has not been sufficiently revealed. In this study, it was observed that GAC addition in an upflow anaerobic sludge blanket (UASB) reactor led to the significantly shortened anammox enrichment time (shortened by 45 days) than the reactor without GAC addition. The nitrogen removal rate was 0.83 kg N/m3/day versus 0.76 kg N/m3/day in GAC and non-GAC reactors, respectively after 255 days’ operation. Acyl-homoserine lactone (AHL) quorum sensing signal molecule C8-HSL had comparable concentrations in both anammox reactors, whereas the signal molecule C12-HSL was more pervasive in the reactor containing GAC than the reactor without GAC. Microbial analysis revealed distinct anammox development in both reactors, with Candidatus Brocadia predominant in the reactor that did not contain GAC, and Candidatus Kuenenia predominant in the reactor that contained GAC. Denitrification bacteria likely supported anammox metabolism in both reactors. The analyses of microbial functions suggested that AHL-dependent quorum sensing was enhanced with the addition of GAC, and that GAC possibly augmented the extracellular electron transfer (EET)-dependent anammox reaction.

Antiactivators prevent self-sensing in Pseudomonas aeruginosa quorum sensing

Quorum sensing is described as a widespread cell density-dependent signaling mechanism in bacteria. Groups of cells coordinate gene expression by secreting and responding to diffusible signal molecules. Theory, however, predicts that individual cells may short-circuit this mechanism by directly responding to the signals they produce irrespective of cell density. In this study, we characterize this self-sensing effect in the acyl-homoserine lactone quorum sensing system of Pseudomonas aeruginosa. We show that antiactivators, a set of proteins known to affect signal sensitivity, function to prevent self-sensing. Measuring quorum-sensing gene expression in individual cells at very low densities, we find that successive deletion of antiactivator genes qteE and qslA produces a bimodal response pattern, in which increasing proportions of constitutively induced cells coexist with uninduced cells. Comparing responses of signal-proficient and -deficient cells in cocultures, we find that signal-proficient cells show a much higher response in the antiactivator mutant background but not in the wild-type background. Our results experimentally demonstrate the antiactivator-dependent transition from group- to self-sensing in the quorum-sensing circuitry of P. aeruginosa. Taken together, these findings extend our understanding of the functional capacity of quorum sensing. They highlight the functional significance of antiactivators in the maintenance of group-level signaling and experimentally prove long-standing theoretical predictions.

Acinetobacter quorum sensing contributes to inflammation-induced inhibition of orthopaedic implant osseointegration

Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1β levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1β induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.

Differential response to prey quorum signals indicates predatory specialization of myxobacteria and ability to predate Pseudomonas aeruginosa.

SummaryMultiomic analysis of transcriptional and metabolic responses from the predatory myxobacteria Myxococcus xanthus and Cystobacter ferrugineus exposed to prey signalling molecules of the acylhomoserine lactone and quinolone quorum signalling classes provided insight into predatory specialization. Acylhomoserine lactone quorum signals elicited a general response from both myxobacteria. We suggest that this is likely due to the generalist predator lifestyles of myxobacteria and ubiquity of acylhomoserine lactone signals. We also provide data that indicates the core homoserine lactone moiety included in all acylhomoserine lactone scaffolds to be sufficient to induce this general response. Comparing both myxobacteria, unique transcriptional and metabolic responses were observed from Cystobacter ferrugineus exposed to the quinolone signal 2‐heptylquinolin‐4(1H)‐one (HHQ) natively produced by Pseudomonas aeruginosa. We suggest that this unique response and ability to metabolize quinolone signals contribute to the superior predation of P. aeruginosa observed from C. ferrugineus. These results further demonstrate myxobacterial eavesdropping on prey signalling molecules and provide insight into how responses to exogenous signals might correlate with prey range of myxobacteria.

A covariation analysis reveals elements of selectivity in quorum sensing systems.

Many bacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). In these systems, a signal synthase produces an AHL to which its paired receptor selectively responds. Selectivity is fundamental to cell signaling. Despite its importance, it has been challenging to determine how this selectivity is achieved and how AHL QS systems evolve and diversify. We hypothesized that we could use covariation within the protein sequences of AHL synthases and receptors to identify selectivity residues. We began by identifying about 6000 unique synthase-receptor pairs. We then used the protein sequences of these pairs to identify covariation patterns and mapped the patterns onto the LasI/R system from Pseudomonas aeruginosa PAO1. The covarying residues in both proteins cluster around the ligand-binding sites. We demonstrate that these residues are involved in system selectivity toward the cognate signal and go on to engineer the Las system to both produce and respond to an alternate AHL signal. We have thus demonstrated that covariation methods provide a powerful approach for investigating selectivity in protein-small molecule interactions and have deepened our understanding of how communication systems evolve and diversify.

Efficacy of Ten Structurally Related Essential Oil Components on Anti-biofilm and Anti-quorum Sensing against Fish Spoilers Pseudomonas and Aeromonas

ABSTRACT Biofilm formation causes persistent contamination of spoilage microorganisms, leading to serious economic losses in the aquatic processing industry. The antibacterial and anti-biofilm activity of ten carvacrol, cinnamaldehyde, and derivatives against two fish spoilers, Pseudomonas fluorescens and Aeromonas salmonicida, were evaluated by minimum inhibitory concentration (MIC), crystal violet staining, exopolysaccharide, adhesion, swimming, and swarming. Ten essential oils (EOs) tested showed different antibacterial activity, in which carvacrol and thymol exhibited the lowest MIC. Biofilms and exopolysaccharide production were repressed by all EOs at one-fourth MIC without affecting cell viability, as well as, adhesion. Swimming and swarming of two spoilage strains significantly decreased. Carvacrol and eugenol exerted the best anti-biofilm of P. fluorescens (42.2%) and A. salmonicida (62.9%), respectively. All tested EOs components showed that the decrease of acyl-homoserine lactone (AHL) quorum sensing (QS) signals of two strains were positively correlated with anti-biofilm based on violacein production. Molecular docking revealed that the most active EO, carvacrol, competed with C4-HSL to bind with receptor LuxR and AsaR by its hydroxyl group, resulting in the repression of biofilm formation. These results indicated that efficacy of antibiofilm for ten EOs was associated with the structure of compounds, and carvacrol exerted the strongest anti-biofilm activity mediated by QS against two spoilage strains.

Co-dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955.

Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. The At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologs of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR is preferentially biased toward its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.

Bacterial quorum sensing quenching activity of Lysobacter leucyl aminopeptidase acts by interacting with autoinducer synthase

Acyl-homoserine lactone (AHL) is the most studied autoinducer in gram-negative bacteria controlling infections of various pathogens. Quenching of AHL signaling by inhibiting AHL synthesis or AHL-receptor binding via small molecular chemicals or enzymatically degrading AHL is commonly used to block bacterial infections. Here, we describe a new quorum-quenching strategy that directly “acquires” bacterial genes/proteins through a defined platform. We artificially expressed a typical AHL synthase gene pcoI from the biocontrol Pseudomonas fluorescens 2P24 in the antifungal bacterium Lysobacter enzymogenes OH11 lacking AHL production. This step led to the discovery of multiple PcoI interacting protein candidates from L. enzymogenes. The individual expression of these candidate genes in 2P24 led to the identification of Le0959, which encodes leucyl aminopeptidase, an effective protein that inhibits AHL synthesis in 2P24. Therefore, we define Le0959 as LqqP (Lysobacterquorum-quenching protein). The expression of pcoI in E. coli could produce AHL, and the introduction of lqqP into E. coli expressing pcoI could prevent the production of AHL. LqqP directly binds to PcoI, and this protein–protein binding reduced the abundance of free PcoI (capable of AHL synthesis) in vivo, thereby blocking PcoI-dependent AHL production. Overall, this study highlights the discovery of LqqP in quenching AHL quorum sensing by binding to AHL synthase via developing a previously-uncharacterized screening technique for bacterial quorum quenching.

The AHL Quorum-Sensing System Negatively Regulates Growth and Autolysis in Lysobacter brunescens.

Lysobacter species are emerging as novel sources of antibiotics, but the regulation of their physiological metabolism is still poorly understood. In this work, we extracted AHL (acyl-homoserine lactone) autoinducers, identified the structures of AHLs and described the AHL quorum-sensing system in Lysobacter brunescens OH23. AHLs were isolated from the supernatant of L. brunescens OH23, and ESI-MS/MS (electrospray ionization mass spectrometry) analysis revealed biosynthesis of three different AHL chemical structures by L. brunescens OH23: N-(3-oxohexanoyl)- homoserine lactone (HSL), 3-OH-C10-HSL and C8-HSL. The growth rate of AHL quorum-sensing knockout mutants was dramatically increased compared to that of wildtype. Sucrose consumptions were also twice as high in AHL quorum-sensing knockout mutants than that in wildtype in early-log phase. Additionally, expression of key genes related to sucrose metabolism α-glucosidase was enhanced in AHL quorum-sensing knockout mutants, which indicated that AHL quorum sensing negatively regulates sucrose uptake and metabolism which further affects the growth rate of L. brunescens. Furthermore, autolysis was strongly induced in AHL quorum-sensing knockout mutants compared to wildtype, suggesting that AHL quorum sensing plays a negative regulatory role in cell autolysis. Moreover, compared to wildtype, XSAC (Xanthomonas-specific antibiotic compound) production was significantly increased in AHL knockout mutants in the early-log and late-log phases, and surface motility capabilities are also enhanced also in AHL knockout mutants; the normalized data of XSAC production and surface motility and expressions of key genes related to these two phenotypes reveal that growth rare and autolysis strongly affects XSAC biosynthesis and surface motility rather than AHL quorum-sensing system. Our results show that the AHL quorum-sensing system negatively regulates cell growth and autolysis, and further maintain nutrition homeostasis and population stability in L. brunescens.

Conformational and docking studies of acyl homoserine lactones as a robust method to investigate bioactive conformations

A method aiming at investigating possible bioactive conformations of acyl homoserine lactone (AHL) quorum sensing (QS) modulators is established. The method relies on the exhaustive conformational analysis of AHLs by varying torsion angles around the amide group then on the selection of the closest conformation to those known from co-crystallized XRD data of AHL-receptor complexes. These latter are then docked as rigid ligand within the receptor binding site, leading to interactions with binding site residues which are highly consistent as compared with the data arising from XRD studies. The method is first validated using AHLs for which XRD data of their complexes with their cognate receptor are available, then extended to examples for which the binding mode is still unknown.Three compounds were used to validate the method: hexanoyl homoserine lactone (HHL) as an example of autoinducer, 3-oxo-butanoyl homoserine lactone (OBHL), as a representative model of 3-oxo-AHLs, and 4-(4-chlorophenoxy)butanoyl homoserine lactone (CPOBHL) as an example of a QS inhibitor. The conformational analysis of these three compounds to their cognate protein (TraR, SdiA, LasR and CviR) provides the data which enable the next rigid docking step. Further rigid docking of the closest conformations compared to the known bioactive ones within the binding sites allows to recover the expected binding mode with high precision (atomic RMSD < 2 Å). This “conformational analysis/torsion angle filter/rigid ligand docking” method was then used for investigating three non-natural AHL-type QS inhibitors without known co-crystallized XRD structures, namely was 2-hexenoyl homoserine lactone (HenHL), 3-oxo-4-phenylbutanoyl homoserine lactone (OPBHL) and 3-(4-bromophenyl)propanoyl homoserine lactone (BPPHL). Results provide insights into their possible binding mode by identifying specific interactions with some key residues within the receptor binding site, allowing discussion of their biological activity.

Spoilage of refrigerated Litopenaeus vannamei: eavesdropping on Acinetobacter acyl-homoserine lactones promotes the spoilage potential of Shewanella baltica.

Shewanella baltica and Acinetobacter are among the predominant spoilage bacteria in refrigerated shrimp (Litopenaeus vannamei). S. baltica are incapable of producing acyl-homoserine lactone (AHL) quorum sensing signals, but can respond to environmental AHLs. In this paper, Acinetobacter was found to produce three AHLs, i.e. N-butanoyl-l-homoserine lactone (C4-HSL), N-(3-oxohexanoyl)-l-homoserine lactone (O-C6-HSL) and N-(3-oxooctanoyl)-l-homoserine lactone (O-C8-HSL), according to thin-layer chromatography using the bioreporter Agrobacterium tumefaciens A136. The agar diffusion and β-galactosidase assays revealed that S. baltica could eavesdrop on these three AHLs from Acinetobacter. Eavesdropping on Acinetobacter AHLs especially C4-HSL was found to boost the growth of S. baltica particularly under nutrient limiting conditions (up to 40-fold increase) in the co-culture experiments. The azocasein assay revealed that S. baltica produced fourfold more extracellular proteases in response to Acinetobacter AHLs. As demonstrated by the biofilm crystal violet staining assay and confocal laser scanning microscopy, eavesdropping also decreased the biofilm-forming capacity of Acinetobacter. By inoculation of S. baltica and Acinetobacter onto surface-sterilized shrimp, eavesdropping was found to endow a growth advantage to S. baltica in vivo, resulting in a 0.5day shortened shelf life of shrimp according to total volatile basic nitrogen levels and sensory analysis. Overall, the AHL-dependent eavesdropping increased the spoilage potential of S. baltica, providing a fresh perspective on the spoilage process of refrigerated L. vannamei, and this may inspire the development of novel preservation techniques in the future to further reduce post-harvest loss of shrimp.

Microfluidics-based liquid chromatography/mass spectrometry multiple reaction monitoring approach for the relative quantification of Burkholderia cenocepacia secreted virulence factors.

RationaleBurkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking.MethodsBased on results of a label‐free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors.ResultsIncrease in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non‐physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF.ConclusionsMicrofluidics‐based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus, Nitrobacter, and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N-decanoyl-l-homoserine lactone (C10-HSL), N-3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C14-HSL), a monounsaturated AHL (C10:1-HSL), and N-octanoyl-l-homoserine lactone (C8-HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria.IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification.

Positive Autoregulation of an Acyl-Homoserine Lactone Quorum-Sensing Circuit Synchronizes the Population Response.

ABSTRACTMany proteobacteria utilize acyl-homoserine lactone quorum-sensing signals. At low population densities, cells produce a basal level of signal, and when sufficient signal has accumulated in the surrounding environment, it binds to its receptor, and quorum-sensing-dependent genes can be activated. A common characteristic of acyl-homoserine lactone quorum sensing is that signal production is positively autoregulated. We have examined the role of positive signal autoregulation in Pseudomonas aeruginosa. We compared population responses and individual cell responses in populations of wild-type P. aeruginosa to responses in a strain with the signal synthase gene controlled by an arabinose-inducible promoter so that signal was produced at a constant rate per cell regardless of cell population density. At a population level, responses of the wild type and the engineered strain were indistinguishable, but the responses of individual cells in a population of the wild type showed greater synchrony than the responses of the engineered strain. Although sufficient signal is required to activate expression of quorum-sensing-regulated genes, it is not sufficient for activation of certain genes, the late genes, and their expression is delayed until other conditions are met. We found that late gene responses were reduced in the engineered strain. We conclude that positive signal autoregulation is not a required element in acyl-homoserine lactone quorum sensing, but it functions to enhance synchrony of the responses of individuals in a population. Synchrony might be advantageous in some situations, whereas a less coordinated quorum-sensing response might allow bet hedging and be advantageous in other situations.

Draft Genome Sequence of Nitrobacter vulgaris Strain Ab1, a Nitrite-Oxidizing Bacterium.

ABSTRACTHere, we present the 3.9-Mb draft genome sequence of Nitrobacter vulgaris strain Ab1, which was isolated from a sewage system in Hamburg, Germany. The analysis of its genome sequence will contribute to our knowledge of nitrite-oxidizing bacteria and acyl-homoserine lactone quorum sensing in nitrifying bacteria.

Effect of small chain N acyl homoserine lactone quorum sensing signals on biofilms of food-borne pathogens.

Quorum sensing or cell to cell communication which includes inter- and intra-cellular communication has been implicated in the production of virulence factor and formation of biofilm in food-borne pathogens. In the present study, the effect of quorum sensing signals on the biofilms of food-borne pathogens has been elucidated. N-butryl homoserine lactone and N-hexanoyl homoserine lactone belonging to acyl homoserine lactone (AHL) family of signaling molecules were investigated for their effect on the biofilm formation (attachment and exopolymeric substance production) in the food-borne pathogens Escherichia coli, Salmonella enterica serovar Typhimurium and Vibrio parahemolyticus. The signaling molecules at a concentration of 1µM were capable of increasing biofilm formation in all the tested pathogens. There was an increase in the attachment of the bacterial cells and biomass as observed by microtiter plate assay and exopolymeric substances production in the biofilms in presence of the AHLs. Further, it needs to be elucidated if the effect of AHLS on the biofilms of E. coli and S. enterica serovar Typhimurium is SdiA dependent.

Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.

Effect of salinity and incubation time of planktonic cells on biofilm formation, motility, exoprotease production, and quorum sensing of Aeromonas hydrophila

The aim of this study was to determine the effect of salinity and age of cultures on quorum sensing, exoprotease production, and biofilm formation by Aeromonas hydrophila on stainless steel (SS) and crab shell as substrates. Biofilm formation was assessed at various salinities, from fresh (0%) to saline water (3.0%). For young and old cultures, planktonic cells were grown at 30 °C for 24 h and 96 h, respectively. Biofilm formation was assessed on SS, glass, and crab shell; viable counts were determined in R2A agar for SS and glass, but Aeromonas-selective media was used for crab shell samples to eliminate bacterial contamination. Exoprotease activity was assessed using a Fluoro™ protease assay kit. Quantification of acyl-homoserine lactone (AHL) was performed using the bioreporter strain Chromobacterium violaceum CV026 and the concentration was confirmed using high-performance liquid chromatography (HPLC). The concentration of autoinducer-2 (AI-2) was determined with Vibrio harveyi BB170. The biofilm structure at various salinities (0–3 %) was assessed using field emission electron microscopy (FESEM). Young cultures of A. hydrophila grown at 0–0.25% salinity showed gradual increasing of biofilm formation on SS, glass and crab shell; swarming and swimming motility; exoproteases production, AHL and AI-2 quorum sensing; while all these phenotypic characters reduced from 0.5 to 3.0% salinity. The FESEM images also showed that from 0 to 0.25% salinity stimulated formation of three-dimensional biofilm structures that also broke through the surface by utilizing the chitin surfaces of crab, while 3% salinity stimulated attachment only for young cultures. However, in marked contrast, salinity (0.1–3%) had no effect on the stimulation of biofilm formation or on phenotypic characters for old cultures. However, all concentrations reduced biofilm formation, motility, protease production and quorum sensing for old culture. Overall, 0–0.25% salinity enhanced biofilm formation and expression of quorum sensing regulatory genes in young cultures, whereas these responses were reduced when salinity was >0.25%. In old cultures, salinity at any concentrations (0.1–3%) induced stress in A. hydrophila. The present study provides insight into the ecology of A. hydrophila growing on fish and crustaceans such as shrimp and crabs in estuarine and seawater.