Introduction to Quorum Sensing Research in Diverse Microbial Systems

Ralstonia solanacearum species complex (RSSC) includes soilborne bacterial plant pathogens with worldwide distribution and wide host ranges. Virulence factors are regulated via four hierarchically organized cell-cell contact independent quorum-sensing (QS) signalling systems: the Phc, which uses as signals (R)-methyl 3-hydroxypalmitate [(R)-3-OH PAME] or (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME], the N-acyl homoserine lactone (AHL)-dependent RasI/R and SolI/R systems, and the recently identified anthranilic acid-dependent system. The unique Phc QS system has been extensively studied; however, the role of the two AHL QS systems has only recently been addressed. In this microreview, we present and discuss current data of the SolI/R and RasI/R QS systems in the RSSC. We also present the distribution and frequency of these AHL QS systems in the RSSC, discuss possible ecological roles and evolutive implications. The complex QS hierarchical networks emphasizes the crucial role of cell-cell signalling in the virulence of the RSSC.

Human pathogen Pseudomonas aeruginosa uses quorum-sensing (QS) signalling systems to synchronize the production of virulence factors. There are two interrelated QS systems, las and rhl, in P. aeruginosa. In addition to this complexity, a number of transcriptional regulators were shown to have complicated interplays with las and rhl central QS components. Here, we describe a novel virulence and QS modulator (VqsM) that positively regulates the QS systems in P. aeruginosa. Mutation in vqsM resulted in much reduced production of N-acylhomoserine lactones (AHLs) and extracellular enzymes. Sequence analysis revealed that vqsM encodes a transcriptional regulator with an AraC-type helix-turn-helix DNA binding domain at the C-terminal of the peptide. Global gene expression profile analysis showed at least a total of 302 genes to be influenced, directly or indirectly, by VqsM. Among the 203 VqsM-promoted genes, 52.2% were known to be QS upregulated. Several genes encoding the key regulators implicated in QS, such as rhlR, rsaL, vqsR, mvfR, pprB and rpoS, and two AHL synthesis genes, lasI and rhlI, were suppressed in the vqsM mutant. Similar to the 'AHL-blind' phenotype of vqsR and pprB mutants, vqsM mutant did not respond to external addition of N-3-oxo-dodecanoyl-homoserine lactone signals. Moreover, overexpression of vqsR in vqsM mutant more or less restored the production of both AHL and virulence factors. The results demonstrate that VqsM, largely through modulation of vqsR expression, plays a vital role in regulation of QS signalling in P. aeruginosa.

Flexibility and Adaptability of Quorum Sensing in Nature.

Synthesis of antimicrobial glucosamides as bacterial quorum sensing mechanism inhibitors

Quorum-sensing (QS) systems based on acyl-homoserine lactones (AHLs) regulate gene expression in response to cell density in many bacteria, including Rhizobium. These systems, typically composed of LuxI-like synthases and LuxR-like regulators, control processes such as plasmid conjugation, biofilm formation, and plant interactions. However, their evolutionary dynamics and genomic distribution in Rhizobium remain poorly understood. We analyzed 142 complete Rhizobium genomes using comparative genomics, phylogenetic reconstruction, and genomic context analysis. LuxI/LuxR homologs were identified based on sequence similarity and Pfam domain architecture, and their genomic contexts were examined. Phylogenetic relationships and coevolution between LuxI/LuxR pairs were assessed using cophylogenetic approaches. QS systems showed a highly heterogeneous distribution across Rhizobium genomes: some strains lacked canonical systems, whereas others encoded one or multiple systems in chromosomes and/or plasmids. Chromosomal QS systems were associated with multiple distinct genomic contexts, supporting at least seven independent acquisition events. In contrast, plasmid-encoded systems exhibited substantially greater diversity in both sequence and genomic organization. Phylogenetic and comparative analyses revealed dynamic gains and losses of QS systems, variable coevolution among LuxI/LuxR pairs, and evidence of partner recruitment. Notably, plasmids appear to act as major reservoirs of QS systems and likely sources of their transfer to chromosomes. These findings indicate that QS systems in Rhizobium evolve through a combination of horizontal gene transfer, genomic rearrangement, and differential retention across replicons. The higher diversity and mobility of plasmid-encoded systems highlight their central role in shaping QS evolution and functional innovation. Overall, this study provides a comprehensive framework for understanding the diversification and evolutionary trajectories of QS systems in complex multipartite bacterial genomes.

ABSTRACTQuorum-sensing (QS) signals are widely employed by bacteria to regulate biological functions in response to cell densities. Previous studies showed that Burkholderia cenocepacia mostly utilizes two types of QS systems, including the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) systems, to regulate biological functions. We demonstrated here that a LysR family transcriptional regulator, Bcal3178, controls the QS-regulated phenotypes, including biofilm formation and protease production, in B. cenocepacia H111. Expression of Bcal3178 at the transcriptional level was obviously downregulated in both the AHL-deficient and BDSF-deficient mutant strains compared to the wild-type H111 strain. It was further identified that Bcal3178 regulated target gene expression by directly binding to the promoter DNA regions. We also revealed that Bcal3178 was directly controlled by the AHL system regulator CepR. These results show that Bcal3178 is a new downstream component of the QS signaling network that modulates a subset of genes and functions coregulated by the AHL and BDSF QS systems in B. cenocepacia.IMPORTANCEBurkholderia cenocepacia is an important opportunistic pathogen in humans that utilizes the BDSF and AHL quorum-sensing (QS) systems to regulate biological functions and virulence. We demonstrated here that a new downstream regulator, Bcal3178 of the QS signaling network, controls biofilm formation and protease production. Bcal3178 is a LysR family transcriptional regulator modulated by both the BDSF and AHL QS systems. Furthermore, Bcal3178 controls many target genes, which are regulated by the QS systems in B. cenocepacia. Collectively, our findings depict a novel molecular mechanism with which QS systems regulate some target gene expression and biological functions by modulating the expression level of a LysR family transcriptional regulator in B. cenocepacia.

Quorum sensing (QS) cell-cell communication systems are utilized by bacteria to coordinate their behaviour according to cell density. Several different types of QS signal molecules have been identified, among which acyl-homoserine lactones (AHLs) produced by Proteobacteria have been studied to the greatest extent. Although QS has been studied extensively in cultured microorganisms, little is known about the QS systems of uncultured microorganisms and the roles of these systems in microbial communities. To extend our knowledge of QS systems and to better understand the signalling that takes place in the natural environment, metagenomic libraries constructed using DNA from activated sludge and soil were screened, using an Agrobacterium biosensor strain, for novel QS synthase genes. Three cosmids (QS6-1, QS10-1 and QS10-2) that encode the production of QS signals were identified and DNA sequence analysis revealed that all three clones encode a novel luxI family AHL synthase and a luxR family transcriptional regulator. Thin layer chromatography revealed that these LuxI homologue proteins are able to synthesize multiple AHL signals. Tandem mass spectrometry analysis revealed that LuxI(QS6-1) directs the synthesis of at least three AHLs, 3-O-C14:1 HSL, 3-O-C16:1 HSL and 3-O-C14 HSL; LuxI(QS10-1) directs the synthesis of at least 3-O-C12 HSL and 3-O-C14 HSL; while LuxI(QS10-2) directs the synthesis of at least C8 HSL and C10 HSL. Two possible new AHLs, C14:3 HSL and (?)-hydroxymethyl-3-O-C14 HSL, were also found to be synthesized by LuxI(QS6-1).

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.

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C(12)-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an alpha-beta-alpha fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-A resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. QS relies on the cell-density-dependent production, accumulation, and receptor-mediated detection of extracellular signaling molecules called autoinducers (AIs). Gram-negative bacteria commonly use N-acyl homoserine lactones (AHLs) as their AIs, and they are detected by LuxR-type receptors. Often, LuxR-type receptors are insoluble when not bound to a cognate AI. In this report, we show that LuxR-type receptors are encoded on phage genomes, and in the cases we tested, the phage LuxR-type receptors bind to and are solubilized specifically by the AHL AI produced by the host bacterium. We do not yet know the viral activities that are controlled by these phage QS receptors; however, our observations, coupled with recent reports, suggest that their occurrence is more widespread than previously appreciated. Using receptor-mediated detection of QS AIs could enable phages to garner information concerning the population density status of their bacterial hosts. We speculate that such information can be exploited by phages to optimize the timing of execution of particular steps in viral infection.IMPORTANCE Bacteria communicate with chemical signal molecules to regulate group behaviors in a process called quorum sensing (QS). In this report, we find that genes encoding receptors for Gram-negative bacterial QS communication molecules are present on genomes of viruses that infect these bacteria. These viruses are called phages. We show that two phage-encoded receptors, like their bacterial counterparts, bind to the communication molecule produced by the host bacterium, suggesting that phages can "listen in" on their bacterial hosts. Interfering with bacterial QS and using phages to kill pathogenic bacteria represent attractive possibilities for development of new antimicrobials to combat pathogens that are resistant to traditional antibiotics. Our findings of interactions between phages and QS bacteria need consideration as new antimicrobial therapies are developed.

Chapter 5.6 - Quorum Sensing Directed Microbial Diversity in Infectious Bacteria

It is now apparent that bacteria utilize regulatory systems called quorum sensing (QS) to sense their population density. Such systems are dependant on the production of signaling molecules that activate specific genes when the signal reaches a critical threshold concentration. Such QS-regulated genes produce phenotypes that require coordinate behavior to convey competitive advantage to the population (such as biofilm formation and pathogenesis). The best-characterized QS system is that driven by acylated homoserine lactone (AHL) molecules. Quorum sensing-regulated phenotypes are diverse; however, their evolutionary selection is based on the competitive advantage conveyed by coordinating gene expression with the establishment of a quorum. Population density and coordinated gene expression are coupled for either (1) the multicellular characteristic of behaviors such as cell differentiation (for example swarming, biofilm formation), or (2) the fitness benefit of many individual cells simultaneously expressing the same phenotype (for example virulence factors, luminescence). QS enables a population to differentiate under favorable conditions where the population is dense enough to support the division and coordination of labor into subpopulations. In undifferentiated populations, QS coordinates gene expression so that it is simultaneous for cells within the population. In both scenarios, having QS regulation provides a competitive advantage for a population to both produce and respond to QS molecules. A selective pressure also exists for non-QS bacteria to sense and respond to QS molecules produced within the community. Examples of QS bacteria and bacteria able to detect and respond to exogenous signals are found in the literature; however, the frequency of QS and QS cheaters in the environment is poorly documented. With the growing number of bacterial genomes sequenced, especially genomes of nonclinically isolated bacteria, it may not be surprising that the number of genomes containing homologs of AHL-QS circuitry is ever growing. In this article, we use all current bacterial genomes to examine the frequency of AHL-QS among these bacteria, and the surprising number of bacteria with the genetic potential for eavesdropping on AHL signals from other bacteria.

Pseudomonas aeruginosa and Vibrio vulnificus are Gram-negative human pathogens, which exert their virulence through quorum sensing (QS) regulation. The infection of these pathogens have been known to be mediated by biofilm formation in many cases and this study carried out the time-course analysis of biofilm formation depending on the QS regulation in P. aeruginosa and V. vulnificus. In P. aeruginosa, our results demonstrated that QS-deficient mutant better attached to surface at initial stage of biofilm formation, but poorly proceeded to the maturation of the biofilm structure, while wild type less attached at initial stage but developed highly structured biofilm at late stage. Because of this, the quantitative comparison of biofilm formation between wild type and the QS mutant showed the reversion; the QS mutant formed more biofilm until 10 h after inoculation than wild type, but wild type formed much more biofilm after 10 h than QS mutant. V. vulnificus has been reported to form more biofilm with the mutation on QS system. When we performed the same time-course analysis of the V. vulnificus biofilm formation, the reversion was not detected even with prolonged culture for 108 h and the QS mutant always forms more biofilm than wild type. These results indicate that the QS regulation negatively affects the attachment at early stage but positively facilitates the biofilm maturation at late stage in P. aeruginosa, while the QS regulation has a negative effect on the biofilm formation throughout the biofilm development in V. vulnificus. Based on our results, we suggest that the developmental stage of biofilm and bacterial species should be considered when the QS system is targeted for biofilm control.

To investigate roles of quorum-sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin-resistant variant (hereinafter DR1R). The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane-degradation and biofilm-formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm-formation and hexadecane-biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild-type cell supernatant and exogenous C(12) -AHL. The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.

Many rhizobial species use complex N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) systems to monitor their population density and regulate their symbiotic interactions with their plant hosts. There are at least three LuxRI-type regulatory systems in Rhizobium etli CFN42: CinRI, RaiRI, and TraRI. In this study, we show that CinI, RaiI, and TraI are responsible for synthesizing all AHLs under the tested conditions. The activation of these AHL synthase genes requires their corresponding LuxR-type counterparts. We further demonstrate that CinRI is at the top of the regulatory cascade that activates RaiRI and TraRI QS systems. Moreover, we discovered that CinR possesses a specific affinity to bind cinI promoter in the absence of its cognate AHL ligand, thereby activating cinI transcription. Addition of AHLs leads to improved binding to the cinI promoter and enhanced cinI expression. Furthermore, we found that compared to the wild type, the cinR mutation displayed reduced nodule formation, and cinR, raiR, and traI mutants show significantly lower levels of nitrogen fixation activity than the wild type. These results suggest that the complex QS regulatory systems in R. etli play an important role in its symbiosis with legume hosts. Many bacteria use quorum sensing (QS) to monitor their cell densities and coordinately regulate a number of physiological functions. Rhizobia often have diverse and complex LuxR/LuxI-type quorum sensing systems that may be involved in symbiosis and N2 fixation. In this study, we identified three LuxR/LuxI-type QS systems in Rhizobium etli CFN42: CinRI, RaiRI, and TraRI. We established a complex network of regulation between these QS components and found that these QS systems played important roles in symbiosis processes.