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The bacterium Pseudomonas aeruginosa is an opportunistic pathogen and it thrives in many different saprophytic habitats. In this bacterium acyl-homoserine lactone quorum sensing (QS) can activate expression of over 100 genes, many of which code for extracellular products. P. aeruginosa has become a model for studies of cell-cell communication and coordination of cooperative activities. We hypothesized that long-term growth of bacteria under conditions where only limited QS-controlled functions were required would result in a reduction in the size of the QS-controlled regulon. To test this hypothesis, we grew P. aeruginosa for about 1000 generations in a condition in which expression of QS-activated genes is required for growth. We compared the QS regulons of populations after about 35 generations to those after about 1000 generations in two independent lineages by using quorum quenching and RNA-seq technology. In one evolved lineage the number of QS-activated genes identified was reduced by about 70% and in the other by about 45%. Our results lend important insights about the variations in the number of QS-activated genes reported for different bacterial strains and, more broadly, about the environmental histories of P. aeruginosa.

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., eLife digest Communication is vital in any community and it is no different for bacteria. Some of the microbes living in bacterial communities are closely related to one another and can help each other survive and grow. They do this by releasing chemical signals that coordinate their behaviors, including those that are damaging to the infected host. A key aspect of this coordination is knowing how many related bacteria are present in a given environment. In a process known as quorum sensing, the bacteria release a chemical signal which neighboring sibling bacteria detect and respond to. The larger the population of bacteria, the more the signal accumulates. At a certain threshold, the signal activates the genes needed to trigger a coordinated action, such as producing toxins or antibiotics. Many bacteria communicate using acylhomoserine lactone signaling systems, which involve different signals depending on the species of bacteria. But it is unclear how this diversity evolved, and how bacteria can distinguish between signals from related and unrelated bacterial cells. To understand this, Wellington Miranda et al. used computational techniques to analyze how the proteins responsible for acylhomoserine lactone signaling coevolved. The analysis identified specific parts of these proteins that determine which signal will be produced and which will trigger a bacterium into action. Wellington Miranda et al. then used these insights to engineer the bacteria Pseudomonas aeruginosa to produce and respond to a signal that is naturally made by another bacterial species. These computational methods could be used to analyze other proteins that have coevolved but do not physically interact. Within the area of quorum sensing, this approach will help to better understand the costs and benefits of signal selectivity. This may help to predict bacterial interactions and therefore behaviors during infections.

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 6,000 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 a new application for covariation methods and have deepened our understanding of how communication systems evolve and diversify.

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.

Certain plant-associated Proteobacteria sense their host environment by detecting an unknown plant signal recognized by a member of a LuxR subfamily of transcription factors. This interkingdom communication is important for both mutualistic and pathogenic interactions. The Populus root endophyte Pseudomonas sp. GM79 possesses such a regulator, named PipR. In a previous study we reported that PipR activates an adjacent gene (pipA) coding for a proline iminopeptidase in response to Populus leaf macerates and peptides and that this activation is dependent on a putative ABC-type transporter [Schaefer AL, et al. (2016) mBio 7:e01101-16]. In this study we identify a chemical derived from ethanolamine that induces PipR activity at picomolar concentrations, and we present evidence that this is the active inducer present in plant leaf macerates. First, a screen of more than 750 compounds indicated ethanolamine was a potent inducer for the PipR-sensing system; however, ethanolamine failed to bind to the periplasmic-binding protein (PBP) required for the signal response. This led us to discover that a specific ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), binds to the PBP and serves as a potent PipR-dependent inducer. We also show that a compound, which coelutes with HEHEAA in HPLC and induces pipA gene expression in a PipR-dependent manner, can be found in Populus leaf macerates. This work sheds light on how plant-associated bacteria can sense their environment and on the nature of inducers for a family of plant-responsive LuxR-like transcription factors found in plant-associated bacteria.

The phytopathogen Dickeya zeae MS2 is a particularly virulent agent of banana soft rot disease. To begin to understand this banana disease and to understand the role of quorum sensing and quorum-sensing-related regulatory elements in D. zeae MS2, we sequenced its genome and queried the sequence for genes encoding LuxR homologs. We identified a canonical LuxR-LuxI homolog pair similar to those in other members of the genus Dickeya. The quorum-sensing signal for this pair was N-3-oxo-hexanoyl-homoserine lactone, and the circuit affected motility, cell clumping, and production of the pigment indigoidine, but it did not affect infections of banana seedlings in our experiments. We also identified a luxR homolog linked to a gene annotated as encoding a proline iminopeptidase. Similar linked pairs have been associated with virulence in other plant pathogens. We show that mutants with deletions in the proline iminopeptidase gene are attenuated for virulence. Surprisingly, a mutant with a deletion in the gene encoding the LuxR homolog shows normal virulence. IMPORTANCEDickeya zeae is an emerging banana soft rot pathogen in China. We used genome sequencing and annotation to create an inventory of potential virulence factors and virulence gene regulators encoded in Dickeya zeae MS2, a particularly virulent strain. We created mutations in several genes and tested these mutants in a banana seedling infection model. A strain with a mutated proline iminopeptidase gene, homologs of which are important for disease in the Xanthomonas species phytopathogens, was attenuated for soft rot symptoms in our model. Understanding how the proline iminopeptidase functions as a virulence factor may lead to insights about how to control the disease, and it is of general importance as homologs of the proline iminopeptidase occur in dozens of plant-associated bacteria.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA . We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa. Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA . Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels. IMPORTANCE Pseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

Multiple species of bacteria oxidize methane in the environment after it is produced by anaerobic ecosystems. These organisms provide reduced carbon substrates for species that cannot oxidize methane themselves, thereby serving a key role in these niches while also sequestering this potent greenhouse gas before it enters the atmosphere. Deciphering the molecular details of how methane-oxidizing bacteria interact in the environment enables us to understand an important aspect that shapes the structures and functions of these communities. Here we show that many members of the Methylomonas genus possess a LuxR-type acyl-homoserine lactone (acyl-HSL) receptor/transcription factor that is highly homologous to MbaR from the quorum-sensing (QS) system of Methylobacter tundripaludum, another methane oxidizer that has been isolated from the same environment. We reconstitute this detection system in Escherichia coli and use mutant and transcriptomic analysis to show that the receptor/transcription factor from Methylomonas sp. strain LW13 is active and alters LW13 gene expression in response to the acyl-HSL produced by M. tundripaludum. These findings provide a molecular mechanism for how two species of bacteria that may compete for resources in the environment can interact in a specific manner through a chemical signal. IMPORTANCE Methanotrophs are bacteria that sequester methane, a significant greenhouse gas, and thereby perform an important ecosystem function. Understanding the mechanisms by which these organisms interact in the environment may ultimately allow us to manipulate and to optimize this activity. Here we show that members of a genus of methane-oxidizing bacteria can be influenced by a chemical signal produced by a possibly competing species. This provides insight into how gene expression can be controlled in these bacterial communities via an exogenous chemical signal.

The opportunistic bacterial pathogen Pseudomonas aeruginosa has a layered acyl-homoserine lactone (AHL) quorum-sensing (QS) system, which controls production of a variety of extracellular metabolites and enzymes. The LasRI system activates genes including those coding for the extracellular protease elastase and for the second AHL QS system, RhlRI. Growth of P. aeruginosa on casein requires elastase production and LasR-mutant social cheats emerge in populations growing on casein. P. aeruginosa colonizes the lungs of individuals with the genetic disease cystic fibrosis (CF), and LasR mutants can be isolated from the colonized lungs; however, unlike laboratory-generated LasR mutants, many of these CF isolates have functioning RhlR-RhlI systems. We show that one such mutant can use the RhlR-RhlI system to activate expression of elastase and grow on casein. We carried out social-evolution experiments by growing this isolate on caseinate and, as with wild-type P. aeruginosa , elastase-negative mutants emerge as cheats, but these are not RhlR mutants; rather, they are mutants that do not produce the non-AHL Pseudomonas quinolone signal (PQS). Furthermore, we generated a RhlRI mutant and showed it had a fitness defect when growing together with the parent. Apparently, RhlR QS and PQS collude to support growth on caseinate in the absence of a functional LasR. Our findings provide a plausible explanation as to why P. aeruginosa LasR mutants, but not RhlR mutants, are common in CF lungs.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA. We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa. Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA. Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels. IMPORTANCE Pseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

Pseudomonas aeruginosa uses two acyl-homoserine lactone signals and two quorum sensing (QS) transcription factors, LasR and RhlR, to activate dozens of genes. LasR responds to N -3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and RhlR to N -butanoyl-homoserine lactone (C4-HSL). There is a third P. aeruginosa acyl-homoserine-lactone-responsive transcription factor, QscR, which acts to dampen or delay activation of genes by LasR and RhlR by an unknown mechanism. To better understand the role of QscR in P. aeruginosa QS, we performed a chromatin immunoprecipitation analysis, which showed this transcription factor bound the promoter of only a single operon of three genes linked to qscR , PA1895 to PA1897. Other genes that appear to be regulated by QscR in transcriptome studies were not direct targets of QscR. Deletion of PA1897 recapitulates the early QS activation phenotype of a QscR-null mutant, and the phenotype of a QscR-null mutant was complemented by PA1895-1897 but not by PA1897 alone. We conclude that QscR acts to modulate quorum sensing through regulation of a single operon, apparently raising the QS threshold of the population and providing a “brake” on QS autoinduction. IMPORTANCE Quorum sensing, a cell-cell communication system, is broadly distributed among bacteria and is commonly used to regulate the production of shared products. An important consequence of quorum sensing is a delay in production of certain products until the population density is high. The bacterium Pseudomonas aeruginosa has a particularly complicated quorum sensing system involving multiple signals and receptors. One of these receptors, QscR, downregulates gene expression, unlike the other receptors in P. aeruginosa . QscR does so by inducing the expression of a single operon whose function provides an element of resistance to a population reaching a quorum. This finding has importance for design of quorum sensing inhibitory strategies and can also inform design of synthetic biological circuits that use quorum sensing receptors to regulate gene expression.

Pseudomonas aeruginosa uses two acyl-homoserine lactone signals and two quorum sensing (QS) transcription factors, LasR and RhlR, to activate dozens of genes. LasR responds to N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and RhlR to N-butanoyl-homoserine lactone (C4-HSL). There is a third P. aeruginosa acyl-homoserine-lactone-responsive transcription factor, QscR, which acts to dampen or delay activation of genes by LasR and RhlR by an unknown mechanism. To better understand the role of QscR in P. aeruginosa QS, we performed a chromatin immunoprecipitation analysis, which showed this transcription factor bound the promoter of only a single operon of three genes linked to qscR, PA1895 to PA1897. Other genes that appear to be regulated by QscR in transcriptome studies were not direct targets of QscR. Deletion of PA1897 recapitulates the early QS activation phenotype of a QscR-null mutant, and the phenotype of a QscR-null mutant was complemented by PA1895-1897 but not by PA1897 alone. We conclude that QscR acts to modulate quorum sensing through regulation of a single operon, apparently raising the QS threshold of the population and providing a “brake” on QS autoinduction., IMPORTANCE Quorum sensing, a cell-cell communication system, is broadly distributed among bacteria and is commonly used to regulate the production of shared products. An important consequence of quorum sensing is a delay in production of certain products until the population density is high. The bacterium Pseudomonas aeruginosa has a particularly complicated quorum sensing system involving multiple signals and receptors. One of these receptors, QscR, downregulates gene expression, unlike the other receptors in P. aeruginosa. QscR does so by inducing the expression of a single operon whose function provides an element of resistance to a population reaching a quorum. This finding has importance for design of quorum sensing inhibitory strategies and can also inform design of synthetic biological circuits that use quorum sensing receptors to regulate gene expression.

The bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that uses a quorum sensing signal cascade to activate expression of dozens of genes when sufficient population densities have been reached. Quorum sensing controls production of several key virulence factors, including secreted proteases such as elastase. Cooperating groups of bacteria growing on protein are susceptible to social cheating by quorum-sensing defective mutants. A possible way to restrict cheater emergence is by policing where cooperators produce costly goods to sanction or punish cheats. The P. aeruginosa LasR-LasI quorum sensing system controls genes including those encoding proteases and also those encoding a second quorum-sensing system, the RhlR-RhlI system, which controls numerous genes including those for cyanide production. By using RhlR quorum sensing mutants and cyanide synthesis mutants, we show that cyanide production is costly and cyanide-producing cooperators use cyanide to punish LasR-null social cheaters. Cooperators are less susceptible to cyanide than are LasR mutants. These experiments demonstrate policing in P. aeruginosa, provide a mechanistic understanding of policing, and show policing involves the cascade organization of the two quorum sensing systems in this bacterium.

Many Proteobacteria synthesize acyl-homoserine lactone (AHL) molecules for use as signals in cell density-dependent gene regulation known as quorum sensing (QS) and response. AHL detection protocols are essential to QS researchers and several techniques are available, including a 14C-AHL radiolabel assay. This assay is based on the uptake of radiolabeled methionine by living cells and conversion of the radiolabel into S-adenosylmethionine (SAM). The radiolabeled SAM is then incorporated into AHL signal by an AHL synthase enzyme. Here we describe a methodology to perform the AHL radiolabel assay, which is unbiased, relatively fast, and very sensitive compared to other AHL detection protocols.

The laboratory strain of Pseudomonas aeruginosa , PAO1, activates genes for catabolism of adenosine using quorum sensing (QS). However, this strain is not well-adapted for growth on adenosine, with doubling times greater than 40 h. We previously showed that when PAO1 is grown on adenosine and casein, variants emerge that grow rapidly on adenosine. To understand the mechanism by which this adaptation occurs, we performed whole-genome sequencing of five isolates evolved for rapid growth on adenosine. All five genomes had a gene duplication-amplification (GDA) event covering several genes, including the quorum-regulated nucleoside hydrolase gene, nuh , and PA0148, encoding an adenine deaminase. In addition, two of the growth variants also exhibited a nuh promoter mutation. We recapitulated the rapid growth phenotype with a plasmid containing six genes common to all the GDA events. We also showed that nuh and PA0148, the two genes at either end of the common GDA, were sufficient to confer rapid growth on adenosine. Additionally, we demonstrated that the variant nuh promoter increased basal expression of nuh but maintained its QS regulation. Therefore, GDA in P. aeruginosa confers the ability to grow efficiently on adenosine while maintaining QS regulation of nucleoside catabolism. IMPORTANCE Pseudomonas aeruginosa thrives in many habitats and is an opportunistic pathogen of humans. In these diverse environments, P. aeruginosa must adapt to use myriad potential carbon sources. P. aeruginosa PAO1 cannot grow efficiently on nucleosides, including adenosine; however, it can acquire this ability through genetic adaptation. We show that the mechanism of adaptation is by amplification of a specific region of the genome and that the amplification preserves the regulation of the adenosine catabolic pathway by quorum sensing. These results demonstrate an underexplored mechanism of adaptation by P. aeruginosa , with implications for phenotypes such as development of antibiotic resistance.

Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Dissecting the molecular details of how these organisms interact in the environment may increase our understanding of how they perform this important ecological role. Many bacterial species use quorum sensing (QS) systems to regulate gene expression in a cell density-dependent manner. We have identified a QS system in the genome of Methylobacter tundripaludum , a dominant methane oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA). We determined that M. tundripaludum produces primarily N -3-hydroxydecanoyl- l -homoserine lactone (3-OH-C 10 -HSL) and that its production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by QS in this methane oxidizer using transcriptome sequencing (RNA-seq) and discovered that this system regulates the expression of a putative nonribosomal peptide synthetase biosynthetic gene cluster. Finally, we detected an extracellular factor that is produced by M. tundripaludum in a QS-dependent manner. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium. IMPORTANCE Aerobic methanotrophs are critical for sequestering carbon from the potent greenhouse gas methane in the environment, yet the mechanistic details of chemical interactions in methane-oxidizing bacterial communities are not well understood. Understanding these interactions is important in order to maintain, and potentially optimize, the functional potential of the bacteria that perform this vital ecosystem function. In this work, we identify a quorum sensing system in the aerobic methanotroph Methylobacter tundripaludum and use both chemical and genetic methods to characterize this system at the molecular level.

Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities. A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. The identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins., IMPORTANCE We describe the transcription factor PipR from a Pseudomonas strain isolated as a cottonwood tree endophyte. PipR is a member of the LuxR family of transcriptional factors. LuxR family members are generally thought of as quorum-sensing signal receptors, but PipR is one of an emerging subfamily of LuxR family members that respond to compounds produced by plants. We found that PipR responds to a peptidelike compound, and we present a model for Pip system signal transduction. A better understanding of plant-responsive LuxR homologs and the compounds to which they respond is of general importance, as they occur in dozens of bacterial species that are associated with economically important plants and, as we report here, they also occur in members of certain root endophyte communities.

Quorum sensing in the bacterium Rhodopseudomonas palustris involves the RpaI signal synthase, which produces p -coumaroyl-homoserine lactone ( p C-HSL) and RpaR, which is a p C-HSL–dependent transcriptional activator. There is also an antisense rpaR transcript (as rpaR ) of unknown function. Recent RNAseq studies have revealed that bacterial antisense RNAs are abundant, but little is known about the function of these molecules. Because as rpaR expression is quorum sensing dependent, we sought to characterize its production and function. We show that as rpaR is approximately 300–600 bases and is produced in response to p C-HSL and RpaR. There is an RpaR-binding site centered 51.5 bp from the mapped as rpaR transcript start site. We show that as rpaR overexpression reduces RpaR levels, rpaI expression, and p C-HSL production. We also generated an as rpaR mutant, which shows elevated RpaR levels, and elevated rpaI expression. Thus, as rpaR inhibits rpaR translation, and this inhibition results in suppression of RpaR-dependent rpaI expression and, thus, p C-HSL production. The R. palustris as rpaR represents an antisense RNA for which an activity can be measured and for which a distinct regulatory circuit related to a function is elucidated. It also represents yet another subtle regulatory layer for acyl-homoserine lactone quorum-sensing signal-responsive transcription factors.

The phenylpropanoid p -coumarate and structurally related aromatic compounds are produced in large amounts by green plants and are excellent carbon sources for many soil bacteria. Aerobic bacteria remove the acyl side chain from phenylpropanoids to leave an aromatic aldehyde, which then enters one of several possible central pathways of benzene ring degradation. We investigated the pathway for the anaerobic degradation of p -coumarate by the phototrophic bacterium Rhodopseudomonas palustris and found that it also follows this metabolic logic. We characterized enzymes for the conversion of p -coumarate to p -hydroxybenzaldehyde and acetyl coenzyme A (acetyl-CoA) encoded by the couAB operon. We also identified a MarR family transcriptional regulator that we named CouR. A couR mutant had elevated couAB expression. In addition, His-tagged CouR bound with high affinity to a DNA fragment encompassing the couAB promoter region, and binding was abrogated by the addition of nanomolar quantities of p -coumaroyl-CoA but not by p -coumarate. Footprinting demonstrated binding of CouR to an inverted repeat sequence that overlaps the −10 region of the couAB promoter. Our results provide evidence for binding of a CoA-modified aromatic compound by a MarR family member. Although the MarR family is widely distributed in bacteria and archaea and includes over 12,000 members, ligands have been identified for relatively few family members. Here we provide biochemical evidence for a new category of MarR ligand.

Many species of Proteobacteria communicate by using LuxI-LuxR–type quorum-sensing systems that produce and detect acyl-homoserine lactone (acyl-HSL) signals. Most of the known signals are straight-chain fatty acyl-HSLs, and evidence indicates that LuxI homologs prefer fatty acid-acyl carrier protein (ACP) over fatty acyl-CoA as the acyl substrate for signal synthesis. Two related LuxI homologs, RpaI and BtaI from Rhodopseudomonas palustris and photosynthetic stem-nodulating bradyrhizobia, direct production of the aryl-HSLs p -coumaroyl-HSL and cinnamoyl-HSL, respectively. Here we report that BjaI from the soybean symbiont Bradyrhizobium japonicum USDA110 is closely related to RpaI and BtaI and catalyzes the synthesis of isovaleryl-HSL (IV-HSL), a branched-chain fatty acyl-HSL. We show that IV-HSL induces expression of bjaI , and in this way IV-HSL functions like many other acyl-HSL quorum-sensing signals. Purified histidine-tagged BjaI was an IV-HSL synthase, which was active with isovaleryl-CoA but not detectably so with isovaleryl-ACP. This suggests that the RpaI-BtaI-BjaI subfamily of acyl-HSL synthases may use CoA- rather than ACP-linked substrates for acyl-HSL synthesis. The bjaI -linked bjaR 1 gene is involved in the response to IV-HSL, and BjaR 1 is sensitive to IV-HSL at concentrations as low as 10 pM. Low but sufficient levels of IV-HSL (about 5 nM) accumulate in B. japonicum culture fluid. The low levels of IV-HSL synthesis have likely contributed to the fact that the quorum-sensing signal from this bacterium has not been described elsewhere.

Quorum-sensing (QS) systems allow organisms, such as the pathogen Pseudomonas aeruginosa , to link gene expression with their population density and the diffusion and flow characteristics of their environment. The leading hypotheses about QS systems' biological functions necessitate that QS-controlled gene expression be suppressed until a threshold culture density (the quorum) is reached. Despite a detailed understanding of QS in P. aeruginosa , known regulatory elements do not fully explain how the quorum threshold for gene activation is produced. Here we investigated the mechanism with a screening approach that used random gene activation. These experiments uncovered a regulator without close homologs in other species that produces the quorum expression threshold. Expression of this regulator (named QteE) reduces LasR protein stability without affecting LasR transcription or translation. QteE also independently reduces RhlR levels. Because QteE can block QS when signal levels are high, it could provide a mechanism for individual cells to exert autonomous control over their QS regulons. This unique regulator governs two central QS control points in P. aeruginosa and shapes the expression pattern thought fundamental to the biological functions of QS.

The bacterial genus Rhodopseudomonas is comprised of photosynthetic bacteria found widely distributed in aquatic sediments. Members of the genus catalyze hydrogen gas production, carbon dioxide sequestration, and biomass turnover. The genome sequence of Rhodopseudomonas palustris CGA009 revealed a surprising richness of metabolic versatility that would seem to explain its ability to live in a heterogeneous environment like sediment. However, there is considerable genotypic diversity among Rhodopseudomonas isolates. Here we report the complete genome sequences of four additional members of the genus isolated from a restricted geographical area. The sequences confirm that the isolates belong to a coherent taxonomic unit, but they also have significant differences. Whole genome alignments show that the circular chromosomes of the isolates consist of a collinear backbone with a moderate number of genomic rearrangements that impact local gene order and orientation. There are 3,319 genes, 70% of the genes in each genome, shared by four or more strains. Between 10% and 18% of the genes in each genome are strain specific. Some of these genes suggest specialized physiological traits, which we verified experimentally, that include expanded light harvesting, oxygen respiration, and nitrogen fixation capabilities, as well as anaerobic fermentation. Strain-specific adaptations include traits that may be useful in bioenergy applications. This work suggests that against a backdrop of metabolic versatility that is a defining characteristic of Rhodopseudomonas , different ecotypes have evolved to take advantage of physical and chemical conditions in sediment microenvironments that are too small for human observation.

The Vibrio fischeri quorum-sensing signal N -3-oxohexanoyl- l -homoserine lactone (3OC6-HSL) activates expression of the seven-gene luminescence operon. We used microarrays to unveil 18 additional 3OC6-HSL-controlled genes, 3 of which had been identified by other means previously. We show most of these genes are regulated by the 3OC6-HSL-responsive transcriptional regulator LuxR directly. This demonstrates that V. fischeri quorum sensing regulates a substantial number of genes other than those involved in light production.

The opportunistic human pathogen Pseudomonas aeruginosa possesses two cell density-dependent genetic regulatory systems that control expression of a number of secreted virulence factors. These two systems, the lasI-lasR and rhII-rhIR gene pairs, are members of the luxI-luxR family of quorum-sensing signal generators and signal receptors. The rhII gene in P. aeruginosa encodes a 201-amino-acid protein that catalyses the synthesis of an autoinducer, butyrylhomoserine lactone. Through a programme of random and site-specific mutagenesis of rhII we have gained a better understanding of how its protein product functions. Eight residues critical to butyrylhomoserine lactone synthesis by RhiI were identified by random mutagenesis, and all mapped to a conserved region that spans residues 24-104. Seven of the eight residues were charged amino acids and the other was a glycine. By using site-specific mutagenesis we showed that an active-site cysteine or serine was not required for butyrylhomoserine lactone synthesis, and that two conserved aromatic amino acids in the postulated active site region could be altered without complete loss of RhII activity. Furthermore, two residues towards the C-terminus that align with critical residues in LuxI can be altered in RhII without loss of activity. These studies suggest that as opposed to the current models for acyl substrate binding to quorum-sensing signal generators, charged amino acid residues participate directly in the catalysis of butyrylhomoserine lactone synthesis rather than cysteines, serines or hydrophobic amino acids.

We are interested in the root microbiome of the fast-growing Eastern cottonwood tree, Populus deltoides . There is a large bank of bacterial isolates from P. deltoides , and there are 44 draft genomes of bacterial endophyte and rhizosphere isolates. As a first step in efforts to understand the roles of bacterial communication and plant-bacterial signaling in P. deltoides , we focused on the prevalence of acyl-homoserine lactone (AHL) quorum-sensing-signal production and reception in members of the P. deltoides microbiome. We screened 129 bacterial isolates for AHL production using a broad-spectrum bioassay that responds to many but not all AHLs, and we queried the available genome sequences of microbiome isolates for homologs of AHL synthase and receptor genes. AHL signal production was detected in 40% of 129 strains tested. Positive isolates included members of the Alpha -, Beta -, and Gammaproteobacteria . Members of the luxI family of AHL synthases were identified in 18 of 39 proteobacterial genomes, including genomes of some isolates that tested negative in the bioassay. Members of the luxR family of transcription factors, which includes AHL-responsive factors, were more abundant than luxI homologs. There were 72 in the 39 proteobacterial genomes. Some of the luxR homologs appear to be members of a subfamily of LuxRs that respond to as-yet-unknown plant signals rather than bacterial AHLs. Apparently, there is a substantial capacity for AHL cell-to-cell communication in proteobacteria of the P. deltoides microbiota, and there are also Proteobacteria with LuxR homologs of the type hypothesized to respond to plant signals or cues.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

Chitin, a polymer of N -acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae (“vibrios”), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes . The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone.

The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p -coumaroyl-homoserine lactone ( p C-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by p C-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His 6 -RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although p C-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is p C-HSL-RpaR-activated antisense transcription of rpaR . These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.

Many Proteobacteria possess LuxI-LuxR–type quorum-sensing systems that produce and detect fatty acyl-homoserine lactone (HSL) signals. The photoheterotroph Rhodopseudomonas palustris is unusual in that it produces and detects an aryl-HSL, p -coumaroyl-HSL, and signal production requires an exogenous source of p -coumarate. A photosynthetic stem-nodulating member of the genus Bradyrhizobium produces a small molecule signal that elicits an R. palustris quorum-sensing response. Here, we show that this signal is cinnamoyl-HSL and that cinnamoyl-HSL is produced by the LuxI homolog BraI and detected by BraR. Cinnamoyl-HSL reaches concentrations on the order of 50 nM in cultures of stem-nodulating bradyrhizobia grown in the presence or absence of cinnamate. Acyl-HSLs often reach concentrations of 0.1–30 μM in bacterial cultures, and generally, LuxR-type receptors respond to signals in a concentration range from 5 to a few hundred nanomolar. Our stem-nodulating Bradyrhizobium strain responds to picomolar concentrations of cinnamoyl-HSL and thus, produces cinnamoyl-HSL in excess of the levels required for a signal response without an exogenous source of cinnamate. The ability of Bradyrhizobium to produce and respond to cinnamoyl-HSL shows that aryl-HSL production is not unique to R. palustris , that the aromatic acid substrate for aryl-HSL synthesis does not have to be supplied exogenously, and that some acyl-HSL quorum-sensing systems may function at very low signal production and response levels.

Mechanisms for controlling symbiont populations are critical for maintaining the associations that exist between a host and its microbial partners. We describe here the transcriptional, metabolic, and ultrastructural characteristics of a diel rhythm that occurs in the symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri. The rhythm is driven by the host’s expulsion from its light-emitting organ of most of the symbiont population each day at dawn. The transcriptomes of both the host epithelium that supports the symbionts and the symbiont population itself were characterized and compared at four times over this daily cycle. The greatest fluctuation in gene expression of both partners occurred as the day began. Most notable was an up-regulation in the host of >50 cytoskeleton-related genes just before dawn and their subsequent down-regulation within 6 h. Examination of the epithelium by TEM revealed a corresponding restructuring, characterized by effacement and blebbing of its apical surface. After the dawn expulsion, the epithelium reestablished its polarity, and the residual symbionts began growing, repopulating the light organ. Analysis of the symbiont transcriptome suggested that the bacteria respond to the effacement by up-regulating genes associated with anaerobic respiration of glycerol; supporting this finding, lipid analysis of the symbionts’ membranes indicated a direct incorporation of host-derived fatty acids. After 12 h, the metabolic signature of the symbiont population shifted to one characteristic of chitin fermentation, which continued until the following dawn. Thus, the persistent maintenance of the squid–vibrio symbiosis is tied to a dynamic diel rhythm that involves both partners.

A recently available transposition system was utilized to isolate a nonmotile mutant of the coral-bleaching pathogen Vibrio coralliilyticus . The mutation was localized to the fhlA gene, and the mutant lacked flagella. The flhA mutant was unable to exhibit chemotaxis toward coral mucus or to adhere to corals and subsequently cause infection.

The bacterial quorum sensing (QS) signal molecule 3-oxo-dodecanoyl-L-homoserine lactone (OdDHL) is produced by the opportunistic pathogen Pseudomonas aeruginosa and controls expression of virulence factors associated with life threatening infections in immune compromised individuals. OdDHL has also demonstrated anticancer activity, yet its ability to enhance pathogenicity of P. aeruginosa compromises further consideration as a potential anticancer agent. In search of acylhomoserine lactones that selectively inhibit cancer cell growth, a library of phenacylhomoserine lactone analogues has been prepared by microwave synthesis and evaluated for cancer growth inhibition and quorum sensing activation. Comparative SAR analysis demonstrates that both anticancer and QS signaling systems require long acyl side chains with a 3-oxo substitution for maximum activity. Compound 12b, 3-oxo-12-phenyldodecanoyl-L-homoserine lactone, was identified as a lead compound with strong cancer growth inhibitory activity that minimizes activation of QS signaling pathways in a P. aeruginosa reporter assay.

We describe the development of a new radioactive assay for acyl-HSL production by bacterial cultures. The assay is based on the uptake of radiolabeled methionine and conversion of the radiolabel into SAM. The radiolabeled SAM is then incorporated into acyl-HSL by an acyl-HSL synthase. This assay is faster than previously used bioassays and shows no bias for the detection of acyl-HSLs of a particular length or side chain substitution. Acyl-HSL production can be monitored over a wide range of growth conditions in liquid culture. This assay can also be used in conjunction with a tube biofilm reactor to monitor acyl-HSL production by biofilm cultures. Ultimately this assay will allow comparison of acyl-HSL production by cells subjected to a variety of physiological conditions.

The Vibrio fischeri luminescence genes are activated by the transcription factor LuxR in combination with a diffusible signal compound, N-(3-oxohexanoyl) homoserine lactone, termed the autoinducer. We have synthesized a set of autoinducer analogs. Many analogs with alterations in the acyl side chain showed evidence of binding to LuxR. Some appeared to bind with an affinity similar to that of the autoinducer, but none showed a higher affinity, and many did not bind as tightly as the autoinducer. For the most part, compounds with substitutions in the homoserine lactone ring did not show evidence of binding to LuxR. The exceptions were compounds with a homocysteine thiolactone ring in place of the homoserine lactone ring. Many but not all of the analogs showing evidence of LuxR binding had some ability to activate the luminescence genes. None were as active as the autoinducer. While most showed little ability to induce luminescence, a few analogs with rather conservative substitutions had appreciable activity. Under the conditions we employed, some of the analogs showing little or no ability to induce luminescence were inhibitors of the autoinducer.