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.
The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19.
Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp).
Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections.
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.
During infection, Streptococcus pneumoniae exists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role of luxS (encoding S-ribosylhomocysteine lyase) on biofilm formation by S. pneumoniae D39. Fe(III) strongly enhanced biofilm formation at concentrations of ≥50 μM, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression of luxS in wild-type D39. A luxS-deficient mutant (D39luxS) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressing luxS (D39luxS+) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39luxS exhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39luxS+ had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related to luxS expression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genes comD, comX, comW, cglA, and dltA and the murein hydrolase cbpD, which is associated with fratricide-dependent DNA release, all were directly related to luxS expression levels and were further upregulated by Fe(III). Moreover, mutagenesis of cbpD blocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and that luxS is a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation of luxS expression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.
A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms.
Comparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated.
As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR.
Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments.
These findings reveal genes associated with the thickness of biofilms of S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.
LuxS/AI-2 quorum sensing (QS) system involves the production of cell signaling molecules via luxS-based autoinducer-2 (AI-2). LuxS has been reported to plays critical roles in regulating various behaviors of bacteria. AI-2 is a byproduct of the catabolism of S-adenosylhomocysteine (SAH) performed by the LuxS and Pfs enzymes. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as an inability to produce AI-2 were observed in bacteria having a luxS mutant gene. In this study, the level of AI-2 activity exhibits a growth-phase dependence with a maximum in late exponential culture in SS. An SS strain that overexpressed luxS was constructed to comprehensively understand the function of AI-2. Overexpressed luxS was not able to increase the level of pfs expression and produce additional AI-2, and the bacteria were slower growing and produced only slightly more biofilm than the wild type. Thus, AI-2 production is not correlated with luxS transcription. luxS expression is constitutive, but the transcription of pfs is perhaps correlated with AI-2 production in SS.
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.
In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits.
The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum–sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study.
V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.
Vibrio scophthalmi; Biofilm formation; Quorum-sensing; AiiA; LuxS; Acyl homoserine lactone
Quorum sensing is involved in the regulation of multicellular behavior through communication via small molecules. Given the high number and diversity of the gastrointestinal microbiota, it is postulated that members of this community communicate to coordinate a variety of adaptive processes. AI-2 is suggested to be a universal bacterial signaling molecule synthesized by the LuxS enzyme, which forms an integral part of the activated methyl cycle. We have previously reported that the well-documented probiotic strain Lactobacillus rhamnosus GG, a human isolate, produces AI-2-like molecules. In this study, we identified the luxS homologue of L. rhamnosus GG. luxS seems to be located in an operon with a yxjH gene encoding a putative cobalamin-independent methionine synthase. In silico analysis revealed a methionine-specific T box in the leader sequence of the putative yxjH-luxS operon. However, transcriptional analysis showed that luxS is expressed mainly as a monocistronic transcript. Construction of a luxS knockout mutant confirmed that the luxS gene is responsible for AI-2 production in L. rhamnosus GG. However, this mutation also resulted in pleiotropic effects on the growth of this fastidious strain. Cysteine, pantothenate, folic acid, and biotin could partially complement growth, suggesting a central metabolic role for luxS in L. rhamnosus GG. Interestingly, the luxS mutant also showed a defect in monospecies biofilm formation. Experiments with chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione, coculture with the wild type, and nutritional complementation suggested that the main cause of this defect has a metabolic nature. Moreover, our data indicate that suppressor mutations are likely to occur in luxS mutants of L. rhamnosus GG. Therefore, results of luxS-related studies should be carefully interpreted.
The luxS gene in Shewanella oneidensis was shown to encode an autoinducer-2 (AI-2)-like molecule, the postulated universal bacterial signal, but the impaired biofilm growth of a luxS deficient mutant could not be restored by AI-2, indicating it might not have a signalling role in this organism.
Here, we provide further evidence regarding the metabolic role of a luxS mutation in S. oneidensis. We constructed a luxS mutant and compared its phenotype to a wild type control with respect to its ability to remove AI-2 from the medium, expression of secreted proteins and biofilm formation. We show that S. oneidensis has a cell-dependent mechanism by which AI-2 is depleted from the medium by uptake or degradation at the end of the exponential growth phase. As AI-2 depletion is equally active in the luxS mutant and thus does not require AI-2 as an inducer, it appears to be an unspecific mechanism suggesting that AI-2 for S. oneidensis is a metabolite which is imported under nutrient limitation. Secreted proteins were studied by iTraq labelling and liquid chromatography mass spectrometry (LC-MS) detection. Differences between wild type and mutant were small. Proteins related to flagellar and twitching motility were slightly up-regulated in the luxS mutant, in accordance with its loose biofilm structure. An enzyme related to cysteine metabolism was also up-regulated, probably compensating for the lack of the LuxS enzyme. The luxS mutant developed an undifferentiated, loosely-connected biofilm which covered the glass surface more homogenously than the wild type control, which formed compact aggregates with large voids in between.
The data confirm the role of the LuxS enzyme for biofilm growth in S. oneidensis and make it unlikely that AI-2 has a signalling role in this organism.
quorum sensing; autoinducer-2; Shewanella oneidensis; luxS; biofilm; secretome
Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.
Streptococcus gordonii and Streptococcus oralis are among the first bacterial species to colonize clean tooth surfaces. Both produce autoinducer-2 (AI-2): a family of inter-convertible cell–cell signal molecules synthesized by the LuxS enzyme. The overall aim of this work was to determine whether AI-2 alters interspecies interactions between S. gordonii DL1 and S. oralis 34 within dual-species biofilms in flowing human saliva. Based upon AI-2 bioluminescence assays, S. gordonii DL1 produced more AI-2 activity than S. oralis 34 in batch culture, and both were able to remove AI-2 activity from solution. In single-species, saliva-fed flowcell systems, S. oralis 34 formed scant biofilms that were similar to the luxS mutant. Conversely, S. gordonii DL1 formed confluent biofilms while the luxS mutant formed architecturally distinct biofilms that possessed twofold greater biovolume than the wild-type. Supplementing saliva with 0.1–10 nM chemically synthesized AI-2 (csAI-2) restored the S. gordonii DL1 luxS biofilm phenotype to that which was similar to the wild-type; above or below this concentration range, biofilms were architecturally similar to that formed by the luxS mutant. In dual-species biofilms, S. gordonii DL1 was always more abundant than S. oralis 34. Compared with dual-species, wild-type biofilms, the biovolume occupied by S. oralis 34 was reduced by greater than sevenfold when neither species produced AI-2. The addition of 1 nM csAI-2 to the dual-species luxS–luxS mutant biofilms re-established the biofilm phenotype to resemble that of the wild-type pair. Thus, this work demonstrates that AI-2 can alter the biofilm structure and composition of pioneering oral streptococcal biofilms. This may influence the subsequent succession of other species into oral biofilms and the ecology of dental plaque.
Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-β-d-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.
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.
Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (Δffh, ΔscRNA, and ΔftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription- and translation-related proteins, the β subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (Δffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.
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.
Autoinducer-2 (AI-2) molecules are one class of signalling molecules involved in gene regulation dependent on population density in a mechanism commonly referred to as quorum sensing (QS). AI-2 is produced by the methylthioadenosine/S-adenosyl-homocysteine nucleosidase LuxS. In the present study, we characterise the function of bifidobacterial LuxS proteins to address the question whether these economically important bacteria are able to perform QS communication. All publically available genome sequences of bifidobacteria harbour putative luxS genes. The deduced amino acid sequences are well conserved in the genus and show good homology to the LuxS protein of the prototypical AI-2 producer Vibrio harveyi. The luxS genes of three bifidobacterial strains were successfully expressed in AI-2-negative Escherichia coli DH5α. Supernatants of these recombinant E. coli strains contained significant AI-2 activity. In initial experiments, we failed to detect AI-2 activity in supernatants of bifidobacteria grown in MRSc. High concentration of glucose as well as acidic pH had strong inhibitory effects on AI-2 activity. AI-2 activity could be detected when lower volumes of supernatants were used in the assay. Homologous overexpression of luxS in Bifidobacterium longum NCC2705 increased AI-2 levels in the supernatant. Furthermore, over-expression of luxS or supplementation with AI-2-containing supernatants enhanced biofilm formation of B. longum NCC2705. Collectively, these results suggest that bifidobacteria indeed harbour functional luxS genes that are involved in the production of AI-2-like molecules. To the best of our knowledge, this represents the first report on AI-2 activity produced by bifidobacteria. Self-produced AI-2 activity as well as AI-2-like molecules of other bacteria of the intestinal tract may have a regulatory function in biofilm formation and host colonization by bifidobacteria.
Vibrio fischeri possesses two acyl-homoserine lactone quorum-sensing systems, ain and lux, both of which are involved in the regulation of luminescence gene expression and are required for persistent colonization of the squid host, Euprymna scolopes. We have previously demonstrated that the ain system induces luminescence at cell densities that precede lux system activation. Our data suggested that the ain system both relieves repression and initially induces the lux system, thereby achieving sequential induction of gene expression by these two systems. Analysis of the V. fischeri genome revealed the presence of a putative third system based on the enzyme LuxS, which catalyzes the synthesis of the Vibrio harveyi autoinducer 2 (AI-2). In this study, we investigated the impact of V. fischeri LuxS on luminescence and colonization competence in comparison to that of the ain system. Similar to the ain system, inactivation of the AI-2 system decreased light production in culture, but not in the squid host. However, while an ainS mutant produces no detectable light in culture, a luxS mutant expressed approximately 70% of wild-type luminescence levels. A mutation in luxS alone did not compromise symbiotic competence of V. fischeri; however, levels of colonization of an ainS luxS double mutant were reduced to 50% of the already diminished level of ainS mutant colonization, suggesting that these two systems regulate colonization gene expression synergistically through a common pathway. Introduction of a luxO mutation into the luxS and ainS luxS background could relieve both luminescence and colonization defects, consistent with a model in which LuxS, like AinS, regulates gene expression through LuxO. Furthermore, while luxS transcription appeared to be constitutive and the AI-2 signal concentration did not change dramatically, our data suggest that ainS transcription is autoregulated, resulting in an over 2,000-fold increase in signal concentration as culture density increased. Taken together, these data indicate that V. fischeri LuxS affects both luminescence regulation and colonization competence; however, its quantitative contribution is small when compared to that of the AinS signal.
We previously reported that LuxS in Streptococcus mutans is involved in stress tolerance and biofilm formation. In this study, flowcells and confocal laser scanning microscopy were used to further examine the effects of LuxS-deficiency on biofilm formation. Similar to the wild-type strain (UA159), a strain deficient in LuxS (TW26D) bound efficiently to the flowcells and formed microcolonies 4 h after inoculation. Unlike UA159, which accumulated and formed compact, evenly distributed biofilms after 28 h, TW26D showed only loose, sporadic, thin biofilms. DNA microarray analysis revealed alterations in transcription of more than 60 genes in TW26D biofilms by at least 1.5-fold (P < 0.001). Among the upregulated genes were those for sugar-specific enzymes II of the phosphotransferase (PTS) system and the atp operon, which codes for the proton-pumping F-ATPase. Of the downregulated genes, several encode proteins with putative functions in DNA repair. Mutation of selected genes caused severe defects in the ability of the mutants to tolerate low pH and oxidative stress. These results provide additional proof that LuxS-deficiency causes global alterations in the expression of genes central to biofilm formation and virulence of S. mutans, including those involved in energy metabolism, DNA repair and stress tolerance.
biofilms; dental caries; DNA array analysis; quorum sensing; Streptococcus mutans
The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS−, a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.
The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic ΔluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p ≤ 0.05 were considered to be significantly expressed differentially between WT and ΔluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic ΔluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of ΔluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.
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.
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 quorum-sensing (QS) signal autoinducer-2 (AI-2) has been proposed to promote interspecies signaling in a broad range of bacterial species. AI-2 is spontaneously derived from 4,5-dihydroxy-2,3-pentanedione that, along with homocysteine, is produced by cleavage of S-adenosylhomocysteine (SAH) and S-ribosylhomocysteine by the Pfs and LuxS enzymes. Numerous phenotypes have been attributed to AI-2 QS signaling using luxS mutants. We have previously reported that the luxS mutation also affects the synthesis of the AI-3 autoinducer that activates enterohemorrhagic Escherichia coli virulence genes. Here we show that several species of bacteria synthesize AI-3, suggesting a possible role in interspecies bacterial communication. The luxS mutation leaves the cell with only one pathway, involving oxaloacetate and l-glutamate, for de novo synthesis of homocysteine. The exclusive use of this pathway for homocysteine production appears to alter metabolism in the luxS mutant, leading to decreased levels of AI-3. The addition of aspartate and expression of an aromatic amino acid transporter, as well as a tyrosine-specific transporter, restored AI-3-dependent phenotypes in an luxS mutant. The defect in AI-3 production, but not in AI-2 production, in the luxS mutant was restored by expressing the Pseudomonas aeruginosa S-adenosylhomocysteine hydrolase that synthesizes homocysteine directly from SAH. Furthermore, phenotype microarrays revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. This study examines how AI-3 production is affected by the luxS mutation and explores the roles of the LuxS/AI-2 system in metabolism and QS.