Autoinducer-2 Production Research Articles

Pfs-dependent regulation of autoinducer 2 production in Salmonella enterica serovar Typhimurium.

Bacterial intercellular communication provides a mechanism for signal-dependent regulation of gene expression to promote coordinated population behavior. Salmonella enterica serovar Typhimurium produces a non-homoserine lactone autoinducer in exponential phase as detected by a Vibrio harveyi reporter assay for autoinducer 2 (AI-2) (M. G. Surette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). The luxS gene product mediates the production of AI-2 (M. G. Surette, M. B. Miller, and B. L. Bassler, Proc. Natl. Acad. Sci. USA 96:1639-1644, 1999). Environmental cues such as rapid growth, the presence of preferred carbon sources, low pH, and/or high osmolarity were found to influence the production of AI-2 (M. G. Surette and B. L. Bassler, Mol. Microbiol. 31:585-595, 1999). In addition to LuxS, the pfs gene product (Pfs) is required for AI-2 production, as well as S-adenosylhomocysteine (SAH) (S. Schauder, K. Shokat, M. G. Surette, and B. L. Bassler, Mol. Microbiol. 41:463-476, 2001). In bacterial cells, Pfs exhibits both 5'-methylthioadenosine (MTA) and SAH nucleosidase functions. Pfs is involved in methionine metabolism, regulating intracellular MTA and SAH levels (elevated levels of MTA and SAH are potent inhibitors of polyamine synthetases and S-adenosylmethionine dependent methyltransferase reactions, respectively). To further investigate regulation of AI-2 production in Salmonella, we constructed pfs and luxS promoter fusions to a luxCDABE reporter in a low-copy-number vector, allowing an examination of transcription of the genes in the pathway for signal synthesis. Here we report that luxS expression is constitutive but that the transcription of pfs is tightly correlated to AI-2 production in Salmonella serovar Typhimurium 14028. Neither luxS nor pfs expression appears to be regulated by AI-2. These results suggest that AI-2 production is regulated at the level of LuxS substrate availability and not at the level of luxS expression. Our results indicate that AI-2-dependent signaling is a reflection of metabolic state of the cell and not cell density.

Novel intercellular communication system in Escherichia coli that confers antibiotic resistance between physically separated populations.

To determine whether intercellular signalling can occur between physically separated populations of Escherichia coli. Intercellular signalling between physically discrete populations of E. coli BL21 was analysed in bi-partite Petri dishes. Transfer of a growth-promoting signal resulted in induction of resistance to the antibiotic ampicillin. Optimal expression of the signal occurred when the signalling population was established as a bacterial lawn for 24 h. This represented an entry into the stationary phase of growth, as indicated by the expression profile of the RNA polymerase subunit sigma38 (sigmaS; sigma S). The growth-promoting effect was also observed when E. coli DH5alpha (luxS-) was used as the signalling population. Preventing passage of air between the two populations resulted in a complete cessation of the growth-promoting effect. A growth-promoting signal occurs between physically separated cultures of E. coli. The exact nature of the signal remains to be determined but does not involve the production of autoinducer-2 from the luxS gene. Signal transmission is likely to involve airborne transfer of a signal species. Intercellular signalling systems exist in bacteria that enable antibiotic resistance to be conferred between physically separated populations.

The luxS gene is involved in cell-cell signalling for toxin production in Clostridium perfringens.

A Gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI-2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell-free culture supernatant from wild-type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI-2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha-, kappa- and theta-toxins. In the luxS mutant, the transcription of the theta-toxin gene (pfoA) was lower at mid-exponential growth phase, whereas alpha- and kappa-toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild-type cells, possibly because of the presence of AI-2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild-type C. perfringens, Escherichia coli DH5alpha carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell-cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell-cell signalling by AI-2 plays an important role in the regulation of toxin production in C. perfringens.

Role ofNeisseria meningitidis luxSin Cell-to-Cell Signaling and Bacteremic Infection

Numerous pathogenic bacteria contain luxS, which is required for autoinducer-2 production. Here, we demonstrate that Neisseria meningitidis contains a functional copy of luxS that is necessary for full meningococcal virulence; strains with a luxS deletion are defective for bacteremia, a prerequisite of meningococcal pathogenesis.

RelA-Independent amino acid starvation response network of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococcus [GAS]), a multiple-amino-acid-auxotrophic human pathogen, may face starvation for essential amino acids during various stages of the infection process. Since the response of GAS to such conditions is likely to influence pathogenetic processes, we set out to identify by transcriptional analyses genes and operons that are responsive to amino acid starvation and examined whether functionally meaningful response patterns can be ascertained. We discovered that GAS are capable of mounting a relA-independent amino acid starvation response that involves transcriptional modulation of a wide array of housekeeping genes as well as accessory and dedicated virulence genes. Housekeeping genes that were upregulated during starvation of both wild-type and relA mutant strains included the newly identified T-box members of the aminoacyl-tRNA synthetase genes, the genes for components of the tmRNA-mediated peptide tagging and proteolysis system for abnormal proteins (ssrA, smpB, clpP, and clpC), and the operons for the dnaK and groE groups of molecular chaperones. In addition to upregulation of the genes for oligopeptide permease (opp), intracellular peptidase (pepB), and the two-component regulator covRS reported previously (K. Steiner and H. Malke, Mol. Microbiol. 38:1004-1016, 2000), amino acid starvation stimulated the transcription of the growth phase-associated, virulence-regulatory fas operon, the streptolysin S operon (sag), and the gene for autoinducer-2 production protein (luxS). A prominent feature of operons exhibiting internal transcriptional termination (opp, fas, and sag) was starvation-promoted full-length transcription, a mechanism that improves the efficacy of these systems by increasing the level of coordinate transcription of functionally related genes. Based on these results, a regulatory network with feedback mechanisms is proposed that counteracts the stringent response, links the levels of key rate-limiting enzymes to virulence gene expression, and enables the organism in a dynamic way to take advantage of protein-rich environments provided by its human host. As several of the affected target genes are controlled by more than one regulator, fine modulation may result in accordance with the demands imposed by ecologically different colonization sites upon the adaptive capacity of the pathogen.

The 1.2 Å structure of a novel quorum-sensing protein, Bacillus subtilis LuxS

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione. The crystal structure of Bacillus subtilis LuxS has been determined at 1.2 Å resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 and 2.3 Å resolution, respectively. These structures show that LuxS is a homodimer with an apparently novel fold based on an eight-stranded β-barrel, flanked by six α-helices. Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits. S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme-bound zinc ion. Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125–129 and those at the N terminus. The structure contains an oxidised cysteine residue in the active site whose role in the biological process of LuxS has not been determined. The autoinducer-2 signalling pathway has been linked to aspects of bacterial virulence and pathogenicity. The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics.

Signaling system in Porphyromonas gingivalis based on a LuxS protein.

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.

The languages of bacteria.

Bacteria communicate with one another using chemical signaling molecules as words. Specifically, they release, detect, and respond to the accumulation of these molecules, which are called autoinducers. Detection of autoinducers allows bacteria to distinguish between low and high cell population density, and to control gene expression in response to changes in cell number. This process, termed quorum sensing, allows a population of bacteria to coordinately control the gene expression of the entire community. Quorum sensing confuses the distinction between prokaryotes and eukaryotes because it allows bacteria to behave as multicellular organisms, and to reap benefits that would be unattainable to them as individuals. Many bacterial behaviors are regulated by quorum sensing, including symbiosis, virulence, antibiotic production, and biofilm formation. Recent studies show that highly specific as well as universal quorum sensing languages exist which enable bacteria to communicate within and between species. Finally, both prokaryotic and eukaryotic mechanisms that interfere with bacterial quorum sensing have evolved. Specifically, the secretion of enzymes that destroy the autoinducers, and the production of autoinducer antagonists, are used by competitor bacteria and susceptible eukaryotic hosts to render quorum sensing bacteria mute and deaf, respectively. Analogous synthetic strategies are now being explored for the development of novel antimicrobial therapies. Bacteria in communities convey their presence to one another by releasing and responding to the accumulation of chemical signaling molecules called autoinducers. This process of intercellular communication, called quorum sensing, was first described in the bioluminescent marine bacterium Vibrio fischeri (Hastings and Nealson 1977; Nealson and Hastings 1979). V. fischeri lives in symbiotic associations with a number of marine animal hosts. In these partnerships, the host uses the light produced by V. fischeri for specific purposes such as attracting prey, avoiding predators, or finding a mate. In exchange for the light it provides, V. fischeri obtains a nutrient-rich environment in which to reside (Ruby 1996; Visick and McFall-Ngai 2000). A luciferase enzyme complex is responsible for light production in V. fischeri. Bioluminescence only occurs when V. fischeri is at high cell number, and this process is controlled by quorum sensing. Specifically, the production and accumulation of, and the response to, a minimum threshold concentration of an acylated homoserine lactone (HSL) autoinducer regulates density-dependent light production in V. fischeri, and enables V. fischeri to emit light only inside the specialized light organ of the host but not when free-living in the ocean. The reason for this is twofold. First, only under the nutrient-rich conditions of the light organ can V. fischeri grow to high population densities, and second, trapping of the diffusible autoinducer molecule in the light organ with the bacterial cells allows it to accumulate to a sufficient concentration that V. fischeri can detect it. Engebrecht and Silverman discovered the regulatory circuit controlling quorum sensing in V. fischeri (Engebrecht et al. 1983; Engebrecht and Silverman 1984, 1987). They showed that two regulatory components are required for the process. The LuxI protein is responsible for production of the HSL autoinducer, and the LuxR protein is responsible for binding the HSL autoinducer and activating transcription of the luciferase structural operon at high cell density (Engebrecht et al. 1983; Engebrecht and Silverman 1984). They showed that, as an autoinducer-producing population of V. fischeri cells grows, the concentration of autoinducer increases as a function of increasing cell-population density. When the autoinducer concentration reaches the micromolar range, it can interact with the LuxR protein, and the LuxR-autoinducer complex binds the luciferase promoter to activate transcription. Therefore, this quorum sensing circuit allows light production to be tightly correlated with the cell population density. For over 10 years the V. fischeri LuxI/LuxR signal–response system was considered a curious, but isolated, example of bacterial communication that had presumably evolved for a specific purpose required for the colonization of a symbiotic host. However, we now understand that most bac1Corresponding author. E-MAIL bbassler@molbio.princeton.edu; FAX (609) 258–6175. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.899601.

Evidence for a signaling system in Helicobacter pylori: detection of a luxS-encoded autoinducer.

Helicobacter pylori possesses a homolog of the luxS gene, initially identified by its role in autoinducer production for the quorum-sensing system 2 in Vibrio harveyi. The genomes of several other species of bacteria, notably Escherichia coli, Salmonella enterica serovar Typhimurium, and Vibrio cholerae, also include luxS homologs. All of these bacteria have been shown to produce active autoinducers capable of stimulating the expression of the luciferase operon in V. harveyi. In this report, we demonstrate that H. pylori also synthesizes a functional autoinducer (AI-2) that can specifically activate signaling system 2 in V. harveyi. Maximal activity is produced during early log phase, and the activity is diminished when cells enter stationary phase. We show that AI-2 is not involved in modulating any of the known or putative virulence factors in H. pylori and that a luxS null mutant has a two-dimensional protein profile identical to that of its isogenic parent strain. We discuss the implications of having an AI-2-like quorum-sensing system in H. pylori and suggest possible roles that it may play in H. pylori infection.

Invasive strains of Pseudomonas aeruginosa are able to cause epithelial cell cytotoxicity that is dependent on bacterial cell density.

The purpose of this study was to investigate the hypothesis that quorum-sensing systems are involved in the ability of invasive Pseudomonas aeruginosa strains to cause corneal epithelial cell death. Two invasive strains, 6294 and PAOI, were co-cultured with human corneal epithelial cells at different bacterial concentrations (10(5), 10(7) and 10(9) CFU/mL). Cytotoxicity was measured using a cytotoxicity assay kit. The levels of autoinducer in the supernatant were examined using a reporter strain of Agrobacterium tumefaciens (A136). Protease production was also monitored. Cytotoxicity of both strains was dependent on bacterial density; a moderate to high concentration of bacterial cells (10(7) and 10(9) CFU/mL) caused 70% to 94% loss of cell variability. Cytotoxicity was significantly correlated with enhanced autoinducer and protease production (r>0.95, P<0.05). These results indicate that the invasive strains regulate the production of virulence factors and, in turn, induce chronic dose-related cytotoxicity.

New signal molecules on the quorum-sensing block

Apart from AHLs and PQS, P. aeruginosa has recently been shown to produce another family of small diffusible signal molecules, the diketopiperazines (DKPs)5xQuorum-sensing crosstalk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria. Holden, M.T. et al. Mol. Microbiol. 1999; 33: 1254–1266Crossref | PubMed | Scopus (306)See all References5. These cyclic dipeptides were identified as a consequence of their ability to activate biosensors previously considered specific for AHLs. Although the DKPs are structurally quite distinct (Fig. 1Fig. 1), at high concentrations they are able to cross-activate AHL-dependent reporter constructs based on several different LuxR homologues5xQuorum-sensing crosstalk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria. Holden, M.T. et al. Mol. Microbiol. 1999; 33: 1254–1266Crossref | PubMed | Scopus (306)See all References5. The detection of these DKPs appears to be an example of fortuitous chemical crosstalk and raises the obvious question as to their origin and biological function(s). In higher plants and animals, DKPs exhibit a remarkable spectrum of biological and pharmacological activities9xBioactive cyclic dipeptides. Prasad, C. Peptides. 1995; 16: 151–164Crossref | PubMed | Scopus (222)See all References9. Therefore, although it is quite feasible that the DKPs do not function as bacteria-to-bacteria signalling molecules per se, they might have a role in modulating prokaryotic–eukaryotic interactions. In this respect, it is worth noting that certain AHLs possess potent immunomodulatory10xThe Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-l-homoserine lactone has immunomodulatory activity. Telford, G. et al. Infect. Immun. 1998; 66: 36–42PubMedSee all References10 and cardiovascular activity11xThe Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone, inhibits porcine arterial smooth muscle contraction. Lawrence, R.N. et al. Br. J. Pharmacol. 1999; 128: 845–848Crossref | PubMed | Scopus (56)See all References11.In bacteria, the production of DKPs is not limited to P. aeruginosa, as other Gram-negative bacteria, including E. coli, produce the same or related molecules5xQuorum-sensing crosstalk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria. Holden, M.T. et al. Mol. Microbiol. 1999; 33: 1254–1266Crossref | PubMed | Scopus (306)See all References5. Moreover, for both Salmonella spp. and E. coli there are several reports of quorum-sensing-type phenomena3xQuorum sensing in Escherichia coli, Salmonella typhimurium and Vibrio harveyi: a new family of genes responsible for autoinducer production. Surette, M.G. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1639–1644Crossref | PubMed | Scopus (588)See all References, 12xQuorum-sensing acts at initiation of chromosomal replication in Escherichia coli. Withers, H.L. and Nordstrom, K. Proc. Natl. Acad. Sci. U. S. A. 1999; 95: 15694–15699Crossref | Scopus (61)See all References, 13xEscherichia coli genes regulated by cell-to-cell signalling. Baca-DeLancey, R.R. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 4610–4614Crossref | Scopus (88)See all References, 14xStimulation of bacterial growth by heat-stable, norepinephrine-induced autoinducers. Freestone, P.P. et al. FEMS Microbiol. Lett. 1999; 172: 53–60Crossref | PubMedSee all References in which the structure(s) of the signal molecules(s) involved has yet to be elucidated. Such molecules may well prove to be DKPs or 4-quinolones.

Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli.

Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora.

Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production.

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows. In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor-autoinducer pair. V. harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E. coli O157:H7, Salmonella typhimurium 14028, and S. typhimurium LT2 produce autoinducers similar or identical to the V. harveyi system 2 autoinducer AI-2. However, the domesticated laboratory strain E. coli DH5alpha does not produce this signal molecule. Here we report the identification and analysis of the gene responsible for AI-2 production in V. harveyi, S. typhimurium, and E. coli. The genes, which we have named luxSV.h., luxSS.t., and luxSE.c. respectively, are highly homologous to one another but not to any other identified gene. E. coli DH5alpha can be complemented to AI-2 production by the introduction of the luxS gene from V. harveyi or E. coli O157:H7. Analysis of the E. coli DH5alpha luxSE.c. gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxSE.c. protein. Our results indicate that the luxS genes define a new family of autoinducer-production genes.

Regulation of autoinducer production in Salmonella typhimurium.

Salmonella typhimurium strain LT2 secretes an organic signalling molecule that can be assayed by its ability to activate one of two specific quorum-sensing systems in Vibrio harveyi. Maximal activity is produced during mid- to late exponential phase when S. typhimurium is grown in the presence of glucose or other preferred carbohydrates. The signal is degraded by the onset of stationary phase or when the carbohydrate is depleted from the medium. Presumably, quorum sensing in S. typhimurium is operational during periods of rapid, nutrient-rich growth. Protein synthesis is required for degradation of the activity, suggesting that a complex regulatory circuitry controls signal production and detection in S. typhimurium. Increased signalling activity is observed if, after growth in the presence of glucose, S. typhimurium is transferred to a high-osmolarity (0.4 M NaCl) or to a low-pH (pH 5.0) environment. Degradation of the signal is induced by conditions of low osmolarity (0.1 M NaCl). High osmolarity and low pH are two conditions encountered by S. typhimurium cells when they undergo the transition to a pathogenic existence inside a host organism, suggesting that quorum sensing may have a role in the regulation of virulence in S. typhimurium.

Promotion of antibiotic production by high ethanol, high NaCl concentration, or heat shock in Pseudomonas fluorescens S272.

A stress imposed by a continuous feed of high ethanol, high NaCl concentration, or a high temperature shock increased antibiotic production by several times in Pseudomonas fluorescens S272. A tentative bioassay showed that the stress caused about 40-fold elevation in the autoinducer activity. Addition of synthetic autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone or N-(3-oxohexanoyl)-L-homoserine lactone at a concentration of more than 100 micrograms/l to a non-stressed culture also increased the antibiotic production by several times. These results suggested that the antibiotic production in P. fluorescens S272 was regulated by N-acyl-homoserine lactone and the promotive effect by stress occurred through any function that increased the autoinducer production.

Evidence for interspecies communication and its potential role in pathogen suppression in a naturally occurring disease suppressive soil

Streptomyces strains isolated from potato scab suppressive (n = 9) and conducive (n = 5) soils were screened for their ability to produce diffusible chemicals that trigger antibiotic production in the pathogen-suppressive Streptomyces diastatochromogenes PonSSII. Using an Agrobacterium detection system, the strains were tested for the ability to produce homoserine lactone autoinducers. In addition, suppressive strain PonSSII was screened for production of an autoinducer for antibiotic production in a chemically defined liquid medium. Interspecies communication was investigated by growing suppressive and pathogenic strains individually in liquid medium and determining whether broth from these strains could induce antibiotic production in PonSSII. No evidence was found for production of homoserine lactones by any of the Streptomyces strains nor for the production of autoinducers by PonSSII. However, addition of conditioned broth from Streptomyces strains to cultures of PonSSII stimulated, suppressed, or had no effect on antibiotic production. Conditioned broth from suppressive strain 23 and pathogenic strain RB4 triggered antibiotic production by PonSSII at earlier times during culture growth and also enhanced antibiotic production levels compared with the control. The results suggest that interspecies communication between these Streptomyces species is occurring and may contribute to pathogen inhibition in the naturally occurring disease suppressive soil.Key words: Streptomyces, suppressive soil, interspecies communication, potato scab, autoinducers.

Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the auto-inducer molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), the synthesis of which is directed by LasI. Previously we have shown that in PAN067, an elastase-negative mutant of PAO1, elastase production can be restored to some extent by addition of exogenous N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Here we report that PAN067 is also defective in the production of alkaline protease, haemolysin, cyanide, pyocyanin and autoinducer(s). As neither addition of exogenous OdDHL nor introduction of lasR restored PAN067 to the parental phenotype, we sought to complement PAN067 with PAO1 DNA. From a cosmid library, a 2 kb DNA fragment was identified which re-established production of autoinducer(s) and exoproducts in PAN067. From the nucleotide sequence of this fragment, two genes termed rhIR and rhII were identified. RhII is responsible for autoinducer synthesis and shares 31% homology with LasI; RhIR has been previously identified in P. aeruginosa strain DSM2659 as a regulator of rhamnolipid biosynthesis and shares 28% identity with LasR. These data provide clear evidence that multiple families of quorum-sensing modulons interactively regulate gene expression in P. aeruginosa.

Detection and quantification of Vibrio fischeri autoinducer from symbiotic squid light organs

Vibrio fischeri is the specific light organ symbiont of the sepiolid squid species Euprymna scolopes and Euprymna morsei. Both species of squid are luminescent by virtue of their bacterial symbionts, but the natural symbionts of E. scolopes do not produce visible luminescence in laboratory culture. The primary cause of this depressed luminescence by E. scolopes symbionts in culture was found to be the production of relatively low levels of V. fischeri autoinducer, a positive transcriptional coregulator of the lux regulon, identified as N-(3-oxohexanoyl) homoserine lactone. Concentrations of autoinducer activity produced by these symbionts in culture were quantified and found to be at least 10-fold lower than those produced by E. morsei isolates (which are visibly luminous outside the association) and perhaps 10,000-fold lower than those of the brightest V. fischeri strains. Despite the differences in their symbiont strains, the intact light organs of the two species of squid contained comparable amounts of extractable autoinducer activity (between 100 and 200 pg per adult animal). The chromatographic behavior of this autoinducer activity on reverse-phase high-performance liquid chromatography was consistent with its presumptive identification as V. fischeri autoinducer. Within the 5-microliter volume of the epithelial core of the light organ in which the symbiotic V. fischeri strains are housed, these amounts would result in an effective autoinducer concentration of at least 100 nM. Because these levels are over 40-fold higher than the concentration needed for the induction of luminescence of bacteria in culture, we conclude that the inherent degree of autoinducer production by strains of V. fischeri may not influence their effectiveness as light organ symbionts. Furthermore, this study provides the first direct evidence that the phenomenon of cell density-dependent autoinduction, discovered and described first for laboratory cultures of V. fischeri but believed to be a general phenomenon in many species of host-associated symbionts and pathogens, is in fact a consequence of bacterial colonizations of host tissues.

Mechanisms of iron regulation of luminescence in Vibrio fischeri

Synthesis of luciferase (an autoinducible enzyme) is repressed by iron in the symbiotic bioluminescent bacterium Vibrio fischeri. Possible mechanisms of iron regulation of luciferase synthesis were tested with V. fischeri and with Escherichia coli clones containing plasmids carrying V. fischeri luminescence genes. Experiments were conducted in complete medium with and without the synthetic iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid). Comparison of the effect of ethylenediamine-di(o-hydroxyphenyl acetic acid) and another growth inhibitor, (2-n-heptyl-4-hydroxyquinoline-N-oxide), showed that iron repression is not due to inhibition of growth. A quantitative bioassay for autoinducer was developed with E. coli HB101 containing pJE411, a plasmid carrying V. fischeri luminescence genes with a transcriptional fusion between luxI and E. coli lacZ. Bioassay experiments showed no effect of iron on either autoinducer activity or production (before induction) or transcription of the lux operon. Ethylenediamine-di(o-hydroxyphenyl acetic acid) did not affect luciferase induction in E. coli strains with wild-type iron assimilation (ED8654) or impaired iron assimilation (RW193) bearing pJE202 (a plasmid with functional V. fischeri lux genes), suggesting that the genes responsible for the iron effect are missing or substituted in these clones. Two models are consistent with the data: (i) iron represses autoinducer transport, and (ii) iron acts through an autoinduction-independent regulatory system (e.g., an iron repressor).

Luminous bacteria synthesize luciferase anaerobically

Four species of luminous bacteria, Photobacterium phosphoreum, P. leiognathi, P. fischeri and Beneckea harveyi (two strains of each), were shown to synthesize luciferase anaerobically. One of these, P. phosphoreum, produced as much luciferase anaerobically as it did aerobically, and all four species were found to grow almost equally rapidly under the two sets of conditions. Previous work with B. harveyi and P. fischeri had shown that aerobic luciferase synthesis can proceed only after an inhibitor in the complex medium has been removed and a species-specific autoinducer secreted. All strains tested also removed the inhibitor and secreted an autoinducer anaerobically. The small amount of luciferase produced anaerobically by some strains is thus apparently not due either to lack of removal of inhibitor or to insufficient production of autoinducer but may involve an oxygen-dependent control mechanism.