Lux Box Research Articles

Redesigning regulatory components of quorum-sensing system for diverse metabolic control

Quorum sensing (QS) is a ubiquitous cell–cell communication mechanism that can be employed to autonomously and dynamically control metabolic fluxes. However, since the functions of genetic components in the circuits are not fully understood, the developed QS circuits are still less sophisticated for regulating multiple sets of genes or operons in metabolic engineering applications. Here, we discover the regulatory roles of a CRP-binding site and the lux box to −10 region within luxR-luxI intergenic sequence in controlling the lux-type QS promoters. By varying the numbers of the CRP-binding site and redesigning the lux box to −10 site sequence, we create a library of QS variants that possess both high dynamic ranges and low leakiness. These circuits are successfully applied to achieve diverse metabolic control in salicylic acid and 4-hydroxycoumarin biosynthetic pathways in Escherichia coli. This work expands the toolbox for dynamic control of multiple metabolic fluxes under complex metabolic background and presents paradigms to engineer metabolic pathways for high-level synthesis of target products.

Characterization of differentiated autoregulation of LuxI/LuxR-type quorum sensing system in Pseudoalteromonas

Gram-negative bacteria usually use acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR-type quorum sensing (QS) systems for cell-cell cooperation and/or bacteria-environment communication. LuxI and LuxR are AHLs synthase and receptor, respectively. These two parts could form a positive regulatory feedback loop, controlling various types of group behaviors. However, the autoregulation mechanisms between them are fragmented and could be highly differentiated in different bacteria. Here, we clarified the autoregulation mechanism between LuxI and LuxR in Pseudoalteromonas sp. R3. YasI (LuxI in strain R3) synthesizes two types of AHLs, C8-HSL and 3-OH-C8-HSL. It is worth noting that YasR (LuxR in strain R3) only responds to C8-HSL rather than 3-OH-C8-HSL. YasR-C8HSL can activate the yasI transcription by recognizing "lux box" at yasI upstream. Interestingly, YasR can directly promote the yasR expression with AHL-independent manner, but AHL absence caused by the yasI-deficiency led to the significant decrease in the yasR expression. Further study demonstrated that the yasI-deficiency can result in the decrease in the yasR mRNA stability. Notably, both yasI-deficiency and yasR-deficiency led to the significant decrease in the expression of hfq encoding RNA chaperone. Therefore, it was speculated that not only YasR itself can directly regulate the yasR transcription, but YasR-C8HSL complex indirectly affects the yasR mRNA stability by regulating Hfq.

PsyR, a transcriptional regulator in quorum sensing system, binds lux box-like sequence in psyI promoter without AHL quorum sensing molecule and activates psyI transcription with AHL in Pseudomonas syringae pv. tabaci 6605

Quorum sensing (QS) is a mechanism for bacterial cell–cell communication using QS signals. N-acyl-homoserine lactones (AHLs), QS signals in Pseudomonas syringae pv. tabaci (Pta) 6605, are synthesized by an AHL synthase (PsyI) and recognized by the cognate transcription factor PsyR. To reveal the role of PsyR in virulence, we generated a ∆psyR mutant and complemented strains of Pta 6605 and found that the ∆psyR mutant is remarkably reduced in AHL production and ability to cause disease and propagate in host tobacco leaves. The phenotypes of complemented strains were restored to that of the wild type (WT). Because the ∆psyR mutant lost nearly all AHL production, we investigated the function of PsyR in the transcription of psyI and production of AHL. Electrophoretic mobility shift assays suggested that the recombinant PsyR protein binds the promoter region of psyI but not psyR without AHL. The addition of AHL did not significantly affect this binding. The binding core sequence of this region was identified as a 20-bp lux box-like sequence. To reveal the function of PsyR and AHL on psyI transcription, we constructed a psyI promoter::lacZYA chimeric reporter gene, and inserted it into the WT and ∆psyI mutant of Pta 6605. β-galactosidase activity increased in a bacterial density-dependent manner in the WT and also in a ∆psyI mutant after the addition of exogenous AHL. These results indicate that the solo PsyR binds the lux box in the psyI promoter and activates transcription in the concomitant presence of AHL.

Quorum-sensing based small RNA regulation for dynamic and tuneable gene expression.

Developing a dynamic regulation strategy is an essential step in establishing an automatic control system for manipulating metabolic fluxes and cellular behaviors. To broaden the extent of the application, a system that can generally control any gene of interest is demanded. Through characterization and optimization, the strategy repressed the immediate expression incrementally from 0 to 90% during culturing. Moreover, by changing single base pair in the lux box of the Plux promoter, the degree of repression of the target genomic gene was tuned to a difference of 70%. This strategy is expected to control metabolic flux without disrupting cell growth. We engineered bacterial small RNA to develop a pathway-independent strategy that can dynamically repress the expression of any gene at the posttranscription level.

Characterization of quorum sensing genes and N-acyl homoserine lactones in Citrobacter amalonaticus strain YG6

In the phylum of Proteobacteria, quorum sensing (QS) system is widely driven by synthesis and response of N-acyl homoserine lactone (AHL) signalling molecules. AHL is synthesized by LuxI homologue and sensed by LuxR homologue. Once the AHL concentration achieves a threshold level, it triggers the regulation of target genes. In this study, QS activity of Citrobacter amalonaticus strain YG6 which was isolated from clams was investigated. In order to characterise luxI/R homologues, the genome of C. amalonaticus strain YG6 (4.95 Mbp in size) was sequenced using Illumina MiSeq sequencer. Through in silico analysis, a pair of canonical luxI/R homologues and an orphan luxR homologue were identified and designated as camI, camR, and camR2, respectively. A putative lux box was identified at the upstream of camI. The camI gene was cloned and overexpressed in E. coli BL21 (DE3)pLysS. High-resolution triple quadrupole liquid chromatography mass spectrometry (LC-MS/MS) analysis verified that the CamI is a functional AHL synthase which produced multiple AHL species, namely N‑butyryl‑l‑homoserine lactone (C4-HSL), N‑hexanoyl‑l‑homoserine lactone (C6-HSL), N‑octanoyl‑l‑homoserine lactone (C8-HSL), N‑tetradecanoyl‑l‑homoserine lactone (C14-HSL) and N‑hexadecanoyl‑l‑homoserine lactone (C16-HSL) in C. amalonaticus strain YG6 and camI gene in recombinant E. coli BL21(DE3)pLysS. To our best knowledge, this is the first functional study report of camI as well as the first report describing the production of C14-HSL by C. amalonaticus.

Comparative analysis reveals regulatory motifs at the ainS/ainR pheromone-signaling locus of Vibrio fischeri

Vibrio fischeri uses the AinS/AinR pheromone-signaling system to control bioluminescence and other symbiotic colonization factors. The Ain system is thought to initiate cell-cell signaling at moderate cell densities and to prime the LuxI/LuxR signaling system. Here we compared and analyzed the ain locus from two V. fischeri strains and a Vibrio salmonicida strain to explore ain regulation. The ainS and ainR genes were predicted to constitute an operon, which we corroborated using RT-PCR. Comparisons between strains revealed a stark area of conservation across the ainS-ainR junction, including a large inverted repeat in ainR. We found that this inverted repeat in cis can affect accumulation of the AinS-generated pheromone N-octanoyl homoserine lactone, which may account for the previously unexplained low-signal phenotype of a ∆ainR mutant, although the mechanism behind this regulation remains elusive. We also extended the previous observation of a possible “lux box” LuxR binding site upstream of ainS by showing the conservation of this site as well as a second putative lux box. Using a plasmid-based reporter we found that LuxR can mediate repression of ainS, providing a negative feedback mechanism in the Ain/Lux signaling cascade. Our results provide new insights into the regulation, expression, and evolution of ainSR.

Engineering Vibrio fischeri transcriptional activator LuxR for diverse transcriptional activities.

To alter DNA binding specificity of Vibrio fischeri LuxR and to expand the toolbox for constructing synthetic networks. A mutation library (about 10,000 individuals) of the DNA binding domain of LuxR were generated. A genetic selection was performed to obtain LuxR mutants that recognize three lux box DNA variants that are not recognized by wild-type LuxR. Six LuxR mutants were identified. The evolved LuxR mutants were further characterized by measuring the transcriptional activities of different combinations of LuxR mutants and lux box variants. Varied transcriptional activities were found in these LuxR-lux box pairs. The background expressions of the evolved LuxR-lux box systems are more tightly regulated than the wild-type LuxR-lux box system. The LuxR transcriptional system was evolved to recognize three lux box DNAs which are not recognized by wild-type LuxR.

In vitro analysis of essential binding sites on the promoter of the Serratia marcescens spn operon with the quorum-sensing receptor SpnR.

The N-acylhomoserine lactone (AHL) receptor SpnR is a LuxR family protein that acts as a negative regulator of AHL-dependent quorum sensing (QS). SpnR binds to DNA in Serratia marcescens AS-1 via the spn box; however, the binding affinity of SpnR with the nucleotides on the spn box has not yet been investigated. In this study, we used an spn-box-modified sensor electrode, and quartz crystal microbalance analysis demonstrated a drastic reduction of the uptake of SpnR. The nucleotides G5 and C16 at the AHL-receptor complex-binding site are conserved in Gram-negative bacteria, including the lux box in Vibrio fischeri, the tra box in Agrobacterium tumefaciens, and the spn box in S. marcescens. Indeed, the affinity of SpnR to DNA was reduced to 8% by G5C substitution of the spn box. The affinity of SpnR tagged with maltose-binding protein to the immobilized gene promoter was reduced in the order of C16G and G5C substitutions, which corresponded with previous reports on the lux box. These results suggest that formation of hydrogen bonds at amino acid residues containing guanine at position 5 on a lux-box-like promoter universally contributes to the stability of the receptor complex, whose interaction initiates a sequential QS process in the LuxR family. Biotechnol. Bioeng. 2016;113: 2513-2517. © 2016 Wiley Periodicals, Inc.

Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli.

The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.

Modeling Analysis of Signal Sensitivity and Specificity by Vibrio fischeri LuxR Variants.

The LuxR protein of the bacterium Vibrio fischeri belongs to a family of transcriptional activators that underlie pheromone-mediated signaling by responding to acyl-homoserine lactones (-HSLs) or related molecules. V. fischeri produces two acyl-HSLs, N-3-oxo-hexanoyl-HSL (3OC6-HSL) and N-octanoyl-HSL (C8-HSL), each of which interact with LuxR to facilitate its binding to a “lux box” DNA sequence, thereby enabling LuxR to activate transcription of the lux operon responsible for bioluminescence. We have investigated the HSL sensitivity of four different variants of V. fischeri LuxR: two derived from wild-type strains ES114 and MJ1, and two derivatives of LuxRMJ1 generated by directed evolution. For each LuxR variant, we measured the bioluminescence induced by combinations of C8-HSL and 3OC6-HSL. We fit these data to a model in which the two HSLs compete with each other to form multimeric LuxR complexes that directly interact with lux to activate bioluminescence. The model reproduces the observed effects of HSL combinations on the bioluminescence responses directed by LuxR variants, including competition and non-monotonic responses to C8-HSL and 3OC6-HSL. The analysis yields robust estimates for the underlying dissociation constants and cooperativities (Hill coefficients) of the LuxR-HSL complexes and their affinities for the lux box. It also reveals significant differences in the affinities of LuxRMJ1 and LuxRES114 for 3OC6-HSL. Further, LuxRMJ1 and LuxRES114 differed sharply from LuxRs retrieved by directed evolution in the cooperativity of LuxR-HSL complex formation and the affinity of these complexes for lux. These results show how computational modeling of in vivo experimental data can provide insight into the mechanistic consequences of directed evolution.

Artificially constructed quorum-sensing circuits are used for subtle control of bacterial population density.

Vibrio fischeri is a typical quorum-sensing bacterium for which lux box, luxR, and luxI have been identified as the key elements involved in quorum sensing. To decode the quorum-sensing mechanism, an artificially constructed cell–cell communication system has been built. In brief, the system expresses several programmed cell-death BioBricks and quorum-sensing genes driven by the promoters lux pR and PlacO-1 in Escherichia coli cells. Their transformation and expression was confirmed by gel electrophoresis and sequencing. To evaluate its performance, viable cell numbers at various time periods were investigated. Our results showed that bacteria expressing killer proteins corresponding to ribosome binding site efficiency of 0.07, 0.3, 0.6, or 1.0 successfully sensed each other in a population-dependent manner and communicated with each other to subtly control their population density. This was also validated using a proposed simple mathematical model.

FimY of Salmonella enterica serovar Typhimurium functions as a DNA-binding protein and binds the fimZ promoter

Salmonella enterica serovar Typhimurium produces type 1 fimbriae with binding specificity to mannose residues. Elements involved in fimbrial structural biosynthesis, transport, and regulation are encoded by the fim gene cluster. FimZ, FimY, FimW, STM0551, and an arginine transfer RNA (fimU) were previously demonstrated to regulate fimbrial expression. The amino acid sequences of the C-terminal portion of FimY revealed similarity with those of LuxR-like proteins. Electrophoretic mobility shift assays indicated that FimY possessed DNA-binding capacity and bound a 605-bp DNA fragment spanning the intergenic region between fimY and fimZ, while a FimY protein harboring a double mutation in the C-terminal helix-turn-helix region containing a glycine (G) to aspartate (D) substitution at residue 189 and isoleucine (I) to lysine (K) substitution at residue 195 lost its ability to bind this DNA fragment. A lux box sequence (5′-TCTGTTATTACATAACAAATACT-3′) within the fimZ promoter was required for binding. None of the DNA fragments derived from the promoters for fimA, fimY, or fimW was shifted by FimY. Pull-down assays showed that there were physical protein/protein interactions between FimY and FimZ. We propose that in the regulatory circuit of type 1 fimbriae, FimY functions as a DNA-binding protein to activate fimZ, and a FimY-FimZ protein complex may form to regulate other fim genes. Confirming these proposals requires further study.

Complex pathways for regulation of pyrimidine metabolism by carbon catabolite repression and quorum sensing in Pseudomonas putida RU-KM3S

Pseudomonads are metabolically versatile microbes that employ complex regulatory networks to control gene expression, particularly with respect to carbon and nitrogen metabolism. The aim of this study was to characterise the regulatory networks that control pyrimidine metabolism (hydantoin-hydrolysing activity) in Pseudomonas putida strain RU-KM3S, focussing on transcriptional activation of dihydropyrimidinase (Dhp) and β-ureidopropionase (Bup), encoding dhp and bup, respectively. The two genes are arranged divergently on the chromosome and are separated by ORF1, encoding a putative transporter, which lies upstream of and in the same orientation as bup. The results from this study reveal that pyrimidine metabolism, as a function of Bup and Dhp activity in P. putida RU-KM3S, is controlled by a complex regulatory network including several global pathways in addition to induction by the substrate. Three major control pathways act at the level of transcriptional and include: (1) induction of transcriptional activation in the presence of hydantoin, (2) carbon catabolite repression mediated via a pathway independent of Crc and (3) quorum sensing that does not require a putative lux box located upstream of the dhp transcriptional start. Finally, the data suggest a minor role for the global regulators Anr, Vfr and Crc, likely through regulation of the activity of transcription factors interacting directly with the bup/ORF1-dhp promoter.

The flhDC gene affects motility and biofilm formation in Yersinia pseudotuberculosis

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mutated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIIIDeltaflhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regulatory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.

Characterization of a luxI/ luxR-type quorum sensing system and N-acyl-homoserine lactone-dependent regulation of exo-enzyme and antibacterial component production in Serratia plymuthica RVH1

Quorum sensing by means of N-acyl- l-homoserine lactones (AHLs) is widespread in Gram-negative bacteria, where diverse AHLs influence a wide variety of functions, even in a single genus such as Serratia. Here we report the identification and characterization of the quorum sensing system of Serratia plymuthica strain RVH1. This strain isolated from a raw vegetable processing line produces at least three AHLs which were identified as N-butanoyl- (C4-HSL), N-hexanoyl- (C6-HSL) and N-(3-oxo-hexanoyl)-homoserine lactone (3-oxo-C6-HSL). The identified LuxI homolog SplI synthesizes 3-oxo-C6-HSL, and influences the production of C4-HSL and C6-HSL, as splI gene inactivation resulted in loss of 3-oxo-C6-HSL production and smaller amounts of C4-HSL and C6-HSL produced. SplI-dependent quorum sensing controls 2,3-butanediol fermentation (previously reported) and the production of an extracellular chitinase, nuclease, protease and antibacterial compound. The identity of the latter is not yet elucidated, but appears to be different from the known antibacterial compounds produced by Serratia strains. SplR, the homolog of the LuxR regulator, appears to act as a repressor of synthesis of extracellular enzymes and antibacterial compound and to autorepress its own expression, probably by binding to a 21 bp lux box sequence.

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

The genome of the acidophilic, proteobacterium Acidithiobacillusferrooxidans, contains linked but divergently oriented genes, termed afel and afeR, whose predicted protein products are significantly similar to the LuxI and LuxR families of proteins. A possible promoter and Lux box are predicted upstream of afel. A cloned copy of afel, expressed in E. coli, encodes an enzyme that catalyzes the production of a diffusible compound identified by gas chromatography and mass spectrometry as an unsubstituted N-acyl homoserine lactone (AHL) of chain length C14. This AHL can be detected by a reporter strain of Sinorhizobium meliloti Rm41 suggesting that it is biologically active. The reporter strain also responds to extracts of the supernatant of A. ferrooxidans grown to early stationary phase in sulfur medium indicating that a diffusible AHL is produced by this microorganism. Semi-quantitative RT-PCR experiments indicate that afeI and afeR are expressed maximally in early stationary phase and are more expressed when A. ferrooxidans is grown in sulfur--rather than iron-containing medium. Given the predicted amino acid sequence and functional properties of AfeI and AfeR it is proposed that A. ferrooxidans has a quorum sensing system similar to the LuxI-LuxR paradigm.

Involvement of region 4 of the σ 70 subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing

Quorum sensing-dependent activation of the luminescence ( lux) genes of Vibrio fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl)- L-homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. In its active conformation, LuxR binds to a site known as the lux box centered at position −42.5 relative to the luxI transcriptional start site and is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli σ 70 subunit of RNAP in LuxR-dependent activation of the luxI promoter has been investigated. Single-round transcription assays were performed in the presence of purified LuxRΔN, the autoinducer-independent C-terminal domain of LuxR, and a variant RNAP which contained a C-terminally truncated σ 70 subunit devoid of region 4. Results indicated that region 4 is essential for LuxRΔN-dependent luxI transcription, therefore 16 single and two triple alanine substitutions in region 4.2 of σ 70 between amino acid residues 590 and 613 were examined for their effects on LuxR- and LuxRΔN-dependent transcription at the luxI promoter. Taken together, the analyses performed on these variants of RpoD suggest that some individual residues in region 4.2 are important to the mechanism of activator-dependent transcription initiation under investigation.

Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis.

Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso-active glycolipids containing one (mono-rhamnolipid) or two (di-rhamnolipid) L-rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono-rhamnolipid from dTDP-L-rhamnose and beta-hydroxydecanoyl-beta-hydroxydecanoate, whereas di-rhamnolipid is produced from mono-rhamnolipid and dTDP-L-rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamnosyltransferase involved in di-rhamnolipid (L-rhamnose-L-rhamnose-beta-hydroxydecanoyl-beta-hydroxydecanoate) production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved in the transfer of L-rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII-2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di-rhamnolipid, whereas mono-rhamnolipid was unaffected. In contrast, a null rhlA mutant (PAO1-rhlA) was incapable of producing both mono- and di-rhamnolipid. Complementation of mutant RTII-2 with plasmid pRTII-26 containing rhlC restored the level of di-rhamnolipid production in the recombinant to a level similar to that of the wild-type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A sigma54-type promoter for the PA1131-rhlC operon was identified, and a single transcriptional start site was mapped. Expression of the PA1131-rhlC operon was dependent on the P. aeruginosa rhl quorum-sensing system, and a well-conserved lux box was identified in the promoter region. The genetic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsible for the biosynthesis of di-rhamnolipid.

Quorum sensing in Vibrio fischeri: elements of the luxl promoter.

Although cell density-dependent regulation of the luminescence genes in Vibrio fischeri is a model for quorum sensing in Gram-negative bacteria, relatively little is known about the promoter of the luminescence operon. The luminescence operon is activated by the LuxR protein, which requires a diffusible acylhomoserine lactone signal. The lux box, a 20 bp inverted repeat, is located in the luxl promoter region and is required for LuxR-dependent induction of the luminescence genes. Using primer extension, we mapped the LuxR-dependent transcriptional start site of the lux operon to 19 bp upstream of the luxl start codon. This indicates that the lux box is centred at -42.5 bp from the start of transcription. To gain evidence about the location of the -10 sequence, we placed a consensus -35 hexamer at different locations relative to the luxl transcriptional start site and measured constitutive levels of luminescence in recombinant Escherichia coli. The strongest constitutive promoter contained a TATAGT hexamer 17 bp from the -35 consensus sequence and 6 bp from the transcriptional start site. We propose that this is the -10 hexamer. Also in recombinant E. coli, both half-sites of the lux box were required for LuxR-dependent gene activation and for activation by an autoinducer-independent, monomeric LuxR deletion protein. LuxR-dependent activation of luminescence was eliminated when the lux box was centred at -47.5, -52.5 and -62.5 with respect to the luxl transcriptional start site. Our evidence, taken together with other information, points to a model in which a LuxR dimer overlaps the -35 region of the luxl promoter and functions as an ambidextrous activator with each LuxR subunit interacting with a different region of RNA polymerase.

Quorum sensing in Vibrio fischeri: essential elements for activation of the luminescence genes.

LuxR is required for cell density-dependent activation of the Vibrio fischeri luminescence (lux) genes. It has not been possible to study full-length LuxR in vitro, but a polypeptide containing the C-terminal transcriptional-activator domain of LuxR (LuxRdeltaN) has been purified, and its binding to lux regulatory DNA has been investigated. By itself, LuxRdeltaN interacts with a region of lux regulatory DNA that is upstream of the lux box, which is a 20-bp element that is required for LuxR activation of the luminescence operon. Individually, neither the purified LuxRdeltaN nor RNA polymerase binds to the lux box region, but together the two proteins bind in synergy to the lux box-luxI promoter region. We show that binding of LuxRdeltaN to the upstream region is not a prerequisite for its synergistic binding with RNA polymerase to the lux box and the luxI promoter region. We also show that LuxRdeltaN and RNA polymerase are both required and sufficient for transcriptional activation of the lux operon. This argues against the hypothesis that LuxR functions to alleviate repression of the lux operon by another cellular factor. Rather, our data support the view that LuxR functions as an accessory factor that enables RNA polymerase to bind to and initiate transcription from the promoter of the lux operon.