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Vfr controls quorum sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa controls several genes in a cell density-dependent manner through a phenomenon termed quorum sensing. The transcriptional activator protein of the las quorum-sensing system is encoded for by the lasR gene, which is at the top of a quorum-sensing hierarchy. The activation of LasR as a transcriptional activator induces the expression of multiple genes that code for factors important for virulence, and rhlR, which encodes the transcriptional activator protein of the P. aeruginosa rhl quorum-sensing system. Elucidating the method of lasR regulation is crucial to understanding P. aeruginosa quorum sensing. In this report, we present studies on the transcriptional control of lasR. We identified two distinct transcriptional start sites for lasR that were located 201 bp (transcript T1) and 231 bp (transcript T2) upstream from the lasR start of translation. With the use of transcriptional lasRp-lacZ fusions, we showed that in P. aeruginosa, lasR expression is cell density dependent. This gene was expressed at a basal level until it was induced during the second half of log-phase growth, with expression becoming maximal during stationary-phase growth. We also showed that lasR expression was regulated through the cyclic AMP receptor protein (CRP)-binding consensus sequence in its promoter region. Our results from P. aeruginosa mutant studies and gel retardation assays indicated that this regulation was mediated by Vfr, a homolog of the Escherichia coli CRP.

Bacterial Proteins↗

Contribution of quorum-sensing systems to virulence of Pseudomonas aeruginosa in an experimental pyelonephritis model.

BACKGROUND AND PURPOSE: Pseudomonas aeruginosa has been reported to monitor its cell density as well as expression of virulence determinants by quorum-sensing signal mechanisms operative through autoinducers. In the present investigation, we studied the contribution of quorum-sensing signals during the course of P. aeruginosa-induced pyelonephritis in mice. METHODS: The standard parent strain of P. aeruginosa (PAO1), possessing functional las and rhl quorum-sensing systems and its isogenic mutant strains, PAO-JP1 (single mutant), harboring a mutated lasI gene and PAO-JP3 (double mutant), harboring mutated lasI and rhlR genes were employed. One uroisolate of P. aeruginosa belonging to serotype O8 and deficient in production of quorum-sensing signals was also used. RESULTS: The parent strain of P. aeruginosa was significantly more virulent compared to its isogenic mutant strains and quorum-sensing negative clinical strain, as assessed by neutrophil influx, malondialdehyde production, renal bacterial load and pathology induced in experimental animals. CONCLUSIONS: Quorum-sensing systems play an important role in the pathogenicity of P. aeruginosa in pyelonephritis. Both the las and rhl quorum-sensing systems are important for the virulence of P. aeruginosa in the development of pyelonephritis.

Animals↗

Mutational analysis of TraR. Correlating function with molecular structure of a quorum-sensing transcriptional activator.

TraR, the quorum-sensing activator of the Agrobacterium tumefaciens Ti plasmid conjugation system, induces gene expression in response to its quormone, N-(3-oxooctanoyl)-L-homoserine lactone. Ligand binding results in dimerization of TraR and is required for its activity. Analysis of N- and C-terminal deletion mutants of TraR localized the quormone-binding domain to a region between residues 39 and 140 and the primary dimerization domain to a region between residues 119 and 156. The dominant-negative properties of these mutants predicted a second dimerization domain at the C terminus of the protein. Analysis of fusions of N-terminal fragments of TraR to lambda cI' confirmed the dimerization activity of these two domains. Fifteen single amino acid substitution mutants of TraR defective in dimerization were isolated. According to the analysis of these mutants, Asp-70 and Gly-113 are essential for quormone binding, whereas Ala-38 and Ala-105 are important, but not essential. Additional residues located within the N-terminal half of TraR, including three located in alpha-helix 9, contribute to dimerization, but are not required for ligand binding. These results and the recently reported crystal structure of TraR are consistent with and complement each other and together define some of the structural and functional relationships of this quorum-sensing activator.

Agrobacterium tumefaciens↗

Quorum sensing in vibrios: complexity for diversification.

N-acylhomoserine lactone-dependent quorum sensing was first discovered in two luminescent marine bacteria, Vibrio fischeri and Vibrio harveyi. The LuxI/R system of V. fischeri is the paradigm of Gram-negative quorum-sensing systems; however, it is not found in all vibrios. A more complex quorum-sensing regulation is found in V. harveyi. Three parallel systems transmit signals via phosphorelays that converge onto one regulatory protein LuxO. Components of the three systems are found only in vibrios. Of the five Vibrio strains analysed, the number and types of signal circuits found in each strain are diverse. The signalling systems have different regulatory responses depending on the type of association the Vibrio strains have with an animal host, which may reflect the diverse roles the vibrios have in structuring and maintaining microniches within the aquatic milieu. Further studies are likely to show that the diversity and complexity of the Vibrio quorum-sensing systems coordinate intraspecies behaviour, niche occupation, and possibly evolution.

Aliivibrio fischeri↗

Bacterial quorum sensing and cell surface electrokinetic properties.

The hypothesis tested in this paper is that quorum sensing influences the microbial surface electrokinetic properties. Escherichia coli MG1655 and MG1655 LuxS- mutant (lacking quorum-sensing gene for Autoinducer synthase AI-2) were used for this study. AI-2 production (or lack of) in both strains was analyzed using the Vibrio harveyi bioassay. The levels of extracellular AI-2 with and without glucose in the growth medium were consistent with previously published work. The surface electrokinetic properties were determined for each strain of E. coli MG1655 by measuring the electrophoretic mobility using a phase amplitude light-scattering (PALS) Zeta potential analyser. The findings show that the surface charge of the cells is dependent upon the stage in the growth phase as well as the ability to participate in quorum sensing. In addition, significant differences in the electrophoretic mobility were observed between both strains of E. coli. These findings suggest that quorum sensing plays a significant role in the surface chemistry of bacteria during their growth.

Bacterial Proteins↗

Promoter specificity elements in Pseudomonas aeruginosa quorum-sensing-controlled genes.

The LasR-dependent and RhlR-dependent quorum-sensing systems are global regulators of gene expression in Pseudomonas aeruginosa. Previous studies have demonstrated that promoter elements of the quorum-sensing-controlled genes lasB and hcnABC are important in density-dependent regulation. We have identified LasR- and RhlR-dependent determinants in promoters of quorum-sensing-controlled genes qsc102, qsc117 (acpP), and qsc131 (phzA to -G) by in silico, deletion, point-mutational, and primer extension analyses. Each of these genes (in addition to lasI and rsaL) is activated by LasR, and qsc117 and qsc131 also respond to RhlR. Point mutations in the promoters of the LasR-specific gene, qsc102, relax specificity so that this promoter can respond to RhlR in addition to LasR. Our findings indicate that quorum-sensing-controlled promoters in P. aeruginosa are either specific for LasR or respond to both LasR and RhlR and that critical bases in the promoter elements determine specificity.

Bacterial Proteins↗

Quorum sensing and starvation: signals for entry into stationary phase.

Quorum sensing occurs at high cell density in many microorganisms. It regulates specialized processes such as genetic competence, bioluminescence, virulence, and sporulation. However, recent evidence suggests that quorum-sensing may play a more central role in the physiology of bacteria, where quorum-sensing pathways converge with starvation-sensing pathways to regulate cell entry into stationary phase.

Amino Acid Sequence↗

The impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and Vibrio harveyi on their virulence towards gnotobiotically cultured Artemia franciscana.

Disruption of quorum sensing, bacterial cell-to-cell communication by means of small signal molecules, has been suggested as a new anti-infective strategy for aquaculture. However, data about the impact of quorum sensing on the virulence of aquatic pathogens are scarce. In this study, a model system using gnotobiotically cultured Artemia franciscana was developed in order to determine the impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and V. harveyi on their virulence. Mutations in the autoinducer 2 (AI-2) synthase gene luxS, the AI-2 receptor gene luxP or the response regulator gene luxO of the dual channel quorum sensing system of V. harveyi abolished virulence of the strain towards Artemia. Moreover, the addition of an exogenous source of AI-2 could restore the virulence of an AI-2 non-producing mutant. In contrast, none of the mutations in either the acylated homoserine lactone (AHL)-mediated component of the V. harveyi system or the quorum sensing systems of Ae. hydrophila and V. anguillarum had an impact on virulence of these bacteria towards Artemia. Our results indicate that disruption of quorum sensing could be a good alternative strategy to combat infections caused by V. harveyi.

Aeromonas hydrophila↗

Lead precipitation by Vibrio harveyi: evidence for novel quorum-sensing interactions.

Three pleiotropic, quorum sensing-defective Vibrio harveyi mutants were observed to precipitate soluble Pb2+ as an insoluble compound. The compound was purified and subjected to X-ray diffraction and elemental analyses. These assays identified the precipitated compound as Pb9(PO4)6, an unusual and complex lead phosphate salt that is produced synthetically at temperatures of ca. 200 degrees C. Regulation of the precipitation phenotype was also examined. Introduction of a luxO::kan allele into one of the mutants abolished lead precipitation, indicating that the well-characterized autoinducer 1 (AI1)-AI2 quorum-sensing system can block lead precipitation in dense cell populations. Interestingly, the V. harveyi D1 mutant, a strain defective for secretion of both AI1 and AI2, was shown to be an effective trans inhibitor of lead precipitation. This suggests that a previously undescribed V. harveyi autoinducer, referred to as AI3, can also negatively regulate lead precipitation. Experiments with heterologous bacterial populations demonstrated that many different species are capable of trans regulating the V. harveyi lead precipitation phenotype. Moreover, one of the V. harveyi mutants in this study exhibited little or no response to intercellular signals from other V. harveyi inocula but was quite responsive to some of the heterologous bacteria. Based on these observations, we propose that V. harveyi carries at least one quorum sensor that is specifically dedicated to receiving cross-species communication.

Bacterial Proteins↗

Quorum sensing by enteric pathogens.

PURPOSE OF REVIEW: This review presents advances in our understanding of how pathogenic, enteric bacteria use quorum sensing to regulate several traits that allow them to establish and maintain infection in their host, including motility, biofilm formation, and virulence-specific genes. RECENT FINDINGS: Quorum sensing in enteric bacteria has been elusive for a long time. Recent data indicate that enteric bacteria use several quorum-sensing mechanisms including the LuxR-I quorum-sensing system, the LuxS/AI-2 system, and the AI-3/epinephrine/norepinephrine system to assess their environment and to recognize the host environment. These systems allow bacteria to communicate across species boundaries, and the AI-3/epinephrine/norepinephrine system is involved in interkingdom signaling. SUMMARY: Recent developments in our understanding of the molecular and biochemical mechanisms involved in quorum sensing as well as the chemical signal(s) to which bacteria respond and their corresponding physiological responses will improve our understanding of bacterial pathogenesis and microbial flora-host interactions, and potentially lead to novel strategies for combating infection.

Animals↗

The effect of the chemical, biological, and physical environment on quorum sensing in structured microbial communities.

As researchers attempt to study quorum sensing in relevant clinical or environmental settings, it is apparent that many factors have the potential to affect signaling. These factors span a range of physical, chemical, and biological variables that can impact signal production, stability and distribution. Optimizing experimental systems to natural or clinical environments may be crucial for defining when and where quorum sensing occurs. These points are illustrated in our case study of S. aureus signaling in biofilms, where signal stability may be affected by the host environment. The basic signaling schemes have been worked out at the molecular level for a few of the major quorum-sensing systems. As these studies continue to refine our understanding of these mechanisms, an emerging challenge is to identify if and when the local environment can affect signaling.

4-Butyrolactone↗

Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production.

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows. In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor-autoinducer pair. V. harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E. coli O157:H7, Salmonella typhimurium 14028, and S. typhimurium LT2 produce autoinducers similar or identical to the V. harveyi system 2 autoinducer AI-2. However, the domesticated laboratory strain E. coli DH5alpha does not produce this signal molecule. Here we report the identification and analysis of the gene responsible for AI-2 production in V. harveyi, S. typhimurium, and E. coli. The genes, which we have named luxSV.h., luxSS.t., and luxSE.c. respectively, are highly homologous to one another but not to any other identified gene. E. coli DH5alpha can be complemented to AI-2 production by the introduction of the luxS gene from V. harveyi or E. coli O157:H7. Analysis of the E. coli DH5alpha luxSE.c. gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxSE.c. protein. Our results indicate that the luxS genes define a new family of autoinducer-production genes.

4-Butyrolactone↗

Evidence for quorum sensing in Clostridium botulinum 56A.

AIMS: Experiments were designed to detect quorum-sensing signals produced by Clostridium botulinum. METHODS AND RESULTS: Clostridium botulinum 56A cell-free supernatants obtained at the end of lag phase, the mid-exponential phase and early stationary phase of growth were assayed for bioluminescence in the Vibrio harveyi quorum-sensing assay system. Twelve and 16-h culture supernatants induced bioluminescence in the auto-inducer 2 (AI-2) but not the auto-inducer 1 (AI-1) assay. Intra-species quorum sensing was also assayed as the ability of the supernatants to promote spore germination and outgrowth in a microtitre plate system. Spore populations exposed to C. botulinum supernatant from the end of lag phase became positive for growth sooner than controls. CONCLUSIONS: The influence of cell-free supernatant on ungerminated spores and detection of bioluminescence in the AI-2 assay are evidence for a signalling molecule(s) and provide a first step in characterizing C. botulinum quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that spores do not behave independently of each other and may explain the inocula size effects observed in challenge studies. Whether AI-2 production in C. botulinum serves as an inter-species signal or as a detoxification mechanism remains to be determined.

4-Butyrolactone↗

Quorum-sensing control in Staphylococci -- a target for antimicrobial drug therapy?

Today, we find ourselves in an urgent need for novel antibacterial drugs, as many important human pathogens have acquired multiple antibiotic resistance factors. Among those, Staphylococcus aureus and S. epidermidis play a major role as the leading sources of nosocomial infections. Recently, it has been suggested to develop therapeutics that attack bacterial virulence rather than kill bacteria. Such drugs are called "antipathogenic" and are believed to reduce the development of antibiotic resistance. Specifically, cell-density-dependent gene regulation (quorum-sensing) in bacteria has been proposed as a potential target. While promising reports exist about quorum-sensing blockers in gram-negative bacteria, the use of the staphylococcal quorum-sensing system as a drug target is now seen in an increasingly critical way. Inhibition of quorum-sensing in Staphylococcus has been shown to enhance biofilm formation. Furthermore, down-regulation or mutation of the Staphylococcus quorum-sensing system increases bacterial persistence in device-related infection, suggesting that interference with quorum-sensing would enhance rather than suppress this important type of staphylococcal disease. The chemical nature and biological function of another proposed staphylococcal quorum-sensing inhibitor, named "RIP", are insufficiently characterized. Targeting quorum-sensing systems might in principle constitute a reasonable way to find novel antibacterial drugs. However, as outlined here, this approach requires careful investigation in every specific pathogen and type of infection.

Anti-Bacterial Agents↗

The Vibrio harveyi quorum-sensing system uses shared regulatory components to discriminate between multiple autoinducers.

The quorum-sensing bacterium Vibrio harveyi produces and responds to three autoinducers (AIs), and this sensory information converges to control the expression of bioluminescence, biofilm formation, type III secretion (TTS), and protease production. The AIs are detected by cognate sensor histidine kinases that all relay phosphate to the shared response regulator LuxO. LuxO indirectly represses the master regulator of quorum sensing, LuxR, through the activation of multiple genes encoding small regulatory RNAs (called qrr genes for Quorum Regulatory RNA). Here we use differential fluorescence induction to identify 50 quorum-sensing-controlled promoters. Some promoters only showed significant responses in the simultaneous presence of all three AIs, while others displayed substantial responses to the individual AIs. A differential response to each AI input state was also observed for qrr and luxR expression and LuxR protein production. Individual cell analyses revealed that, in each case, all the bacteria in the population respond in unison to the various AI inputs. We propose that the V. harveyi quorum-sensing transition is not switch-like but rather operates in a graded manner, and that this signaling arrangement, which uses shared regulatory proteins, nonetheless provides V. harveyi a mechanism to respond uniquely to different AI input states.

Bacterial Proteins↗

Communications blackout? Do N-acylhomoserine-lactone-degrading enzymes have any role in quorum sensing?

A number of bacteria, including some significant pathogens, utilize N-acylhomoserine lactones (AHLs) as quorum sensing signals. There is considerable interest in the therapeutic potential of disrupting quorum sensing. Recently, a number of bacteria have been identified which are capable of enzymic inactivation of AHLs. These enzymes show considerable promise as 'quenchers' of quorum sensing. However, the assumption that the natural function of these enzymes is to disrupt or modulate quorum sensing has yet to be established. This review surveys the progress made to date in this field and examines what implications these findings have for our understanding of the role played by these enzymes in vivo.

4-Butyrolactone↗

Role of quorum sensing and antimicrobial component production by Serratia plymuthica in formation of biofilms, including mixed biofilms with Escherichia coli.

We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.

4-Butyrolactone↗

The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses intercellular signals to control the density-dependent expression of many virulence factors. The las and rhl quorum-sensing systems function, respectively, through the autoinducers N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone (C(4)-HSL), which are known to positively regulate the transcription of the elastase-encoding gene, lasB. Recently, we reported that a second type of intercellular signal is involved in lasB induction. This signal was identified as 2-heptyl-3-hydroxy-4-quinolone and designated the Pseudomonas quinolone signal (PQS). PQS was determined to be part of the quorum-sensing hierarchy since its production and bioactivity depended on the las and rhl quorum-sensing systems, respectively. In order to define the role of PQS in the P. aeruginosa quorum-sensing cascade, lacZ gene fusions were used to determine the effect of PQS on the transcription of the quorum-sensing system genes lasR, lasI, rhlR, and rhlI. We found that in P. aeruginosa, PQS caused a major induction of rhlI'-lacZ and had lesser effects on the transcription of lasR'-lacZ and rhlR'-lacZ. We also observed that the transcription of both rhlI'-lacZ and lasB'-lacZ was cooperatively effected by C(4)-HSL and PQS. Additionally, we present data indicating that PQS was not produced maximally until cultures reached the late stationary phase of growth. Taken together, our results imply that PQS acts as a link between the las and rhl quorum-sensing systems and that this signal is not involved in sensing cell density.

4-Butyrolactone↗