Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression
2003; Springer Nature; Volume: 22; Issue: 4 Linguagem: Inglês
10.1093/emboj/cdg085
ISSN1460-2075
Autores Tópico(s)Vibrio bacteria research studies
ResumoArticle17 February 2003free access Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression Kenny C. Mok Kenny C. Mok Department of Molecular Biology, Princeton University, Princeton, NJ, 08544-1014 USA Search for more papers by this author Ned S. Wingreen Ned S. Wingreen NEC Laboratories America, Inc., 4 Independence Way, Princeton, NJ, 08540 USA Search for more papers by this author Bonnie L. Bassler Corresponding Author Bonnie L. Bassler Department of Molecular Biology, Princeton University, Princeton, NJ, 08544-1014 USA Search for more papers by this author Kenny C. Mok Kenny C. Mok Department of Molecular Biology, Princeton University, Princeton, NJ, 08544-1014 USA Search for more papers by this author Ned S. Wingreen Ned S. Wingreen NEC Laboratories America, Inc., 4 Independence Way, Princeton, NJ, 08540 USA Search for more papers by this author Bonnie L. Bassler Corresponding Author Bonnie L. Bassler Department of Molecular Biology, Princeton University, Princeton, NJ, 08544-1014 USA Search for more papers by this author Author Information Kenny C. Mok1, Ned S. Wingreen2 and Bonnie L. Bassler 1 1Department of Molecular Biology, Princeton University, Princeton, NJ, 08544-1014 USA 2NEC Laboratories America, Inc., 4 Independence Way, Princeton, NJ, 08540 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:870-881https://doi.org/10.1093/emboj/cdg085 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In a process called quorum sensing, bacteria communicate with one another by exchanging chemical signals called autoinducers. In the bioluminescent marine bacterium Vibrio harveyi, two different auto inducers (AI-1 and AI-2) regulate light emission. Detection of and response to the V.harveyi autoinducers are accomplished through two two-component sensory relay systems: AI-1 is detected by the sensor LuxN and AI-2 by LuxPQ. Here we further define the V.harveyi quorum-sensing regulon by identifying 10 new quorum-sensing-controlled target genes. Our examination of signal processing and integration in the V.harveyi quorum-sensing circuit suggests that AI-1 and AI-2 act synergistically, and that the V.harveyi quorum-sensing circuit may function exclusively as a 'coincidence detector' that discriminates between conditions in which both autoinducers are present and all other conditions. Introduction Quorum sensing is a process that allows bacteria to communicate using secreted chemical signaling molecules called autoinducers (Nealson and Hastings, 1979; Miller and Bassler, 2001). This process enables a population of bacteria collectively to regulate gene expression and, therefore, behavior. Using quorum sensing, bacteria assess population density by detecting a particular autoinducer whose concentration is correlated with cell density (Schauder and Bassler, 2001). This 'census-taking' enables the group to express specific genes only at particular population densities. In general, processes controlled by quorum sensing are ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, sporulation and conjugation. Thus, quorum sensing allows bacteria to act as multicellular organisms (de Kievit and Iglewski, 2000; Miller and Bassler, 2001). Quorum sensing regulates bioluminescence (Lux) in Vibrio harveyi, a free-living Gram-negative marine bacterium. When associated with eukaryotes, V.harveyi commonly is a member of the commensal microflora; however, V.harveyi is also a potent shrimp pathogen (Alvarez et al., 1998). Most Gram-negative bacterial quorum-sensing systems are composed of a LuxI-dependent acyl homoserine lactone (HSL) signal molecule and a LuxR-type autoinducer-binding transcriptional regulator protein. In contrast, the V.harveyi quorum-sensing circuit consists of a multichannel two-component phosphorelay signal transduction pathway (Bassler et al., 1993, 1994a). This intricate circuit is proposed to facilitate intra- and interspecies cell–cell communication and to provide V.harveyi with a mechanism to monitor both the population density and species composition of the bacterial community (Bassler, 1999). In V.harveyi, the expression of bioluminescence depends on the production and detection of two different autoinducers, AI-1 and AI-2 (Figure 1). The autoinducer synthase LuxLM produces AI-1, which is 4-hydroxyl C4 HSL (Cao and Meighen, 1989; Bassler et al., 1993). AI-2, synthesized by the LuxS enzyme, is the furanosyl borate diester 3A-methyl-5,6-dihydro-furo [2,3-D][1,3,2] dioxa borole-2,2,6,6A tetraol (Bassler et al., 1993; Surette et al., 1999; Schauder et al., 2001; Chen et al., 2002). AI-1 and AI-2 are detected via their cognate sensors LuxN and LuxPQ, respectively (Bassler et al., 1993, 1994a; Freeman et al., 2000). LuxP is homologous to the Escherichia coli periplasmic ribose-binding protein. LuxP binds AI-2 in the periplasm, and our data suggest that the LuxP–AI-2 complex interacts with LuxQ to transduce the AI-2 signal (Bassler et al., 1994a; Chen et al., 2002). LuxN and LuxQ are hybrid two-component sensor kinases, containing periplasmic sensory domains, and cytoplasmic histidine kinase and response regulator domains. Figure 1.The V.harveyi quorum-sensing system. The low and high cell density states of the V.harveyi quorum-sensing system are shown (A and B, respectively). The components and their putative interactions are described in the text. H, D, IM, OM and H-T-H denote histidine, aspartate, inner membrane, outer membrane and helix–turn–helix, respectively. The 'P' in the circle signifies that signal transduction occurs by phosphorelay. Phosphate flow in the forward direction goes from histidine (H1) to aspartate (D1) to histidine (H2) to aspartate (D2). AI-1 and AI-2 are depicted as pentagons and triangles, respectively. Download figure Download PowerPoint At low cell density (Figure 1A), LuxN and LuxQ act as kinases and transfer phosphate to the shared phosphotransferase protein LuxU (Freeman and Bassler, 1999b). LuxU transmits the phosphate to the response regulator protein LuxO (Bassler et al., 1994b; Freeman and Bassler, 1999a). Together with the alternative sigma factor σ54, LuxO is hypothesized to activate the expression of an as yet unidentified repressor 'X', which negatively regulates luxCDABE (luciferase) expression, and V.harveyi does not make light (Lilley and Bassler, 2000). At high cell density (Figure 1B), the sensors LuxN and LuxPQ detect their cognate autoinducers, AI-1 and AI-2, respectively, which convert LuxN and LuxQ from kinases to phosphatases (Freeman et al., 2000). This action reverses the flow of phosphate through the pathway, from LuxO to LuxU, and finally to LuxN and LuxQ, where the phosphoryl group is hydrolyzed. Dephosphorylation of LuxO inactivates it and terminates the expression of the repressor X. A transcriptional activator, LuxR (not similar to other LuxR-type quorum-sensing proteins) binds the luxCDABE promoter, activates transcription, and light is produced (Martin et al., 1989). The use of two autoinducers to control quorum sensing in V.harveyi is intriguing, as one autoinducer is sufficient for density-dependent gene regulation. We suggested previously that the two autoinducers play distinct roles in V.harveyi quorum sensing. This assertion stems from two findings: (i) AI-1 is highly specific and its known production thus far is limited to V.harveyi and the closely related Vibrio species Vibrio parahaemolyticus; and (ii) AI-2 production and the AI-2 synthase, LuxS, are widespread in bacteria and to date have been shown to exist in >40 Gram-negative and Gram-positive bacterial species (Bassler et al., 1997; Surette et al., 1999; Miller and Bassler, 2001). Thus, we proposed that AI-1 and AI-2 are used by V.harveyi for intra- and interspecies communication, respectively, which allows V.harveyi to distinguish between situations when it exists predominately as a mono-culture versus situations in which it exists in a consortium (Bassler et al., 1997; Surette et al., 1999; Schauder and Bassler, 2001). To understand further how intra- and interspecies communication is achieved in V.harveyi, in the present work, we identify genes in addition to luxCDABE that are members of the V.harveyi quorum-sensing regulon, and we determine the mechanism of their regulation. We investigate the individual roles of AI-1 and AI-2 in quorum-sensing gene regulation and show that AI-1 and AI-2 act synergistically. Because the V.harveyi quorum-sensing circuit responds to two signals, AI-1 and AI-2, there are at least four different input states: no autoinducer, AI-1 only, AI-2 only, and AI-1 + AI-2. Our measurements of light production show that the circuit has the potential to distinguish between all four states. However, under the conditions of our experiments, the V.harveyi quorum-sensing circuit appears to function primarily as a coincidence detector that differentiates between the presence of both autoinducers and all other input states. Results A screen for genes regulated by AI-2 We have shown that, in addition to Lux, quorum sensing regulates siderophore production and colony morphology (Lilley and Bassler, 2000). We suspected that quorum sensing controls additional behaviors and, further, that AI-1 and AI-2 could have distinct functions in cell–cell communication. To extend our definition of the quorum-sensing regulon and to investigate the precise roles of AI-1 and AI-2, we performed screens to identify genes specifically regulated by AI-1 and AI-2. Here we describe the experiments designed to identify and characterize AI-2-regulated genes; the AI-1 experiments will be reported elsewhere (Henke,J. and Bassler,B.L., in preparation). Random transposon Mini-MulacZ insertion mutagenesis was performed on the V.harveyi luxS (i.e. AI-1+, AI-2−) strain MM30. A total of 6500 insertion mutants were arrayed onto agar grids, allowed to grow into colonies and subsequently stamped to agar plate grids supplemented with 10% (v/v) cell-free culture fluids prepared from either an AI-1−, AI-2+ or an AI-1−, AI-2− V.harveyi strain. We compared the activity of the lacZ transcriptional fusions on the two sets of plates using X-gal, and identified 10 fusions that are regulated specifically by AI-2. Nine of the fusions showed reduced β-galactosidase (β-gal) activity on the Petri plates containing AI-2 compared with plates lacking AI-2. To verify and quantify the activities of the fusions, β-gal assays were performed (Figure 2A). Prior to analysis, the strains were grown in broth containing either 10% (v/v) AI-1−, AI-2− or AI-1−, AI-2+ cell-free culture fluid (white and black bars, respectively). The fusions displayed a wide range of basal activities and extent of regulation by AI-2. Specifically, the fusion in KM87 was induced 25-fold in the presence of AI-2, and the other targets were repressed from 9- to 120-fold. To verify that the fusions were regulated by AI-2 and not by some unidentified component present in the cell-free culture fluid, the wild-type luxS gene was restored in each of the 10 insertion mutant strains. The β-gal activity of each lacZ fusion was measured in the corresponding luxS+ and luxS− strains and compared. These results (Figure 2B) show a similar pattern to that obtained by adding AI-2+ and AI-2− cell-free culture fluids. Figure 2.Regulation of target gene expression by AI-2. β-Gal activities for the 10 lacZ target gene fusions listed in Table I are shown. (A) Activities following addition of 10% (v/v) V.harveyi stationary phase cell-free culture fluid lacking (white bars) and containing (black bars) AI-2. (B) Activities in luxS-null (i.e. AI-2−) and luxS wild-type (i.e. AI-2+) strain backgrounds (white and black bars, respectively). β-Gal units are defined as Vmax × 0.2/[OD550 of cells × volume (ml)]. Download figure Download PowerPoint Identification of the AI-2-regulated genes The Mini-MulacZ transposon used in the above screen contains inverted repeat sequences at its ends that interfere with PCR and preclude amplification and sequencing of the chromosome–transposon fusion junctions (Casadaban and Cohen, 1979; Metcalf et al., 1990). We therefore attempted to clone the transposon–chromosome junction from each strain, and we successfully cloned and sequenced six of the 10 fusion junctions. We have not been able to clone the other four targets so they remain unidentified. The clones are listed in Table I along with the predicted function of the genes based on database analysis. The AI-2-regulated genes encode a variety of putative functions including a secreted metalloprotease, three putative type III secretion system components and a conserved hypothetical protein. One open reading frame has no significant homology to any protein in the database. The most notable of these targets are the putative type III components as, to our knowledge, Vibrios are not known to possess such secretory systems. We have cloned additional genes encoding components of the secretory apparatus and currently are investigating their functions. Table 1. Autoinducer-regulated target genes Strain Putative function Gene name Accession No. % Similarity KM87 Metalloprotease proAC BAA82875 63 KM92 Putative type III secretion proteina NA NA NA KM93 No sequence homology NA NA NA KM94 Type III secretion protein pscT AAG05080 82 KM96 Unknownb KM100 Unknownb KM108 Conserved hypothetical protein VC2647c E82049 87 KM114 Type III secretion protein popB AAC45937 42 KM118 Unknownb KM121 Unknownb NA = not available. a This gene is inferred to encode a poorly conserved type III secretion protein due to its specific location in a type III secretion operon. b Unknown denotes that these fusions were not sequenced (see text). c VC numbers refer to V.cholerae genome annotations. The Lux circuit regulates the target genes The above screen was designed to identify genes specifically controlled by AI-2. Importantly, in this experiment, we used a V.harveyi strain that was incapable of producing AI-2 but produced wild-type levels of AI-1. Therefore, this screen allowed us to identify genes whose expression remained controllable by AI-2 in the presence of high levels of AI-1. We considered two possible mechanisms that could account for this pattern of gene regulation. First, an AI-2 quorum-sensing detection–response mechanism that is distinct from, or diverges from the one we have already identified (Figure 1) could control some or all of the 10 target genes. If so, the presence of AI-1 is irrelevant for control of these genes. Secondly, the known quorum-sensing pathway (Figure 1) could control the targets. In this case, the presence of AI-1 must not cause the complete inactivation (i.e. dephosphorylation) of LuxO, or the targets could not respond to the addition of AI-2. To test if the V.harveyi quorum-sensing circuit controls the targets, we introduced a dominant allele of LuxO and measured its effect on expression of the fusions. This protein, LuxO D47E, contains a missense mutation at the phosphorylation site that 'locks' it into a form mimicking active, LuxO-P (Freeman and Bassler, 1999a). Cells containing LuxO D47E are Lux− because the circuit is fixed in the low cell density state (Figure 1A). Additionally, the activity of LuxO D47E is not modulated by the autoinducers. If an unidentified circuit controls the 10 targets, introduction of LuxO D47E into the lacZ fusion strains should have no effect on their expression. If, on the other hand, the known quorum-sensing system controls the newly identified targets, then introduction of LuxO D47E would lock the expression of the targets into the low cell density state, and render the targets unresponsive to AI-2. To assess whether regulation of the target fusions depends on LuxO, strains containing LuxO D47E were grown in the presence of 10% (v/v) cell-free culture fluid from an AI-1−, AI-2− strain or an AI-1−, AI-2+ strain. Subsequently, β-gal assays were performed, and Figure 3 shows the results for the AI-2 activated target gene (KM87; encoding a metalloprotease) and for one representative AI-2 repressed target gene (KM114; encoding the type III secretion PopB homolog). Introduction of LuxO D47E eliminated AI-2 regulation of the transcriptional fusions. Specifically, the left panel of Figure 3 shows that, in the presence of the vector (denoted pLAFR), lacZ transcription in strain KM87 was induced 5-fold upon addition of AI-2. However, when LuxO D47E was present (denoted D47E), transcription of lacZ was very low in both the absence and presence of AI-2. The right panel of Figure 3 shows that in the absence of AI-2, high lacZ activity was produced by strain KM114 containing the empty vector (pLAFR), and the lacZ activity decreased 10-fold when AI-2 was present. Following introduction of LuxO D47E, KM114 displayed high lacZ transcription in both the absence and presence of AI-2. The other eight negatively regulated targets behaved identically to KM114 when LuxO D47E was introduced (data not shown). We conclude that the known quorum-sensing circuit controls the fusions because each depends on LuxO for regulation. These and earlier results show that at low cell density, LuxO-P induces target genes such as those required for siderophore production (Lilley and Bassler, 2000), popB in KM114 and the eight additional targets listed in Table I. Additionally, at low cell density, LuxO-P activates expression of X that represses targets such as luxCDABE and the metalloprotease gene in KM87. Figure 3.The Lux signal transduction circuit controls the AI-2-regulated target genes. β-Gal activities of two representative AI-2-regulated lacZ fusions strains are shown. KM87 (A) has a fusion to a metalloprotease gene, and KM114 (B) has a fusion to a putative type III secretion popB-like gene. White and black bars denote the −AI-2 and +AI-2 conditions, respectively. AI-2 was supplied in 10% (v/v) cell-free culture fluids. pLAFR2 is the vector control, and D47E shows the result when the constitutively active luxO D47E allele was introduced on the pLAFR2 cosmid. Download figure Download PowerPoint Both AI-1 and AI-2 control the target genes The results of the LuxO D47E experiment were puzzling because it was not clear how the activity/phosphorylation state of LuxO could be modulated by AI-2 in the presence of high levels of AI-1. We wanted to verify that addition of AI-2 impacts LuxO activity in the presence of AI-1, and to show that the above results are not a consequence of the presence of a constitutive gain-of-function allele. Since both AI-1 and AI-2 channel information to LuxO, we reasoned that, in addition to AI-2, AI-1 should regulate the expression of the 10 targets. To test if AI-1 affects expression of the AI-2 regulated lacZ fusions, we needed to assay LacZ activity in V.harveyi strains that did not produce AI-1 so that we could add it exogenously. To do this, we constructed an in-frame deletion of the AI-1 synthase luxLM on the chromosome of each fusion strain. Thus, we engineered 10 strains that produced neither AI-1 nor AI-2, and each strain contained a different AI-2-regulated lacZ fusion (see Figure 2; Table I). Subsequently, we measured lacZ transcription following the addition of cell-free culture fluids containing no autoinducer, only AI-1, only AI-2, or both AI-1 and AI-2. Figure 4 shows the results for the AI-1−, AI-2− derivative of the activated fusion KM87 (this strain is called KM314, Figure 4A) and the AI-1−, AI-2− derivative of the representative repressed fusion KM114 (this strain is called KM321, Figure 4B). Again, the eight additional repressed fusions behaved identically to KM321. As expected, transcription of the fusion in KM314 was low in the absence of autoinducers and high in the presence of both autoinducers (1 and 10 U, respectively). The fusion in KM321 was regulated by the autoinducers in a reciprocal manner (550 U in the absence and 40 U in the presence of both autoinducers). However, in both cases, fluids containing only AI-1 or only AI-2 did not significantly impact expression of the lacZ fusions, showing that the autoinducers act synergistically to control gene expression. Figure 4.AI-1 and AI-2 act synergistically. β-Gal activities of the fusions in the luxS, luxLM derivatives of strains KM87 (KM314) and KM114 (KM321), and light production of the luxLM, luxS (AI-1−, AI-2−) strain KM135 are shown in (A), (B) and (C), respectively. The following V.harveyi cell-free culture fluids were added at 10% (v/v): MM77 (no AI), MM30 (AI-1), BB152 (AI-2), BB120 (AI-1 + AI-2). Note the logarithmic scale in (C); the inset shows the identical data plotted on a linear scale. Relative light units (RLU) are defined as c.p.m. × 103/c.f.u./ml. Download figure Download PowerPoint The finding that the target genes are only induced or repressed when both autoinducers are present together, while striking, should be considered in light of the method used in their initial identification. As discussed earlier, the parent strain used in the screen has an AI-1+, AI-2− phenotype. The bank of insertion strains was screened for differential LacZ activity on plates with and without added AI-2. Thus, the conditions on the screening plates were AI-1+, AI-2− versus AI-1+, AI-2+. Two possible classes of target genes could be identified in this experiment: (i) genes requiring only AI-2 for regulation; and (ii) genes requiring both AI-1 and AI-2 for regulation. We only identified target genes in the latter class. This screen is not saturated, so it is possible that additional genes exist requiring both AI-1 and AI-2 for their control as well as genes requiring only AI-2 for their regulation. In an experiment similar to those in Figure 4A and B, we measured light production in an AI-1−, AI-2− strain (KM135) following the addition of neither, one or both autoinducers. We observed (Figure 4C) <1 RLU (light production/cell) in the absence of both autoinducers, and 106 RLU when both autoinducers were added. When only AI-1 was present, 104 RLU were observed, and the addition of fluids with AI-2 alone resulted in 102 RLU. Our measurements thus distinguish four discrete levels of light output. However, these differences may not reflect distinct physiological states of V.harveyi. It is important to consider that alone, AI-1 and AI-2 stimulate only 1 and 0.01%, respectively, of the amount of light production that both autoinducers together stimulate. To depict this situation more clearly, we have plotted the Lux data on a linear scale in the inset to Figure 4C. Possibly, the low levels of light induced by AI-1 or AI-2 alone represent 'leakage' from a system that is designed to detect only the simultaneous presence of both autoinducers. AI-1 and AI-2 act synergistically To better understand how AI-1 and AI-2 jointly regulate gene expression, we analyzed the effect of varying the levels of AI-1 and AI-2 on expression of the target genes. Our goal was to understand if the autoinducers play equivalent roles or if one autoinducer plays a major role and one a minor role in gene regulation. We varied the ratio of AI-1 to AI-2 present in the experiments by adding different mixtures of cell-free culture fluids from AI-1+, AI-2− and AI-1−, AI-2+ strains. We held constant the total amount of cell-free culture fluids supplied to the growth medium at 10% (v/v) so that we could compare these results with those in Figures 2,3,4. Figure 5A and B shows the results of β-gal assays for KM314 and KM321, respectively, and Figure 5C shows the luxCDABE expression data from KM135. In each panel, the two left-most bars show the controls when cell-free culture fluid containing no autoinducers (denoted no AI) and wild-type culture fluid containing both autoinducers at their native ratio (denoted AI-1 + AI-2) were added. These controls show the minimal and maximal levels of expression of the lacZ fusions and of luxCDABE. As in Figure 4, fluid with AI-1 alone or fluid with AI-2 alone did not dramatically alter gene expression, whereas every combination of the two autoinducers significantly changed transcription of the genes. In the case of the lacZ fusions, each was regulated appreciably (activated or repressed) when as little as 1% (v/v) cell-free culture fluid containing either autoinducer was added in combination with 9% (v/v) of the fluid containing the other autoinducer. In the case of luxCDABE expression, exactly as in Figure 4C, each autoinducer alone stimulated only a small amount of light production compared with both autoinducers together, although AI-1 alone had a stronger inducing effect than AI-2 alone. Figure 5.Non-native ratios of AI-1 and AI-2 properly regulate target gene expression. β-gal activity (KM314 and KM321) and light output (KM135) are shown, respectively, in (A), (B) and (C) (logarithmic scale), for conditions in which different mixtures of cell-free culture fluids were added at total concentrations of 10% (v/v). The AI-1-containing culture fluid was prepared from MM30, the AI-2 fluid from BB152, the AI-1 + AI-2 fluid from BB120 and the no AI fluid from MM77. Download figure Download PowerPoint Low levels of autoinducers control gene expression We wanted to determine how gene expression is modulated when we maintained the native ratio of AI-1:AI-2 but varied the total amount of signal present and, in so doing, to determine the amount of autoinducer required for half-maximal activation (KM314), repression (KM321) or luxCDABE expression (KM135). To do this, we added different amounts of cell-free culture fluid prepared from wild-type V.harveyi (i.e. AI-1+, AI-2+) to the various strains. Half-maximal response was achieved when 4% (v/v) wild-type cell-free culture fluid was added to KM314 (Figure 6A), and half-maximal repression (KM321, Figure 6B) occurred following the addition of 2% (v/v) wild-type culture fluid. Less autoinducer was required for luxCDABE regulation, as significant activation occurred at 0.5% (v/v) and full activation occurred when 2% (v/v) culture fluid was added (Figure 6C). Together, the results shown in Figures 5 and 6 suggest first, that both AI-1 and AI-2 play major roles in quorum-sensing gene regulation in V.harveyi, and, secondly, that even if the amount of one signal is much lower than the other signal, or only low levels of both signals are present, the combination of the two signals is effective for quorum-sensing gene regulation. Figure 6.Low concentrations of the autoinducers are sufficient for gene regulation. β-Gal activity was measured for KM314 (A) and KM321 (B), and light emission for KM135 (C; logarithmic scale) following the addition of various amounts (v/v) of V.harveyi BB120 (wild-type; AI-1+, AI-2+) culture fluid. Download figure Download PowerPoint Both AI-1 and AI-2 are required for gene regulation Figures 4,5,6 show that low levels of the autoinducers have significant effects on V.harveyi gene expression. However, in each of the above experiments, the total autoinducer added was equivalent to that present in ≤10% (v/v) V.harveyi cell-free culture fluids. Much more autoinducer could be present in 100% V.harveyi stationary phase culture fluids. We could not add 100% fluids to V.harveyi because these preparations have toxic effects, presumably from stationary phase by-products that inhibit growth. To test the effects of higher concentrations of AI-1 and AI-2 on V.harveyi gene regulation, we added AI-1 and AI-2 that we had prepared by in vitro procedures. AI-1 was prepared by the method of Cao and Meighen (1989) and AI-2 by the method of Schauder et al. (2001). AI-1 and AI-2 are estimated to be present in V.harveyi stationary phase fluids at 10–20 μM (AI-1) and 20–50 μM (AI-2). We added the autoinducers at 20 μM (1× wild-type concentration) and 100 μM (5× wild-type concentration), and monitored lacZ and luxCDABE expression (Figure 7). As observed above, individually, each autoinducer had only a minor impact on lacZ expression. Even at ∼5-fold higher concentration of autoinducer than that estimated to be present at stationary phase, neither AI-1 nor AI-2 alone dramatically modulated lacZ or luxCDABE gene transcription. Specifically, in KM314, 5-fold excess AI-1 or AI-2 had a <10% effect on the activation of lacZ transcription (Figure 7A), and in KM321, the highest concentration of autoinducer caused 50% repression of lacZ (Figure 7B). Individually, 1× or 5× AI-1 induced 33%, and 1× or 5× AI-2 induced 0.01% of the level of light production induced by the autoinducers combined (Figure 7C). Figure 7.High concentrations of the individual autoinducers do not regulate gene expression. (A, B and C) The β-gal activity (KM314 and KM321) and light production (KM135; logarithmic scale), respectively, in the absence and presence of AI-1 and/or AI-2 prepared by in vitro methods. 1× and 5× denote the addition of 20 and 100 μM of the specified synthetic autoinducer. Download figure Download PowerPoint A model for Lux signal transduction We have suggested that LuxO-P activates the expression of a putative function X, which represses (directly or indirectly) the expression of the luciferase operon luxCDABE (Figure 1). If repress
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