Bonnie L. Bassler

Structural identification of a bacterial quorum-sensing signal containing boron

Cell–cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. Community cooperation probably enhances the effectiveness of processes such as bioluminescence, virulence factor expression, antibiotic production and biofilm development. Unlike other autoinducers, which are specific to a particular species of bacteria, a recently discovered autoinducer (AI-2) is produced by a large number of bacterial species. AI-2 has been proposed to serve as a ‘universal’ signal for inter-species communication. The chemical identity of AI-2 has, however, proved elusive. Here we present the crystal structure of an AI-2 sensor protein, LuxP, in a complex with autoinducer. The bound ligand is a furanosyl borate diester that bears no resemblance to previously characterized autoinducers. Our findings suggest that addition of naturally occurring borate to an AI-2 precursor generates active AI-2. Furthermore, they indicate a potential biological role for boron, an element required by a number of organisms but for unknown reasons.

Bacterial Quorum-Sensing Network Architectures

Quorum sensing is a cell-cell communication process in which bacteria use the production and detection of extracellular chemicals called autoinducers to monitor cell population density. Quorum sensing allows bacteria to synchronize the gene expression of the group, and thus act in unison. Here, we review the mechanisms involved in quorum sensing with a focus on the Vibrio harveyi and Vibrio cholerae quorum-sensing systems. We discuss the differences between these two quorum-sensing systems and the differences between them and other paradigmatic bacterial signal transduction systems. We argue that the Vibrio quorum-sensing systems are optimally designed to precisely translate extracellular autoinducer information into internal changes in gene expression. We describe how studies of the V. harveyi and V. cholerae quorum-sensing systems have revealed some of the fundamental mechanisms underpinning the evolution of collective behaviors.

The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule.

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram‐negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram‐negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI‐1 and AI‐2). AI‐1 is a homoserine lactone. The structure of AI‐2 is not known. We have suggested previously that V. harveyi uses AI‐1 for intraspecies communication and AI‐2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram‐negative and Gram‐positive bacteria produce AI‐2 and, in every case, production of AI‐2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI‐2 synthase and that AI‐2 is produced from S‐adenosylmethionine in three enzymatic steps. The substrate for LuxS is S‐ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI‐2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI‐2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI‐2 is a unique, ‘universal’ signal that could be used by a variety of bacteria for communication among and between species.

The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae

Quorum-sensing bacteria communicate with extracellular signal molecules called autoinducers. This process allows community-wide synchronization of gene expression. A screen for additional components of the Vibrio harveyi and Vibrio cholerae quorum-sensing circuits revealed the protein Hfq. Hfq mediates interactions between small, regulatory RNAs (sRNAs) and specific messenger RNA (mRNA) targets. These interactions typically alter the stability of the target transcripts. We show that Hfq mediates the destabilization of the mRNA encoding the quorum-sensing master regulators LuxR (V. harveyi) and HapR (V. cholerae), implicating an sRNA in the circuit. Using a bioinformatics approach to identify putative sRNAs, we identified four candidate sRNAs in V. cholerae. The simultaneous deletion of all four sRNAs is required to stabilize hapR mRNA. We propose that Hfq, together with these sRNAs, creates an ultrasensitive regulatory switch that controls the critical transition into the high cell density, quorum-sensing mode.

How bacteria talk to each other: regulation of gene expression by quorum sensing

Quorum sensing, or the control of gene expression in response to cell density, is used by both gram-negative and gram-positive bacteria to regulate a variety of physiological functions. In all cases, quorum sensing involves the production and detection of extracellular signalling molecules called autoinducers. While universal signalling themes exist, variations in the design of the extracellular signals, the signal detection apparatuses, and the biochemical mechanisms of signal relay have allowed quorum sensing systems to be exquisitely adapted for their varied uses. Recent studies show that quorum sensing modulates both intra- and inter-species cell-cell communication, and it plays a major role in enabling bacteria to architect complex community structures.

Bacterial Small-Molecule Signaling Pathways

Bacteria use diverse small molecules for extra- and intracellular signaling. They scan small-molecule mixtures to access information about both their extracellular environment and their intracellular physiological status, and based on this information, they continuously interpret their circumstances and react rapidly to changes. Bacteria must integrate extra- and intracellular signaling information to mount appropriate responses to changes in their environment. We review recent research into two fundamental bacterial small-molecule signaling pathways: extracellular quorum-sensing signaling and intracellular cyclic dinucleotide signaling. We suggest how these two pathways may converge to control complex processes including multicellularity, biofilm formation, and virulence. We also outline new questions that have arisen from recent studies in these fields.

Quorum-sensing regulators control virulence gene expression in Vibrio cholerae

The production of virulence factors including cholera toxin and the toxin-coregulated pilus in the human pathogen Vibrio cholerae is strongly influenced by environmental conditions. The well-characterized ToxR signal transduction cascade is responsible for sensing and integrating the environmental information and controlling the virulence regulon. We show here that, in addition to the known components of the ToxR signaling circuit, quorum-sensing regulators are involved in regulation of V. cholerae virulence. We focused on the regulators LuxO and HapR because homologues of these two proteins control quorum sensing in the closely related luminous marine bacterium Vibrio harveyi. Using an infant mouse model, we found that a luxO mutant is severely defective in colonization of the small intestine. Gene arrays were used to profile transcription in the V. cholerae wild type and the luxO mutant. These studies revealed that the ToxR regulon is repressed in the luxO mutant, and that this effect is mediated by another negative regulator, HapR. We show that LuxO represses hapR expression early in log-phase growth, and constitutive expression of hapR blocks ToxR-regulon expression. Additionally, LuxO and HapR regulate a variety of other cellular processes including motility, protease production, and biofilm formation. Together these data suggest a role for quorum sensing in modulating expression of blocks of virulence genes in a reciprocal fashion in vivo.

Quorum sensing controls biofilm formation in Vibrio cholerae

Multiple quorum‐sensing circuits function in parallel to control virulence and biofilm formation in Vibrio cholerae. In contrast to other bacterial pathogens that induce virulence factor production and/or biofilm formation at high cell density in the presence of quorum‐sensing autoinducers, V. cholerae represses these behaviours at high cell density. Consistent with this, we show here that V. cholerae strains ‘locked’ in the regulatory state mimicking low cell density are enhanced for biofilm production whereas mutants ‘locked’ in the regulatory state mimicking high cell density are incapable of producing biofilms. The quorum‐sensing cascade we have identified in V. cholerae regulates the transcription of genes involved in exopolysaccharide production (EPS), and variants that produce EPS and form biofilms arise at high frequency from non‐EPS, non‐biofilm producing strains. Our data show that spontaneous mutation of the transcriptional regulator hapR is responsible for this effect. Several toxigenic strains of V. cholerae possess a naturally occurring frameshift mutation in hapR. Thus, the distinct environments occupied by this aquatic pathogen presumably include niches where cell‐cell communication is crucial, as well as ones where loss of quorum sensing via hapR mutation confers a selective advantage. Bacterial biofilms could represent a complex habitat where such differentiation occurs.

Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence

Density‐dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two–component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal–response system controlling density‐dependent expression of luminescence remains to be identified.

Multiple signalling systems controlling expression of luminescence in Vibrio harveyi : sequence and function of genes encoding a second sensory pathway

Density‐dependent expression of luminescence in Vibrio harveyl is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal‐response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably β‐hydroxybutryl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal‐response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density‐sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two‐component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal‐response pathways which converge to regulate expression of luminescence in V. harveyl.