Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, uses a cell-cell signaling system called quorum sensing to coordinate group behaviors. Quorum sensing in P. aeruginosa is a model to study cooperative behaviors in populations. Several studies of cooperation have been conducted using strain PAO1. Wild-type PAO1 harbors a mutation in mexS, which encodes a negative regulator of the transcription factor MexT, which, in turn, activates many genes, including the efflux pump MexEF-OprN. We hypothesized that the PAO1 mexS mutation might affect cooperative behaviors. When P. aeruginosa is passaged daily on casein as a sole carbon source, quorum sensing is required to induce synthesis of the extracellular proteases needed to acquire carbon and energy. When PAO1 is grown on casein, individuals with inactivating mutations in the quorum-sensing regulator LasR are reproducibly enriched in the population. These LasR mutants are cheaters that benefit from cooperatively produced proteases and have a fitness advantage over cooperators. We passaged wild-type PAO1, PAO1 with a gene-corrected version of mexS, or PAO1 with a null mutant of mexT on casein as the sole carbon and energy source. We found that correcting the mexS mutation resulted in unstable cooperation: bacterial cultures failed to propagate after about 15 days, whereas the wild type propagated for the duration of our 30-day experiment. The MexS-corrected and MexT-deficient populations also reproducibly exhibited emergence of a particular quorum-sensing variant, LasR-V226I. This variant activated a subset of quorum-sensing regulated genes, suggesting an evolutionary pathway to alter P. aeruginosa quorum-sensing regulons.IMPORTANCEQuorum sensing governs cooperative activities and has been used as a model for studying cooperative behaviors in bacterial populations. Pseudomonas aeruginosa has two interlinked acyl-homoserine lactone quorum-sensing circuits, LasR-I and RhlR-I; the relationship between these circuits is influenced by the regulator MexT. In the well-studied strain PAO1, inactivation of MexT results in enhanced activity of the quorum-sensing transcription factor, RhlR, and we found that it also destabilized cooperative behaviors in this bacterium. MexT-inactive strains also reproducibly support the emergence of a specific LasR variant, V226I, of P. aeruginosa, offering insight into evolutionary pressures that select for mutations in quorum-sensing transcription factors and, by extension, a method for bacteria to alter the cohort of genes that are quorum-controlled.

Acyl-homoserine lactone (AHL) quorum sensing enables many species of proteobacteria to coordinate collective behaviors. In such systems, a synthase produces an AHL signal, which is sensed by a LuxR-family receptor. Despite extensive genomic annotation of LuxR homologs, preferred AHLs for most receptors remain unknown, limiting functional understanding of quorum sensing across diverse bacteria. Here, we present the COPAL (combining ordered predictions of audited ligands) pipeline, which integrates multiple protein-ligand co-folding models to identify preferred AHLs for a specific LuxR. Benchmarking on a leakage-controlled subset of 96 experimentally characterized LuxR-AHL pairs shows that COPAL places the preferred AHL within the top-6 candidates (out of 58) for 68% of receptors, outperforming every individual co-folding model. Further, inter-model agreement correlates with ranking accuracy, offering an indication of confidence. We show that COPAL resolves the specificity shift induced by three-point mutations in LasR and correctly nominates C8-HSL as the preferred ligand for the previously uncharacterized Mesorhizobium sp. NJ3 receptor, which we verified experimentally. Finally, we release the Ranked AHL-LuxR Prediction Hub (RALPH), comprising precomputed rankings for about 10,000 unique LuxR homologs. More broadly, COPAL shows that unweighted rank aggregation of complementary co-folding models offers a general strategy for predicting receptor-ligand specificity in data-scarce biological systems.

Some bacteria use acyl-homoserine lactone (AHL) signals in quorum sensing, a type of cell-cell communication. Here, we present "RefAHL," an updated, curated collection of LuxI-type AHL synthases with their AHL products and associated metadata. RefAHL is publicly available as a community resource to help catalog LuxI-type diversity encoded in (meta) genomic data.

Cationic polymers have emerged as promising next-generation antimicrobial agents, albeit with inherent limitations such as low potency and limited biocompatibility. Classical cationic polymers kill bacteria via physical membrane disruption. We propose a non-classical mechanism of crossing the bacterial plasma membrane barrier, a step required for subsequent inhibition of intracellular targets, by cationic polymers which are carbon acids. Oligoimidazolium (OIM) carbon acids, instead of lysing bacteria, transiently deprotonate in water to form hydrophobic N-heterocyclic carbenes (NHCs) and exhibit efficient plasma membrane translocation. Only OIMs that are carbon acids have potent antibacterial activities against even colistin- and multidrug-resistant bacteria. OIM amide derivatives exhibit excellent antibacterial efficacy in murine sepsis and thigh infection models, while a polymeric version acts as a prophylactic agent against bovine mastitis, which is a global agricultural problem. This study unveils a promising path for the development of an alternative class of potent antimicrobial agents.

Pseudomonas aeruginosa uses quorum sensing (QS) to coordinate cooperative behaviors. In the model strain PAO1, QS activates dozens of genes, including those for cyanide production and cyanide resistance. The induction of cyanide synthesis and resistance mechanisms serves to constrain the emergence of QS mutants. Strain CI27 does not possess the cyanide synthesis genes. Here, we ask if this isolate can constrain emergence of QS mutants. In daily transfer experiments, where PAO1 cyanide synthesis mutants are incapable of QS mutant restraint, we found that CI27 constrained emergence of QS mutants. Genome sequencing revealed that a temperate phage, which we call RC5, in evolved QS mutants had copied itself at new chromosomal locations. This led to investigations of the relationship between QS and temperate phages in CI27. We found seven prophage genomes in the CI27 chromosome (RC1-7), one of which occurred in two copies (RC3). Upon mitomycin C induction of CI27, we found four different phage genomes in lysates with RC7 predominating. The lysates of a QS mutant contained three times as many phages as lysates. Without mitomycin C induction, phage numbers in culture fluid from either the wild-type or a CI27 QS mutant were low, but upon daily transfer, phage numbers increased dramatically in the QS mutant. The increase was due to RC3 specifically, and after several transfers, the mutant cultures underwent a massive lysis. Further experiments indicated that RC3 protects P. aeruginosa from QS mutant invasion. Protection might result at least in part from the fact that LasR mutants are more susceptible to RC3 superinfection than the wild type.IMPORTANCEQuorum sensing (QS) enables bacteria such as Pseudomonas aeruginosa to coordinate cooperative activities. How bacteria in cooperating groups can resist infiltration by non-cooperating variants is an emerging area of interest in sociobiology and molecular biology. There have been several recent reports on how QS and certain bacteriophage interact. In some strains of P. aeruginosa, QS can activate phage defense systems. At least one bacteriophage can repress P. aeruginosa QS. Here, we show that a previously undescribed bacteriophage can help cooperating groups of P. aeruginosa resist infiltration by non-cooperating QS mutants. This represents a mutualism in which both the bacteriophage and the P. aeruginosa host benefit at least under certain conditions.

Drug resistance in bacteria is a major problem that calls for new classes of antimicrobial drugs. We report a biodegradable poly(imidazolium ester) (PIE), P8, with excellent broad-spectrum antibacterial activity, high therapeutic selectivity, and an unexploited mechanism of action. P8, a short oligomer, translocates across bacterial membrane and phase separates with intracellular nucleic acids, forming biomolecular condensates. P8 binds the DNA minor groove and intercalates with DNA, interacting with it via electrostatic and hydrogen-bonding interactions. The phase separation of nucleic acids modulated by P8 inhibits in vitro transcription, thereby impeding translation and potentially leading to cell death. Bacterial cytological profiling indicates that the antimicrobial mechanism of P8 differs from those of conventional antibiotics, though it also suggests that P8 may inhibit RNA synthesis. P8 is safe and effective against drug-resistant bacteria in murine models of systemic, intramuscular, and lung infections. This study shows the great potential of intracellular biomolecular condensate formation for combating drug-resistant bacteria.

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa has complex quorum sensing (QS) circuitry, which involves two acylhomoserine lactone (AHL) systems, the LasI AHL synthase and LasR AHL-dependent transcriptional activator system and the RhlI AHL synthase-RhlR AHL-responsive transcriptional activator. There is also a quinoline signaling system (the Pseudomonas quinolone signal, PQS, system). Although there is a core set of genes regulated by the AHL circuits, there is substantial strain-to-strain variation in the non-core QS regulated genes. Reductive evolution of the QS regulon, and variation in specific genes activated by QS, occurs in laboratory evolution experiments with the model strain PAO1. We used a transcriptomics approach to test the hypothesis that reductive evolution in the PAO1 QS regulon can in large part be explained by a simple null mutation in pqsR , the gene encoding the transcriptional activator of the pqs operon. We found that PqsR had very little influence on the AHL QS regulon. This was a surprising finding because the last gene in the PqsR-dependent pqs operon, pqsE , codes for a protein, which physically interacts with RhlR and this interaction is required for RhlR-dependent activation of some genes. We used comparative transcriptomics to examine the influence of a pqsE mutation on the QS regulon and identified only three transcripts, which were strictly dependent on PqsE. By using reporter constructs we showed that the PqsE influence on other genes was dependent on experimental conditions and we have gained some insight about those conditions. This work adds to our understanding of the plasticity of the P. aeruginosa QS regulon and to the role PqsE plays in RhlR-dependent gene activation.

The opportunistic pathogen Pseudomonas aeruginosa has complex quorum sensing (QS) circuitry, which involves two acylhomoserine lactone (AHL) systems, the LasI AHL synthase and LasR AHL-dependent transcriptional activator system and the RhlI AHL synthase-RhlR AHL-responsive transcriptional activator. There is also a quinoline signaling system [the Pseudomonas quinolone signal (PQS) system]. Although there is a core set of genes regulated by the AHL circuits, there is strain-to-strain variation in the non-core QS regulon. A size reduction of the QS regulon occurs in laboratory evolution experiments with the model strain PAO1. We used transcriptomics to test the hypothesis that reductive evolution in the PAO1 QS regulon can in large part be explained by a null mutation in pqsR, the gene encoding the transcriptional activator of the pqs operon. We found that PqsR had very little influence on the AHL QS regulon. This was a surprising finding because the last gene in the PqsR-dependent pqs operon, pqsE, codes for a protein, which physically interacts with RhlR, and this interaction is required for RhlR-dependent activation of some genes. We used comparative transcriptomics to examine the influence of a pqsE mutation on the QS regulon and identified only three transcripts, which were strictly dependent on PqsE. By using reporter constructs, we showed that the PqsE influence on other genes was dependent on experimental conditions and we have gained some insight about those conditions. This work adds to our understanding of the plasticity of the P. aeruginosa QS regulon and to the role PqsE plays in RhlR-dependent gene activation.IMPORTANCEOver many generations of growth in certain conditions, Pseudomonas aeruginosa undergoes a large reductive evolution in the number of genes activated by quorum sensing. Here, we rule out one plausible route of the reductive evolution: that a mutation in a transcriptional activator PqsR or the PqsR activation of pqsE, which codes for a chaperone for the quorum sensing signal-responsive transcription factor RhlR, explains the finding. We further provide information about the influence of PqsR and PqsE on quorum sensing in P. aeruginosa.

A few decades ago, scientists believed that bacteria were very basic creatures that did not communicate with each other and were only good at multiplying. Recently, we have realized that this is far from the truth! Bacteria communicate with one another using a language called quorum sensing. You can think of bacterial quorum sensing as the first-ever social network! In this article, we will tell you about the discovery of quorum sensing and how it radically changed our understanding of the microbial world. We will also tell you how our new knowledge of quorum sensing might help doctors to treat dangerous bacterial infections in humans. Join us in this journey exploring the fascinating language of bacteria and how it could benefit human health.

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.

ABSTRACT Quorum sensing (QS) regulates expression of many virulence genes in the opportunistic human pathogen Pseudomonas aeruginosa . There are two acyl-homoserine lactone (AHL) QS circuits, LasI-R and RhlI-R, which together activate expression of hundreds of genes in a cell density-dependent manner. There is also an inter-related non-AHL QS circuit, the Pseudomonas Quinolone Signal (PQS) circuit. Studies of the model strain PAO1 show that AHL QS is hierarchical, with the LasI-R system required for full activity of RhlI-R. We show that the LysR-type transcription factor MexT is an important modulator of the RhlI-R and PQS circuits. MexT delays the expression of genes activated by PQS and by the RhlI-R circuits but not genes regulated by the LasI-R circuit. In PAO1, MexT is constitutively active because of a mutation in mexS , a gene involved in maintaining redox homeostasis. Consistent with other reports, we show that in another model strain, PA14, MexT is quiescent but is activated by disulfide stress, and we show that this is true for all clinical isolates we tested. Compared with strain PA14, strain PAO1 has a virulence defect in a Caenorhabditis elegans infection model. We show that PAO1 mutants with inactive MexT are more virulent than their parent during C. elegans infection. Conversely, a PA14 mutant with a constitutively active MexT is attenuated for virulence. Our findings point to an important role for MexT as a governor of P. aeruginosa virulence and show that MexT limits virulence though its inhibition of the RhlI-R system. IMPORTANCE Pseudomonas aeruginosa is an opportunistic bacterial pathogen. Many of its virulence genes are regulated by quorum sensing (QS), a form of cell-to-cell communication. P. aeruginosa QS consists of three interlinked circuits, LasI-R, Rhl-R, and Pseudomonas quinolone signal (PQS). Additionally, its QS system is interconnected with other regulatory networks, which help optimize gene expression under variable conditions. The numbers of genes regulated by QS differ substantially among P. aeruginosa strains. We show that a regulatory factor MexT, which is activated in response to certain antibiotics, downregulates the RhlI-R circuit and in turn measurably lowers virulence in a nematode worm infection model. Our findings help understand how existing and future therapeutic interventions for P. aeruginosa infections may impact this bacterium’s gene regulation and physiology.

ABSTRACT Many bacterial species communicate and cooperate with kin using a form of cell-cell signaling called quorum sensing. Sensitivity and selectivity are fundamental properties of quorum sensing, and of cell signaling more generally, as they dictate the ability of cells to accurately detect and interpret signals. In this study, we investigate the impact of signal sensitivity on gene regulation and on bacterial fitness using two variants of the LasR quorum sensing receptor in the opportunistic pathogen Pseudomonas aeruginosa . Our studies reinforce that altering LasR signal-binding residues tends to result in decreased signal selectivity. We also find that LasR hyper-sensitivity results in earlier and stronger expression of LasR-regulated genes, whereas hypo-sensitivity results in delayed transcription from both LasR- and RhlR-regulated promoters. Furthermore, strains expressing either the hyper- or hypo-sensitive LasR polypeptide exhibit reduced production of pyocyanin, a key antimicrobial that impacts intra- and interspecies competition as well as pathogenicity. These changes to gene expression underscore the complex regulatory network by which P. aeruginosa controls group behaviors. Ultimately, altered signal sensitivity results in a fitness defect for LasR hyper-sensitive cells during kin competition against wild-type P. aeruginosa and for hypo-sensitive cells during interspecies competition against the opportunistic pathogen Burkholderia multivorans . Our findings highlight the delicate balance between group behaviors regulated by quorum sensing and bacterial fitness and contribute to a better understanding of the evolutionary pressures that may tune sensitivity in cell-cell signaling. IMPORTANCE Quorum sensing (QS) is a widespread form of cell-cell signaling that regulates group behaviors important for competition and cooperation within bacterial communities. The QS systems from different bacterial species have diverse properties, but the functional consequences of this diversity are largely unknown. Taking advantage of hyper- and hypo-sensitive QS receptor variants in the opportunistic pathogen Pseudomonas aeruginosa , we examine the costs and benefits of altered signal sensitivity. We find that the sensitivity of a model QS receptor, LasR, impacts the timing and level of quorum gene expression, and fitness during intra- and interspecies competition. These findings suggest competition with kin and with other bacterial species work together to tune signal sensitivity.

Quorum sensing is a term describing bacterial cell-to-cell communication systems for monitoring and responding to changes in population density. This primer serves as an introduction to the canonical LuxR-LuxI-type quorum sensing circuits common to many species of Gram-negative bacteria. Quorum sensing can synchronize behaviours across a community. Different species employ quorum sensing strategies to control specific behaviours such as bioluminescence, virulence factor production, secondary metabolite production, and biofilm formation.

Pseudomonas aeruginosa, like many bacteria, uses chemical signals to communicate between cells in a process called quorum sensing (QS). QS allows groups of bacteria to sense population density and, in response to changing cell densities, to coordinate behaviors. The P. aeruginosa QS system consists of two complete circuits that involve acyl-homoserine lactone signals and a third system that uses quinolone signals. Together, these three QS circuits regulate the expression of hundreds of genes, many of which code for virulence factors. P. aeruginosa has become a model for studying the molecular biology of QS and the ecology and evolution of group behaviors in bacteria. In this chapter, we recount the history of discovery of QS systems in P. aeruginosa, discuss how QS relates to virulence and the ecology of this bacterium, and explore strategies to inhibit QS. Finally, we discuss future directions for research in P. aeruginosa QS.

In the opportunistic pathogenic bacterium Pseudomonas aeruginosa acylhomoserine lactone quorum sensing (QS) can activate expression of dozens to hundreds of genes depending on the strain under investigation. Many QS-activated genes code for extracellular products. P. aeruginosa has become a model for studies of cellcell communication and coordination of cooperative activities, which result from production of extracellular products. We hypothesized that strain variation in the size of the QS regulon might reflect the environmental history of an isolate. We tested the hypothesis by performing long-term growth experiments with the well-studied strain PAO1, which has a relatively large QS regulon, under conditions where only limited QScontrolled functions are required. We grew P. aeruginosa for about 1000 generations in a condition where expression of QS-activated genes was required, and emergence of QS mutants was constrained and compared the QS regulons of populations after 35 generations to those after about 1000 generations in two independent lineages by using quorum quenching and RNA-seq technology. In one lineage the number of QSactivated genes identified was reduced by over 60% and in the other by about 30% in 1000-generation populations compared to 35-generation populations. Our results provide insight about the variations in the number of QS-activated genes reported for different P. aeruginosa environmental and clinical isolates and, about how environmental conditions might influence social evolution. IMPORTANCE Pseudomonas aeruginosa uses quorum sensing (QS) to activate expression of dozens of genes (the QS regulon). Because there is strain-to-strain variation in the size and content of the QS regulon, we asked how the regulon might evolve during long-term P. aeruginosa growth when cells require some but not all the functions activated by QS. We demonstrate that the P. aeruginosa QS-regulon can undergo a reductive adaptation in response to continuous QS-dependent growth. Our results provide insights into why there is strain-to-strain variability in the size and content of the P. aeruginosa QS regulon.

A number of plant-associated proteobacteria have LuxR family transcription factors that we refer to as PipR subfamily members. PipR proteins play roles in interactions between bacteria and their plant hosts, and some are important for bacterial virulence of plants. We identified an ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), as a potent effector of PipR-mediated gene regulation in the plant endophyte Pseudomonas GM79. HEHEAA-dependent PipR activity requires an ATP-binding cassette-type active transport system, and the periplasmic substrate-binding protein (SBP) of that system binds HEHEAA. To begin to understand the molecular basis of PipR system responses to plant factors we crystallized a HEHEAA-responsive SBP in the free- and HEHEAA-bound forms. The SBP, which is similar to peptide-binding SBPs, was in a closed conformation. A narrow cavity at the interface of its two lobes is wide enough to bind HEHEAA, but it cannot accommodate peptides with side chains. The polar atoms of HEHEAA are recognized by hydrogen-bonding interactions, and additional SBP residues contribute to the binding site. This binding mode was confirmed by a structure-based mutational analysis. We also show that a closely related SBP from the plant pathogen Pseudomonas syringae pv tomato DC3000 does not recognize HEHEAA. However, a single amino acid substitution in the presumed effector-binding pocket of the P. syringae SBP converted it to a weak HEHEAA-binding protein. The P. syringae PipR depends on a plant effector for activity, and our findings imply that different PipR-associated SBPs bind different effectors.

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.