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Multiple signalling systems controlling expression 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. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably beta-hydroxybutyl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyi.
... V. harveyi bioluminescence, as well as biofilm, virulence and flagellar production, is controlled by QS. V. harveyi is thought to respond to at least three different autoinducers through the binding of three separate QS receptors ( Bassler et al., 1993Bassler et al., , 1994Henke and Bassler, 2004). These receptors, LuxN, LuxQ, and CsqS are transmembrane hybrid histidine kinases. ...
... These receptors, LuxN, LuxQ, and CsqS are transmembrane hybrid histidine kinases. LuxQ also utilizes an accessory protein, LuxP, to sense its cognate autoinducer ( Bassler et al., 1994;Neiditch et al., 2006). When cell density is low, the receptors LuxN, LuxQ, and CsqS autophosphorylate and subsequently transfer phosphate to a single histidine phosphotransfer protein LuxU (Figure 1). ...
The emerging threat of drug resistant bacteria has prompted the investigation into bacterial signaling pathways responsible for pathogenesis. One such mechanism by which bacteria regulate their physiology during infection of a host is through a process known as quorum sensing (QS). Bacteria use QS to regulate community-wide gene expression in response to changes in population density. In order to sense these changes in population density, bacteria produce, secrete and detect small molecules called autoinducers. The most common signals detected by Gram-negative and Gram-positive bacteria are acylated homoserine lactones and autoinducing peptides (AIPs), respectively. However, increasing evidence has supported a role for the small molecule nitric oxide (NO) in influencing QS-mediated group behaviors like bioluminescence, biofilm production, and virulence. In this review, we discuss three bacteria that have an established role for NO in influencing bacterial physiology through QS circuits. In two Vibrio species, NO has been shown to affect QS pathways upon coordination of hemoprotein sensors. Further, NO has been demonstrated to serve a protective role against staphylococcal pneumonia through S-nitrosylation of a QS regulator of virulence.
... To test the function of putative QS circuit homologs in Vcor, we focused on strain OCN008 and deleted a subset of the putative Vcor-encoded QS genes identified by bioinformatics and assayed their function using bioluminescence. As observed in other Vibrio species, deletion of luxO resulted in a constitutive bioluminescent phenotype ( Fig. 3B) (41,47,48,69,70). We next assessed the phenotypes of strains harboring deletions of cqsS, luxN, or luxP. ...
The bacterial pathogen Vibrio coralliilyticus causes disease in coral species worldwide. The mechanisms of V. coralliilyticus coral colonization, coral microbiome interactions, and virulence factor production are understudied. In other model Vibrio species, virulence factors like biofilm formation, toxin secretion, and protease production are controlled through a density-dependent communication system called quorum sensing (QS). Comparative genomics indicated that V. coralliilyticus genomes share high sequence identity for most of the QS signaling and regulatory components identified in other Vibrio species. Here, we identify an active QS signaling pathway in two V. coralliilyticus strains with distinct infection etiologies: type strain BAA-450 and coral isolate OCN008. In V. coralliilyticus, the inter-species AI-2 autoinducer signaling pathway in both strains controls expression of the master QS transcription factor and LuxR/HapR homolog VcpR to regulate >300 genes, including protease production, biofilm formation, and two conserved type VI secretion systems (T6SSs). Activation of T6SS1 by QS results in the secretion of effectors and enables interbacterial competition and killing of prey bacteria. We conclude that the QS system in V. coralliilyticus is functional and controls the expression of genes involved in relevant bacterial behaviors typically associated with host infection.
IMPORTANCE
Vibrio coralliilyticus infects many marine organisms, including multiple species of corals, and is a primary causative agent of tissue loss diseases and bacterial-induced bleaching. Here, we investigated a common cell-cell communication mechanism called quorum sensing, which is known to be intimately connected to virulence in other Vibrio species. Our genetic and chemical studies of V. coralliilyticus quorum sensing uncovered an active pathway that directly regulates the following key virulence factors: proteases, biofilms, and secretion systems. These findings connect bacterial signaling in communities to the infection of corals, which may lead to novel treatments and earlier diagnoses of coral diseases in reefs.
... The discovery of bacterial communication by means of diffusible signal molecules known as quorum sensing [74,90,91] revolutionized the way scientists see bacterial populations (for a review of this theme, see [92]). Although not being required for all cooperative interactions, communication among neighbouring individuals is considered a fundamental mechanism to coordinate cooperative strategies [10]. ...
Social interactions impact microbial communities and these relationships are mediated by small molecules. The chemical ecology of bacteria on the phylloplane environment is still little explored. The harsh environmental conditions found on leaf surface require high metabolic performances of the bacteria in order to survive. That is interesting both for scientific fields of prospecting natural molecules and for the ecological studies. Important queries about the bacterial lifestyle on leaf surface remain not fully comprehended. Does the hostility of the environment increase the populations’ cellular altruism by the production of molecules, which can benefit the whole community? Or does the reverse occur and the production of molecules related to competition between species is increased? Does the phylogenetic distance between the bacterial populations influence the chemical profile during social interactions? Do phylogenetically related bacteria tend to cooperate more than the distant ones? The phylloplane contains high levels of yet uncultivated microorganisms, and understanding the molecular basis of the social networks on this habitat is crucial to gain new insights on the ecology of the mysterious community members due to interspecies molecular dependence. Here, we review and discuss what is known about bacterial social interactions and their chemical lifestyle on leaf surface.
... The experimental studies showed that the QS circuit in different species shows the production of different autoinducers which is responsible for the expression and the regulation of different phenotypic characters via the same QS outline circuit. This was explained by the use of marine Gram negative bacteria Vibrio fischeri and Vibrio harveyi, these two bacteria use the same LuxR/I gene system but the autoinducers in both bacterium are different; the former uses acyl-Homoserine lactone as the signaling molecule for the expression of bioluminescence character whereas the latter uses furanosyl borate diester as the signaling molecule for the expression of virulence character [8][9][10][11]. Another characteristic feature of the quorum sensing signaling pathway is the presence of LuxS gene in many species of the bacterial population which was explained to be the reason for the expression of autoinducers which aid in the interkingdom and interspecies communication. LuxS gene system in many bacteria has been found to have same biosynthetic pathways, but the targeted gene by the autoinducers produced by it has been attributed to different areas like few target the respiratory track of the host like Clostridium perfringens by producing toxins in that region; whereas in case pathogen like Vibrio cholerae and Streptococcus typhimurium , the LuxS gene system is responsible in the expression and establishment of the virulence cascade [12][13]. ...
... A breakthrough in QS research was made by Eberhard et al. (1981), who for the first time identified the structure of signaling factors involved in the microbial communication, i.e., acyl-homoserine lactones (AHLs), now often named as autoinducer-1 (AI-1). In 1994, the presence of other substances controlling bioluminescence was shown ( Bassler et al., 1994). They were called autoinducer-2 (AI-2), and their structure was identified at the beginning of the twenty-first century (Chen et al., 2002). ...
Quorum sensing (QS) is a mechanism allowing microorganisms to sense population density and synchronously control genes expression. It has been shown that QS supervises the activity of many processes important for microbial pathogenicity, e.g., sporulation, biofilm formation, and secretion of enzymes or membrane vesicles. This contributed to the concept of anti-QS therapy [also called quorum quenching (QQ)] and the opportunity of its application in fighting against various types of pathogens. In recent years, many published articles reported promising results indicating the possibility of reducing pathogenicity of tested microorganisms and their easier eradication when co-treated with antibiotics. The aim of the present article is to point to the opposite, negative side of the QQ therapy, with particular emphasis on three fundamental properties attributed to anti-QS substances: the selectivity, virulence reduction, and lack of resistance against QQ. This point of view may highlight new directions of research, which should be taken into account in the future before the widespread introduction of QQ therapies in the treatment of people.
... The experimental studies showed that the QS circuit in different species shows the production of different autoinducers which is responsible for the expression and the regulation of different phenotypic characters via the same QS outline circuit. This was explained by the use of marine Gram negative bacteria Vibrio fischeri and Vibrio harveyi, these two bacteria use the same LuxR/I gene system but the autoinducers in both bacterium are different; the former uses acyl-Homoserine lactone as the signaling molecule for the expression of bioluminescence character whereas the latter uses furanosyl borate diester as the signaling molecule for the expression of virulence character [8][9][10][11]. Another characteristic feature of the quorum sensing signaling pathway is the presence of LuxS gene in many species of the bacterial population which was explained to be the reason for the expression of autoinducers which aid in the interkingdom and interspecies communication. LuxS gene system in many bacteria has been found to have same biosynthetic pathways, but the targeted gene by the autoinducers produced by it has been attributed to different areas like few target the respiratory track of the host like Clostridium perfringens by producing toxins in that region; whereas in case pathogen like Vibrio cholerae and Streptococcus typhimurium , the LuxS gene system is responsible in the expression and establishment of the virulence cascade [12][13]. ...
Quorum sensing is a cell-to-cell communication, which is extensively observed in bacteria. This process allows the cell to detect, analyze, share and act upon various environmental stimuli based on cell density. The molecular aspect of this process is the secretion and detection of chemical signaling molecules called autoinducers (AIs), which act upon the gene expression. The quorum sensing signaling pathway is specifically observed only bulk population or in other words, the quorum sensing is effective only in high cell density. The quorum sensing circuit in the bacterial population is widely studied under the following heading; quorum sensing in Gram positive bacterium, Quorum sensing in Gram negative bacterium and the Quorum sensing with respect to Interkingdom communication. These models are studied using the widely studied models like Vibrio fischeri in Gram negative QS circuit, Staphylococcus aureus in Gram positive QS circuit and Vibrio harveyi. This review paper details the introduction of quorum sensing and their gene level explanation and how they effect on the virulence of a particular species of bacteria. This paper also throws light on the realization that the bacteria has the capable of performing coordinated activities that was so long contributed to the eukaryotic cell performanc
(PDF) Quorum sensing: A molecular cell communication in bacterial cells. Available from: https://www.researchgate.net/publication/332539333_Quorum_sensing_A_molecular_cell_communication_in_bacterial_cells [accessed Aug 22 2020].
... By "eavesdropping" on AI-2 produced by other species, the strain was capable of interfering with AI-2-regulated behaviors such as virulence. Although AI-2 has not been detected in marine particles, it is very likely to occur due to the prevalence of AI-2-producing Vibrio species (Bassler et al., 1994) in marine particles (Hmelo et al., 2011;Jatt et al., 2015; Table 1). The AI-2 pathway might facilitate Rm01 competing against antagonistic species in the microflora. ...
Quorum sensing (QS) promotes in situ extracellular enzyme (EE) activity via the exogenous signal N-acylhomoserine lactone (AHL), which facilitates marine particle degradation, but the species that engage in this regulatory mechanism remain unclear. Here, we obtained AHL-producing and AHL-degrading strains from marine particles. The strain Ruegeria mobilis Rm01 of the Roseobacter group (RBG), which was capable of both AHL producing and degrading, was chosen to represent these strains. We demonstrated that Rm01 possessed a complex QS network comprising AHL-based QS and quorum quenching (QQ) systems and autoinducer-2 (AI-2) perception system. Rm01 was able to respond to multiple exogenous QS signals through the QS network. By applying self-generated AHLs and non-self-generated AHLs and AI-2 QS signal molecules, we modulated biofilm formation and lipase production in Rm01, which reflected the coordination of bacterial metabolism with that of other species via eavesdropping on exogenous QS signals. These results suggest that R. mobilis might be one of the participators that could regulate EE activities by responding to QS signals in marine particles.
... By "eavesdropping" on AI-2 produced by other species, the strain was capable of interfering with AI-2-regulated behaviors such as virulence. Although AI-2 has not been detected in marine particles, it is very likely to occur due to the prevalence of AI-2-producing Vibrio species ( Bassler et al., 1994) in marine particles (Hmelo et al., 2011;Jatt et al., 2015; Table 1). The AI-2 pathway might facilitate Rm01 competing against antagonistic species in the microflora. ...
Quorum sensing (QS) promotes in situ extracellular enzyme (EE) activity via the exogenous signal N-acylhomoserine lactone (AHL), which facilitates marine particle degradation, but the species that engage in this regulatory mechanism remain unclear. Here, we obtained AHL-producing and AHL-degrading strains from marine particles. The strain Ruegeria mobilis Rm01 of the Roseobacter group (RBG), which was capable of both AHL producing and degrading, was chosen to represent these strains. We demonstrated that Rm01 possessed a complex QS network comprising AHL-based QS and quorum quenching (QQ) systems and autoinducer-2 (AI-2) perception system. Rm01 was able to respond to multiple exogenous QS signals through the QS network. By applying self-generated AHLs and non-self-generated AHLs and AI-2 QS signal molecules, we modulated biofilm formation and lipase production in Rm01, which reflected the coordination of bacterial metabolism with that of other species via eavesdropping on exogenous QS signals. These results suggest that R. mobilis might be one of the participators that could regulate EE activities by responding to QS signals in marine particles.
... These include LuxP (Vibrio harveyi), LsrB (Salmonella enterica serovar Typhimurium str. 14028, Escherichia coli MG1655, Sinorhizobium meliloti, and Aggregatibacter actinomycetecommitans HK1651), and RbsB (A. actinomycetecommitans HK1651) ( Bassler et al., 1994;Taga et al., 2001Taga et al., , 2003Xavier and Bassler, 2005;Shao et al., 2007a,b;Pereira et al., 2008). AI-2 production has been reported in the literature to be regulated in a positive feedback loop: as AI-2 is transported into the cell in a cell density-depended manner, luxS expression is upregulated, leading to increased production of AI- 2 to achieve quorum followed by rapid internalization during stationary phase to activate the QS operon and downstream regulated genes ( Taga et al., 2001Taga et al., , 2003Wang et al., 2005). ...
The ultra-small, obligate parasitic epibiont, TM7x, the first and only current member of the long-elusive Saccharibacteria (formerly the TM7 phylum) phylum to be cultivated, was isolated in co-culture with its bacterial host, Actinomyces odontolyticus subspecies actinosynbacter, XH001. Initial phenotypic characterization of the TM7x-associated XH001 co-culture revealed enhanced biofilm formation in the presence of TM7x compared to XH001 as monoculture. Genomic analysis and previously published transcriptomic profiling of XH001 also revealed the presence of a putative AI-2 quorum sensing (QS) operon, which was highly upregulated upon association of TM7x with XH001. This analysis revealed that the most highly induced gene in XH001 was an lsrB ortholog, which encodes a putative periplasmic binding protein for the auto inducer (AI)-2 QS signaling molecule. Further genomic analyses suggested the lsrB operon in XH001 is a putative hybrid AI-2/ribose transport operon as well as the existence of a luxS ortholog, which encodes the AI-2 synthase. In this study, the potential role of AI-2 QS in the epibiotic-parasitic relationship between XH001 and TM7x in the context of biofilm formation was investigated. A genetic system for XH001 was developed to generate lsrB and luxS gene deletion mutants in XH001. Phenotypic characterization demonstrated that deletion mutations in either lsrB or luxS did not affect XH001’s growth dynamic, mono-species biofilm formation capability, nor its ability to associate with TM7x. TM7x association with XH001 induced lsrB gene expression in a luxS-dependent manner. Intriguingly, unlike wild type XH001, which displayed significantly increased biofilm formation upon establishing the epibiotic-parasitic relationship with TM7x, XH001ΔlsrB, and XH001ΔluxS mutants failed to achieve enhanced biofilm formation when associated with TM7x. In conclusion, we demonstrated a significant role for AI-2 QS in modulating dual-species biofilm formation when XH001 and TM7x establish their epibiotic-parasitic relationship.
... The complex regulates the expression of multiple downstream target genes, such as the master regulating gene luxR of V. harveyi (Van Kessel et al., 2013), the elastase coding gene lasR of P. aeruginosa ( Gambello and Iglewski, 1991), the curvature coding gene crvA of V. cholerae ( Bartlett et al., 2017), and the QS regulon coding gene esaR of Pantoea stewartii (Ramachandran et al., 2014). Thus, those aforementioned regulations ultimately initiate or silence RNA transcription and protein translation to express related functions ( Bassler et al., 1994;Anetzberger et al., 2009; Figure 5). ...
N-Acyl Homoserine Lactones (N-AHLs) are an important group of small quorum-sensing molecules generated and released into the surroundings by Gram-negative bacteria. N-AHLs play a crucial role in various infection-related biological processes of marine Vibrio species, including survival, colonization, invasion, and pathogenesis. With the increasing problem of antibiotic abuse and subsequently the emergence of drug-resistant bacteria, studies on AHLs are therefore expected to bring potential new breakthroughs for the prevention and treatment of Vibrio infections. This article starts from AHLs generation in marine Vibrio, and then discusses the advantages, disadvantages, and trends in the future development of various detection methods for AHLs characterization. In addition to a detailed classification of the various marine Vibrio-derived AHL types that have been reported over the years, the regulatory mechanisms of AHLs and their roles in marine Vibrio biofilms, pathogenicity and interaction with host cells are also highlighted. Intervention measures for AHLs in different stages are systematically reviewed, and the prospects of their future development and application are examined.
... The autoinducer-2 (AI-2) QS molecule is one of the most extensively studied, and AI-2 has been recognized as an intra-and inter-species communication signal. The AI-2 synthase, LuxS, is encoded by luxS homologues found in several different bacterial species [4][5][6]. The substrate of LuxS is S-Ribosylhomocysteine, which is cleaved to yield homocysteine and 4, 5-dihydroxy-2, 3-pentandione (DPD). ...
Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli.
... 4-6 V. harveyi detects the three autoinducers using the cognate membrane-bound receptors LuxN, CqsS, and LuxPQ, respectively. [7][8][9] At low cell density (LCD), autoinducer concentrations are low, and the unliganded receptors act as kinases, funneling phosphate to the phosphorelay protein LuxU. 10 LuxU transfers the phosphoryl group to the response regulator protein LuxO, which activates transcription of genes encoding ve homologous quorum regulatory small RNAs (qrr sRNAs). 11,12 The Qrr sRNAs post-transcriptionally activate production of the transcription factor AphA and repress production of the transcription factor LuxR. AphA and LuxR are the two master quorum-sensing regulators that promote global changes in gene expression in response to population density changes. ...
Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed BONCAT to characterize the time-dependent transition of V. harveyi from individual- to group-behaviors. We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86 known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.
... The prevailing rationalization is that the bacteria benefit by operating collectively, although the emergence of parsed sub-populations that exhibit altruistic or even aberrant social behavior (that is, cheating on the collective good) has stimulated new hypotheses of sociomicrobiology including the suggestion that microbial QS serves as a surrogate for human sociobiology (Ben-Jacob et al., 2004; Parsek and Greenberg, 2005; Gardner et al., 2007; Davidson and Surette, 2008; Ben-Jacob and Schultz, 2010; Connell et al., 2012; Dandekar et al., 2012; Pradhan and Chatterjee, 2014; Smith et al., 2014). Certainly, among the beneficial phenotypes attributed to QS, which could also be referred to as the 'currency' of the public good, are bioluminescence (Bassler et al., 1994), virulence (Zhu et al., 2002), biofilm formation (Balestrino et al., 2005; Gonzalez Barrios et al., 2006; Li et al., 2007), cell division, motility and cooperativity (Dandekar et al., 2012). We note that intentional rewiring these QS-regulated systems also benefits biotechnological applications, if not the microbes themselves (Fernandes et al., 2010; Tsao et al., 2010; Wu et al., 2013; Swofford et al., 2015; Thompson et al., 2015). ...
Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can 'call' groups neighboring cells that 'travel' and establish a QS-mediated phenotype upon reaching the new locale.The ISME Journal advance online publication, 5 June 2015; doi:10.1038/ismej.2015.89.
... The autoindicer-2 bioassay was conducted according to an established method with slight modifications 29 . In brief, Vibrio harveyi BB170 was grown in Autoinducer Bioassay (AB) medium 30 for 16 h and then diluted 5,000-fold in fresh AB medium. ...
We developed a high-throughput mass spectrometry method, pLink-SS (http://pfind.ict.ac.cn/software/pLink/2014/pLink-SS.html), for precise identification of disulfide-linked peptides. Using pLink-SS, we mapped all native disulfide bonds of a monoclonal antibody and ten standard proteins. We performed disulfide proteome analyses and identified 199 disulfide bonds in Escherichia coli and 568 in proteins secreted by human endothelial cells. We discovered many regulatory disulfide bonds involving catalytic or metal-binding cysteine residues.
Objectives
The bacteriostatic effects of a citral nanoemulsion against Shewanella putrefaciens CN-32 (SHP CN-32) were investigated using in vitro culture and gene expression analysis, for building a potential application in spoilage microorganism control and aquatic products quality maintenance.
Materials and Methods
The SHP CN-32 was treated by prepared citral nanoemulsion when the minimal inhibitory concentration (MIC) was verified. The growth curve, membrane integrity, scanning electron microscope (SEM) observation, biofilm formation and quorum sensing (QS) signaling molecule AI-2 content were evaluated in different MIC treatment groups (0 MIC to 1.00 MIC). The gene expression status of SHP CN-32 in 0 MIC group and 0.50 MIC group were compared using transcriptome sequencing and quantitative PCR.
Results
The in vitro culture revealed that the citral nanoemulsion could inhibit the growth of SHP CN-32 with MIC of about 200 μg/ml. Images from membrane integrity, SEM and biofilm formation suggested significant biological structure damage in bacteria after treatment. Meanwhile, the quorum sensing (QS) signaling molecule AI-2 content showed a decline following the rise of treatment concentration. Transcriptome sequencing and quantitative PCR revealed that the majority genes related diversified functional metabolic pathways of SHP CN-32 were down-regulated at varying degree.
Conclusion
A significant bacteriostasis of citral nanoemulsion against Shewanella putrefaciens CN-32 (SHP CN-32) were verified via the results of growth inhibition, structural destruction, signal molecular decrease and gene expression down-regulation of strains. These synergies significantly affect the characteristic expression of SHP CN-32, revealing the application potential as bacteriostat, QS inhibitor and preservative in aquatic products.
The Genetics Society of America (GSA) Medal recognizes researchers who have made outstanding contributions to the field of genetics in the past 15 years. The 2019 GSA Medal is awarded to Bonnie L. Bassler of Princeton University and the Howard Hughes Medical Institute in recognition of her groundbreaking studies of bacterial chemical communication and regulation of group behaviors.
Although aquaculture is a growing productive sector, economic losses because of infectious diseases and public health concern of what antibiotic resistance represents have become factors that limit its development. Thus, the control of infectious diseases through antibacterial alternatives is a field of intense research. In this sense, antimicrobial proteins and peptides might represent a safe and effective alternative because their site of action is on microbial components; they are biodegradable; their diversity in terms of mechanisms of action is broad; and some of them can be highly specific. Because this antibacterial alternative is still in research, this review provides a critical analysis of the challenges and opportunities of protein drugs for the treatment of bacterial diseases in the field of aquaculture health; contextualizes the different types of antibacterial protein and peptides, their mechanisms of action, potential sources, as well as the state of the art on assessment of antibacterial proteins and peptides in bioassays with aquatic organisms; and discusses the challenges proteinaceous drugs face regarding their stability for oral delivery and the perspectives for their administration in aquaculture.
Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl-homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta-bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification.
It is generally assumed that each organism has evolved to possess a unique ribosomal RNA (rRNA) species optimal for its physiological needs. However, some organisms express divergent rRNAs, the functional roles of which remain unknown. Here, we show that ribosomes containing the most variable rRNAs, encoded by the rrnI operon (herein designated as I-ribosomes), direct the preferential translation of a subset of mRNAs in Vibrio vulnificus, enabling the rapid adaptation of bacteria to temperature and nutrient shifts. In addition, genetic and functional analyses of I-ribosomes and target mRNAs suggest that both I-ribosomal subunits are required for the preferential translation of specific mRNAs, the Shine–Dalgarno sequences of which do not play a critical role in I-ribosome binding. This study identifies genome-encoded divergent rRNAs as regulators of gene expression at the ribosome level, providing an additional level of regulation of gene expression in bacteria in response to environmental changes. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
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.
Middle ear infections (or otitis media [OM]) are highly prevalent among children worldwide and present a tremendous socioeconomic challenge for health care systems. More importantly, this disease diminishes the quality of life of young children. OM is often chronic and recurrent, due to the presence of highly antibiotic-resistant communities of bacteria (called biofilms) that persist within the middle ear space. To combat these recalcitrant infections, new and powerful biofilm-directed approaches are needed. Here, we describe the ability to disrupt a biofilm formed by the two most common bacteria that cause chronic and recurrent OM in children, via an approach that combines the power of vaccines with that of traditional antibiotics. An outcome of this strategy is that antibiotics can more easily kill the bacteria that our vaccine-induced antibodies have released from the biofilm. We believe that this approach holds great promise for both the prevention and treatment of OM.
The conserved CsrB sRNAs are an example of sibling sRNAs, i.e., sRNAs which are present in multiple copies in genomes. This report illustrates how new copies arise through gene duplication events and highlights two evolutionary advantages of having such multiple copies: differential regulation of the multiple copies allows integration of different input signals into the regulatory network of which they are parts, and the high redundancy that they provide confers a strong robustness to the system.
Bacterial pathogens are highly adaptable organisms, a quality that enables them to overcome changing hostile environments. For example, Vibrio cholerae, the causative agent of cholera, is able to colonize host small intestines and combat host-produced reactive oxygen species (ROS) during infection. To dissect the molecular mechanisms utilized by V. cholerae to overcome ROS in vivo, we performed a whole-genome transposon sequencing analysis (Tn-seq) by comparing gene requirements for colonization using adult mice with and without the treatment of the antioxidant, N-acetyl cysteine. We found that mutants of the methyl-directed mismatch repair (MMR) system, such as MutS, displayed significant colonization advantages in untreated, ROS-rich mice, but not in NAC-treated mice. Further analyses suggest that the accumulation of both catalase-overproducing mutants and rugose colony variants in NAC⁻ mice was the leading cause of mutS mutant enrichment caused by oxidative stress during infection. We also found that rugose variants could revert back to smooth colonies upon aerobic, in vitro culture. Additionally, the mutation rate of wildtype colonized in NAC⁻ mice was significantly higher than that in NAC⁺ mice. Taken together, these findings support a paradigm in which V. cholerae employs a temporal adaptive strategy to battle ROS during infection, resulting in enriched phenotypes. Moreover, ΔmutS passage and complementation can be used to model hypermuation in diverse pathogens to identify novel stress resistance mechanisms.
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.
The luxS gene is responsible for the synthesis of AI-2 (autoinducer-2), a signaling molecule that participates in quorum sensing regulation in a large number of bacteria. In this work, we investigated which phenotypes are regulated by luxS gene in Serratia proteamaculans 94, psychrotrophic strain isolated from spoiled refrigerated meat. AI-2 was identified in S. proteamaculans 94, and the luxS gene involved in its synthesis was cloned and sequenced. A mutant with the inactivated luxS gene was constructed. Inactivation of the luxS gene was shown to lead to the absence of AI-2 synthesis, chitinolytic activity, swimming motility, suppression of the growth of fungal plant pathogens Rhizoctonia solani and Helminthosporium sativum by volatile compounds emitted by S. proteamaculans 94 strain, and to a decrease of extracellular proteolytic activity. The knockout of the luxS gene did not affect synthesis of N-acyl-homoserine lactones, lipolytic, and hemolytic activities of S. proteamaculans 94.
Colibacillosis caused by avian pathogenic E. coli (APEC), is an economically important bacterial disease of poultry. APEC are a subgroup of extra intestinal pathogenic E. coli (ExPEC) and poultry are considered potential sources of foodborne ExPEC to humans. Currently, APEC infections in poultry are controlled by antibiotics and/or vaccination; however, their effect is limited due to emergence of antibiotic resistant strains and infections with heterologous serotypes. Therefore, novel approaches are needed. Here, using the bioluminescent quorum sensing (QS) autoinducer 2 (AI-2) indicator Vibrio harveyi BB170, we screened the cell free culture supernatant of APEC O78 prepared from cultures grown in the presence of 4,182 small molecules (SMs; 100 μM). A total of 69 SMs inhibited > 75% of APEC O78 AI-2 activity in the indicator bacteria. Ten SMs that showed highest AI-2 inhibition were selected for further studies. Most of these SMs inhibited the AI-2 activity of other APEC serotypes and significantly reduced APEC O78 biofilm formation and motility. Most compounds showed minimal toxicity on human intestinal cells (Caco-2), chicken macrophage (HD-11), and chicken and sheep red blood cells, and reduced APEC survival in HD-11 and THP-1 macrophages. The SMs induced no or minimal toxicity and conferred protection against APEC in wax moth larval model. SMs affected the expression of APEC O78 QS, virulence, biofilm and motility associated genes providing insight on their potential mode(s) of action. Further testing in chickens will facilitate development of these SMs as novel therapeutics to control APEC in poultry and thereby also reduce zoonotic transmission.
Quorum sensing (QS) is an important protective mechanism that allows bacteria to adapt to its environment. A limited number of signal molecules play the key role of transmitting information in this mechanism. Signals are transmitted between individual bacterium through QS systems, resulting in the expression of specific genes. QS plays an important role in a variety of bacterial processes, including drug resistance, biofilm formation, motility, adherence, and virulence. Most Gram-positive and Gram-negative bacteria possess QS systems, mainly the LuxS/AI-2-mediated QS system. Evidence has been brought that LuxS/AI-2 system controls major virulence determinants in Streptococcus suis and, as such, the ability of this bacterial species to cause infections in humans and pigs. Understanding the S. suis LuxS/AI-2 system may open up novel avenues for decreasing the drug resistance and infectivity of S. suis. This article focuses on the progress made to date on the S. suis LuxS/AI-2-mediated QS system.
Quorum sensing (QS) is a cell-to-cell communication system that regulates gene expression as a result of the production and perception of signal molecules called autoinducers (AIs). AI-2 is a QS autoinducer produced by both Gram-negative and Gram-positive bacteria, in which it regulates intraspecies and interspecies communication. The identification of QS inhibitors is considered a promising strategy for the development of anti-virulence drugs with reduced selective pressure for resistance. Here we describe a high-throughput virtual screening approach to identify AI-2 quorum sensing inhibitors on the basis of Vibrio harveyi LuxPQ crystal structure. Seven potent inhibitors with IC50 values in the micromolar range were selected with no effect or low effect on V. harveyi growth rate.
Intercellular small-molecular-weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum-sensing molecules. These molecules mediate a variety of population-dependent responses including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules have important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused to the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. Ligand-insensitive LuxP mutant FRET protein sensors were also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2 compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of BAI-2.
The luxS gene is required for autoinducer-2 (AI-2) synthesis in many bacterial species. AI-2 is taken up by a specific receptor to regulate multiple bacterial activities. However, the lack of methods to identify AI-2 receptors has impeded investigations into the roles of AI-2. Here, a luxS mutant of Escherichia coli strain BL21 (DE3) was constructed (named BL21∆luxS), and the recombinant LsrB protein of Salmonella enterica was expressed in BL21∆luxS and BL21 cells, which were named LsrB (BL21∆luxS) and LsrB (BL21), respectively. The results of the activity of recombinant LsrB binding showed that LsrB (BL21) bound to endogenous AI-2 (produced from BL21 strain), while LsrB (BL21∆luxS) did not (as BL21∆luxS cannot produce AI-2). However, the results of recombinant LsrB binding showed that LsrB (BL21∆luxS) can bind exogenous AI-2, which was released from LsrB (BL21∆luxS) at 55 °C for 10 min, while LsrB (BL21) could not bind exogenous AI-2 (due to binding of endogenous AI-2 before). Furthermore, analysis of the thermal stability of AI-2 showed that that AI-2 activity was relatively high at incubation temperatures below 65 °C. These findings will be beneficial for screening of new AI-2 receptors in different bacterial species.
Background:
Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population.
Results:
For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control.
Conclusions:
The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.
Quorum sensing is a process of bacterial communication system wherein the production and secretion of small signaling molecules known as autoinducers enables the bacteria to express specific genes at particular population densities. Quorum quenching (QQ) can be used as an alternative approach to regulate pathogenicity. Well-established QQ strategies include amide bond hydrolysis, lactone hydrolysis, paraoxonase enzymes, and QQ modification of acyl chain. Plants in general lack advanced immune systems, and may have evolved to produce QQ compounds to combat with plant invading pathogens. Most common sources of QQ compounds in marine environment are bacteria, fungi, algae, bryozoan, corals, and sponges. Marine cyanobacteria have become one among the best source for obtaining biologically active and structurally unique QQ natural products. QQ compounds are being innovated as alternatives of antibiotics to treat pathogenic infections. Marine ecosystem is a unique and unexplored hotspot for the development of new derivatives of potential QQ compounds.
Quorum sensing (QS) is a mechanism of chemical communication that bacteria use to monitor cell-population density and coordinate group behaviors. QS relies on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. Vibrio cholerae employs parallel QS circuits that converge into a shared signaling pathway. At high cell density, the CqsS and LuxPQ QS receptors detect the intra-genus and inter-species autoinducers CAI-1 and AI-2, respectively, to repress virulence factor production and biofilm formation. We show that positive feedback, mediated by the QS pathway, increases CqsS but not LuxQ levels during the transition into QS-mode, which amplifies the CAI-1 input into the pathway relative to the AI-2 input. Asymmetric feedback on CqsS enables responses exclusively to the CAI-1 autoinducer. Because CqsS exhibits the dominant QS signaling role in V. cholerae, agonism of CqsS with synthetic compounds could be used to control pathogenicity and host dispersal. We identify nine compounds that share no structural similarity to CAI-1, yet potently agonize CqsS via inhibition of CqsS autokinase activity.
The resistance of bacterial biofilms to antibiotic treatment has been attributed to the emergence of structurally heterogeneous microenvironments containing metabolically inactive cell populations. In this study, we use a three-dimensional individual-based cellular automata model to investigate the influence of nutrient availability and quorum sensing on microbial heterogeneity in growing biofilms. Mature biofilms exhibited at least three structurally distinct strata: a high-volume, homogeneous region sandwiched between two compact sections of high heterogeneity. Cell death occurred preferentially in layers in close proximity to the substratum, resulting in increased heterogeneity in this section of the biofilm; the thickness and heterogeneity of this lowermost layer increased with time, ultimately leading to sloughing. The model predicted the formation of metabolically dormant cellular microniches embedded within faster-growing cell clusters. Biofilms utilizing quorum sensing were more heterogeneous compared to their non-quorum sensing counterparts, and resisted sloughing, featuring a cell-devoid layer of EPS atop the substratum upon which the remainder of the biofilm developed. Overall, our study provides a computational framework to analyze metabolic diversity and heterogeneity of biofilm-associated microorganisms and may pave the way toward gaining further insights into the biophysical mechanisms of antibiotic resistance.
In addition to the flow of metabolites and energy that drives the distribution of populations of a wide variety of micro-organisms. These cells respond to specific signals from their environment and from other organisms. The responses allow them to adapt to changing conditions and to generate stable ecological interactions. The stimuli vary enormously and include changes in light, temperature, osmolality, nutrient availability and changes in the presence of adherable surfaces. The organisms generally respond to these changes by modulating their metabolic potential and gene expression. They have mechanisms that allow them to sense the density of members of their own species or in the case of parasites and symbionts of appropriate host organisms. They can adapt by inducing the expression of previously repressed genes and gaining new catalytic capacity, eg the adaptation to anaerobiosis and nitrate utilization (Stewart et al., 1989). In addition, micro-organisms can undergo extensive morphological and physiological change, eg sporulation or lateral flagella formation and they can modify their own environment, for example, by growing in microbial mats to allow for efficient nutrient utilization. How do micro-organisms detect environmental change? How do they integrate environmental information to generate an appropriate response? And how do these mechanisms evolve so that they can be adapted to the ecological strategy of specific organisms?
One of the major concerns in bacterial communication is to understand and to decode the language spoken by microorganisms. The intraspecies languages spoken by Gram-negative and -positive bacteria, which are used for interspecies communication (universal chemical lexicon), and the relatively new bacterial signal that seems to be involved in cross-talk between bacteria and the human host (e.g., interkingdom communication) are reviewed in this chapter. Several examples are given for species that are predominantly relevant in foods.
Sueharu Horinouchi graduated from the Department of Agricultural Chemistry, University of Tokyo in 1974. He received his Ph.D. in 1979 from the University of Tokyo. He spent 2 years from 1979 to 1981 as project associate in Bernard Weisblum’s laboratory, Pharmacology Department, University of Wisconsin, where he revealed a posttranslational regulation of the macrolide resistance genes in pathogenic bacteria. He became an assistant professor in 1981, associate professor (1987), and professor (1994) in the Department of Biotechnology, University of Tokyo. He was the Director of Biotechnology Research Center, University of Tokyo from 2003 to 2005. His research field is basic and applied microbiology.
Bacteria utilize small signaling molecules, or autoinducers, to regulate their gene expression in tandem by a process termed quorum sensing. The gene encoding the synthase for autoinducer-2 (AI-2), luxS, is conserved in dozens of diverse bacteria. Behaviors controlled by AI-2 include virulence, motility, toxin production, and biofilm formation. The development of therapies that interfere with AI-2 quorum sensing are attractive for targeting biofilms, which exhibit inherent resistance to most antibiotics and biocidal agents. In this study, in vitro synthesized AI-2, LuxS inhibitors, and (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone were screened for their effect on biofilm formation in Escherichia coli, Bacillus cereus, and Listeria innocua. The LuxS inhibitors were found to have no influence on biofilm formation in any of the screened species, but reduced exponential phase AI-2 production in Listeria innocua. The brominated furanone significantly inhibited growth in B. cereus and L. innocua, and under certain conditions preferentially inhibited biofilm formation independently from growth.
Luminescent vibrios are amongst the most important pathogens in aquaculture. As a result of the massive (mis)use of antibiotics in order to treat infections, these pathogens have acquired resistance to most of the antibiotics that are used. Consequently, antibiotics are not effective anymore to cure luminescent vibriosis disease and in addition, the transfer of resistance determinants constitutes a threat to public health. Therefore, alternative methods to control the disease are urgently needed. In the first part of this work, the disruption of quorum sensing, bacterial cell-to-cell communication, was studied as a possible new strategy to control luminescent vibriosis. First of all, it was shown that quorum sensing regulates the virulence of Vibrio harveyi towards gnotobiotic brine shrimp (Artemia franciscana) nauplii. Subsequently, halogenated furanones were shown to disrupt quorum sensing in these bacteria by decreasing the DNA-binding activity of the quorum sensing master regulator protein LuxR. Interestingly, the addition of 20 mg l-1 of the furanone to the culture water of gnotobiotic Artemia nauplii resulted in a significantly increased survival in challenge tests with different pathogenic luminescent vibrio isolates. Unfortunately, the furanone also appeared to be toxic to the brine shrimp, as high mortality was observed after the addition of 50 mg l-1 to the culture water. The second part of this work describes the application of short-chain fatty acids and polyhydroxyalkanoates. First, it was shown that formic, acetatic, propionic, butyric and valeric acid inhibit growth of luminescent vibrios in vitro in a pH-dependent way. Furthermore, the addition of 20 mM of the short-chain fatty acids to the culture water of gnotobiotic Artemia nauplii resulted in a significantly increased survival in challenge tests with a pathogenic luminescent Vibrio campbellii strain. Subsequently, the application of the homopolymer of the short-chain fatty acid β-hydroxybutyrate, the well-known bacterial storage compound poly-β-hydroxybutyrate, was shown to offer a more efficient protection. Finally, it was shown that poly-β-hydroxybutyrate containing bacteria can be used to protect brine shrimp from luminescent vibriosis. The addition of poly-β-hydroxybutyrate containing Brachymonas bacteria (yielding a poly-β-hydroxybutyrate concentration of 10 mg l-1) resulted in a complete protection from the pathogenic Vibrio campbellii strain.
Bacteria use quorum sensing (QS) for cell-cell communication to carry out group behaviors. This intercellular signaling process relies on cell density-dependent production and detection of chemical signals called autoinducers (AIs). Vibrio cholerae, the causative agent of cholera, detects two AIs, CAI-1 and AI-2, with two histidine kinases, CqsS and LuxQ, respectively, to control biofilm formation and virulence factor production. At low cell density, these two signal receptors function in parallel to activate the key regulator LuxO, which is essential for virulence of this pathogen. At high cell density, binding of AIs to their respective receptors leads to deactivation of LuxO and repression of virulence factor production. However, mutants lacking CqsS and LuxQ maintain a normal LuxO activation level and remain virulent, suggesting that LuxO is activated by additional, unidentified signaling pathways. Here we show that two other histidine kinases, CqsR (formerly known as VC1831) and VpsS, act upstream in the central QS circuit of V. cholerae to activate LuxO. V. cholerae strains expressing any one of these four receptors are QS proficient and capable of colonizing animal hosts. In contrast, mutants lacking all four receptors are phenotypically identical to LuxO-defective mutants. Importantly, these four functionally redundant receptors act together to prevent premature induction of a QS response caused by signal perturbations. We suggest that the V. cholerae QS circuit is composed of quadruple sensory inputs and has evolved to be refractory to sporadic AI level perturbations.
Vibrio harveyi, a luminescent Gram-negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non-luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence-related phenotypic traits of the wild-type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non-luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non-luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.
© 2015 John Wiley & Sons Ltd.
Unlabelled:
Quorum sensing (QS) is a communication process that enables a bacterial population to coordinate and synchronize specific behaviors. The bioluminescent marine bacterium Vibrio harveyi integrates three autoinducer (AI) signals into one quorum-sensing cascade comprising a phosphorelay involving three hybrid sensor kinases: LuxU; LuxO, an Hfq/small RNA (sRNA) switch; and the transcriptional regulator LuxR. Using a new set of V. harveyi mutants lacking genes for the AI synthases and/or sensors, we assayed the activity of the quorum-sensing cascade at the population and single-cell levels, with a specific focus on signal integration and noise levels. We found that the ratios of kinase activities to phosphatase activities of the three sensors and, hence, the extent of phosphorylation of LuxU/LuxO are important not only for the signaling output but also for the degree of noise in the system. The pools of phosphorylated LuxU/LuxO per cell directly determine the amounts of sRNAs produced and, consequently, the copy number of LuxR, generating heterogeneous quorum-sensing activation at the single-cell level. We conclude that the ability to drive the heterogeneous expression of QS-regulated genes in V. harveyi is an inherent feature of the architecture of the QS cascade.
Importance:
V. harveyi possesses one of the most complex quorum-sensing (QS) cascades known, using three different autoinducers (AIs) to control the induction of, e.g., bioluminescence, virulence factors, and biofilm and exoprotease production. We constructed various V. harveyi mutants to study the impact of each component and subsystem of the QS signaling cascade on QS activation at the population and single-cell levels. We found that the output was homogeneous only in the presence of all AIs. In the absence of any one AI, QS activation varied from cell to cell, resulting in phenotypic heterogeneity. This study elucidates a molecular design principle which enables a tightly integrated signaling cascade to control the expression of diverse phenotypes within a genetically homogeneous population.
Bacteria are able to sense an increase in population density and can respond to it by coordinated regulation of the expression of certain sets of genes in the total population of bacteria. This specific mode of regulation is known as Quorum Sensing (QS). The QS systems include low-molecular-weight signaling molecules of different chemical nature and the regulatory proteins that interact with the signaling molecules. The QS systems are global regulators of bacterial gene expression. They play an important role in controlling metabolic processes in bacteria. This review describes QS systems in members of the bacterial family Enterobacteriaceae functioning with the involvement of various signaling molecules, including N-acyl-homoserine lactones, AI-2, AI-3, peptides, and indole. The differences of the QS system in these bacteria from those in other taxonomic groups of bacteria are discussed. Data on the role of different types of QS systems in the regulation of different cellular processes in bacteria, i.e., their virulence, the synthesis of enzymes and antibiotics, biofilm formation, apoptosis, etc. are presented.
The Lyme disease spirochete, Borrelia burgdorferi, controls protein expression patterns during its tick-mammal infection cycle. Earlier studies demonstrated that B. burgdorferi synthesizes 4,5-dihydroxy-2,3-pentanedione (autoinducer-2, AI-2) and responds to AI-2 by measurably changing production of several infection-associated proteins. A luxS mutant, which is unable to produce AI-2, exhibits altered production of several proteins. B. burgdorferi cannot utilize the other product of LuxS, homocysteine, indicating that phenotypes of luxS mutants are not due to absence of that molecule. Although a previous study found that a luxS mutant was capable of infecting mice, a critical caveat to those results is that bacterial loads were not quantified. To more precisely determine whether LuxS serves a role in mammalian infection, mice were simultaneously inoculated with congenic wild-type and luxS strains, then bacterial numbers were assessed using quantitative PCR. The wild type bacteria substantially out-competed the mutant, suggesting that LuxS performs a significant function during mammalian infection. These data also provide further evidence that non-quantitative infection studies do not necessarily provide conclusive results, and that regulatory factors may not make all-or-none, black-or-white contributions to infectivity.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Chemosynthetic Epsilonproteobacteria from deep-sea hydrothermal vents colonize substrates exposed to steep thermal and redox gradients. In many bacteria, substrate attachment, biofilm formation, expression of virulence genes and host colonization are partly controlled via a cell density-dependent mechanism involving signal molecules, known as quorum sensing. Within the Epsilonproteobacteria, quorum sensing has been investigated only in human pathogens that use the luxS/autoinducer-2 (AI-2) mechanism to control the expression of some of these functions. In this study we showed that luxS is conserved in Epsilonproteobacteria and that pathogenic and mesophilic members of this class inherited this gene from a thermophilic ancestor. Furthermore, we provide evidence that the luxS gene is expressed-and a quorum-sensing signal is produced-during growth of Sulfurovum lithotrophicum and Caminibacter mediatlanticus, two Epsilonproteobacteria from deep-sea hydrothermal vents. Finally, we detected luxS transcripts in Epsilonproteobacteria-dominated biofilm communities collected from deep-sea hydrothermal vents. Taken together, our findings indicate that the epsiloproteobacterial lineage of the LuxS enzyme originated in high-temperature geothermal environments and that, in vent Epsilonproteobacteria, luxS expression is linked to the production of AI-2 signals, which are likely produced in situ at deep-sea vents. We conclude that the luxS gene is part of the ancestral epsilonproteobacterial genome and represents an evolutionary link that connects thermophiles to human pathogens.The ISME Journal advance online publication, 14 November 2014; doi:10.1038/ismej.2014.214.
Quorum sensing is a process of bacterial cell-cell communication that relies on the production, release, and receptor-driven detection of extracellular signal molecules called autoinducers. The quorum-sensing bacterium Vibrio harveyi exclusively detects the autoinducer N-((R)-3-hydroxybutanoyl)-L-homoserine lactone (3OH-C4 HSL) via the two-component receptor LuxN. To discover the principles underlying the exquisite selectivity LuxN has for its ligand, we identified LuxN mutants with altered specificity. LuxN uses three mechanisms to verify that the bound molecule is the correct ligand: In the context of the overall ligand-binding site, His210 validates the C3 modification, Leu166 surveys the chain-length, and a strong steady-state kinase bias imposes an energetic hurdle for inappropriate ligands to elicit signal transduction. Affinities for the LuxN Kinaseon and Kinaseoff states underpin whether a ligand will act as an antagonist or an agonist. Mutations that bias LuxN to the agonized, Kinaseoff, state are clustered in a region adjacent to the ligand-binding site, suggesting that this region acts as the switch that triggers signal transduction. Together, our analyses illuminate how a histidine sensor kinase differentiates between ligands and exploits those differences to regulate its signaling activity.
Two controls of the phosphate (PHO) regulon require sensor proteins that are protein kinases that phosphorylate the regulator, PhoB, which in turn activates transcription only when phosphorylated. Pi control requires the Pi sensor PhoR; the other control is Pi independent and requires the sensor CreC (formerly called PhoM). Here we describe an additional control of the PHO regulon which is Pi independent and requires neither PhoR nor CreC. This control is regulated by a two-step pathway in carbon metabolism in which acetyl coenzyme A, Pi, and ADP are converted into acetate, coenzyme A, and ATP via the enzymes phosphotransacetylase (Pta) and acetate kinase (AckA). It responds to the synthesis of acetyl phosphate, an intermediate in the Pta-AckA pathway. Since the synthesis of acetyl phosphate via this pathway leads to the incorporation of Pi into ATP, the primary phosphoryl donor in metabolism, we propose that a regulatory coupling(s) may exist between the PHO regulon, which encodes genes for Pi uptake, and genes for enzymes in central metabolism for incorporation of Pi into ATP. Regulatory interactions of this sort may be important in global control. Further, it provides a functional basis for the concept of cross-regulation in the PHO regulon. This is also the first evidence that acetyl phosphate may have a role as an effector of gene regulation.
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.
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.
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.
The vir gene products of Agrobacterium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.
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 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 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.
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.
Pseudomonas aeruginosa is an opportunistic human pathogen that causes a variety of infections in immunocompromised hosts and
individuals with cystic fibrosis. Expression of elastase, one of the virulence factors produced by this organism, requires
the transcriptional activator LasR. Experiments with gene fusions show that gene lasl is essential for high expression of
elastase. The lasl gene is involved in the synthesis of a diffusible molecule termed Pseudomonas autoinducer (PAI). PAI provides
P. aeruginosa with a means of cell-to-cell communication that is required for the expression of virulence genes and may provide
a target for therapeutic approaches.