Quorum‐sensing autoinducer molecules produced by members of a multispecies biofilm promote horizontal gene transfer to Vibrio cholerae
TL;DR: It is demonstrated that comEA transcription and the horizontal acquisition of DNA by V. cholerae are induced in response to purified CAI-1 and AI-2, and also by autoinducers derived from other Vibrios co-cultured with V. Cholerae within a mixed-species biofilm, suggesting that autoinducer communication within a consortium may promote DNA exchange among VibRIos.
Abstract: Vibrio cholerae, the causative agent of cholera and a natural inhabitant of aquatic environments, regulates numerous behaviors using a quorum-sensing (QS) system conserved among many members of the marine genus Vibrio. The Vibrio QS response is mediated by two extracellular autoinducer (AI) molecules: CAI-I, which is produced only by Vibrios, and AI-2, which is produced by many bacteria. In marine biofilms on chitinous surfaces, QS-proficient V. cholerae become naturally competent to take up extracellular DNA. Because the direct role of AIs in this environmental behavior had not been determined, we sought to define the contribution of CAI-1 and AI-2 in controlling transcription of the competence gene, comEA, and in DNA uptake. In this study we demonstrated that comEA transcription and the horizontal acquisition of DNA by V. cholerae are induced in response to purified CAI-1 and AI-2, and also by autoinducers derived from other Vibrios co-cultured with V. cholerae within a mixed-species biofilm. These results suggest that autoinducer communication within a consortium may promote DNA exchange among Vibrios, perhaps contributing to the evolution of these bacterial pathogens.
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TL;DR: Genetic and biochemical methods, coupled with computational and genomics approaches, are being used to validate known sRNAs and also to identify many additional putative sRNAAs that may play a role in the pathogenic lifestyle of V. cholerae.
Abstract: Vibrio cholerae is the waterborne bacterium responsible for worldwide outbreaks of the acute, potentially fatal cholera diarrhea. The primary factors this human pathogen uses to cause the disease are controlled by a complex regulatory program linking extracellular signaling inputs to changes in expression of several critical virulence genes. Recently it has been uncovered that many non-coding regulatory sRNAs are important components of the V. cholerae virulence regulon. Most of these sRNAs appear to require the RNA-binding protein, Hfq, to interact with and alter the expression of target genes, while a few sRNAs appear to function by an Hfq-independent mechanism. Direct base-pairing between the sRNAs and putative target mRNAs has been shown in a few cases but the extent of each sRNAs regulon is not fully known. Genetic and biochemical methods, coupled with computational and genomics approaches, are being used to validate known sRNAs and also to identify many additional putative sRNAs that may play a role in the pathogenic lifestyle of V. cholerae.
60 citations
TL;DR: Four environmental factors promote genetic competence and natural transformation in Vibrio cholerae by co‐ordinating expression of the regulators CRP, CytR, HapR and TfoX respectively.
Abstract: Natural transformation is a major mechanism of horizontal gene transfer in bacteria. By incorporating exogenous DNA elements into chromosomes, bacteria are able to acquire new traits that can enhance their fitness in different environments. Within the past decade, numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae, including the pathogen Vibrio cholerae. Four environmental factors: (i) nutrient limitation, (ii) availability of extracellular nucleosides, (iii) high cell density and (iv) the presence of chitin, promote genetic competence and natural transformation in Vibrio cholerae by co-ordinating expression of the regulators CRP, CytR, HapR and TfoX respectively. Studies of other Vibrionaceae members highlight the general importance of natural transformation within this bacterial family.
54 citations
Cites background from "Quorum‐sensing autoinducer molecule..."
...Numerous studies have shown that various environmental and physiological factors impact competence and natural transformation in V. cholerae (Meibom et al., 2005; Antonova and Hammer, 2011; Antonova et al., 2012; Blokesch, 2012; Lo Scrudato and Blokesch, 2012)....
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...Accordingly, both transformation frequency and comEA expression are affected by AI levels, with CAI-1 eliciting a stronger response than AI-2 (Antonova and Hammer, 2011; Suckow et al., 2011)....
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...Remarkably, polymeric chitin in the form of crab-shell tiles, which can induce natural competence in V. cholerae (Meibom et al., 2005; Antonova and Hammer, 2011; Antonova et al., 2012), fails to do so in V. fischeri (Pollack-Berti et al., 2010)....
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...located within the biofilm, suggesting that quorum sensing may facilitate DNA exchange among members of the genus (Antonova and Hammer, 2011)....
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...At low cell density, i.e. when CAI-1 and AI-2 levels are low, their unbound cognate receptors CqsS and LuxP/Q complex, respectively, behave as kinases and initiate a phosphorylation cascade via LuxU that phosphorylates the response regulator LuxO (Fig....
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TL;DR: It is experimentally demonstrated that Vibrio cholerae can acquire new T6SS effector genes via horizontal transfer and utilize them to kill neighboring cells, the first direct empirical support for the hypothesis that T6 genes are exchanged horizontally and functionally deployed to compete with neighboring cells.
Abstract: Horizontal gene transfer (HGT) can have profound effects on bacterial evolution by allowing individuals to rapidly acquire adaptive traits that shape their strategies for competition. One strategy for intermicrobial antagonism often used by Proteobacteria is the genetically encoded contact-dependent type VI secretion system (T6SS), a weapon used to kill heteroclonal neighbors by direct injection of toxic effectors. Here, we experimentally demonstrate that Vibrio cholerae can acquire new T6SS effector genes via horizontal transfer and utilize them to kill neighboring cells. Replacement of one or more parental alleles with novel effectors allows the recombinant strain to dramatically outcompete its parent. Using spatially explicit modeling, we examine how this process could affect the ecology and evolution of surface-attached microbial populations. HGT of T6SS effector-immunity pairs is risky: transformation brings a cell into conflict with its former clone mates but can be adaptive when superior T6SS alleles are acquired. More generally, we find that these costs and benefits are not symmetric and that high rates of HGT can act as a hedge against competitors with unpredictable T6SS efficacy. We conclude that antagonism and horizontal transfer drive successive rounds of weapon optimization and selective sweeps, dynamically shaping the composition of microbial communities.IMPORTANCE The contact-dependent type VI secretion system (T6SS) is frequently used by Proteobacteria to kill adjacent competitors. While DNA released by T6 killing can be horizontally acquired, it remains untested whether T6 genes themselves can be horizontally acquired and then utilized to compete with neighboring cells. Using naturally transformable Vibrio cholerae, we provide the first direct empirical support for the hypothesis that T6 genes are exchanged horizontally (e.g., from dead competitors) and functionally deployed to compete with neighboring cells. Using computational simulations, we also demonstrate that high rates of HGT can be adaptive, allowing V. cholerae to improve upon existing T6 weaponry and survive direct encounters with otherwise superior competitors. We anticipate that our evolutionary results are of broad microbiological relevance, applying to many bacteria capable of HGT that utilize the T6SS or similar antagonistic systems, and highlight the profound impact of HGT in shaping microbial community structure.
53 citations
TL;DR: Detailed insights are provided into the function of a key regulator of natural competence and type VI secretion in V. cholerae by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing and demonstrating that Qstr is a transcription factor that binds upstream of the up- and down-regulated genes.
Abstract: During growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is a transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall, these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.
52 citations
Cites background or methods from "Quorum‐sensing autoinducer molecule..."
...High cell density, measured through the local concentration of secreted autoinducers (Vibrio species specific cholera autoinducer 1 [CAI-1] and autoinducer 2 [AI-2]), leads to the accumulation of the master regulator of quorum sensing (QS) HapR (12)....
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...cholerae strains C6706str El Tor biotype, O1; HapR + (217) EA305 tfoX * (tfoX controlled by tac promoter) This study SLS349 ΔluxO (232) EA281 ΔluxO, tfoX* This study EA349 ΔluxO, ΔlacZ::hapR, tfoX* This study EA407 ΔluxO, ΔlacZ::hapR, tfoX*, cytR::Tn5 This study BH1543 ΔhapR (8) EA307 ΔhapR, tfoX* This study EA408 ΔcytR This study EA410 ΔcytR, tfoX* This study EA415 ΔluxO, ΔcytR This study EA636 ΔluxO, ΔcytR, tfoX* This study EA517 ΔcytR, ΔlacZ::cytR, tfoX* This study EA605 cytR-L161A This study EA606 cytR-L161A, tfoX* This study EA680 cytR-D273N This study EA682 cytR-D273N, tfoX* This study EA577 Δcrp::Kan R This study EA601 Δcrp::Kan R , tfoX* This study MN171 Δcrp::Kan R , ΔcytR This study MN173 Δcrp::Kan R , ΔcytR, tfoX* This study EA090 ΔlacZ::Kan R (8) Plasmids Features Reference pBBRlux Cloning vector, Cm R (128) pEA209 comEA-lux, pBBRlux-based, Cm R (8) pEA493 pilA-lux, pBBRlux-based, Cm R This study pEA495 chiA-1-lux, pBBRlux-based, Cm R This study pEA603 udp-lux, pBBRlux-based, Cm R This study pBBRlux-hap hapA-lux, pBBRlux-based, Cm R (16) pEA500 p-tac-cytR, pEVS143-based, Kan R This study...
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...cholerae strain unable to produce either AI only expresses comEA and takes up DNA when provided exogenous AIs (8, 210)....
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...Plasmids carrying the luciferase-based transcriptional reporters (comEA–lux, pilA–lux, chiA-1–lux and udp– lux) were constructed as previously described (8)....
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...transformation assays as described previously (8)....
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TL;DR: This work indirectly visualized the transfer of the external DNA from outside the cell into the periplasm followed by the shuttling of the DNA into the cytoplasm of V. cholerae and provided evidence that the DNA translocation across the membranes is spatially but not temporally coupled.
Abstract: The physiological state of natural competence for transformation allows certain bacteria to take up free DNA from the environment and to recombine such newly acquired DNA into their chromosomes. However, even though conserved components that are required to undergo natural transformation have been identified in several naturally competent bacteria, our knowledge of the underlying mechanisms of the DNA uptake process remains very limited. To better understand these mechanisms, we investigated the competence-mediated DNA transport in the naturally transformable pathogen Vibrio cholerae. Previously, we used a cell biology-based approach to experimentally address an existing hypothesis, which suggested the competence protein ComEA plays a role in the DNA uptake process across the outer membrane of Gram-negative bacteria. Here, we extended this knowledge by investigating the dynamics of DNA translocation across both membranes. More precisely, we indirectly visualized the transfer of the external DNA from outside the cell into the periplasm followed by the shuttling of the DNA into the cytoplasm. Based on these data, we conclude that for V. cholerae, the DNA translocation across the outer and inner membranes is spatially but not temporally coupled. IMPORTANCE As a mode of horizontal gene transfer, natural competence for transformation has contributed substantially to the plasticity of genomes and to bacterial evolution. Natural competence is often a tightly regulated process and is induced by diverse environmental cues. This is in contrast to the mechanistic aspects of the DNA translocation event, which are most likely conserved among naturally transformable bacteria. However, the DNA uptake process is still not well understood. We therefore investigated how external DNA reaches the cytosol of the naturally transformable bacterium V. cholerae. More specifically, we provide evidence that the DNA translocation across the membranes is spatially but not temporally coupled. We hypothesize that this model also applies to other competent Gram-negative bacteria and that our study contributes to the general understanding of this important biological process.
52 citations
References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments.
Abstract: Biofilms--matrix-enclosed microbial accretions that adhere to biological or non-biological surfaces--represent a significant and incompletely understood mode of growth for bacteria. Biofilm formation appears early in the fossil record (approximately 3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages, including the 'living fossils' in the most deeply dividing branches of the phylogenetic tree. It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments. Recent advances show that biofilms are structurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments and also disperse to colonize new niches. The implications of these survival and propagative mechanisms in the context of both the natural environment and infectious diseases are discussed in this review.
6,170 citations
"Quorum‐sensing autoinducer molecule..." refers background in this paper
...We reasoned that a mixed-species consortium may more closely reflect conditions in environmental biofilms that are unlikely to be mono-species in composition (Hall-Stoodley et al., 2004; Wintermute & Silver, 2010)....
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TL;DR: The evolution of quorum sensing systems in bacteria could, therefore, have been one of the early steps in the development of multicellularity.
Abstract: ▪ Abstract Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species. Furthermore, there is mounting data suggesting that ba...
4,449 citations
"Quorum‐sensing autoinducer molecule..." refers background in this paper
...…molecules has been studied in many laboratory systems that relied exclusively on cell-free culture fluids or monocultures (Bassler et al., 1997; Miller & Bassler, 2001; Henke & Bassler, 2004a), single-species co-cultures (Hammer & Bassler, 2007), or cocultures of Vibrios with other bacteria…...
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TL;DR: How the marine luminescent bacterium V. fischeri uses the LuxR and LuxI proteins for intercellular communication is reviewed and a newly discovered family of LuxRand LuxI homologs in diverse bacterial species is described.
Abstract: It has long been appreciated that certain groups of bacteria exhibit cooperative behavioral patterns. For example, feeding and sporulation of both myxobacteria and actinomycetes seem optimized for large populations of cells behaving almost as a single multicellular organism. The swarming motility of microorganisms such as Vibrio parahaemolyticus and Proteus mirabilis provides another excellent example of multicellular behavior among bacteria (2). Intercellular communication likewise has been appreciated for several years in Vibrio fischeri, Myxococcus xanthus, Bacillus subtilis, Streptomyces spp., the eukaryotic slime mold Dictyostelium discoideum, and other species (44). Here we first review how the marine luminescent bacterium V. fischeri uses the LuxR and LuxI proteins for intercellular communication and then describe a newly discovered family of LuxR and LuxI homologs in diverse bacterial species.
2,693 citations
"Quorum‐sensing autoinducer molecule..." refers background in this paper
...…bacterium and the causative agent of the disease cholera, produces and then responds to extracellular small molecules called autoinducers (AIs) to collectively control gene expression and coordinate group behaviors, a process called quorum sensing (QS) (Fuqua et al., 1994; Ng & Bassler, 2009)....
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TL;DR: The emergence of toxigenic V. cholerae involves horizontal gene transfer that may depend on in vivo gene expression, and is shown here to be encoded by a filamentous bacteriophage (designated CTXΦ), which is related to coliphage M13.
Abstract: Vibrio cholerae, the causative agent of cholera, requires two coordinately regulated factors for full virulence: cholera toxin (CT), a potent enterotoxin, and toxin-coregulated pili (TCP), surface organelles required for intestinal colonization. The structural genes for CT are shown here to be encoded by a filamentous bacteriophage (designated CTXphi), which is related to coliphage M13. The CTXphi genome chromosomally integrated or replicated as a plasmid. CTXphi used TCP as its receptor and infected V. cholerae cells within the gastrointestinal tracts of mice more efficiently than under laboratory conditions. Thus, the emergence of toxigenic V. cholerae involves horizontal gene transfer that may depend on in vivo gene expression.
1,744 citations
"Quorum‐sensing autoinducer molecule..." refers background in this paper
...Transduction of the cholera toxin genes encoded within a filamentous phage (CTXF) permits exchange of virulence factors among V. cholerae (Waldor & Mekalanos, 1996)....
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