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Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi
Abstract
Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density-dependent response. Transposon Tn5 insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild-type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins. The constitutive phenotype of LuxO- mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.
... Twenty years ago, it has been observed that S. Typhimurium and E. coli produced and secreted a small, soluble, heat-labile signaling molecule called autoinducer-2 [40]. Some years before, the presence of a second quorum sensing system in bacteria had been proposed [41]. Thus, it was observed that an AHL-blind reporter strain was able to produce bioluminescence only upon induction of the AI-2-dependent second system. ...
... The most interesting fact was that this strain responded to culture fluids from several unrelated bacteria ( Figure 4). These data indicated that the production of AI-2 was not restricted to a single bacterial species and that its production could be extended through bacteria [41]. The presence of homologues of the gene luxS, responsible for AI-2 activity, has been observed in several sequence genomes [42], and every luxS-containing species could be correlated with the detection of AI-2 activity in the extracellular media, suggesting that bacterial cells use AI-2 to communicate with cells of other bacterial species [40,41]. ...
... These data indicated that the production of AI-2 was not restricted to a single bacterial species and that its production could be extended through bacteria [41]. The presence of homologues of the gene luxS, responsible for AI-2 activity, has been observed in several sequence genomes [42], and every luxS-containing species could be correlated with the detection of AI-2 activity in the extracellular media, suggesting that bacterial cells use AI-2 to communicate with cells of other bacterial species [40,41]. In S. Typhimurium, the production of AI-2 is linked to the exponential growth phase, with a maximum concentration at the mid-exponential phase. ...
Salmonella spp. is one of the main foodborne pathogens around the world. It has a cyclic lifestyle that combines host colonization with survival outside the host, implying that Salmonella has to adapt to different conditions rapidly in order to survive. One of these environments outside the host is the food production chain. In this environment, this foodborne pathogen has to adapt to different stress conditions such as acidic environments, nutrient limitation, desiccation, or biocides. One of the mechanisms used by Salmonella to survive under such conditions is biofilm formation. Quorum sensing plays an important role in the production of biofilms composed of cells from the same microorganism or from different species. It is also important in terms of food spoilage and regulates the pathogenicity and invasiveness of Salmonella by regulating Salmonella pathogenicity islands and flagella. Therefore, in this review, we will discuss the genetic mechanism involved in Salmonella quorum sensing, paying special attention to small RNAs and their post-regulatory activity in quorum sensing. We will further discuss the importance of this cell-to-cell communication mechanism in the persistence and spoilage of Salmonella in the food chain environment and the importance in the communication with microorganisms from different species. Subsequently, we will focus on the role of quorum sensing to regulate the virulence and invasion of host cells by Salmonella and on the interaction between Salmonella and other microbial species. This review offers an overview of the importance of quorum sensing in the Salmonella lifestyle.
... Thus, one possibility is that SbcB is important for regulating the expression of genes required for NT. Regulatory control of NT can be bypassed by ectopically expressing the master regulator of competence TfoX (10) and by constitutively activating quorum sensing via deletion of LuxO (1,11,12). So to test this hypothesis, we assessed the impact of sbcB on NT in a background where tfoX is ectopically expressed (P tac -tfoX) and luxO is deleted. ...
Natural transformation (NT) is a conserved mechanism of horizontal gene transfer in bacterial species. During this process, DNA is taken up into the cytoplasm where it can be integrated into the host genome by homologous recombination. We have previously shown that some cytoplasmic exonucleases inhibit NT by degrading ingested DNA prior to its successful recombination. However, one exonuclease, SbcB, counterintuitively promotes NT in Vibrio cholerae. Here, through a systematic analysis of the distinct steps of NT, we show that SbcB acts downstream of DNA uptake into the cytoplasm, but upstream of recombinational branch migration. Through mutational analysis, we show that SbcB promotes NT in a manner that does not rely on its exonuclease activity. Finally, we provide genetic evidence that SbcB directly interacts with the primary bacterial recombinase, RecA. Together, these data advance our molecular understanding of horizontal gene transfer in V. cholerae and reveal that SbcB promotes homologous recombination during NT in a manner that does not rely on its canonical exonuclease activity.
IMPORTANCE
Horizontal gene transfer by natural transformation contributes to the spread of antibiotic resistance and virulence factors in bacterial species. Here, we study how one protein, SbcB, helps facilitate this process in the facultative bacterial pathogen Vibrio cholerae. SbcB is a well-known for its exonuclease activity (i.e., the ability to degrade the ends of linear DNA). Through this study, we uncover that while SbcB is important for natural transformation, it does not facilitate this process using its exonuclease activity. Thus, this work helps further our understanding of the molecular events required for this conserved evolutionary process and uncovers a function for SbcB beyond its canonical exonuclease activity.
... Much of the early work uncovering the bacterial cell-cell communication process called quorum sensing was performed in two Vibrio species: Vibrio fischeri and Vibrio harveyi (54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67). Of note, the strain V. harveyi BB120 (with which most studies referred to herein were performed) was later re-classified as Vibrio campbellii (68). ...
Species of the Vibrio genus occupy diverse aquatic environments ranging from brackish water to warm equatorial seas to salty coastal regions. More than 80 species of Vibrio have been identified, many of them as pathogens of marine organisms, including fish, shellfish, and corals, causing disease and wreaking havoc on aquacultures and coral reefs. Moreover, many Vibrio species associate with and thrive on chitinous organisms abundant in the ocean. Among the many diverse Vibrio species, the most well-known and studied is Vibrio cholerae, discovered in the 19th century to cause cholera in humans when ingested. The V. cholerae field blossomed in the late 20th century, with studies broadly examining V. cholerae evolution as a human pathogen, natural competence, biofilm formation, and virulence mechanisms, including toxin biology and virulence gene regulation. This review discusses some of the historic discoveries of V. cholerae biology and ecology as one of the fundamental model systems of bacterial genetics and pathogenesis.
... Vibrio QS signaling pathways are composed of the QS signal synthases (LuxM, CqsA, and LuxS producing AI-1, CAI-1, and AI-2 respectively), the membrane receptors (LuxN, CqsS, and LuxPQ detecting AI-1, CAI-1, and AI-2 respectively), a two-component phosphorelay protein (LuxU/LuxO) (Milton, 2006), and the master regulators (Girard, 2019). The signal transduction of QS signals that bind their membrane sensors converges intracellularly via a shared two-component LuxU/LuxO phosphorelay signal transduction cascade to activate the master regulators that exert the regulation of a broad variety of physiological processes (Bassler et al., 1994;Freeman & Bassler, 1999;Lilley & Bassler, 2000). ...
... The unique positioning of the R-C linker into the active site is exclusively witnessed in LuxO but not with another member of the bEBP family proteins [8]. We learn from the previously reported σ 54 associated transcriptional regulators that it binds with enhancer sequences upstream of the σ 54 promoter [19,20]. Hence, it can be said that the transcriptional regulations through σ 54 promoter region unwinding is mainly initiated by LuxO's C-terminal DNA binding domain. ...
Quorum sensing plays a vital role in the environmental and host life cycles of Vibrio cholerae. The quorum-sensing circuit involves the consorted action of autoinducers, small RNAs, and regulatory proteins to control a plethora of physiological events in this bacterium. Among the regulatory proteins, LuxO is considered a low-cell-density master regulator. It is a homolog of NtrC, a two-component response regulator. NtrC belongs to an evolving protein family that works with the alternative sigma factor σ⁵⁴ to trigger gene transcription. Structurally, these proteins comprise 3 domains: a receiver domain, a central AAA+ATPase domain, and a C-terminal DNA-binding domain (DBD). LuxO communicates with its cognate promoters by employing its DNA binding domain. In the present study, we desired to identify the critical residues in the DBD of LuxO. Our combined mutagenesis and biochemical assays resulted in the identification of eleven residues that contribute significantly to LuxO regulatory function.
... aphA, which has been described as a major regulator of natural competence in Vibrio cholerae [63], also plays a similar role in Vibrio alginolyticus after the acquisition of phage resistance. The transcriptional status of many QS-related genes, such as LuxQ [57], LuxU [64], and LuxO [65], has been correlated with fitness costs under various ecological habitats. Furthermore, in addition to studying the transcriptional interplay of aphA and LuxR genes in populations with acquired bacteriophage resistance, we investigated the transcriptional levels of genes involved in virulence and controlled by aphA. ...
Bacteria employ a wide range of molecular mechanisms to confer resistance to bacteriophages, and these mechanisms are continuously being discovered and characterized. However, there are instances where certain bacterial species, despite lacking these known mechanisms, can still develop bacteriophage resistance through intricate metabolic adaptation strategies, potentially involving mutations in transcriptional regulators or phage receptors. Vibrio species have been particularly useful for studying the orchestrated metabolic responses of Gram-negative marine bacteria in various challenges. In a previous study, we demonstrated that Vibrio alginolyticus downregulates the expression of specific receptors and transporters in its membrane, which may enable the bacterium to evade infection by lytic bacteriophages. In our current study, our objective was to explore how the development of bacteriophage resistance in Vibrio species disrupts the quorum-sensing cascade, subsequently affecting bacterial physiology and metabolic capacity. Using a real-time quantitative PCR (rt-QPCR) platform, we examined the expression pattern of quorum-sensing genes, auto-inducer biosynthesis genes, and cell density regulatory proteins in phage-resistant strains. Our results revealed that bacteriophage-resistant bacteria downregulate the expression of quorum-sensing regulatory proteins, such as LuxM, LuxN, and LuxP. This downregulation attenuates the normal perception of quorum-sensing peptides and subsequently diminishes the expression of cell density regulatory proteins, including LuxU, aphA, and LuxR. These findings align with the diverse phenotypic traits observed in the phage-resistant strains, such as altered biofilm formation, reduced planktonic growth, and reduced virulence. Moreover, the transcriptional depletion of aphA, the master regulator associated with low cell density, was linked to the downregulation of genes related to virulence. This phenomenon appears to be phage-specific, suggesting a finely tuned metabolic adaptation driven by phage–host interaction. These findings contribute to our understanding of the role of Vibrio species in microbial marine ecology and highlight the complex interplay between phage resistance, quorum sensing, and bacterial physiology.
... Vibrio QS signaling pathways are composed of the QS signal synthases (LuxM, CqsA, and LuxS producing AI-1, CAI-1, and AI-2 respectively), the membrane receptors (LuxN, CqsS, and LuxPQ detecting AI-1, CAI-1, and AI-2 respectively), a two-component phosphorelay protein (LuxU/LuxO) (Milton, 2006), and the master regulators (Girard, 2019). The signal transduction of QS signals that bind their membrane sensors converges intracellularly via a shared two-component LuxU/LuxO phosphorelay signal transduction cascade to activate the master regulators that exert the regulation of a broad variety of physiological processes (Bassler et al., 1994;Freeman & Bassler, 1999;Lilley & Bassler, 2000). ...
Vibrio parahaemolyticus is a significant food‐borne pathogen that is found in diverse aquatic habitats. Quorum sensing (QS), a signaling system for cell–cell communication, plays an important role in V. parahaemolyticus persistence. We characterized the function of three V. parahaemolyticus QS signal synthases, CqsAvp, LuxMvp, and LuxSvp, and show that they are essential to activate QS and regulate swarming. We found that CqsAvp, LuxMvp, and LuxSvp activate a QS bioluminescence reporter through OpaR. However, V. parahaemolyticus exhibits swarming defects in the absence of CqsAvp, LuxMvp, and LuxSvp, but not OpaR. The swarming defect of this synthase mutant (termed Δ3AI) was recovered by overexpressing either LuxOvpD47A, a mimic of dephosphorylated LuxOvp mutant, or the scrABC operon. CqsAvp, LuxMvp, and LuxSvp inhibit lateral flagellar (laf) gene expression by inhibiting the phosphorylation of LuxOvp and the expression of scrABC. Phosphorylated LuxOvp enhances laf gene expression in a mechanism that involves modulating c‐di‐GMP levels. However, enhancing swarming requires phosphorylated and dephosphorylated LuxOvp which is regulated by the QS signals that are synthesized by CqsAvp, LuxMvp, and LuxSvp. The data presented here suggest an important strategy of swarming regulation by the integration of QS and c‐di‐GMP signaling pathways in V. parahaemolyticus.
... www.nature.com/scientificreports/ (AI-2) and homologues of LuxU and LuxO involved in a phospho-relay in V. harveyi [15][16][17][18][19] have been identified. The AI-2 signal converges to LuxO, a nitrogen regulatory protein (NtrC) homolog, and is transduced to the master regulator SmcR [20][21][22] . ...
Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB -deletion isotype. RyhB binds directly to the 5′-UTR (untranslated region) of the luxS transcript to form a heteroduplex, which not only stabilizes luxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to luxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.
... At low cell density (LCD) and low AIs concentration, the receptors CqsS and LuxQ act as kinases, channeling phosphate to the phosphotransfer protein LuxU, which passes it to the response regulator LuxO (Bassler et al, 1994). Phosphorylated LuxO, together with the sigma factor σ 54 induces the transcription of four homologous sRNAs Qrr1-4 (2-5 copies in other vibrios) (Lilley & Bassler, 2000). ...
Bacterial regulatory small RNAs, usually 50-300 nt long, act by base-pairing with specific mRNA targets, affecting their translation and/or stability, are important elements which regulate a variety of processes. V. tasmaniensis LGP32 is a facultative oyster pathogen. A sRNA Vsr217 was found to be conserved within vibrios and highly upregulated during oyster infection. I found that vsr217 and the downstream gene malK (encoding a subunit of the major maltose transporter) are both expressed from an upstream promoter regulated by the maltose activator MalT with Vsr217 being generated from the long 5' UTR of the malK mRNA. Beside a cis-effect on malK expression, which decreases in the Δvsr217 mutant, we found that the absence of this sRNA resulted, when cells grown in maltose, in the increase of two important enzymes involved in the glycolysis/neoglucogenesis pathway, Fbp and PpsA and that fbp mRNA was a direct target of Vsr217. I also explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs: Leucine, Valine and Isoleucine) in V. alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. We found that the ilvGMEDA operon (encoding the main pathway for biosynthesis of BCAAs) is regulated by a translated leader peptide. Thus, the translation of a BCAA rich peptide encoded upstream of the structural genes provides an adaptive response by a mechanism similar to the E. coli canonical model. This study with a non-model Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.
... Quorum sensing has been heavily studied in marine Vibrio species, owing to easily observable behaviours such as bioluminescence and biofilm formation (Bassler et al., 1997). Vibrio campbellii BB120 was one of the first Vibrio species to have its quorum-sensing circuit discovered (Bassler et al., 1993(Bassler et al., , 1994Freeman et al., 2000). V. campbellii BB120 was historically called Vibrio harveyi BB120 (also known as strain ATCC BAA-1116) until it was recently reclassified (Lin et al., 2010). ...
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 >30 isogenic strains with deletions of genes encoding BB120 quorum‐sensing system homologs. Our results show that the quorum‐sensing circuit of DS40M4 is distinct from BB120 in three ways: 1) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, 2) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signaling through the response regulator LuxO, and 3) 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. This article is protected by copyright. All rights reserved.
... They regulate bioluminescence, metalloproteinases, iron carrier, and exopolysaccharide production and negative type III secretion in a cell density-dependent manner [50]. LuxM/LuxN system is made up of the autoinducer signal N-(3-hydroxybutanoyl)-L-homoserine lactone (HAI-1, 1) [51] and its two-component sensor LuxN [50]. HAI-1 is an acylated homoserine lactone, and produced by the LuxM synthase [52e54]. ...
Vibrio harveyi is a marine bacterial pathogen which infects a wide range of marine organisms and results in severe loss. Antibiotics have been used for prophylaxis and treatment of V. harveyi infection. However, antibiotic resistance is a major public health threat to both human and animals. Therefore, there is an urgent need for novel antimicrobial agents with new modes of action. In V. harveyi, many virulence factors production and bioluminescence formation depend on its quorum sensing (QS) network. Therefore, the QS system has been widely investigated as an effective potential target for the treatment of V. harveyi infection. This perspective focuses on the quorum sensing inhibitors (QSIs) of V. harveyi QS systems (LuxM/N, LuxS/PQ, and CqsA/S) and evaluates medicinal chemistry strategies.
... It is the only AI that is produced and identified by a wide range of diverse bacteria and controls gene expression (Ismail 2016). V. harveyi uses two AI signals (Bassler et al. 1994;Lilley and Bassler, 2000), one for intraspecies communication (AI-1) and the other for interspecies communication (AI-2) (Federle and Bassler 2003). LuxLM protein synthesizes AI-1 (N-(3-hydroxybutanoyl)-homoserine lactone), and LuxS protein synthesizes AI-2 (furanosyl borate diester) (Schauder et al. 2001). ...
Biofilms are structured microbial communities of single or multiple populations in which microbial cells adhere to a surface and get embedded in extracellular polymeric substances (EPS). This review attempts to explain biofilm architecture, development phases, and forces that drive bacteria to promote biofilm mode of growth. Bacterial chemical communication, also known as Quorum sensing (QS), which involves the production, detection, and response to small molecules called autoinducers, is highlighted. The review also provides a brief outline of interspecies and intraspecies cell–cell communication. Additionally, we have performed docking studies using Discovery Studio 4.0, which has enabled our understanding of the prominent interactions between autoinducers and their receptors in different bacterial species while also scoring their interaction energies. Receptors, such as LuxN (Phosphoreceiver domain and RecA domain), LuxP, and LuxR, interacted with their ligands (AI-1, AI-2, and AHL) with a CDocker interaction energy of − 31.6083 kcal/mole; − 34.5821 kcal/mole, − 48.2226 kcal/mole and − 41.5885 kcal/mole, respectively. Since biofilms are ideal for the remediation of contaminants due to their high microbial biomass and their potential to immobilize pollutants, this article also provides an overview of biofilm-mediated bioremediation.
... The overall regulatory functions of TepR in B. glumae are reminiscent of LuxO in Vibrio spp., although limited sequence similarity is shared between them (32% amino acid identity). LuxO suppresses luminescence of Vibrio harveyi or Vibrio fischeri that is under the positive regulation of the luxI/luxR QS system (Bassler et al., 1994;Freeman and Bassler, 1999;Miyamoto et al., 2000Miyamoto et al., , 2003. luxO mutants in several Vibrio spp. ...
The pathogenesis of the rice pathogenic bacterium Burkholderia glumae is under the tight regulation of the tofI/tofR quorum‐sensing (QS) system. tepR, encoding a group I bacterial enhancer‐binding protein, negatively regulates the production of toxoflavin, the phytotoxin acting as a major virulence factor in B. glumae. In this study, through a transcriptomic analysis, we identified the genes that were modulated by tepR and/or the tofI/tofR QS system. More than half of the differentially expressed genes, including the genes for the biosynthesis and transport of toxoflavin, were significantly more highly expressed in the ΔtepR mutant but less expressed in the ΔtofI‐tofR (tofI/tofR QS‐defective) mutant. In consonance with the transcriptome data, other virulence‐related functions of B. glumae, extracellular protease activity and flagellum‐dependent motility, were also negatively regulated by tepR, and this negative regulatory function of tepR was dependent on the IclR‐type transcriptional regulator gene qsmR. Likewise, the ΔtepR mutant exhibited a higher level of heat tolerance in congruence with the higher transcription levels of heat shock protein genes in the mutant. Interestingly, tepR also exhibited its positive regulatory function on a previously uncharacterized type VI secretion system (denoted as BgT6SS‐1). The survival of the both ΔtepR and ΔtssD (BgT6SS‐1‐defective) mutants was significantly compromised compared to the wild‐type parent strain 336gr‐1 in the presence of the natural rice‐inhabiting bacterium, Pantoea sp. RSPAM1. Taken together, this study revealed pivotal regulatory roles of tepR in orchestrating multiple biological functions of B. glumae, including pathogenesis, heat tolerance, and bacterial interspecies competition. We characterized the multiple regulatory functions of tepR, which include negative regulation of bacterial virulence and positive regulation of interspecies competition through qsmR and a type VI secretion system, respectively.
... Using chitin-independent natural transformation via tfoX induction, we generated ΔluxO and ΔluxR mutations in both the DS40M4 and NBRC 15631 strain backgrounds. In the model strain BB120, a ΔluxO mutant results in a constitutively expressed master quorum-sensing transcription factor, LuxR, producing high levels of bioluminescence and mimicking a high cell density phenotype (27). Conversely, a ΔluxR mutant of BB120 is unable to produce bioluminescence (28). ...
Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus .
... All four receptors act as kinases at LCD in their unliganded states [21][22][23]. They funnel phosphate through the phosphotransfer protein LuxU to the response regulator LuxO, which, via a set of small regulatory RNAs (sRNAs) called the Qrr sRNAs, drives the production of the LCD master regulator AphA and represses production of the HCD master regulator HapR [24][25][26][27]. Under these conditions, behaviors including biofilm formation and virulence factor production are undertaken (Fig 1A, left) [3,11]. ...
Vibrio cholerae possesses multiple quorum-sensing (QS) systems that control virulence and biofilm formation among other traits. At low cell densities, when QS autoinducers are absent, V. cholerae forms biofilms. At high cell densities, when autoinducers have accumulated, biofilm formation is repressed, and dispersal occurs. Here, we focus on the roles of two well-characterized QS autoinducers that function in parallel. One autoinducer, called cholerae autoinducer-1 (CAI-1), is used to measure Vibrio abundance, and the other autoinducer, called autoinducer-2 (AI-2), is widely produced by different bacterial species and presumed to enable V. cholerae to assess the total bacterial cell density of the vicinal community. The two V. cholerae autoinducers funnel information into a shared signal relay pathway. This feature of the QS system architecture has made it difficult to understand how specific information can be extracted from each autoinducer, how the autoinducers might drive distinct output behaviors, and, in turn, how the bacteria use QS to distinguish kin from nonkin in bacterial communities. We develop a live-cell biofilm formation and dispersal assay that allows examination of the individual and combined roles of the two autoinducers in controlling V. cholerae behavior. We show that the QS system works as a coincidence detector in which both autoinducers must be present simultaneously for repression of biofilm formation to occur. Within that context, the CAI-1 QS pathway is activated when only a few V. cholerae cells are present, whereas the AI-2 pathway is activated only at much higher cell density. The consequence of this asymmetry is that exogenous sources of AI-2, but not CAI-1, contribute to satisfying the coincidence detector to repress biofilm formation and promote dispersal. We propose that V. cholerae uses CAI-1 to verify that some of its kin are present before committing to the high-cell–density QS mode, but it is, in fact, the broadly made autoinducer AI-2 that sets the pace of the V. cholerae QS program. This first report of unique roles for the different V. cholerae autoinducers suggests that detection of kin fosters a distinct outcome from detection of nonkin.
... LuxO, which, via a set of small regulatory RNAs (sRNAs) called the Qrr sRNAs, drives the 65 production of the LCD master regulator AphA and represses production of the HCD master 66 regulator HapR [23][24][25][26]. Under these conditions, behaviors including biofilm formation and 67 virulence factor production are undertaken (Fig 1A, left) [3,10]. ...
Vibrio cholerae possesses multiple quorum-sensing systems that control virulence and biofilm formation among other traits. At low cell densities, when quorum-sensing autoinducers are absent, V. cholerae forms biofilms. At high cell densities, when autoinducers have accumulated, biofilm formation is repressed and dispersal occurs. Here, we focus on the roles of two well-characterized quorum-sensing autoinducers that function in parallel. One autoinducer, called CAI-1, is used to measure vibrio abundance, and the other autoinducer, called AI-2, is a broadly-made universal autoinducer that is presumed to enable V. cholerae to assess the total bacterial cell density of the vicinal community. The two V. cholerae autoinducers funnel information into a shared signal relay pathway. This feature of the quorum-sensing system architecture has made it difficult to understand how specific information can be extracted from each autoinducer, how the autoinducers might drive distinct output behaviors, and in turn, how the bacteria use quorum sensing to distinguish self from other in bacterial communities. We develop a live-cell biofilm formation and dispersal assay that allows examination of the individual and combined roles of the two autoinducers in controlling V. cholerae behavior. We show that the quorum-sensing system works as a coincidence detector in which both autoinducers must be present simultaneously for repression of biofilm formation to occur. Within that context, the CAI-1 quorum-sensing pathway is activated when only a few V. cholerae cells are present, whereas the AI-2 pathway is activated only at much higher cell density. The consequence of this asymmetry is that exogenous sources of AI-2, but not CAI-1, contribute to satisfying the coincidence detector to repress biofilm formation and promote dispersal. We propose that V. cholerae uses CAI-1 to verify that some of its kin are present before committing to the high-cell-density quorum-sensing mode, but it is, in fact, the universal autoinducer AI-2, that sets the pace of the V. cholerae quorum-sensing program. This first report of unique roles for the different V. cholerae autoinducers suggests that detection of self fosters a distinct outcome from detection of other.
... Además, se ha usado para PCR en V. parahaemolyticus (Venkateswaran et al., 1998). De la misma forma es factible la idea de trabajar con los genes luxO que codifican para LuxO; que es una proteína represora de la luminiscencia en V. harveyi (Bassler et al., 1994) y a la fecha no se ha detectado en otras especies de bacterias luminiscentes (Dunlap, 1997), por lo que es probable que tenga regiones conservadas para esta especie. ...
En diversos laboratorios de producción de larvas de camarón del país se han presentado eventos de luminiscencia asociados con mortalidades en los estadíos Zoea, Mysis y Postlarva. En México el único trabajo formal enfocado a su estudio es sobre larvas de Penaeus vannamei, aun cuando en Asia, Ecuador y Venezuela está ampliamente documentada su importancia desde hace más de una década debido a las mortalidades masivas registradas.
Corroboramos la presencia de Vibrio harveyi en el sistema de cultivo larvario de Penaeus stylirostris en dos laboratorios mexicanos productores de postlarvas, uno ubicado en Sinaloa y el otro en el norte de Sonora. Durante los muestreos realizados, no hubo eventos de luminiscencia ni mortalidades masivas de larvas. La identificación de la especie y otras bacterias luminiscentes marinas se basó en una clave taxonómica desarrollada a partir de datos de la literatura. Las cepas aisladas en los laboratorios de producción fueron resistentes hasta 30 ppm de Argentyne y 30 ppm de formol, casi el doble que la cepa de colección de la ATCC; a densidades celulares en el orden de 102 cel/mL. Esta resistencia varía según el estadío del que fueron aisladas.
Los reproductores, importados de Venezuela, parecen ser la fuente de V. harveyi al sistema de producción. Se evidenció su presencia en larvas de Nauplio, Zoea, Mysis y Postlarva, así como en agua y paredes del estanque de reproductores y en la fosa de oxidación.
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There have been luminescence events in different shrimp hatcheries of México related with larvae mortalities, mainly in Zoea, Mysis and Postlarvae stages. The only formal work focused to their study in Mexico was due on Penaeus vannamei larvae, even that their pathogenic importance has been reported a decade ago in Asia, Ecuador and Venezuela (LaVilla-Pitogo et al., 1998; Alvarez et al., 1998; Robertson et al., 1998).
The presence of Vibrio harveyi was confirmed in two Mexican shrimp hatcheries, one located in Sinaloa and the other in the north of Sonora. There was no mortalities or luminescence event during our period of sampling. The identification of this specie and other other luminiscence bacteria, was based on a taxonomy key developed from literature data. The colonies isolated from hatcheries production systems were resistant until 30 ppm of Argentyne and 30 ppm of formaline, almost a double concentration than reference strains. This resistance changes according to the larval stage.
The source of V. harveyi into production system, was the broodstock imported from Venezuela. The bacteria were isolated from water and walls of stock tanks, and their incidence was also detected in Nauplii, Zoea, Mysis, Postlarvae and seawater sewage system.
... [88][89][90][91][92] Ultimately, the cellular activities controlled by quorum-sensing molecules are diverse and vary significantly by species. These activities can include toxin production, 93 biofilm formation, 94 bioluminescence, 95 protein secretion, 96 motility, 97 and cell division. 98 In the same way, a variety of cellular readouts and outputs could easily be engineered into these regulatory systems for therapeutic purposes. ...
Small molecules have many important roles across the tree of life: they regulate processes from metabolism to transcription, they enable signaling within and between species, and they serve as the biochemical building blocks for cells. They also represent valuable phenotypic endpoints that are promising for use as biomarkers of disease states. In the context of engineering cell‐based therapeutics, they hold particularly great promise for enabling finer control over the therapeutic cells and allowing them to be responsive to extracellular cues. The natural signaling and regulatory functions of small molecules can be harnessed and rewired to control cell activity and delivery of therapeutic payloads, potentially increasing efficacy while decreasing toxicity. To that end, this review considers small molecule‐mediated regulation and signaling in bacteria. We first discuss some of the most prominent applications and aspirations for responsive cell‐based therapeutics. We then describe the transport, signaling, and regulation associated with three classes of molecules that may be exploited in the engineering of therapeutic bacteria: amino acids, fatty acids, and quorum‐sensing signaling molecules. We also present examples of existing engineering efforts to generate cells that sense and respond to levels of different small molecules. Finally, we discuss future directions for how small molecule‐mediated regulation could be harnessed for therapeutic applications, as well as some critical considerations for the ultimate success of such endeavors. WIREs Syst Biol Med 2018, 10:e1405. doi: 10.1002/wsbm.1405
This article is categorized under: Biological Mechanisms > Cell Signaling
Biological Mechanisms > Metabolism
Translational, Genomic, and Systems Medicine > Therapeutic Methods
... Unlabeled Thr: M9 + 0.4% glucose + 0.4 mM [ 12 Overnight cultures of wild-type V. cholerae were divided into aliquots and back-diluted into unlabeled medium or medium containing a labeled amino acid. Cell-free culture fluids were prepared as above and loaded onto a Thermo Scientific Hypersep Hypercarb column (100-700 mg resin) that had been washed with 5 column volumes (CV) of MeOH, 5 CV of MeCN, and equilibrated with 10 CV of water. ...
Quorum sensing (QS) is a cell–cell communication process that enables bacteria to track cell population density and orchestrate collective behaviors. QS relies on the production and detection of, and the response to, extracellular signal molecules called autoinducers. In Vibrio cholerae, multiple QS circuits control pathogenesis and biofilm formation. Here, we identify and characterize a new QS autoinducer–receptor pair. The autoinducer is 3,5-dimethylpyrazin-2-ol (DPO). DPO is made from threonine and alanine, and its synthesis depends on threonine dehydrogenase (Tdh). DPO binds to and activates a transcription factor, VqmA. The VqmA–DPO complex activates expression of vqmR, which encodes a small regulatory RNA. VqmR represses genes required for biofilm formation and toxin production. We propose that DPO allows V. cholerae to regulate collective behaviors to, among other possible roles, diversify its QS output during colonization of the human host.
... LuxO has been well characterized for its role in quorum sensing (34,41) (for reviews, see references 42 and 43). The regulatory activity of LuxO has been proposed to be linked in a contrary manner to HapR, where at low cell densities, high levels of phosphorylated LuxO reduce the accumulation of the HapR message (32,34,37). ...
Vibrio cholerae is the etiological agent of the infectious disease cholera, which is characterized by vomiting and severe watery diarrhea. Recently, V. cholerae clinical isolates have demonstrated increased virulence capabilities, causing more severe symptoms with a much higher rate of disease progression than previously observed. We have identified single nucleotide polymorphisms (SNPs) in four virulence-regulatory genes (hapR, hns, luxO, and vieA) of a hypervirulent V. cholerae clinical isolate, MQ1795. Herein, all SNPs and SNP combinations of interest were introduced into the prototypical El Tor reference strain N16961, and the effects on the production of numerous virulence-related factors, including cholera toxin (CT), the toxin-coregulated pilus (TCP), and ToxT, were analyzed. Our data show that triple- SNP (hapR hns luxO and hns luxO vieA) and quadruple-SNP combinations produced the greatest increases in CT, TCP, and ToxT production. The hns and hns luxO SNP combinations were sufficient for increased TCP and ToxT production. Notably, the hns luxO vieA triple-SNP combination strain produced TCP and ToxT levels similar to those of MQ1795. Certain SNP combinations (hapR and hapR vieA) had the opposite effect on CT, TCP, and ToxT expression. Interestingly, the hns vieA double-SNP combination strain increased TCP production while decreasing CT production. Our findings suggest that SNPs identified in the four regulatory genes, in various combinations, are associated with increased virulence capabilities observed in V. cholerae clinical isolates. These studies provide insight into the evolution of highly virulent strains.
... However, in a well-studied V. vulnificus strain, MO6-24/O, whose genome has been completely sequenced (GenBank accession number: CP002469.1 for chromosome I and CP002470.1 for chromosome II) (28), there are no genes for the biosynthesis of either AI-1 or cholera autoinducer-1 (CAI-1). Homologues of LuxPQ, the cognate receptor for AI-2 in V. harveyi, and LuxU and LuxO, which are involved in a phospho-relay, were identified in V. vulnificus (29)(30)(31)(32)(33). The autoinducer signal converges on LuxO, a nitrogen regulatory protein (NtrC) homologue, which in turn regulates the master regulator SmcR (34)(35)(36). ...
Vibrio vulnificus is a marine bacterium that causes human infections resulting in high mortality. This pathogen harbors five
quorum-regulatory RNAs (Qrrs 1-5) that affect the expression of pathogenicity genes by modulating the expression of the master
regulator SmcR. The qrr genes are activated by phosphorylated LuxO to different degrees: qrr2 is strongly activated, qrr3
and qrr5 are moderately activated, and qrr1 and qrr4 are marginally activated and are the only two that do not respond to
cell density-dependent regulation. Qrrs function redundantly to inhibit SmcR at low cell density, and fully repress when all
five are activated. In this study, we found that iron inhibits qrr expression in three distinct ways. First, the iron-ferric
uptake regulator (Fur) complex directly binds to qrr promoter regions, inhibiting LuxO activation by competing with LuxO for
cis-acting DNA elements. Second, qrr transcription is repressed by iron independently of Fur. Third, LuxO expression is repressed
by iron independently of Fur. We also found that, under iron-limiting conditions, the five Qrrs functioned additively, not
redundantly, to repress SmcR, suggesting that cells lacking iron enter a high cell density mode earlier and could thereby
modulate expression of virulence factors sooner. This study suggests that iron and quorum sensing, along with their cognate
regulatory circuits, are linked together in the coordinated expression of virulence factors.
... The new assay involves the development of a non-growth bioluminescence based assay for soil AOC measurements using starved cells of the naturally occurring luminous microorganism, Vibrio harveyi BB721 [25]. V. harveyi is a Gram-negative, bioluminescent, rod-shaped, motile, facultative anaerobic, halophilic marine bacterium capable of fermentative and respiratory metabolism. ...
Microbial growth can be a significant issue for reverse osmosis (RO) and microfiltration (MF) processes in the water industry. Microbial growth or regrowth can affect plant performance and increases plant operation costs. Assimilable Organic Carbon (AOC) is the fraction of the total organic carbon (TOC) that bacteria can use for growth and other metabolic processes. Several published bioassays for measuring AOC are available to water utilities; however, AOC concentrations are not routinely measured due to the complexity and difficulty of the methods. The objective of this study was to design a rapid bioluminescent assay that is reliable and sensitive for monitoring low concentrations of AOC. The rapid bioluminescent assay was evaluated and compared with the standard growth-based AOC assay. The performances of both assays are presented. It was determined that the bioluminescent based AOC assay was rapid, reduced monitoring costs, increased sensitivity, and reliably measured low concentrations of AOC in surface water samples. 2011 © American Water Works Association AWWA WQTC Conference Proceedings All Rights Reserved.
The area of chemical communication in bacteria has grown explosively since the end of the 20 th century. Among a number of key individuals and seminal findings that broke open this area of microbiology, the contributions of Bonnie Bassler and her colleagues are immense and multi‐layered. In this short and informal review, I provide perspective on my own entry into this research field, my introduction to Dr. Bassler and her early findings, followed by the founding of the Bassler lab and the flood of brilliant experimentation and public outreach that has done so much to propel the field of bacterial chemical communication.
Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P. aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P. aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthesized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical interaction with the enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand-bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR-mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor production in P. aeruginosa.
Autoinducer-2 (AI-2), a quorum-sensing signal molecule from the human pathogen Vibrio vulnificus, was assessed for its effect on the gut microbiome of mice. For this, we employed 16S rRNA sequencing to compare the gut microbiome of mice infected with either wild-type V. vulnificus or with the isotype ΔluxS that has a deletion in luxS which encodes the biosynthetic function of AI-2. The relative ratio of wild-type Vibrio species in the jejunum and ileum of mice infected with the wild type was significantly higher than that in mice infected with ΔluxS, suggesting that AI-2 plays an important role in the colonization of V. vulnificus in the small intestine. The bacterial composition in the gut of mice infected with ΔluxS comprises a higher proportion of Firmicutes, composed mainly of Lactobacillus, compared to the mice infected with wild-type cells. In the large intestine, Vibrio species were barely detected regardless of genetic background. Three Lactobacillus spp. isolated from fecal samples from mice infected with ΔluxS manifested significant antibacterial activities against V. vulnificus. Culture supernatants from these three species were dissolved by HPLC, and a substance in fractions showing inhibitory activity against V. vulnificus was determined to be lactic acid. Our results suggest that luxS in V. vulnificus affects not only the ability of the species to colonize the host gut but also its susceptibility to the growth-inhibiting activity of commensal bacteria including Lactobacillus.
Key points
• Gut microbiomes of ΔluxS-infected and WT Vibrio-infected mice differed greatly.
• Difference was most prominent in the jejunum and ileum compared to the duodenum or large intestine.
• In the small and large intestines of mice, the relative proportions of Vibrio and Lactobacillus species showed a negative relationship.
• Effector molecules produced by Lactobacillus in mouse gut inhibit Vibrio growth.
In many natural and clinical settings, bacteria are associated with some type of biotic or abiotic surface that enables them to form biofilms, a multicellular lifestyle with bacteria embedded in an extracellular matrix. Staphylococcus aureus and Staphylococcus epidermidis , the most frequent causes of biofilm-associated infections on indwelling medical devices, can switch between an existence as single free-floating cells and multicellular biofilms. During biofilm formation, cells first attach to a surface and then multiply to form microcolonies. They subsequently produce the extracellular matrix, a hallmark of biofilm formation, which consists of polysaccharides, proteins, and extracellular DNA. After biofilm maturation into three-dimensional structures, the biofilm community undergoes a disassembly process that leads to the dissemination of staphylococcal cells. As biofilms are dynamic and complex biological systems, staphylococci have evolved a vast network of regulatory mechanisms to modify and fine-tune biofilm development upon changes in environmental conditions. Thus, biofilm formation is used as a strategy for survival and persistence in the human host and can serve as a reservoir for spreading to new infection sites. Moreover, staphylococcal biofilms provide enhanced resilience toward antibiotics and the immune response and impose remarkable therapeutic challenges in clinics worldwide. This review provides an overview and an updated perspective on staphylococcal biofilms, describing the characteristic features of biofilm formation, the structural and functional properties of the biofilm matrix, and the most important mechanisms involved in the regulation of staphylococcal biofilm formation. Finally, we highlight promising strategies and technologies, including multitargeted or combinational therapies, to eradicate staphylococcal biofilms.
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 >30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system homologs. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120 in three ways: 1) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, 2) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signaling through the response regulator LuxO, and 3) 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.
Originality-Significance Statement
Wild bacterial isolates yield important information about traits that vary within species. Here, we compare environmental isolate Vibrio campbellii DS40M4 to its close relative, the model strain BB120 that has been a fundamental strain for studying quorum sensing for >30 years. We examine several phenotypes that define this species, including quorum sensing, bioluminescence, and biofilm formation. Importantly, DS40M4 is naturally transformable with exogenous DNA, which allows for the rapid generation of mutants in a laboratory setting. By exploiting natural transformation, we genetically dissected the functions of BB120 quorum-sensing system homologs in the DS40M4 strain, including two-component signaling systems, transcriptional regulators, and small RNAs.
Although the gut microbiome is generally symbiotic or commensal, some microbiome members become pathogenic under certain circumstances. However, the factors driving this pathogenic switch are largely unknown. Pathogenic bacteria can generate uracil that triggers host dual oxidase (DUOX) to produce antimicrobial reactive oxygen species (ROS). We show that pathogens generate uracil and ribose upon nucleoside catabolism of gut luminal uridine, which triggers not only host defenses but also inter-bacterial communication and pathogenesis in Drosophila. Uridine-derived uracil triggers DUOX-dependent ROS generation, whereas ribose induces bacterial quorum sensing (QS) and virulence gene expression. Genes implicated in nucleotide metabolism are found in pathogens but not commensal bacteria, and their genetic ablation blocks QS and the commensal-to-pathogen transition in vivo. Furthermore, commensal bacteria lack functional nucleoside catabolism, which is required to achieve gut-microbe symbiosis, but can become pathogenic by enabling nucleotide catabolism. These findings reveal molecular mechanisms governing the commensal-to-pathogen transition in different contexts of host-microbe interactions.
Chemical halogenation is an important transformation in organic synthesis. Halogenations involve the use of reactive and toxic halogens (X2) or hydrogen halides (HX). Chemical halogenations are fast and exothermic, but with little regio- or stereoselectivity. By doing halogenation reactions with halide anions, O2 or hydrogen peroxide (H2O2) as oxidants and vanadium- or iron-dependent haloperoxidases (HPOs) or FAD-dependent halogenases (HOs) as catalysts biosynthesis provides this specificity and selectivity. In nature, halogenation is a strategy to increase the biological activity of secondary metabolites with antibacterial, antiviral, antiprotozoal, and antifungal properties. Since halogenated secondary metabolites prevent the formation of bacterial biofilms and combat biofouling, halogenating enzymes have been proposed as alternative to traditional antifouling compounds. Feedback inhibition of HPO synthesis in bacteria is caused by the halogenated metabolites, which limits the concentration of halogenating agent (biocide) that can be produced. This review classifies these enzymes according to their catalytic functions and in view the current knowledge about the chemistry of settlement and adhesion of fouling organisms. It highlights molecular enzyme analogues and transition metal-based nanoparticles as functional enzyme mimics for the catalytic production of repellents in situ. The validity of the various modes of action is evaluated, and enzyme mimics with the highest potential are showcased.
Microorganisms employ a precised communication pattern among themselves in order to coordinate between various processes during their growth. Both unicellular and multicellular microbes are found to show cell density-driven gene expression. This phenomenon of density-dependent cell regulation used for survival, prevalence and colonization of specific host is generally termed as quorum sensing (QS). Microorganisms respond to this stimulus once the signalling molecule reaches its threshold concentration. Since they are found to be able to regulate their own production, they are termed as autoinducers (quorum sensing molecules). These molecules function by sensing their own population with respect to their density and distribution pattern in the prevailing environment. Hence, microorganisms use such environmental sensing mechanisms to get adapted as well as for their survival in the existing conditions in their habitat, thereby maintaining healthy cell population. The autoinducers occur widespread in several microorganisms and differ from each other in their molecular structures. Acyl homoserine lactones (AHL), Autoinducer (AI), i.e., AI-2, AI-3 and quinolones are the common QS signalling molecules in Gram-negative bacteria, while cyclic peptides, AI-2 and butyrolactones are observed as signalling molecules in Gram-positive bacteria. In the case of actinomycetes, small diffusible molecules called autoregulators, A-factor and 2-iso-octanoyl-(3R)-hydroxymethyl-γ-butyrolactone act as QS signalling molecules. Understanding the connection between genomes, gene expression and the molecules in complex environment is considered to be a tough task. Increasing interest towards studying the underlying mechanisms has led to the development of various model systems. Among them, plant-microbe symbiotic system is considered to be the best one to study the inter-kingdom molecular cross-talk. During the process of evolution, plants started to respond to the external stimuli in different and more specific ways. One such way includes production of AHL-like molecules to regulate the QS of plant-associated microorganisms. In view of this, the present chapter will be focused on quorum sensing molecules and their role in plant-microbe interaction.
Quorum sensing (QS) regulates major bacterial behaviors such as virulence, antibiotic resistance biofilm formation, and bioluminescence when the population reaches high density. The role of QS in biofilm formation and virulence is an extreme problem for food safety, biofilm-related infectious diseases, etc. Food spoilage is a consequence of degrading enzymatic activities, viz., proteolytic, lipolytic, chitinolytic, and pectinolytic, of some food-associated bacteria. Several activities associated with the deterioration of goods are regulated by QS, suggesting a potential role of such cell-to-cell communication in food spoilage. Therefore, interrupting QS mechanism might be an alternative strategy to develop novel QS-based antibacterial/anti-biofilm drugs. QS-based antibacterial/anti-biofilm agents can be used to manage foodborne pathogens and biofilm formation in food industries. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. Production and bioactivity of mushroom polysaccharide was enhanced by using microbial QS molecules. Plant food extracts and phytochemicals were found to have anti-QS properties. Organic acids can act as effective potential sanitizers in reducing microbial load associated with fresh fruits and vegetables. Moreover, organic acids are known to be used as food preservatives due to their antimicrobial potential. Most approaches to use QQ as an anti-virulence strategy are still in initial phase; the increasing number of organisms and enzymes known to interfere with QS opens new perspectives for the development of innovative antibacterial strategies to prevent food spoilage.
Bacteria respond to host immunity for their proliferation and survival by cell-cell communications such as biofilm formation, bioluminescence, and secreting virulence factors. In the biofilm form, bacteria are more resistant to various antimicrobial treatments and withstand the host's immune system. The approaches of deciphering biofilm formation for treating bacterial infections are therefore highly desirable. Recently, we have reported that the ethanolic extract of the red seaweed Gracilaria fisheri (G. fisheri) enhanced immune activities and inhibited growth of the luminescent bacteria Vibrio harveyi in shrimp. We undertook the present research study in order to evaluate and compare the effectiveness of the ethanolic extract from G. fisheri and furanone, a known biofilm inhibitor, in inhibiting the formation of clinically important Vibrio biofilms. The results showed that sub-lethal concentrations of both the ethanolic extracts (5, 10 and 100 μg ml ⁻¹ ) and furanone (5 μM) inhibited biofilm formation by V. harveyi and Vibrio parahaemolyticus and also light production (luminescence) in V. harveyi. It is known that V. harveyi mediated light production via autoinducer AI-2 pathway, we further determined whether the inhibitory effect of the extract was involved the AI-2 signaling. The bioluminescence assay was conducted in an AI-2 deletion mutant V. harveyi. Supplementation of the AI-2 containing media with the extract or furanone impaired the light production in the mutant V. harveyi suggesting that the extract interfered AI-2 mediated light production similar to furanone. In vivo challenge study showed that the low concentrations (Sub MICs) of the ethanolic extract and furanone decreased bacterial adhesion and colonization in the surfaces of stomach lumen, down-regulated expression of a virulence factor, and protected shrimp against mortality from V. harveyi and V. parahaemolyticus infection. In conclusion, the present results suggest a potential application of the low concentrations of the ethanolic extract of G. fisheri as an efficient approach for treating biofilm-associated Vibrio diseases in aquacultures.
Bacteria communicate and collectively regulate gene expression using a process called quorum sensing (QS). QS relies on group-wide responses to signal molecules called autoinducers. Here, we show that QS activates a new program of multicellularity in Vibrio cholerae. This program, which we term aggregation, is distinct from the canonical surface-biofilm formation program, which QS represses. Aggregation is induced by autoinducers, occurs rapidly in cell suspensions, and does not require cell-division, features strikingly dissimilar from those characteristic of V. cholerae biofilm formation. Extracellular DNA limits aggregate size, but is not sufficient to drive aggregation. A mutagenesis screen identifies genes required for aggregate formation, revealing proteins involved in V. cholerae intestinal colonization, stress response, and a protein that distinguishes the current V. cholerae pandemic strain from earlier pandemic strains. We suggest that QS-controlled aggregate formation is important for V. cholerae to successfully transit between the marine niche and the human host.
Vibrio campbellii is a major pathogen in aquaculture. It is a causative agent of the so-called “luminescent vibriosis,” a life-threatening condition caused by bioluminescent Vibrio spp. that often involves mass mortality of farmed shrimps. The emergence of multidrug resistant Vibrio strains raises a concern and poses a challenge for the treatment of this infection in the coming years. Inhibition of bacterial cell-to-cell communication or quorum sensing (QS) has been proposed as an alternative to antibiotic therapies. Aiming to identify novel QS disruptors, the 9H-fluroen-9yl vinyl ether derivative SAM461 was found to thwart V. campbellii bioluminescence, a QS-regulated phenotype. Phenotypic and gene expression analyses revealed, however, that the mode of action of SAM461 was unrelated to QS inhibition. Further evaluation with purified Vibrio fischeri and NanoLuc luciferases revealed enzymatic inhibition at micromolar concentrations. In silico analysis by molecular docking suggested binding of SAM461 in the active site cavities of both luciferase enzymes. Subsequent in vivo testing of SAM461 with gnotobiotic Artemia franciscana nauplii demonstrated naupliar protection against V. campbellii infection at low micromolar concentrations. Taken together, these findings suggest that suppression of luciferase activity could constitute a novel paradigm in the development of alternative anti-infective chemotherapies against luminescent vibriosis, and pave the ground for the chemical synthesis and biological characterization of derivatives with promising antimicrobial prospects.
Quorum sensing is the response-regulation system of genes expression in accordance with the variation of cell population density. In this communication system, the response-regulation system induces group behaviors via signal molecules, which enabling cell acts actively to external environmental fluctuation. From practical point of view, these group behaviors ubiquitously occurred in a myriad of environments threatening human health and causing unrecoverable deterioration of manufacturing processes. Therefore, the mechanism of quorum sensing involves producing, releasing, detecting, and responding to small hormone-like chemical compounds called as autoinducers. For intraspecies communications, Gram-negative bacteria representatively utilize acyl-homoserine lactones, and Gram-positive bacteria typically use modified peptides as autoinducer. In case of a eukaryote, fungi generate a variety of autoinducers, such as farnesol, tyrosol, and aromatic alcohols. The intraspecies quorum sensing allows bacteria themselves to recognize the same species in the mixed culture system, but it could not use to detect the presence of other species and identify and act on their proportions in the overall environmental system. Thus, bacteria produce furanone-based signal molecules to build an interspecies communication and maintain a balance in the entire ecosystem. This type of bacterial Esperanto, called autoinducer-2, is synthesized by LuxS protein and its associated genes have been found in over 70 different microorganisms to date. Meanwhile, recent studies also identified that interkingdom quorum sensing between eukaryote and prokaryote, such as a bacteria-to-fungi, bacteria-to-plant, and bacteria-to-mammalian cell. This chapter focuses on the history of quorum sensing and the signal molecules chemistry; how response-regulation system in bacteria is developed; how the definition of quorum sensing is determined; how the signal molecule is generated; and the recent studies related to the exploration of new quorum sensing systems.
Bacteria interact chemically and physically with each other to coordinate their group behavior. Chemical interactions are well characterized in cell?cell communication where bacteria communicate with each other through small molecules. Physical interactions are observed in the formation of biofilms where cell aggregates are embedded in self-produced extracellular polymeric substances (EPS). In addition, recent findings in membrane vesicles (MVs), which pinch off from the cellular membrane, suggest the great potential of physical interactions. These events are biologically regulated, allowing bisacteria to alter their phenotypes in response to the environment. This chapter is intended to provide general knowledge of these fascinating bacterial interactions with a brief review of how physicochemical approaches have contributed in explaining such processes.
Cholera is a potentially mortal, infectious disease caused by Vibrio cholerae bacterium. Current treatment methods of cholera still have limitations. Beneficial microbes that could sense and kill the V. cholerae could offer potential alter-native to preventing and treating cholera. However, such V. cholerae targeting microbe is still not available. This mi-crobe requires a sensing system to be able to detect the presence of V. cholera bacterium. To this end, we designed and created a synthetic genetic sensing system using nonpathogenic Escherichia coli as the host. To achieve the system, we have moved proteins used by V. cholerae for quorum sensing into E. coli. These sensor proteins have been further lay-ered with a genetic inverter based on CRISPRi technology. Our design process was aided by computer models simulat-ing in vivo behavior of the system. Our sensor shows high sensitivity to presence of V. cholerae supernatant with tight control of expression of output GFP protein.
The integration of signals from the bacterial environment, through a network of cellular transduction mechanisms, determines the profile of genes expressed and thereby the bacterial phenotype. Quorum sensing transmits one such signal, i.e. population density, by relying on the accumulation of a small extracellular signal molecule to modulate transcription of target operons.
The study of prokaryotic small RNAs is one of the most important directions in modern molecular biology. In the last decade, multiple short regulatory transcripts have been found in prokaryotes, and for some of them functional roles have been elucidated. Bacterial small RNAs are implicated in the regulation of transcription and translation, and they affect mRNA stability and gene expression via different mechanisms, including changes in mRNA conformation and interaction with proteins. Most small RNAs are expressed in response to external factors, and they help bacteria to adapt to changing environmental conditions. Bacterial infections of various origins remain a serious medical problem, despite significant progress in fighting them. Discovery of mechanisms that bacteria employ to survive in infected organisms and ways to block these mechanisms is promising for finding new treatments for bacterial infections. Regulation of pathogenesis with small RNAs is an attractive example of such mechanisms. This review considers the role of bacterial small RNAs in adaptation to stress conditions. We pay special attention to the role of small RNAs in Mycobacterium tuberculosis infection, in particular during establishment and maintenance of latent infection.
Bacteria living in communities convey their presence to each other by producing, releasing, and subsequently responding to the accumulation of a minimal threshold concentration of chemical signaling molecules termed autoinducers. This method of cell-cell communication is called quorum sensing. It allows a population of bacteria to coordinate behavior, and thus acquire some of the characteristics of multicellular organisms. The V. harveyi and M. xanthus systems are two of the most complex quorum sensing circuits. These two systems serve to exemplify the diversity of signals and the range of activities that are regulated by cell-cell communication in bacteria. Additionally, the V. harveyi and M. Xanthus multichannel two-component circuits enable these bacteria to collect, integrate, and process multiple sensory inputs. The use of two-component circuits that enable the influx and outflow of phosphate at multiple locations in the signal transduction circuit presumably allows very precise modulation of the output responses.. In V. fischeri, quorum sensing controls the celldensity dependent production of light. Other simpler quorum sensing circuits, such as LuxI-LuxR circuits, may not possess the inherent plasticity to integrate multiple signaling inputs nor the fine control that can be incorporated into multichannel two-component relays.
Importance:
Our results provide novel insight into the function of LuxO, which is a key component of pheromone signaling (PS) cascades in several members of the Vibrionaceae. Our results also contribute to an increasingly appreciated aspect of bacterial behavior and evolution whereby mutants that do not respond to a signal from like cells have a selective advantage. In this case, although "antisocial" mutants locked in the PS signal-off mode can outcompete parents, their survival advantage does not require wild-type cells to exploit. Finally, this work strikes a note of caution for those conducting or interpreting experiments in V. fischeri, as it illustrates how pleiotropic mutants could easily and inadvertently be enriched in this bacterium during prolonged culturing.
Biological processes such as control of bioluminescence in Vibrio fisherii and Vibrio harveyi, expression of virulence factors in Pseudomonas aeruginosa and Erwinia carotovora or conjugative transfer of the Ti plasmid in Agrobacterium tumefaciens are dependent on the production and sensing of specific secondary metabolites derived from the structure of N-acyl homoserine lactone. These metabolites are released and sensed by bacterial populations which in turn modify their metabolism accordingly. In certain bacterial species, virulence is under the control of these metabolites, in genomes of Salmonella typhimuirum and E.coli homologous systems with so far unknown function have been identified. This shows that communication by secondary metabolites is widespread in bacteria. Furthemore, host organism have been shown to respond to these metabolites by non-specific immune reactions. Understanding the biological role of this communication could enable specific modification of behaviour of bacterial populations and thus open a way to new therapeutic measures. All of this becomes even more important in a time when antibiotic resistance in bacteria increases and new ways of prevention and control of infectious diseases are sought.
A brighter variant of a lab cultured strain of Vibrio harveyi was observed under routine lab culturing procedures and was investigated for differences in luminescence characteristics and protein profile as compared to the wild type parent. A lysate of the variant and wild type strain was subjected to BCA protein analysis and proteins were separated on PAGE to elucidate differences. Luminescence studies revealed a faster growing, 20% brighter variant that had a clear difference in protein profiles when subjected to quantitative and qualitative protein analyses. The bright variant of N2 had 0.21μgm/μl more total protein content as compared to the wild type and exhibited darker protein bands for some of the proteins separated on PAGE.
Luminous bacteria are those bacteria that carry the lux genes, genes that code for proteins involved in light production. Many luminous bacteria emit light at high, easily visible levels in laboratory culture and in nature, and the phenomenon of light emission has generated interest in these bacteria for over 125 years. Luminous bacteria are especially common in ocean environments where they colonize a variety of habitats, but some species are found in brackish, freshwater, and terrestrial environments. This chapter, which begins with an historical perspective, summarizes current understanding of the biochemistry and genetics of bacterial light emission, the taxonomy and phylogenetics of light-emitting bacteria, the evolutionary origins and hypothesized physiological and ecological functions of bacterial luminescence, the distributions and activities of these bacteria in nature, their symbiotic interactions with animals and especially with marine fishes, and the quorum sensing regulatory circuitry controlling light production at the operon level. This chapter concludes with information on the isolation, cultivation, storage, and identification of luminous bacteria.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry.
An autoinducer required for the growth-dependent development of luminescence in Vibrio harveyi has been purified, structurally identified, and chemically synthesized. The autoinducer, which is excreted by the cells, was extracted with chloroform from conditioned media in which V. harveyi cells had been grown. The concentrated extract was separated on a silica gel column and the autoinducer activity further purified by thin layer, paper, and high performance liquid chromatography. The structure of the partially purified autoinducer was identified by 1H NMR and mass spectrometry as N-(beta-hydroxybutyryl)homoserine lactone. This compound was chemically synthesized by condensation of beta-hydroxybutyrate with alpha-amino-gamma-butyrolactone hydrobromide using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a carboxyl group activator. The pure synthetic autoinducer gave the characteristic NMR and mass spectra, co-migrated with the natural autoinducer on thin layer plates, and specifically stimulated induction of luminescence of V. harveyi. Light emission of a regulatory dark mutant of V. harveyi could be stimulated over 1000-fold by the addition of N-(beta-hydroxybutyryl)homoserine lactone, reaching intensities comparable to that of the native strain. The similarity in structure of the autoinducer of V. harveyi to that of Vibrio fischeri suggests that the regulation of luminescence induction in these bacteria may be related in spite of their differences in lux gene organization.
This chapter describes the biochemistry and physiology of bioluminescent bacteria. The function of bioluminescent bacteria is to emit light in biological systems. The chapter explains the variety of habitats in which these bacteria are found, the light-emitting system may play an important role in their ecology or physiology. The luciferase in bacteria, unlike that of any other luminous group (except, perhaps, the fungi), is related to the respiratory pathway, functioning as a shunt for electrons directly to oxygen at the level of reduced flavin. This luciferase is an external flavin mono-oxygenase or mixed-function oxidase, electrons for reduction of flavin mononucleotide (FMN) are provided by the reducing power derived from the electron-transport pathway. The light-emitting reaction then proceeds via the reaction of molecular oxygen with reduced flavin to form an intermediate luciferase-flavin peroxy species, whose breakdown provides energy sufficient to leave one of the products in an electronically excited singlet state, with subsequent light emission. The bacterial (luciferase-bound) peroxide chromophore, which has been isolated and characterized, provides a model in this respect for the different bioluminescent reactions.
With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.
In bioluminescent bacteria growing in shake flasks, the enzyme luciferase has been shown to be synthesized in a relatively short burst during the period of exponential growth. The luciferase gene appears to be completely inactive in a freshly inoculated culture; the pulse of preferential luciferase synthesis which occurs later is the consequence of its activation at the level of deoxyribonucleic acid transcription which is attributed to an effect of a "conditioning" of the medium by the growing of cells. Although cells grown in a minimal medium also exhibit a similar burst of synthesis of the luminescent system, the amount of synthesis is quantitatively less, relative to cell mass. Under such conditions, added arginine results in a striking stimulation of bioluminescence. This is attributed to a stimulation of existing patterns of synthesis and not to induction or derepression per se.
Conjugal transfer of Ti plasmids from Agrobacterium donors to bacterial recipients is controlled by two types of diffusible signal molecules. Induction is mediated by novel compounds, called opines, that are secreted by crown gall tumours. These neoplasias result from infection of susceptible plants by virulent agrobacteria. The second diffusible signal, called conjugation factor, is synthesized by the donor bacteria themselves. Production of this factor is induced by the opine. Here we show that conjugation is regulated directly by a transcriptional activator, TraR, which requires conjugation factor as a coinducer to activate tra gene expression. TraR is a homologue of LuxR, the lux gene activator from Vibrio fischeri which also requires an endogenously synthesized diffusible coinducer. The two regulatory systems are related; the two activator proteins show amino-acid sequence similarities and the lux system cofactor, autoinducer, will substitute for conjugation factor in the TraR-dependent activation of Ti plasmid tra genes.
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry.
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
Expression of the lux operon from the marine bacterium Vibrio harveyi is dependent on cell density and requires an unlinked regulatory gene, luxR, and other cofactors for autoregulation. Escherichia coli transformed with the lux operon emits very low levels of light, and this deficiency can be partially alleviated by coexpression of luxR in trans. The V. harveyi lux promoter was analyzed in vivo by primer extension mapping to examine the function of luxR. RNA isolated from E. coli transformed with the Vibrio harveyi lux operon was shown to have a start site at 123 bp upstream of the first ATG codon of luxC. This is in sharp contrast to the start site found for lux RNA isolated from V. harveyi, at 26 bp upstream of the luxC initiation codon. However, when E. coli was cotransformed with both the lux operon and luxR, the start site of the lux mRNA shifted from -123 to -26. Furthermore, expression of the luxR gene caused a 350-fold increase in lux mRNA levels. The results suggest that LuxR of V. harveyi is a transcriptional activator stimulating initiation at the -26 lux promoter.
It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.
We have compiled and analyzed 263 promoters vith known transcriptional start points for E. coll genes. Promoter elements (-35
hexamer, -10 hexamer, and spacing between these regions) were aligned by a program which selects the arrangement consistent
with the start point and statistically most homologous to a reference list of promoters. The initial reference list was that
of Hawley and McClure (Nucl. Acids Res. 11, 2237–2255, 1983). Alignment of the complete list was used for reference until
successive analyses did not alter the structure of the list. In the final compilation, all bases in the -35 (TTGACA) and -10
(TATAAT) hexamers were highly conserved, 92% of promoters had inter-region spacing of 17±1 bp, and 75% of the uniquely defined
start points initiated 7±1 bases downstream of the -10 region. The consensus sequence of promoters with inter-region spacing
of 16, 17, or 18 bp did not differ. This compilation and analysis should be usaful for studies of promoter structure and function
and for programs which identify potential promoter sequences
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Expression of the lux operon from the marine bacterium Vibrio harveyi is dependent on cell density and requires an unlinked regulatory gene, luxR, and other cofactors for autoregulation. Escherichia coli transformed with the lux operon emits very low levels of light, and this deficiency can be partially alleviated by coexpression of luxR in trans. The V. harveyi lux promoter was analyzed in vivo by primer extension mapping to examine the function of luxR. RNA isolated from E. coli transformed with the Vibrio harveyi lux operon was shown to have a start site at 123 bp upstream of the first ATG codon of luxC. This is in sharp contrast to the start site found for lux RNA isolated from V. harveyi, at 26 bp upstream of the luxC initiation codon. However, when E. coli was cotransformed with both the lux operon and luxR, the start site of the lux mRNA shifted from -123 to -26. Furthermore, expression of the luxR gene caused a 350-fold increase in lux mRNA levels. The results suggest that LuxR of V. harveyi is a transcriptional activator stimulating initiation at the -26 lux promoter.
In Escherichia coli, the expression of a group of operons involved in aerobic metabolism is regulated by a two-component signal transduction system in which the arcB gene specifies the membrane sensor protein and the arcA gene specifies the cytoplasmic regulator protein. ArcB is a large protein belonging to a subclass of sensors that have both a transmitter domain (on the N-terminal side) and a receiver domain (on the C-terminal side). In this study, we explored the essential structural features of ArcB by using mutant analysis. The conserved His-292 in the transmitter domain is indispensable, indicating that this residue is the autophosphorylation site, as shown for other homologous sensor proteins. Compression of the range of respiratory control resulting from deletion of the receiver domain and the importance of the conserved Asp-533 and Asp-576 therein suggest that the domain has a kinetic regulatory role in ArcB. There is no evidence that the receiver domain enhances the specificity of signal transduction by ArcB. The defective phenotype of all arcB mutants was corrected by the presence of the wild-type gene. We also showed that the expression of the gene itself is not under respiratory regulation.
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.
Mutagenesis with transposon mini-Mulac was used to identify loci containing genes for bioluminescence (lux) in the marine bacterium Vibrio harveyi. Transposon insertions which resulted in a Lux- phenotype were mapped to two unlinked regions of the genome. Region I contained the luxCDABE operon which was previously shown to encode the enzymes luciferase and fatty acid reductase, which are required for light production. The other locus, region II, which was identified for the first time in this study, appeared to have a regulatory function. In Northern blot analysis of mRNA from mutants with defects in this region, no transcription from the luxCDABE operon could be detected. Strains with transposon-generated lux::lacZ gene fusions were used to analyze control of the transcription of these regions. Expression of luminescence in the wild type was strongly influenced by the density of the culture, and in strains with the lacZ indicator gene coupled to the luxCDABE operon, beta-galactosidase synthesis was density dependent. So, transcription of this operon is responsive to a density-sensing mechanism. However, beta-galactosidase synthesis in strains with lacZ fused to the region II transcriptional unit did not respond to cell density. The organization and regulation of the lux genes of V. harveyi are discussed, particularly with regard to the contrasts observed with the lux system of the fish light-organ symbiont Vibrio fischeri.
The vir region of Agrobacterium tumefaciens spans at least six transcriptional loci required for crown gall tumorigenesis. The transcriptional induction of two of these vir loci in response to cocultivation with tobacco suspension cells was measured by using bacteria containing mutations in each of the six vir loci located on the Ti plasmid. Induction of these vir genes occurred only in bacteria that had functional copies of virA and virG. The nucleic acid sequence of a 1.25-kilobase clone encompassing virG contains one open reading frame capable of coding for a protein of about 30,000 daltons. The amino acid sequence of the predicted virG product is homologous to that of eight bacterial proteins, including that of the ompR gene of Escherichia coli. Most, although not all, of these proteins, like VirG, are positive regulatory elements.
The nucleotide sequence of the luxC gene (1431 bp) and the upstream DNA (1049 bp) of the luminescent bacterium Vibrio harveyi has been determined. Ihe luxC gene can be translated into a polypeptide of 55 kDa in excellent agreement with the molecular mass of the reductase polypeptide
required for synthesis of the aldehyde substrate for the bioluminescent reaction. Analyses of codon usage showed a high frequency
(1.9%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA. B and D genes. The low G/C content of the luxC gene and upstream DNA (38–39%) compared to that found in the other lux genes of V. harvevi (45%) was primarily due to a stretch of 500 nucleotides with only a 24% G/C content, extending from 200 bp inside lux C to 300 bp upstream. Moreover, an open reading frame did not extend far more than 48 codons between the luxC gene and 600 bp upstream at which point a gene transcribed in the opposite direction started. As the lux system in the luminescent bacterium, V. fischeri, contains a regulatory gene immediately upstream of luxC transcribed in the same direction, these results show that the organization and regulation of the lux genes have diverged in different luminescent bacteria.
We have compiled and analyzed 263 promoters with known transcriptional start points for E. coli genes. Promoter elements (-35 hexamer, -10 hexamer, and spacing between these regions) were aligned by a program which selects the arrangement consistent with the start point and statistically most homologous to a reference list of promoters. The initial reference list was that of Hawley and McClure (Nucl. Acids Res. 11, 2237-2255, 1983). Alignment of the complete list was used for reference until successive analyses did not alter the structure of the list. In the final compilation, all bases in the -35 (TTGACA) and -10 (TATAAT) hexamers were highly conserved, 92% of promoters had inter-region spacing of 17 +/- 1 bp, and 75% of the uniquely defined start points initiated 7 +/- 1 bases downstream of the -10 region. The consensus sequence of promoters with inter-region spacing of 16, 17 or 18 bp did not differ. This compilation and analysis should be useful for studies of promoter structure and function and for programs which identify potential promoter sequences.
The virulence (vir) region of Agrobacterium tumefaciens mediates the transfer of a defined segment of plasmid DNA (the T-DNA) into the plant genome. The vir genes are specifically induced by molecules produced by wounded plant cells, and virA is required for this induction. We have determined the nucleotide sequence of virA loci from limited (pTiAg162) and wide (pTiA6) host range tumor-inducing (Ti) plasmids, each of which encodes a single protein of 92,000 daltons. Using antibody directed against the virA gene product, we have localized the VirA protein to the bacterial inner membrane. VirA is homologous to at least four bacterial proteins which play a role in the transcriptional regulation of diverse families of genes. Based on its role in vir gene induction, homology to transcriptional regulators and membrane localization, we propose that VirA acts as an environmental sensor of plant-derived inducer molecules and transmits this information to the level of vir gene expression. The pTiAg162 virA locus was shown to be ineffective at directing vir gene induction, suggesting that this may in part contribute to the narrow host range conferred by this plasmid.
Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation analysis and the minicell programing experiments. These genes, in the order of their position on a linear map, and the apparent molecular weights of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli containing mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light production and luxR and luxI encode regulatory functions.
Recombinant E. coli that produce light were found in a clone library of hybrid plasmids containing DNA from the marine bacterium Vibrio fischeri. All luminescent clones had a 16 kb insert that encoded enzymatic activities for the light reaction as well as regulatory functions necessary for expression of the luminescence phenotype (Lux). Mutants generated by transposons Tn5 and mini-Mu were used to define Lux functions and to determine the genetic organization of the lux region. Regulatory and enzymatic functions were assigned to regions of two lux operons. With transcriptional fusions between the lacZ gene or transposon mini-Mu and the target gene, expression of lux operons could be measured in the absence of light production. The direction of transcription of lux operons was deduced from the orientation of mini-Mu insertions in the fusion plasmids. Induction of transcription of one lux operon required a function encoded by that operon (autoregulation). From these and other regulatory relationships, we propose a model for genetic control of light production.
Synthesis of bacterial luciferase in some strains of luminous bacteria requires a threshold concentration of an autoinducer synthesized by the bacteria and excreted into the medium. Autoinducer excreted by Photobacterium fischeri strain MJ-1 was isolated from the cell-free medium by extraction with ethyl acetate, evaporation of solvent, workup with ethanol-water mixtures, and silica gel chromatography, followed by normal-phase and then reverse-phase high-performance liquid chromatography. The final product was greater than 99% pure. The structure of the autoinducer as determined by high-resolution 1H nuclear magnetic resonance spectroscopy, infrared spectroscopy, and high-resolution mass spectrometry was N-(3-oxohexanoly)-3-aminodihydro-2(3H)-furanone [or N-(beta-ketocaproyl)homoserine lactone]. The formation of homoserine by hydrolysis of the autoinducer was consistent with this structure. Synthetic autoinducer, obtained as a racemate, was prepared by coupling homoserine lactone to the ethylene glycol ketal of sodium 3-oxohexanoate, followed by mildly acidic removal of the protecting group; this synthetic material showed the appropriate biological activity.
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.
Biochemistry and physiology of bioluminescent bacteria Mutational analysis of signal transduction by ArcB. a membrane sensor protein respon-sible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coii
- J W Hastings
- C J Potrikas
- S C Gupta
- M Kurfurst
- J C Makemson
Hastings, J.W.. Potrikas, C.J., Gupta, S.C, Kurfurst. M., and Makemson, J.C. (1985) Biochemistry and physiology of bioluminescent bacteria. Adv Microbiai Physioi 26: 235-291. luchi. S., and Lin. E.C.C. (1992) Mutational analysis of signal transduction by ArcB. a membrane sensor protein respon-sible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coii. J Bacteriol 174: 3972-3980.
Regulation of luminescence in marine bacteria Protein phosphorylation and regulation of adaptive responses in bacteria
- M Silverman
- M Martin
- J Engebrecht
Silverman, M., Martin, M., and Engebrecht, J. (1989) Regulation of luminescence in marine bacteria. In Genet-ics of Bacterial Diversity. Hopwood, D.A., and Chater, K.F. (eds). London: Academic Press, pp. 71-85, Stock, J.B.. Ninfa, A.J., and Stock, A.M. (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbioi Rev 53: 450-490.
Bacterial bioiuminescence: isolation and genetic analysis of functions from Vibrio fischeri Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria
- J Engebrecht
- K Nealson
- M Silverman
- E P Greenberg
- J W Hastings
- S Ulitzur
Engebrecht, J,. Nealson, K.. and Silverman. M. (1983) Bacterial bioiuminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell 32: 773-781, Greenberg. E.P.. Hastings, J.W., and Ulitzur, S. (1979) Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria. Arch Microbioi 120: 87-91.
Moiecular Cloning: A Laboratory Manuai Cold Spring Harbor
- T Maniatis
- E F Fritsch
- Sambrook
Maniatis, T,. Fritsch. E.F.. and Sambrook, J-(1982) Moiecular Cloning: A Laboratory Manuai Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.