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Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

Quorum-sensing systems mediate chemical communication between bacterial cells, coordinating cell-density-dependent processes like biofilm formation and virulence-factor expression. In the proteobacterial LuxI/LuxR quorum sensing paradigm, a signaling molecule generated by an enzyme (LuxI) diffuses between cells and allosterically stimulates a transcriptional regulator (LuxR) to activate its cognate promoter (pR). By expressing either LuxI or LuxR in positive feedback from pR, these versatile systems can generate smooth (monostable) or abrupt (bistable) density-dependent responses to suit the ecological context. Here we combine theory and experiment to demonstrate that the promoter logic of pR – its measured activity as a function of LuxI and LuxR levels – contains all the biochemical information required to quantitatively predict the responses of such feedback loops. The interplay of promoter logic with feedback topology underlies the versatility of the LuxI/LuxR paradigm: LuxR and LuxI positive-feedback systems show dramatically different responses, while a dual positive/negative-feedback system displays synchronized oscillations. These results highlight the dual utility of promoter logic: to probe microscopic parameters and predict macroscopic phenotype.

Author Summary

Bacterial cells constantly communicate with one another by exchanging chemical signals, which constitute a rich source of information about the proximity of friends or foes in the environment. These signals can be used to coordinate the actions of cells across a population. For example, pathogenic bacteria infecting a host can remain quiescent, only becoming virulent once they attain a sufficient cell density. Such coordination, regulated by so-called quorum-sensing systems, works on the following principle: every cell in the population secretes a specific chemical signal; the more cells there are, the more signal is generated; when individual cells sense that the signal has crossed some threshold, they launch a response. The nature of the response depends on the detailed molecular wiring of the secretion and sensing system, which can vary from species to species. It is often impossible to determine all these molecular details for any given system. Borrowing ideas from control theory, we show that this internal wiring can be largely ignored, and these systems can be considered as ‘black boxes’. Our experiments demonstrate that the measured input-output logic of the black box, which we term ‘promoter logic’, is sufficient to predict the diverse responses of different quorum-sensing systems.

Autoinducer 2 (AI-2) is the only species-nonspecific autoinducer known in bacteria and is produced by both gram-negative and gram-positive organisms. Consequently, it is proposed to function as a universal quorum-sensing signal for interaction between bacterial species. AI-2 is produced as the by-product of a metabolic transformation carried out by the LuxS enzyme. To separate the metabolic function of the LuxS enzyme from the signaling role of AI-2, we carried out a global transcriptome analysis of a luxS null mutant culture of Streptococcus mutans UA159, an important cariogenic bacterium and a crucial component of the dental plaque biofilm community, in comparison to a luxS null mutant culture supplemented with chemically pure 4,5-dihydroxy-2,3-pentanedione, the precursor of AI-2. The data revealed fundamental changes in gene expression affecting 585 genes (30% of the genome) which could not be restored by the signal molecule AI-2 and are therefore not caused by quorum sensing but by lack of the transformation carried out by the LuxS enzyme in the activated methyl cycle. All functional classes of enzymes were affected, including genes known to be important for biofilm formation, bacteriocin synthesis, competence, and acid tolerance. At the same time, 59 genes were identified whose transcription clearly responded to the addition of AI-2. Some of them were related to protein synthesis, stress, and cell division. Three membrane transport proteins were upregulated which are not related to any of the known AI-2 transporters. Three transcription factors were identified whose transcription was stimulated repeatedly by AI-2 addition during growth. Finally, a global regulatory protein, the δ subunit of the RNA polymerase (rpoE), was induced 147-fold by AI-2, representing the largest differential gene expression observed. The data show that many phenotypes related to the luxS mutation cannot be ascribed to quorum sensing and have identified for the first time regulatory proteins potentially mediating AI-2-based signaling in gram-positive bacteria.

Streptococcus pneumoniae is the leading cause of death in children worldwide and forms highly organized biofilms in the nasopharynx, lungs, and middle ear mucosa. The luxS-controlled quorum-sensing (QS) system has recently been implicated in virulence and persistence in the nasopharynx, but its role in biofilms has not been studied. Here we show that this QS system plays a major role in the control of S. pneumoniae biofilm formation. Our results demonstrate that the luxS gene is contained by invasive isolates and normal-flora strains in a region that contains genes involved in division and cell wall biosynthesis. The luxS gene was maximally transcribed, as a monocistronic message, in the early mid-log phase of growth, and this coincides with the appearance of early biofilms. Demonstrating the role of the LuxS system in regulating S. pneumoniae biofilms, at 24 h postinoculation, two different D39ΔluxS mutants produced ∼80% less biofilm biomass than wild-type (WT) strain D39 did. Complementation of these strains with luxS, either in a plasmid or integrated as a single copy in the genome, restored their biofilm level to that of the WT. Moreover, a soluble factor secreted by WT strain D39 or purified AI-2 restored the biofilm phenotype of D39ΔluxS. Our results also demonstrate that during the early mid-log phase of growth, LuxS regulates the transcript levels of lytA, which encodes an autolysin previously implicated in biofilms, and also the transcript levels of ply, which encodes the pneumococcal pneumolysin. In conclusion, the luxS-controlled QS system is a key regulator of early biofilm formation by S. pneumoniae strain D39.

Despite the fact that the regulatory system sensing density of cell population and its signaling molecule have been identified in Salmonella enterica, the biological significance of this phenomenon termed as quorum sensing remains unknown. In this report, we provide evidence that the luxS gene is necessary for Salmonella virulence phenotypes. Transcription assays showed that the cell-density-dependent induction of the invF gene was abolished in a Salmonella strain with the luxS gene deleted. The effect of the luxS deletion was also investigated in other InvF-regulated genes expressed from Salmonella pathogenicity island 1 (SPI-1). The decreased expression of SPI-1 genes in the strain with luxS deleted could be restored by either the addition of a synthetic signal molecule or the introduction of a plasmid copy of the luxS gene. Thus, the reduced expression of invF and its regulated genes in Salmonella cells lacking quorum sensing resulted in the attenuation of virulence phenotypes both in vitro and in vivo.

Nosocomial infections caused by Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. Quorum-sensing regulation plays a major role in the biofilm development of many bacterial pathogens. Here, we describe luxS, a quorum-sensing system in staphylococci that has a significant impact on biofilm development and virulence. We constructed an isogenic ΔluxS mutant strain of a biofilm-forming clinical isolate of S. epidermidis and demonstrated that luxS signaling is functional in S. epidermidis. The mutant strain showed increased biofilm formation in vitro and enhanced virulence in a rat model of biofilm-associated infection. Genetic complementation and addition of autoinducer 2-containing culture filtrate restored the wild-type phenotype, demonstrating that luxS repressed biofilm formation through a cell-cell signaling mechanism based on autoinducer 2 secretion. Enhanced production of the biofilm exopolysaccharide polysaccharide intercellular adhesin in the mutant strain is presumably the major cause of the observed phenotype. The agr quorum-sensing system has previously been shown to impact biofilm development and biofilm-associated infection in a way similar to that of luxS, although by regulation of different factors. Our study indicates a general scheme of quorum-sensing regulation of biofilm development in staphylococci, which contrasts with that observed in many other bacterial pathogens.

The role of LuxS in Shewanella oneidensis MR-1 has been examined by transcriptomic profiling, biochemical, and physiological experiments. The results indicate that a mutation in luxS alters biofilm development, not by altering quorum-sensing abilities but by disrupting the activated methyl cycle (AMC). The S. oneidensis wild type can produce a luminescence response in the AI-2 reporter strain Vibrio harveyi MM32. This luminescence response is abolished upon the deletion of luxS. The deletion of luxS also alters biofilm formations in static and flowthrough conditions. Genetic complementation restores the mutant biofilm defect, but the addition of synthetic AI-2 has no effect. These results suggest that AI-2 is not used as a quorum-sensing signal to regulate biofilm development in S. oneidensis. Growth on various sulfur sources was examined because of the involvement of LuxS in the AMC. A mutation in luxS produced a reduced ability to grow with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and to recycle homocysteine. These data suggest that LuxS is important to metabolizing methionine and the AMC in S. oneidensis.

Bacterial cell-to-cell communication, termed quorum sensing (QS), controls bacterial behavior by using various signal molecules. Despite the fact that the LuxS/autoinducer-2 (AI-2) QS system is necessary for normal expression of Salmonella pathogenicity island-1 (SPI-1), the mechanism remains unknown. Here, we report that the LsrR protein, a transcriptional regulator known to be involved in LuxS/AI-2-mediated QS, is also associated with the regulation of SPI-1-mediated Salmonella virulence. We determined that LsrR negatively controls SPI-1 and flagella gene expressions. As phosphorylated AI-2 binds to and inactivates LsrR, LsrR remains active and decreases expression of SPI-1 and flagella genes in the luxS mutant. The reduced expression of those genes resulted in impaired invasion of Salmonella into epithelial cells. Expression of SPI-1 and flagella genes was also reduced by overexpression of the LsrR regulator from a plasmid, but was relieved by exogenous AI-2, which binds to and inactivates LsrR. These results imply that LsrR plays an important role in selecting infectious niche of Salmonella in QS dependent mode.

Acyl-homoserine lactone (acyl-HSL) quorum-sensing signaling is common to many Proteobacteria. Acyl-HSLs are synthesized by the LuxI family of synthases, and the signal response is mediated by members of the LuxR family of transcriptional regulators. Burkholderia thailandensis is a member of a closely related cluster of three species, including the animal pathogens Burkholderia mallei and Burkholderia pseudomallei. Members of this group have similar luxI and luxR homologs, and these genes contribute to B. pseudomallei and B. mallei virulence. B. thailandensis possesses three pairs of luxI-luxR homologs. One of these pairs, BtaI2-BtaR2, has been shown to produce and respond to 3OHC10-HSL and to control the synthesis of an antibiotic. By using a markerless-exhange method, we constructed an assortment of B. thailandensis quorum-sensing mutants, and we used these mutants to show that BtaI1 is responsible for C8-HSL production and BtaI3 is responsible for 3OHC8-HSL production. We also show that a strain incapable of acyl-HSL production is capable of growth on the same assortment of carbon and nitrogen sources as the wild type. Furthermore, this mutant shows no loss of virulence compared to the wild type in mice. However, the wild type self-aggregates in minimal medium, whereas the quorum-sensing mutant does not. The wild-type aggregation phenotype is recovered by addition of the BtaI1-R1 HSL signal C8-HSL. We propose that the key function of the BtaR1-BtaI1 quorum-sensing system is to cause cells to gather into aggregates once a sufficient population has been established.

Vibrio fischeri quorum sensing involves the LuxI and LuxR proteins. The LuxI protein generates the quorum-sensing signal N-3-oxohexanoyl-l-homoserine lactone (3OC6-HSL), and LuxR is a signal-responsive transcriptional regulator which activates the luminescence (lux) genes and 17 other V. fischeri genes. For activation of the lux genes, LuxR binds to a 20-base-pair inverted repeat, the lux box, which is centered 42.5 base pairs upstream of the transcriptional start of the lux operon. Similar lux box-like elements have been identified in only a few of the LuxR-activated V. fischeri promoters. To better understand the DNA sequence elements required for LuxR binding and to identify binding sites in LuxR-regulated promoters other than the lux operon promoter, we have systematically mutagenized the lux box and evaluated the activity of many mutants. By doing so, we have identified nucleotides that are critical for promoter activity. Interestingly, certain lux box mutations allow a 3OC6-HSL-independent LuxR activation of the lux operon promoter. We have used the results of the mutational analysis to create a consensus lux box, and we have used this consensus sequence to identify LuxR binding sites in 3OC6-HSL-activated genes for which lux boxes could not be identified previously.

Cell-to-cell communication in bacteria is mediated by quorum-sensing systems (QSS) that produce chemical signal molecules called autoinducers (AI). In particular, LuxS/AI-2-dependent QSS has been proposed to act as a universal lexicon that mediates intra- and interspecific bacterial behavior. Here we report that the model organism Bacillus subtilis operates a luxS-dependent QSS that regulates its morphogenesis and social behavior. We demonstrated that B. subtilis luxS is a growth-phase-regulated gene that produces active AI-2 able to mediate the interspecific activation of light production in Vibrio harveyi. We demonstrated that in B. subtilis, luxS expression was under the control of a novel AI-2-dependent negative regulatory feedback loop that indicated an important role for AI-2 as a signaling molecule. Even though luxS did not affect spore development, AI-2 production was negatively regulated by the master regulatory proteins of pluricellular behavior, SinR and Spo0A. Interestingly, wild B. subtilis cells, from the undomesticated and probiotic B. subtilis natto strain, required the LuxS-dependent QSS to form robust and differentiated biofilms and also to swarm on solid surfaces. Furthermore, LuxS activity was required for the formation of sophisticated aerial colonies that behaved as giant fruiting bodies where AI-2 production and spore morphogenesis were spatially regulated at different sites of the developing colony. We proposed that LuxS/AI-2 constitutes a novel form of quorum-sensing regulation where AI-2 behaves as a morphogen-like molecule that coordinates the social and pluricellular behavior of B. subtilis.

Quorum-sensing (QS), the regulation of bacterial gene expression in response to changes in cell density, involves pathways that synthesize signaling molecules (auto-inducers). The luxS/AI-2-mediated QS system has been identified in both Gram-positive and Gram-negative bacteria. Bacillus anthracis, the etiological agent of anthrax, possesses genes involved in luxS/AI-2-mediated QS, and deletion of luxS in B. anthracis Sterne strain 34F2 results in inhibition of AI-2 synthesis and a growth defect. In the present study, we created a ΔluxS B. anthracis strain complemented in trans by insertion of a cassette, including luxS and a gene encoding erythromycin resistance, into the truncated plcR regulator locus. The complemented ΔluxS strain has restored AI-2 synthesis and wild-type growth. A B. anthracis microarray study revealed consistent differential gene expression between the wild-type and ΔluxS strain, including downregulation of the B. anthracis S-layer protein gene EA1 and pXO1 virulence genes. These data indicate that B. anthracis may use luxS/AI-2-mediated QS to regulate growth, density-dependent gene expression and virulence factor expression.

The moderately halophilic bacterium Halobacillus halophilus carries a homologue of LuxS, a protein involved in the activated methyl cycle and the production of autoinducer-2, which mediates quorum sensing between certain species. luxS of H. halophilus is part of an operon that encodes an S-adenosylmethionine-dependent methyltransferase, a cysteine synthase, and a β-cystathionine lyase. Expression of luxS was growth phase dependent, with maximal expression in the mid-exponential growth phase. In addition, transcription of luxS was strictly salt dependent; maximal mRNA concentrations were observed with 2.0 M NaCl in the growth medium. Chloride ions stimulated luxS transcription by a factor of three. Western blot analyses demonstrated a growth phase- and salinity-dependent production of LuxS. Moreover, cellular LuxS levels were strictly chloride dependent. Maximal accumulation of LuxS was observed at 0.5 to 1.0 M Cl− and depended on the salinity.

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxSSm) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.

Background

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.

Results

Analysis of additional S. Typhimurium luxS mutants indicated that the LuxS enzyme itself is not a prerequisite for a wild type mature biofilm. However, in close proximity of the luxS coding sequence, a small RNA molecule, MicA, is encoded on the opposite DNA strand. Interference with the MicA expression level showed that a balanced MicA level is essential for mature Salmonella biofilm formation. Several MicA targets known to date have previously been reported to be implicated in biofilm formation in Salmonella or in other bacterial species. Additionally, we showed by RT-qPCR analysis that MicA levels are indeed altered in some luxS mutants, corresponding to their biofilm formation phenotype.

Conclusions

We show that the S. Typhimurium biofilm formation phenotype of a luxS mutant in which the complete coding region is deleted, is dependent on the sRNA molecule MicA, encoded in the luxS adjacent genomic region, rather than on LuxS itself. Future studies are required to fully elucidate the role of MicA in Salmonella biofilm formation.

The LuxS protein is required for the biosynthesis of the type 2 autoinducer (AI-2), which is involved in quorum sensing in a wide range of bacterial species. We have determined the effects of a defined luxS mutation on the virulence of Streptococcus pneumoniae. Although the luxS mutant displayed reduced virulence relative to its wild-type parent, the type 2 strain D39, it was by no means avirulent in a mouse model. After intranasal administration, the luxS mutant was able to colonize the nasopharynx of the mouse as efficiently as the wild type. However, it was less able to spread from the nasopharynx to the lungs or the blood. Intraperitoneal coadministration studies indicated that the luxS mutant was less fit and was readily outcompeted by wild-type D39. However, when administered on its own by this route, the mutant was able to proliferate and cause fatal systemic disease, albeit at a lower rate than the wild type. Western blot analysis of whole-cell lysates of the mutant and its parent did not reveal any differences in the levels of several well-characterized virulence proteins. However, analysis of Coomassie blue-stained protein profiles after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that mutation of luxS had pleiotropic effects on protein expression in all cellular compartments. This is consistent with the product of luxS having a regulatory role in S. pneumoniae. This is the first report of a direct role for luxS (and by inference, AI-2) in the virulence of a gram-positive pathogen. However, the fact that mutagenesis of luxS does not completely attenuate S. pneumoniae has implications for the possible use of AI-2 antagonists for treatment of pneumococcal infections.

Streptococcus pneumoniae causes several diseases, including otitis media, pneumonia, and meningitis. Although little is known about the regulation of or how individual pneumococcal factors contribute to these disease states, there is evidence suggesting that some factors are regulated by a cell-density-dependent mechanism (quorum sensing). Quorum sensing allows bacteria to couple transcription with changes in cell density; bacteria achieve this by sensing and responding to small diffusible signaling molecules. We investigated how the LuxS signaling system impacts the biology of S. pneumoniae. An analysis of the transcriptional profiles of a serotype 2 strain and an isogenic luxS deletion strain utilizing an S. pneumoniae-specific microarray indicated that LuxS regulates gene expression involved in discrete cellular processes, including pneumolysin expression. Contrary to the paradigm for quorum sensing, we observed pronounced effects on transcription in early log phase, where gene expression was repressed in the mutant. Assessing the mutant for its ability to infect and cause disease in animals revealed a profound defect in ability to persist in the nasopharyngeal tissues. Our analysis of an S. pneumoniae transcriptome revealed a function for LuxS in gene regulation that is not dependent upon high cell density and is likely involved in the maintenance of pneumococcal load in susceptible hosts.

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.

Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P ≤ 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.

AI-2–mediated quorum sensing has been identified in various bacteria, including both Gram-negative and Gram-positive species, and numerous phenotypes have been reported to be regulated by this mechanism, using the luxS-mutant strain. But the AI-2 production process confused this regulatory function; some considered this regulation as the result of a metabolic change, which refers to an important metabolic cycle named activated methyl cycle (AMC), caused by luxS-mutant simultaneously with the defect of AI-2. Herein we hypothesized that the quorum sensing system—not the metabolic aspect—is responsible for such a regulatory function. In this study, we constructed plasmids infused with sahH and induced protein expression in the luxS-mutant strain to make the quorum-sensing system and metabolic system independent. The biofilm-related genes were investigated by real-time polymerase chain reaction (PCR), and the results demonstrated that the quorum-sensing completed strain restored the gene expression of the defective strain, but the metabolically completed one did not. This evidence supported our hypothesis that the autoinducer-2-mediated, quorum-sensing system, not the AMC, was responsible for luxS mutant regulation.

The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with ς70 RNA polymerase it can activate transcription of the lux operon. Both the truncated LuxR and RNA polymerase are required for binding to lux regulatory DNA in vitro. We have constructed an artificial lacZ promoter with the lux box positioned between and partially overlapping the consensus −35 and −10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli. Furthermore, multiple lux boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to lux boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a native lux operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in E. coli. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA.

The genome of the gastric pathogen Helicobacter pylori contains a homologue of the gene luxS, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the luxS gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the luxS gene and by the addition of cell-free supernatant containing AI-2. The flagella of the luxS mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators—the two-component sensor kinase flgS, the sigma factor σ28 (also called fliA), and the anti-sigma factor flgM—loss of luxS altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the luxS+ phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of luxS caused a decrease in transcript levels of the flagellar regulator flhA. Addition of DPD to luxS cells induced flhA transcription in a dose-dependent manner. Deletion of flhA in a wild-type or luxS mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in H. pylori.

Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.

The gram-positive pathogen Streptococcus pyogenes was recently reported to possess a homologue of the luxS gene that is responsible for the production of autoinducer 2, which participates in quorum sensing of both gram-positive and gram-negative bacteria. To test the effect of LuxS on streptococcal internalization, a LuxS mutant was constructed in strain SP268, an invasive M3 serotype. Functional analysis of the mutant revealed that it was internalized by HEp-2 cells with higher efficiency than the wild type (wt). Several genes, including hasA (hyaluronic acid synthesis), speB (streptococcal pyrogenic exotoxin B), and csrR (capsule synthesis regulator), a part of a two-component regulatory system, are known to affect the internalization of strain SP268 (J. Jadoun, O. Eyal, and S. Sela, Infect. Immun. 70:462-469, 2002). Therefore, the expression of these genes in the mutant and in the wt was examined. LuxS mutation significantly reduced the mRNA level of speB and increased the mRNA level of emm3. No substantial effect was observed on transcription of hasA and csrR. Yet less hyaluronic acid capsule was expressed in the mutant. Further analysis revealed that luxS is under the regulation of the two-component global regulator CsrR. Our results indicate that LuxS activity in strain SP268 plays an important role in the expression of virulence factors associated with epithelial cell internalization.

The luxS gene, present in many bacterial genera, encodes the autoinducer 2 (AI-2) synthase. AI-2 has been implicated in bacterial signaling, and this study investigated its role in biofilm formation by Streptococcus gordonii, an organism that colonizes human tooth enamel within the first few hours after professional cleaning. Northern blotting and primer extension analyses revealed that S. gordonii luxS is monocistronic. AI-2 production was dependent on nutritional conditions, and maximum AI-2 induction was detected when S. gordonii was grown in the presence of serum and carbonate. In planktonic cultures, AI-2 production rose sharply during the transition from exponential to stationary phase, and the AI-2 concentration peaked approximately 4 h into stationary phase. An S. gordonii luxS mutant that did not produce AI-2 was constructed by homologous recombination. Complementation of the mutant by insertion of an intact luxS gene into the chromosome in tandem with the disrupted gene restored AI-2 production to a level similar to that of the wild-type strain. In planktonic culture, no growth differences were observed between the mutant and wild-type strains when five different media were used. However, when grown for 4 h as biofilms in 25% human saliva under flow, the luxS mutant formed tall microcolonies that differed from those formed by the wild-type and complemented mutant strains. Biofilms of the luxS mutant exhibited finger-like projections of cells that extended into the flow cell lumen. Thus, the inability to produce AI-2 is associated with altered microcolony architecture within S. gordonii biofilms formed in saliva during a time frame consistent with initial colonization of freshly cleaned enamel surfaces.