Quorum Sensing Mutants Research Articles

Lethality and cooperation of Pseudomonas aeruginosa quorum-sensing mutants in Drosophila melanogaster infection models

The virulence profiles of Pseudomonas aeruginosa quorum-sensing (QS) mutants were assessed in Drosophila melanogaster feeding and nicking infection models. Functional RhlIR and LasIR QS systems were required for killing in the fly feeding infection model but were not essential in the fly nicking infection model. Mixed infections between PAO1 and strains harbouring mutations in lasR, rhlI and lasI rhlI resulted in increased lethality in the fly feeding model compared with either isolate alone. These results suggested that the parental strain could cooperate with QS mutants in the Drosophila feeding infection model. Finally, the mixed infection between PAO1 and an rhlR mutant resulted in spiteful behaviour and reduced pathogenicity of the mixed culture.

Diffusible Signal Factor-Mediated Quorum Sensing Plays a Central Role in Coordinating Gene Expression of Xanthomonas citri subsp. citri

Diffusible signal factor (DSF) family signal-mediated quorum sensing (QS) has been identified in many gram-negative bacteria. This QS pathway of Xanthomonas spp. consists of three major QS components: RpfF, RpfC, and RpfG. The rpfF gene encodes a putative enoyl-CoA hydratase that catalyzes the synthesis of the signal molecule. RpfC and RpfG serve as a two-component system for the perception and transduction of the extracellular DSF family signals. In order to further characterize the QS regulatory network in Xanthomonas citri subsp. citri, we investigated the RpfF, RpfC, and RpfG regulons by using transcriptome analyses. Comparison of the transcriptomes of the QS mutants (rpfF, rpfC, and rpfG) with that of the wild-type strain revealed a core group of genes controlled by all three QS components, suggesting that the RpfC-RpfG two-component system is a major and conserved signal perception and transduction system for DSF family signal-mediated QS in X. citri subsp. citri. The unique genes controlled by RpfF alone indicate the complexity of the QS pathway and the involvement of additional sensory mechanisms in X. citri subsp. citri. The unique genes controlled by RpfC and RpfG, respectively, support the possibility that RpfC and RpfG play broader roles in gene regulation other than transduction of DSF signals.

The Sociomicrobiology of Antivirulence Drug Resistance: a Proof of Concept

ABSTRACTAntivirulence drugs disarm rather than kill pathogens and are thought to alleviate the problem of resistance, although there is no evidence to support this notion. Quorum sensing (QS) often controls cooperative virulence factor production and is therefore an attractive antivirulence target, for which inhibitors (QSI) have been developed. We designed a proof-of-principle experiment to investigate the impact of bacterial social interactions on the evolution of QSI resistance. We cocultured Pseudomonas aeruginosa QS-deficient mutants with small proportions of the QS-proficient wild type, which in the absence of QSI mimic QSI-sensitive and -resistant variants, respectively. We employed two different QS-dependent nutrients that are degraded by extracellular (public) and cell-associated (private) enzymes. QS mutants (QSI-sensitive mimics) behaved as social cheaters that delayed population growth and prevented enrichment of wild-type cooperators (QSI-resistant mimics) only when nutrient acquisition was public, suggesting that QSI resistance would not spread. This highlights the potential for antivirulence strategies that target cooperative behaviors and provides a conceptual framework for future studies.

Expression of Quorum Sensing and Virulence Factors Are Interlinked in Pseudomonas aeruginosa: an in vitro Approach

Quorum sensing (QS) has been shown to be important for the pathogenesis of Pseudomonas aeruginosa in number of infections and for the expression of various virulence genes. Although QS has been shown to regulate the expression of virulence factors such as elastase, protease and rhamnolipid but link to others such as lipopolysaccharide (LPS) or exo-polysaccharide has not been studied in detail yet. Present study investigated the contribution of QS to the phenotypic expression of multiple virulence factors in vitro. Multiple virulence factors were estimated among wild type P. aeruginosa and its isogenic QS mutant strains by standard methods. Results indicated that QS also regulates the expression of LPS, haemolysin, siderophores and polysaccharides in addition to well-known virulence factors such as elastase, protease and rhamnolipids. Role in biofilm formation and motilities was further established. It may be concluded that QS regulates the expression of multiple virulence factors in P. aeruginosa. It also depicts that QS may regulates more than it is speculated. Therefore, role of QS signal molecules can also be explored further for other regulation possibilities, as futuristic drug target for the development of preventive strategies and important virulence factor per se.

Cooperation and cheating in Pseudomonas aeruginosa: the roles of the las, rhl and pqs quorum-sensing systems

Pseudomonas aeruginosa coordinates the transcription of hundreds of genes, including many virulence genes, through three hierarchically arranged quorum-sensing (QS) systems, namely las, rhl and pqs. Each system consists of genes involved in autoinducer synthesis, lasI, rhlI and pqsABCDH, as well as cognate-regulatory genes, lasR, rhlR and pqsR. In this study, we analyzed the social behavior of signal-blind (ΔlasR, ΔrhlR, ΔpqsR) and signal-negative (ΔlasI, ΔrhlI, ΔpqsA) mutants from each QS system. As each system controls extracellular common goods but differs in the extent of regulatory control, we hypothesized that all signal-blind mutants can behave as cheaters that vary in their ability to invade a QS-proficient population. We found that lasR and pqsR, but not rhlR, mutants evolve from a wild-type ancestor in vitro under conditions that favor QS. Accordingly, defined lasR and pqsR mutants enriched in wild-type co-culture, whereas rhlR and all signal-negative mutants did not. Both lasR and pqsR mutants enriched with negative frequency dependence, suggesting social interactions with the wild type, although the pqsR mutant also grew well on its own. Taken together, the lasR mutant behaved as a typical cheater, as reported previously. However, the pqsR and rhlR mutants exhibited more complex behaviors, which can be sufficiently explained by positive and negative pleiotropic effects through differential regulation of pqs gene expression in the interconnected QS network. The evolutionary approach adopted here may account for the prevalence of naturally occurring QS mutants.

Functional Quorum Sensing Systems are Maintained during Chronic Burkholderia cepacia Complex Infections in Patients with Cystic Fibrosis

Quorum sensing (QS) contributes to the virulence of Pseudomonas aeruginosa and Burkholderia cepacia complex lung infections. P. aeruginosa QS mutants are frequently isolated from patients with cystic fibrosis. The objective of this study was to determine whether similar adaptations occur over time in B. cepacia complex isolates. Forty-five Burkholderia multivorans and Burkholderia cenocepacia sequential isolates from patients with cystic fibrosis were analyzed for N-acyl-homoserine lactone activity. All but one isolate produced N-acyl-homoserine lactones. The B. cenocepacia N-acyl-homoserine lactone-negative isolate contained mutations in cepR and cciR. Growth competition assays were performed that compared B. cenocepacia clinical and laboratory defined wild-type and QS mutants. Survival of the laboratory wild-type and QS mutants varied, dependent on the mutation. The clinical wild-type isolate demonstrated a growth advantage over its QS mutant. These data suggest that there is a selective advantage for strains with QS systems and that QS mutations do not occur at a high frequency in B. cepacia complex isolates.

Biofilm Development on Caenorhabditis elegans by Yersinia Is Facilitated by Quorum Sensing-Dependent Repression of Type III Secretion

Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS.

P. aeruginosa Quorum Sensing Sistemi ve Deneysel Kronik Akciğer Enfeksiyonu Modeli

Opportunistic pathogen Pseudomonas aeruginosa is a model microorganism used in explaining cell-to-cell signaling system, named Quorum Sensing (QS). In chronic P. aeruginosa infections, bacteria survive in biofilm and defend themselves against host defense mechanisms by different virulence factors. Three known QS systems, las, rhl and quinolones are basically responsible for the production of virulence factors. Experimental animal models are commonly used in examining in vitro and in vivo behaviors of the bacteria, and explaining interactions between host, bacteria and antibacterials. Among these, chronic pulmonary infection model, applied by administration of agar-impreagnated bacterial suspensions into trachea, is special because it mimicks the biofilm, a virulence factor. Comparative studies conducted particulary with QS wild-type and QS mutant strains, provide important information for understanding the pathogenesis of infection. In this article, the role of P. aeruginosa QS system in the experimental model of chronic lung infection is focused.

Active regulation of receptor ratios controls integration of quorum‐sensing signals in Vibrio harveyi

Quorum sensing is a chemical signaling mechanism used by bacteria to communicate and orchestrate group behaviors. Multiple feedback loops exist in the quorum-sensing circuit of the model bacterium Vibrio harveyi. Using fluorescence microscopy of individual cells, we assayed the activity of the quorum-sensing circuit, with a focus on defining the functions of the feedback loops. We quantitatively investigated the signaling input-output relation both in cells with all feedback loops present as well as in mutants with specific feedback loops disrupted. We found that one of the feedback loops regulates receptor ratios to control the integration of multiple signals. Together, the feedback loops affect the input-output dynamic range of signal transmission and the noise in the output. We conclude that V. harveyi employs multiple feedback loops to simultaneously control quorum-sensing signal integration and to ensure signal transmission fidelity.

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:ΔLasI, PAO1:ΔLasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P<0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung.

Co-Swarming and Local Collapse: Quorum Sensing Conveys Resilience to Bacterial Communities by Localizing Cheater Mutants in Pseudomonas aeruginosa

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

Effects of iron depletion on antimicrobial activities against planktonic and biofilm Pseudomonas aeruginosa

Abstract Objectives Iron plays an important role in the development of Pseudomonas aeruginosa biofilm. Here we evaluated effects of iron depletion on the antimicrobial activity of ceftazidime, tobramycin and ciprofloxacin against planktonic and biofilm Pseudomonas aeruginosa. Methods We tested the sensitivities of wild-type PAO1, type-IV pilus mutant PAO-ΔpilHIJK and the quorum-sensing mutant PAO-JP2 P. aeruginosa planktonic cultures and biofilms to antibiotics under iron-depleted conditions. Key findings In planktonic bacteria, the minimum concentration that inhibited visible growth (MIC) of ciprofloxacin was increased slightly in an iron-depleted environment in all three strains, whereas the MIC of tobramycin was similar in iron-depleted and control environments. The MIC of ceftazidime increased in the PAO-JP2 strain when iron was depleted. Tobramycin achieved the best bactericidal effect in biofilms. Viable counts were reduced by one log under iron-depleted conditions in all three strains when tobramycin reached 4 MIC and when ceftazidime and ciprofloxacin reached 8 MIC. Conclusions This study suggests that once the biofilm is formed, iron depletion may only slightly promote the bactericidal effect of antibiotics on PAO1, PAO-ΔpilHIJK and PAO-JP2. Although these changes were relatively small, iron as one of the environmental factors should not be ignored when evaluating bactericidal effect of antibiotics. The combination of an iron chelator and antibiotics may have therapeutic value under certain bacterial growth conditions.

Quorum-sensing-regulated virulence factors in Pseudomonas aeruginosa are toxic to Lucilia sericata maggots

Maggot debridement therapy (MDT) is widely used for debridement of chronic infected wounds; however, for wounds harbouring specific bacteria limited effect or failure of the treatment has been described. Here we studied the survival of Lucilia sericata maggots encountering Pseudomonas aeruginosa PAO1 in a simple assay with emphasis on the quorum-sensing (QS)-regulated virulence. The maggots were challenged with GFP-tagged P. aeruginosa wild-type (WT) PAO1 and a GFP-tagged P. aeruginosa ΔlasR rhlR (ΔRR) QS-deficient mutant in different concentrations. Maggots were killed in the presence of WT PAO1 whereas the challenge with the QS mutant showed a survival reduction of ∼25 % compared to negative controls. Furthermore, bacterial intake by the maggots was lower in the presence of WT PAO1 compared to the PAO1 ΔRR mutant. Maggot excretions/secretions (ES) were assayed for the presence of QS inhibitors; only high doses of ES showed inhibition of QS in P. aeruginosa. Thus P. aeruginosa was shown to be toxic to L. sericata maggots. This, coupled to the preferential feeding by the maggots and reduced ingestion of P. aeruginosa, could explain MDT failure in wounds colonized by P. aeruginosa. Wounds heavily colonized with P. aeruginosa should be a counterindication for MDT unless used in combination with a pre-treatment with other topical therapeutics targeting P. aeruginosa.

AI-2 quorum-sensing inhibitors affect the starvation response and reduce virulence in several Vibrio species, most likely by interfering with LuxPQ

The increase of disease outbreaks caused by Vibrio species in aquatic organisms as well as in humans, together with the emergence of antibiotic resistance in Vibrio species, has led to a growing interest in alternative disease control measures. Quorum sensing (QS) is a mechanism for regulating microbial gene expression in a cell density-dependent way. While there is good evidence for the involvement of auto-inducer 2 (AI-2)-based interspecies QS in the control of virulence in multiple Vibrio species, only few inhibitors of this system are known. From the screening of a small panel of nucleoside analogues for their ability to disturb AI-2-based QS, an adenosine derivative with a p-methoxyphenylpropionamide moiety at C-3' emerged as a promising hit. Its mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of Vibrio harveyi AI-2 QS mutants. Our results indicate that this compound, as well as a truncated analogue lacking the adenine base, block AI-2-based QS without interfering with bacterial growth. The active compounds affected neither the bioluminescence system as such nor the production of AI-2, but most likely interfered with the signal transduction pathway at the level of LuxPQ in V. harveyi. The most active nucleoside analogue (designated LMC-21) was found to reduce the Vibrio species starvation response, to affect biofilm formation in Vibrio anguillarum, Vibrio vulnificus and Vibrio cholerae, to reduce pigment and protease production in V. anguillarum, and to protect gnotobiotic Artemia from V. harveyi-induced mortality.

Effects of iron depletion on antimicrobial activities against planktonic and biofilm &lt;I&gt;Pseudomonas aeruginosa&lt;/I&gt;

Iron plays an important role in the development of Pseudomonas aeruginosa biofilm. Here we evaluated effects of iron depletion on the antimicrobial activity of ceftazidime, tobramycin and ciprofloxacin against planktonic and biofilm Pseudomonas aeruginosa. We tested the sensitivities of wild-type PAO1, type-IV pilus mutant PAO-DeltapilHIJK and the quorum-sensing mutant PAO-JP2 P. aeruginosa planktonic cultures and biofilms to antibiotics under iron-depleted conditions. In planktonic bacteria, the minimum concentration that inhibited visible growth (MIC) of ciprofloxacin was increased slightly in an iron-depleted environment in all three strains, whereas the MIC of tobramycin was similar in iron-depleted and control environments. The MIC of ceftazidime increased in the PAO-JP2 strain when iron was depleted. Tobramycin achieved the best bactericidal effect in biofilms. Viable counts were reduced by one log under iron-depleted conditions in all three strains when tobramycin reached 4 MIC and when ceftazidime and ciprofloxacin reached 8 MIC. This study suggests that once the biofilm is formed, iron depletion may only slightly promote the bactericidal effect of antibiotics on PAO1, PAO-DeltapilHIJK and PAO-JP2. Although these changes were relatively small, iron as one of the environmental factors should not be ignored when evaluating bactericidal effect of antibiotics. The combination of an iron chelator and antibiotics may have therapeutic value under certain bacterial growth conditions.

Quorum sensing is necessary for the virulence of Pseudomonas aeruginosa during urinary tract infection

Quorum sensing is a signaling pathway used by bacteria to monitor their population density by detecting small diffusible molecules. To understand the role of quorum sensing in pathogenesis of urinary tract infections, wild type Pseudomonas aeruginosa, having both functional las and rhl quorum sensing systems, and its isogenic single and double mutants were used in a mouse model of ascending urinary tract infection. Mice were evaluated on the basis of bacteriological and histopathological examinations, detection of acyl homoserine lactone, and the pathologic index factors of renal and bladder tissue. Single as well as double mutant strains were able to reach renal tissue, but they were not able to multiply and cause tissue damage. Reduced rates of infection, mild histopathologic lesions, and decreased production of pathologic index factors like myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates were found in these groups of mice as compared to mice infected with a standard strain (PAO1) possessing both functional sensing systems. Loss of any one of the quorum-sensing components significantly reduced the in vivo virulence of P. aeruginosa in this mouse model of ascending urinary tract infection. Our results suggests that quorum-sensing signals may act as virulence factors and are essential for P. aeruginosa multiplication and virulence during the course of urinary tract infection.

Mutational Analysis ofBurkholderia thailandensisQuorum Sensing and Self-Aggregation

Acyl-homoserine lactone (acyl-HSL) quorum-sensing signaling is common to many Proteobacteria. Acyl-HSLs are synthesized by the LuxI family of synthases, and the signal response is mediated by members of the LuxR family of transcriptional regulators. Burkholderia thailandensis is a member of a closely related cluster of three species, including the animal pathogens Burkholderia mallei and Burkholderia pseudomallei. Members of this group have similar luxI and luxR homologs, and these genes contribute to B. pseudomallei and B. mallei virulence. B. thailandensis possesses three pairs of luxI-luxR homologs. One of these pairs, BtaI2-BtaR2, has been shown to produce and respond to 3OHC(10)-HSL and to control the synthesis of an antibiotic. By using a markerless-exhange method, we constructed an assortment of B. thailandensis quorum-sensing mutants, and we used these mutants to show that BtaI1 is responsible for C(8)-HSL production and BtaI3 is responsible for 3OHC(8)-HSL production. We also show that a strain incapable of acyl-HSL production is capable of growth on the same assortment of carbon and nitrogen sources as the wild type. Furthermore, this mutant shows no loss of virulence compared to the wild type in mice. However, the wild type self-aggregates in minimal medium, whereas the quorum-sensing mutant does not. The wild-type aggregation phenotype is recovered by addition of the BtaI1-R1 HSL signal C(8)-HSL. We propose that the key function of the BtaR1-BtaI1 quorum-sensing system is to cause cells to gather into aggregates once a sufficient population has been established.

Production of the antifungal compound pyrrolnitrin is quorum sensing‐regulated in members of the Burkholderia cepacia complex

Members of the genus Burkholderia are known for their ability to suppress soil-borne fungal pathogens by the production of various antibiotic compounds. In this study we investigated the role of N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) in the expression of antifungal traits. Using a quorum quenching approach, that is, by heterologous expression of the Bacillus sp. AiiA lactonase, we show that expression of antifungal activities is AHL-dependent in the large majority of the investigated strains belonging to various Burkholderia species. We demonstrate that in certain strains of Burkholderia ambifaria, Burkholderia pyrrocinia and Burkholderia lata, one of the QS-regulated antifungal agents is pyrrolnitrin (prn), a common broad-spectrum antibiotic that is also produced by some Pseudomonas and Serratia species. To investigate the underlying molecular mechanisms of AHL-dependent prn production in better detail, we inactivated the AHL synthase cepI as well as cepR, which encodes the cognate AHL receptor protein, in B. lata 383. Both QS mutants no longer produced prn as assessed by gas chromatography-mass spectrometry analysis and as a consequence were unable to inhibit growth of Rhizoctonia solani. Using fusions of the lacZ gene to the promoter of the prnABCD operon, which directs the synthesis of prn, we demonstrate that expression of prn is positively regulated by CepR at the level of transcription.

Do We Have a Quorum?

MICROBIOLOGY Bacteria can communicate with one another and act cooperatively as a group—a process known as quorum sensing (QS). However, such communication and cooperation can be exploited by “cheaters,” who benefit from the shared metabolic or otherwise beneficial activities without contributing to them. In the case of the pathogenic bacterium Pseudomonas aeruginosa , the signal molecules that facilitate population-wide QS regulate important disease-related processes, such as biofilm formation, swarming motility, and the production of secreted virulence factors. Rumbaugh et al. demonstrate that this potentially deadly cocktail of activities can be undermined by mutant bacteria that cheat. In a burn injury model in mice, infection with QS P. aeruginosa leads to rapid death of the host; infection with QS mutant bacteria resulted in significantly less mortality. The mortality caused by QS bacteria was substantially diminished in the presence of the mutant bacteria, which, through their exploitation of the QS bacteria, were able to flourish at their benefactors' expense, reducing the overall virulence of the infection. Furthermore, the mutant bacteria, which cannot normally mount an effective systemic infection, were able to spread to the liver in the presence of the QS bacteria, presumably by piggy-backing on the social activities of the QS bacteria. QS mutants are known to arise in clinical settings and may thus affect the virulence and course of infections. — GR Curr. Biol. 19 , 341 (2009).

ThecinandraiQuorum-Sensing Regulatory Systems inRhizobium leguminosarumAre Coordinated by ExpR and CinS, a Small Regulatory Protein Coexpressed with CinI

To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.