RESUMO
Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.
Assuntos
Percepção de Quorum , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Vibrio/metabolismo , Sequência de Bases , Escherichia coli/genética , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Vibrio/genéticaRESUMO
Shah et al. (2021) uncover phage-encoded protein Aqs1 that tactically blocks Pseudomonas aeruginosa quorum-sensing receptor LasR immediately upon infection to counteract the host's quorum-sensing program, a defense strategy that is likely conserved in other phages.
Assuntos
Bacteriófagos , Fagos de Pseudomonas , Proteínas de Bactérias/genética , Bacteriófagos/genética , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/genética , Percepção de Quorum , TransativadoresRESUMO
CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.
Assuntos
Vibrio vulnificus , Vibrio , Vibrio/genética , Vibrio vulnificus/genética , Vibrio parahaemolyticus/genética , Regulação Bacteriana da Expressão Gênica , Sistemas CRISPR-Cas , Vibrio cholerae/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Silenciamento de Genes , Aliivibrio fischeri/genéticaRESUMO
Quorum sensing gene expression in vibrios is regulated by the LuxR/HapR family of transcriptional factors, which includes Vibrio vulnificus SmcR. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic but highly degenerate with sequence variations at each promoter. To examine the mechanism by which SmcR recognizes diverse DNA sites, we generated SmcR separation-of-function mutants that either repress or activate transcription but not both. SmcR N55I is restricted in recognition of single base-pair variations in DNA binding site sequences and thus is defective at transcription activation but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R and N142D substitutions disrupt the interaction with RNAP alpha but retain functional DNA binding activity. X-ray crystallography and small angle X-ray scattering data show that the SmcR DNA binding domain exists in two conformations (wide and narrow), and the protein complex forms a mixture of dimers and tetramers in solution. The three RNAP interaction-deficient variants also have two DNA binding domain conformations, whereas SmcR N55I exhibits only the wide conformation. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNAP and a multi-conformational DNA binding domain that permits recognition of variable DNA sites.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Vibrio vulnificus/química , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Dimerização , Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Modelos Moleculares , Mutação , Regiões Promotoras Genéticas , Conformação Proteica , Percepção de Quorum/genética , Proteínas Recombinantes , Proteínas Repressoras/química , Proteínas Repressoras/genética , Espalhamento a Baixo Ângulo , Fatores de Transcrição/genética , Vibrio vulnificus/genéticaRESUMO
The quorum-sensing signaling systems in Vibrio bacteria converge to control levels of the master transcription factors LuxR/HapR, a family of highly conserved proteins that regulate gene expression for bacterial behaviors. A compound library screen identified 2-thiophenesulfonamide compounds that specifically inhibit Vibrio campbellii LuxR but do not affect cell growth. We synthesized a panel of 50 thiophenesulfonamide compounds to examine the structure-activity relationship effects on Vibrio quorum sensing. The most potent molecule identified, PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole), inhibits quorum sensing in multiple strains of V. vulnificus, V. parahaemolyticus, and V. campbellii at nanomolar concentrations. However, thiophenesulfonamide inhibition efficacy varies significantly among Vibrio species: PTSP is most inhibitory against V. vulnificus SmcR, but V. cholerae HapR is completely resistant to all thiophenesulfonamides tested. Reverse genetics experiments show that PTSP efficacy is dictated by amino acid sequence in the putative ligand-binding pocket: F75Y and C170F SmcR substitutions are each sufficient to eliminate PTSP inhibition. Further, in silico modeling distinguished the most potent thiophenesulfonamides from less-effective derivatives. Our results revealed the previously unknown differences in LuxR/HapR proteins that control quorum sensing in Vibrio species and underscore the potential for developing thiophenesulfonamides as specific quorum sensing-directed treatments for Vibrio infections.
Assuntos
Percepção de Quorum/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Vibrio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Especificidade da Espécie , Relação Estrutura-Atividade , Sulfonamidas/química , Transativadores/química , Vibrio/química , Vibrio/genéticaRESUMO
Bacteria coordinate cellular behaviors using a cell-cell communication system termed quorum sensing. In Vibrio harveyi, the master quorum sensing transcription factor LuxR directly regulates >100 genes in response to changes in population density. Here, we show that LuxR derepresses quorum sensing loci by competing with H-NS, a global transcriptional repressor that oligomerizes on DNA to form filaments and bridges. We first identified H-NS as a repressor of bioluminescence gene expression, for which LuxR is a required activator. In an hns deletion strain, LuxR is no longer necessary for transcription activation of the bioluminescence genes, suggesting that the primary role of LuxR is to displace H-NS to derepress gene expression. Using RNA-seq and ChIP-seq, we determined that H-NS and LuxR co-regulate and co-occupy 28 promoters driving expression of 63 genes across the genome. ChIP-PCR assays show that as autoinducer concentration increases, LuxR protein accumulates at co-occupied promoters while H-NS protein disperses. LuxR is sufficient to evict H-NS from promoter DNA in vitro, which is dependent on LuxR DNA binding activity. From these findings, we propose a model in which LuxR serves as a counter-silencer at H-NS-repressed quorum sensing loci by disrupting H-NS nucleoprotein complexes that block transcription.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Percepção de Quorum/genética , Proteínas Repressoras/genética , Transativadores/genética , Vibrio/genética , Carga Bacteriana , DNA Bacteriano , Proteínas de Ligação a DNA/deficiência , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Análise de Sequência de RNA , Transativadores/metabolismo , Transcrição Gênica , Vibrio/metabolismoRESUMO
Swimming motility is a critical virulence factor in pathogenesis for numerous Vibrio species. Vibrio campbellii DS40M4 is a wild-type isolate that has been recently established as a highly tractable model strain for bacterial genetics studies. We sought to exploit the tractability and relevance of this strain for characterization of flagellar gene regulation in V. campbellii. Using comparative genomics, we identified homologs of V. campbellii flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core regulators of the flagellar hierarchy established in other vibrios: RpoN (σ54), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multitiered regulatory hierarchy similar to other motile Vibrio species. However, there are several critical differences in V. campbellii: (i) the σ54-dependent regulator FlrA is dispensable for motility; (ii) the flgA, fliEFGHIJ, flrA, and flrBC operons do not require σ54 for expression; and (iii) FlrA and FlrC coregulate class II genes. Our model proposes that the V. campbellii flagellar transcriptional hierarchy has three classes of genes, in contrast to the four-class hierarchy in Vibrio cholerae. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae. IMPORTANCE Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments and is an important global pathogen of fish and shellfish. Disruption of the flagellar motor significantly decreases host mortality of V. campbellii, suggesting that motility is a key factor in pathogenesis. Using this model organism, we identified >60 genes that encode proteins with predicted structural, mechanical, or regulatory roles in function of the single polar flagellum in V. campbellii. We systematically tested strains containing single deletions of each gene to determine the impact on motility and flagellum production. Our studies have uncovered differences in the regulatory network and function of several genes in V. campbellii compared to established systems in Vibrio cholerae and Vibrio parahaemolyticus.
Assuntos
Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Transcrição Gênica/fisiologia , Vibrio/metabolismo , Sequência de Aminoácidos , Evolução Biológica , Quimiotaxia , Deleção de Genes , Modelos Biológicos , Movimento , Vibrio/genéticaRESUMO
Vibrio campbellii BB120 (previously classified as Vibrio harveyi) is a fundamental model strain for studying quorum sensing in vibrios. A phylogenetic evaluation of sequenced Vibrio strains in Genbank revealed that BB120 is closely related to the environmental isolate V. campbellii DS40M4. We exploited DS40M4's competence for exogenous DNA uptake to rapidly generate greater than 30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system homologues. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120 in three ways: (i) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, (ii) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signalling through the response regulator LuxO and (iii) the DS40M4 quorum-sensing regulon is much smaller than BB120 (~100 genes vs. ~400 genes, respectively). Using comparative genomics to expand our understanding of quorum-sensing circuit diversity, we observe that conservation of LuxM/LuxN proteins differs widely both between and within Vibrio species. These strains are also phenotypically distinct: DS40M4 exhibits stronger interbacterial cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. These results underscore the need to examine wild isolates for a broader view of bacterial diversity in the marine ecosystem.
Assuntos
Percepção de Quorum , Vibrio , Proteínas de Bactérias/genética , Ecossistema , Filogenia , Percepção de Quorum/genética , Vibrio/genéticaRESUMO
In vibrios, quorum sensing controls hundreds of genes that are required for cell density-specific behaviors including bioluminescence, biofilm formation, competence, secretion, and swarming motility. The central transcription factor in the quorum-sensing pathway is LuxR/HapR, which directly regulates â¼100 genes in the >400-gene regulon of Vibrio harveyi Among these directly controlled genes are 15 transcription factors, which we predicted would comprise the second tier in the hierarchy of the LuxR regulon. We confirmed that LuxR binds to the promoters of these genes in vitro and quantified the extent of LuxR activation or repression of transcript levels. Transcriptome sequencing (RNA-seq) indicates that most of these transcriptional regulators control only a few genes, with the exception of MetJ, which is a global regulator. The genes regulated by these transcription factors are predicted to be involved in methionine and thiamine biosynthesis, membrane stability, RNA processing, c-di-GMP degradation, sugar transport, and other cellular processes. These data support a hierarchical model in which LuxR directly regulates 15 transcription factors that drive the second level of the gene expression cascade to influence cell density-dependent metabolic states and behaviors in V. harveyiIMPORTANCE Quorum sensing is important for survival of bacteria in nature and influences the actions of bacterial groups. In the relatively few studied examples of quorum-sensing-controlled genes, these genes are associated with competition or cooperation in complex microbial communities and/or virulence in a host. However, quorum sensing in vibrios controls the expression of hundreds of genes, and their functions are mostly unknown or uncharacterized. In this study, we identify the regulators of the second tier of gene expression in the quorum-sensing system of the aquaculture pathogen Vibrio harveyi Our identification of regulatory networks and metabolic pathways controlled by quorum sensing can be extended and compared to other Vibrio species to understand the physiology, ecology, and pathogenesis of these organisms.
Assuntos
Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Vibrio/fisiologia , Regiões Promotoras Genéticas , Regulon , Proteínas Repressoras/genética , Transativadores/genética , Transcrição Gênica , Vibrio/genéticaRESUMO
In Vibrio species, quorum sensing controls gene expression for numerous group behaviors, including bioluminescence production, biofilm formation, virulence factor secretion systems, and competence. The LuxR/HapR master quorum-sensing regulators activate expression of hundreds of genes in response to changes in population densities. The mechanism of transcription activation by these TetR-type transcription factors is unknown, though LuxR DNA binding sites that lie in close proximity to the -35 region of the promoter are required for activation at some promoters. Here, we show that Vibrio harveyi LuxR directly interacts with RNA polymerase to activate transcription of the luxCDABE bioluminescence genes. LuxR interacts with RNA polymerase in vitro and in vivo and specifically interacts with both the N- and C-terminal domains of the RNA polymerase α-subunit. Amino acid substitutions in the RNAP interaction domain on LuxR decrease interactions between LuxR and the α-subunit and result in defects in transcription activation of quorum-sensing genes in vivo. The RNAP-LuxR interaction domain is conserved in Vibrio cholerae HapR and is required for activation of the HapR-regulated gene hapA. Our findings support a model in which LuxR/HapR bind proximally to RNA polymerase to drive transcription initiation at a subset of quorum-sensing genes in Vibrio species.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Percepção de Quorum , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Vibrio cholerae/genética , Vibrio/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Repressoras/genética , Transativadores/genéticaRESUMO
The marine facultative pathogen Vibrio cholerae forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. V. cholerae-chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors that influence chitin-induced expression of one locus, the chitobiose utilization operon (chb), which is required for the uptake and catabolism of the chitin disaccharide. Through a series of genetic screens, we identified that the master regulator of quorum sensing, HapR, is a direct repressor of the chb operon. We also found that the levels of HapR in V. cholerae are regulated by the ClpAP protease. Furthermore, we show that the canonical quorum sensing cascade in V. cholerae regulates chb expression in an HapR-dependent manner. Through this analysis, we found that signaling via the species-specific autoinducer CAI-1, but not the interspecies autoinducer AI-2, influences chb expression. This phenomenon of species-specific regulation may enhance the fitness of this pathogen in its environmental niche.IMPORTANCE In nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen Vibrio cholerae is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. V. cholerae interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that V. cholerae uses QS to regulate the expression of one locus required for V. cholerae-chitin interactions.
Assuntos
Proteínas de Bactérias/genética , Dissacarídeos/metabolismo , Óperon , Percepção de Quorum , Vibrio cholerae/genética , Proteínas de Bactérias/metabolismo , Especificidade da Espécie , Vibrio cholerae/metabolismoRESUMO
In this issue, Bru et al. connect Pseudomonas aeruginosa PQS signaling secretion during stress response to swarming behavior (J.-L. Bru, B. Rawson, C. Trinh, K. Whiteson, et al., J Bacteriol 201:e00383-19, 2019, https://doi.org/10.1128/JB.00383-19). Phage-infected or antibiotic-treated bacterial cells secrete PQS to repel healthy, unexposed cells away from the source of the stress. Thus, the collective stress response mechanism driven by PQS signaling influences spatial organization and population dynamics in P. aeruginosa that may provide competitive advantages in certain niches.
Assuntos
Bacteriófagos , Quinolonas , Antibacterianos , Pseudomonas aeruginosa , Percepção de QuorumRESUMO
Bacteria cycle between periods when they perform individual behaviors and periods when they perform group behaviors. These transitions are controlled by a cell-cell communication process called quorum sensing, in which extracellular signal molecules, called autoinducers (AIs), are released, accumulate, and are synchronously detected by a group of bacteria. AI detection results in community-wide changes in gene expression, enabling bacteria to collectively execute behaviors such as bioluminescence, biofilm formation, and virulence factor production. In this study, we show that the transcription factor AphA is a master regulator of quorum sensing that operates at low cell density (LCD) in Vibrio harveyi and Vibrio cholerae. In contrast, LuxR (V. harveyi)/HapR (V. cholerae) is the master regulator that operates at high cell density (HCD). At LCD, redundant small noncoding RNAs (sRNAs) activate production of AphA, and AphA and the sRNAs repress production of LuxR/HapR. Conversely, at HCD, LuxR/HapR represses aphA. This network architecture ensures maximal AphA production at LCD and maximal LuxR/HapR production at HCD. Microarray analyses reveal that 300 genes are regulated by AphA at LCD in V. harveyi, a subset of which is also controlled by LuxR. We propose that reciprocal gradients of AphA and LuxR/HapR establish the quorum-sensing LCD and HCD gene expression patterns, respectively.
Assuntos
Percepção de Quorum/genética , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Vibrio/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Sequência de Bases , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Análise em Microsséries , Modelos Biológicos , Dados de Sequência Molecular , Percepção de Quorum/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Transativadores/genética , Transativadores/metabolismo , Vibrio/genética , Vibrio/metabolismoRESUMO
Experimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating the construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of the luxCDABE bioluminescence operon and the betIBA-proXWV osmotic stress operon in Vibrio harveyi We describe a new method called rapid arbitrary PCR insertion libraries (RAIL) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of the luxCDABE and betIBA-proXWV operons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method to gfp, mCherry, and lacZ reporters and multiple promoters in V. harveyi We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications.IMPORTANCE Gene reporter constructs have long been essential tools for studying gene regulation in bacteria, particularly following the recent advent of fluorescent gene reporters. We developed a new method that enables efficient construction of promoter fusions to reporter genes to study gene regulation. We demonstrate the versatility of this technique in the model bacterium Vibrio harveyi by constructing promoter libraries for three bacterial promoters using three reporter genes. These libraries can be used to determine the DNA sequences required for gene expression, revealing regulatory elements in promoters. This method is applicable to various model systems and reporter genes for assaying gene expression.
Assuntos
Proteínas de Bactérias/genética , Óperon/genética , Pressão Osmótica , Regiões Promotoras Genéticas/genética , Vibrio/genética , Biblioteca Gênica , Genes Reporter , Reação em Cadeia da Polimerase , Vibrio/fisiologiaRESUMO
The coordination of group behaviors in bacteria is accomplished via the cell-cell signaling process called quorum sensing. Vibrios have historically been models for studying bacterial communication due to the diverse and remarkable behaviors controlled by quorum sensing in these bacteria, including bioluminescence, type III and type VI secretion, biofilm formation, and motility. Here, we discuss the Vibrio LuxR/HapR family of proteins, the master global transcription factors that direct downstream gene expression in response to changes in cell density. These proteins are structurally similar to TetR transcription factors but exhibit distinct biochemical and genetic features from TetR that determine their regulatory influence on the quorum sensing gene network. We review here the gene groups regulated by LuxR/HapR and quorum sensing and explore the targets that are common and unique among Vibrio species.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Percepção de Quorum/genética , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Vibrio/genética , DNA Bacteriano/genética , Carboidratos da Dieta , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Sequência de DNA , Transdução de Sinais , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Vibrio cholerae/genética , Vibrio cholerae/fisiologiaRESUMO
The cell-cell signaling process called quorum sensing allows bacteria to control behaviors in response to changes in population density. In Vibrio harveyi, the master quorum-sensing transcription factor LuxR is a member of the TetR family of transcription factors that both activates and represses genes to coordinate group behaviors, including bioluminescence. Here, we show that integration host factor (IHF) is a key coactivator of the luxCDABE bioluminescence genes that is required together with LuxR for precise timing and expression levels of bioluminescence during quorum sensing. IHF binds to multiple sites in the luxCDABE promoter and bends the DNA in vitro. IHF and LuxR synergistically bind luxCDABE promoter DNA at overlapping, essential binding sites that are required for maximal gene expression in vivo. RNA-seq analysis demonstrated that IHF regulates 300 genes in V. harveyi, and among these are a core set of 19 genes that are also directly bound and regulated by LuxR. We validated these global analyses by demonstrating that both IHF and LuxR are required for transcriptional activation of the osmotic stress response genes betIBA-proXWV. These data suggest that IHF plays an integral role in one mechanism of transcriptional activation by the LuxR-type family of quorum-sensing regulators in vibrios.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Percepção de Quorum/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transativadores/metabolismo , Vibrio/genética , Vibrio/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Fatores Hospedeiros de Integração/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.
Assuntos
Pressão Osmótica/fisiologia , Percepção de Quorum/fisiologia , Vibrio/fisiologia , Betaína/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Transativadores/genética , Transativadores/metabolismo , Vibrio/genéticaRESUMO
Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.
Assuntos
Percepção de Quorum , Transativadores , Vibrio , Percepção de Quorum/efeitos dos fármacos , Vibrio/efeitos dos fármacos , Vibrio/química , Vibrio/metabolismo , Vibrio/genética , Transativadores/antagonistas & inibidores , Transativadores/genética , Transativadores/metabolismo , Transativadores/química , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/química , Sulfonamidas/farmacologia , Sulfonamidas/química , Tiofenos/química , Tiofenos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/químicaRESUMO
CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn 7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus , and Vibrio campbellii . We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.