Biology subjects

Bassler, B. L.

Publications and source records attributed to Bassler, B. L..

30 records · Page 2Linked to original sources

bioRxiv2021

LuxT is a Global Regulator of Low-Cell Density Behaviors Including Type III Secretion, Siderophore Production, and Aerolysin Secretion in Vibrio harveyi

Quorum sensing (QS) is a chemical communication process in which bacteria produce, release, and detect extracellular signaling molecules called autoinducers. Via combined transcriptional and post-transcriptional regulatory mechanisms, QS allows bacteria to collectively alter gene expression on a population-wide scale. Recently, the TetR-family transcriptional regulator LuxT was shown to control V. harveyi qrr1, encoding the Qrr1 small RNA that functions at the core of the QS regulatory cascade. Here, we use RNA-Sequencing to reveal that, beyond control of qrr1, LuxT is a global regulator of 414 V. harveyi genes including those involved in type III secretion, siderophore production, and aerolysin toxin biosynthesis. Importantly, LuxT directly represses swrZ, encoding a GntR-family transcriptional regulator, and LuxT control of type III secretion, siderophore, and aerolysin genes occurs by two mechanisms, one that is SwrZ-dependent and one that is SwrZ-independent. All of these target genes specify QS-controlled behaviors that are enacted when V. harveyi is at low cell density. Thus, LuxT and SwrZ function in parallel with QS to drive particular low cell density behaviors. Phylogenetic analyses reveal that luxT is highly conserved among Vibrionaceae, but swrZ is less well conserved. In a test case, we find that in Aliivibrio fischeri, LuxT also represses swrZ. SwrZ is a repressor of A. fischeri siderophore production genes. Thus, LuxT repression of swrZ drives activation of A. fischeri siderophore gene expression. Our results indicate that LuxT is a major regulator among Vibrionaceae, and, in the species that also possess swrZ, LuxT functions with SwrZ to control gene expression. ImportanceBacteria precisely tune gene expression patterns to successfully react to changes that occur in the environment. Defining the mechanisms that enable bacteria to thrive in diverse and fluctuating habitats, including in host organisms, is crucial for a deep understanding of the microbial world and also for development of effective applications to promote or to combat particular bacteria. In this study, we show that a regulator called LuxT controls over 400 genes in the marine bacterium Vibrio harveyi and, moreover, that LuxT is highly conserved among Vibrionaceae species, ubiquitous marine bacteria that often cause disease. We characterize the mechanisms by which LuxT controls genes involved in virulence and nutrient acquisition. We show that LuxT functions in parallel with a set of regulators of the bacterial cell-to-cell communication process called quorum sensing to promote V. harveyi behaviors at low cell density.

microbiology↗

bioRxiv2021

Quantitative input-output dynamics of a c-di-GMP signal-transduction cascade in Vibrio cholerae

Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second-messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae. Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which in turn, drives alterations specifically to biofilm gene expression. A longstanding question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V. cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input-output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies.

microbiology↗

bioRxiv2021

Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven exclusively by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain as necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA-binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter. AUTHOR SUMMARYBacteria use a chemical communication process called quorum sensing (QS) to orchestrate collective behaviors. Recent studies demonstrate that bacteria-infecting viruses, called phages, also employ chemical communication to regulate collective activities. Phages can encode virus-specific QS-like systems, or they can harbor genes encoding QS components resembling those of bacteria. The latter arrangement suggests the potential for chemical communication across domains, i.e., between bacteria and phages. Ramifications stemming from such cross-domain communication are not understood. Phage VP882 infects the global pathogen Vibrio cholerae, and "eavesdrops" on V. cholerae QS to optimize the timing of its transition from living as a parasite to killing the host, and moreover, to manipulate V. cholerae biology. To accomplish these feats, phage VP882 relies on VqmAPhage, the phage-encoded homolog of the V. cholerae VqmAVc QS receptor and transcription factor. VqmAVc, by contrast, is constrained to the control of only V. cholerae genes and is incapable of regulating phage biology. Here, we discover the molecular mechanism underpinning the asymmetric transcriptional preferences of the phage-encoded and bacteria-encoded VqmA proteins. We demonstrate how VqmA transcriptional regulation is crucial to the survival and persistence of both the pathogen V. cholerae, and the phage that preys on it.

microbiology↗

bioRxiv2021

LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that redundant QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors. Author SummaryBacteria communicate and count their cell numbers using a process called quorum sensing (QS). In response to changes in cell density, QS bacteria alternate between acting as individuals and participating in collective behaviors. Vibrio harveyi is used as a model organism to understand QS-mediated communication. Five small RNAs lie at the heart of the V. harveyi QS system, and they regulate the target genes that underlie the QS response. The small RNAs function redundantly making it difficult to understand why V. harveyi requires five of them. Here, we discover a regulator, called LuxT, that exclusively represses the gene encoding one of the QS small RNAs. LuxT regulation of one QS small RNA enables unique control of a specific subset of QS target genes. LuxT is broadly conserved among Vibrionaceae. Our findings show how redundant regulatory components can possess both common and unique roles that provide bacteria with plasticity in their behaviors.

microbiology↗

bioRxiv2020

Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals

The global pathogen Vibrio cholerae undergoes cycles of biofilm formation and dispersal in the environment and the human host. Little is understood about biofilm dispersal. Here, we show that MbaA, a periplasmic polyamine sensor, and PotD1, a polyamine importer, regulate V. cholerae biofilm dispersal. Spermidine, a commonly produced polyamine, drives V. cholerae dispersal, whereas norspermidine, an uncommon polyamine produced by vibrios, inhibits dispersal. Spermidine and norspermidine differ by one methylene group. Both polyamines function to control dispersal via periplasmic detection by MbaA and subsequent signal relay. Biofilm dispersal fails in the absence of PotD1 because reuptake of endogenously produced norspermidine does not occur, so it accumulates in the periplasm where it stimulates MbaA. These results suggest that V. cholerae uses MbaA to monitor environmental polyamines, blends of which potentially provide information about numbers of self and other. This information is used to dictate whether or not to disperse from biofilms.

microbiology↗

bioRxiv2020

Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal

Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyper-infectious and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well-studied, almost nothing is known about biofilm dispersal. Here, we conduct an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we name DbfS/DbfR for Dispersal of Biofilm Sensor/Regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharide. Reorientations in swimming direction, mediated by CheY3, are necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion and, subsequently, cell motility permits escape from biofilms. This study lays the groundwork for interventions that modulate V. cholerae biofilm dispersal to ameliorate disease. Significance statementThe pathogen Vibrio cholerae alternates between the free-swimming state and existing in sessile multicellular communities known as biofilms. Transitioning between these lifestyles is key for disease transmission. V. cholerae biofilm formation is well studied, however, almost nothing is known about how V. cholerae cells disperse from biofilms, precluding understanding of a central pathogenicity step. Here, we conducted a high-content imaging screen for V. cholerae mutants that failed to disperse. Our screen revealed three classes of components required for dispersal: signal transduction, matrix degradation, and motility factors. We characterized these components to reveal the sequence of molecular events that choreograph V. cholerae biofilm dispersal. Our report provides a framework for developing strategies to modulate biofilm dispersal to prevent or treat disease.

microbiology↗

bioRxiv2020

The Vibrio cholerae quorum-sensing protein VqmA integrates cell density, environmental, and host-derived cues into the control of virulence

Scientific AbstractQuorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile. We show that these two stimuli differentially affect quorum sensing function and, in turn, V. cholerae pathogenicity. The quorum-sensing receptor-transcription factor called VqmA, that detects the autoinducer called DPO, also detects the lack of oxygen and the presence of bile. Detection occurs via DPO-, oxygen-, bile-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum- sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output. Lay AbstractQuorum sensing (QS) is a process of chemical communication bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called autoinducers. QS regulates virulence in Vibrio cholerae, the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine.

microbiology↗

bioRxiv2019

Mechanism underlying autoinducer recognition in the Vibrio cholerae DPO-VqmA quorum-sensing pathway

Quorum sensing is a bacterial communication process whereby bacteria produce, release and detect the accumulation of extracellular signaling molecules called autoinducers to coordinate collective behaviors. In Vibrio cholerae, the quorum-sensing autoinducer, DPO (3,5-dimethyl-pyrazin-2-ol), binds the receptor-transcription factor, VqmA. In response, the DPO-VqmA complex activates transcription of the vqmR gene encoding the VqmR small RNA. VqmR represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA has DPO-dependent and DPO-independent activity. We solved the DPO-VqmA crystal structure and compared it to existing structures to understand the conformational changes the protein undergoes upon DNA binding. Analysis of DPO analogs reveals that a hydroxyl or carbonyl group at the 2 position is critical for binding. The proposed DPO precursor, a linear molecule, Ala-AA (N-alanyl-aminoacetone), also binds and activates VqmA. DPO and Ala-AA occupy the same binding site as judged by site-directed mutagenesis and competitive ligand binding analyses.

microbiology↗

bioRxiv2019

Separating Functions of the Phage-Encoded Quorum-Sensing-Activated Antirepressor Qtip

Quorum sensing is a process of chemical communication that bacteria use to track cell density and coordinate gene expression across a population. Bacteria-infecting viruses, called phages, can encode quorum-sensing components that enable them to integrate host cell density information into the lysis-lysogeny decision. Vibriophage VP882 is one such phage, and activation of its quorum-sensing pathway leads to the production of an antirepressor called Qtip. Qtip interferes with the prophage repressor (cIVP882), leading to host-cell lysis. Here, we show that Qtip interacts with the N-terminus of cIVP882, inhibiting both cIVP882 DNA-binding and cIVP882 autoproteolysis. Qtip also sequesters cIVP882, localizing it to the poles. Qtip can localize to the poles independently of cIVP882. Alanine-scanning mutagenesis of Qtip shows that its localization and interference with cIVP882 activities are separable. Comparison of Qtip to a canonical phage antirepressor reveals that, despite both proteins interacting with their partner repressors, only Qtip drives polar localization.

microbiology↗

bioRxiv2019

Photo sensing and quorum sensing are integrated to control bacterial collective behaviors.

Pseudomonas aeruginosa transitions between the free-swimming state and the sessile biofilm mode during its pathogenic lifestyle. We show that quorum sensing represses P. aeruginosa biofilm formation and virulence by activating expression of genes encoding the KinB-AlgB two-component system. Phospho-AlgB represses biofilm and virulence genes, while KinB dephosphorylates, and thereby, inactivates AlgB. We discover that the photoreceptor BphP is the kinase that, in response to light, phosphorylates and activates AlgB. Indeed, exposing P. aeruginosa to light represses biofilm formation and virulence gene expression. To our knowledge, P. aeruginosa was not previously known to detect light. The KinB-AlgB-BphP module is present in all Pseudomonads, and we demonstrate that AlgB is the cognate response regulator for BphP in diverse bacterial phyla. We propose that KinB-AlgB-BphP constitutes a \"three-component\" system and AlgB is the node at which varied sensory information is integrated. This study sets the stage for light-mediated control of P. aeruginosa infectivity.

microbiology↗

bioRxiv2019

Phage-Encoded LuxR-Type Receptors Responsive to Host-Produced Bacterial Quorum-Sensing Autoinducers

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. QS relies on the cell-density-dependent production, accumulation, and receptor-mediated detection of extracellular signaling molecules called autoinducers (AIs). Gram-negative bacteria commonly use N-acyl homoserine lactones (AHLs) as their AIs and they are detected by LuxR-type receptors. Often, LuxR-type receptors are insoluble when not bound to a cognate AI. In this report, we show that LuxR-type receptors are encoded on phage genomes and, in the cases we tested, the phage LuxR-type receptors bind to and are solubilized specifically by the AHL AI produced by the host bacterium. We do not yet know the viral activities that are controlled by these phage QS receptors, however, our observations, coupled with recent reports, suggest that their occurrence is more widespread than previously appreciated. Using receptor-mediated detection of QS AIs could enable phages to garner information concerning the population density status of their bacterial hosts. We speculate that such information can be exploited by phages to optimize the timing of execution of particular steps in viral infection.\n\nImportanceBacteria communicate with chemical signal molecules to regulate group behaviors in a process called quorum sensing (QS). In this report, we find that genes encoding receptors for Gram-negative bacterial QS communication molecules are present on genomes of viruses that infect these bacteria. These viruses are called phages. We show that two phage-encoded receptors, like their bacterial counterparts, bind to the communication molecule produced by the host bacterium, suggesting that phages can \"listen in\" on their bacterial hosts. Interfering with bacterial QS and using phages to kill pathogenic bacteria represent attractive possibilities for development of new antimicrobials to combat pathogens that are resistant to traditional antibiotics. Our findings of interactions between phages and QS bacteria need consideration as new antimicrobial therapies are developed.

microbiology↗

bioRxiv2018

The Pseudomonas aeruginosa LasR quorum-sensing receptor balances ligand selectivity and sensitivity

Quorum sensing is a cell-cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by extracellular signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use the Pseudomonas aeruginosa LasR quorum-sensing receptor to explore receptor sensitivity and selectivity. Alteration of LasR amino acid S129 increases ligand selectivity and decreases ligand sensitivity. Conversely, the L130F mutation enhances LasR sensitivity while reducing selectivity. We solve crystal structures of LasR ligand binding domains complexed with non-cognate autoinducers. Comparison to existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop adjacent to the ligand binding site. We show that P. aeruginosa harboring LasR variants with modified selectivity or sensitivity exhibit altered quorum-sensing responses. We suggest that an evolutionary trade-off between ligand selectivity and sensitivity enables LasR to optimally regulate quorum-sensing traits.

microbiology↗