LuxS-based signaling affects Streptococcus mutans biofilm formation

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.     

Signaling system in Porphyromonas gingivalis based on a LuxS protein

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

Autoinducer 2 production by Streptococcus gordonii DL1 and the biofilm phenotype of a luxS mutant are influenced by nutritional conditions

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 ycf27 genes from cyanobacteria and eukaryotic algae: distribution and implications for chloroplast evolution

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxS(M.h.) was able to complement AI-2 production in the Escherichia coli DH5alpha luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions.     

Chemical synthesis of (S)-4,5-dihydroxy-2,3-pentanedione, a bacterial signal molecule precursor, and validation of its activity in Salmonella typhimurium

We describe an original, short, and convenient chemical synthesis of enantiopure (S)-4,5-dihydroxy-2,3-pentanedione (DPD), starting from commercial methyl (S)-(-)-2,2-dimethyl-1,3-dioxolane-4-carboxylate. DPD is the precursor of autoinducer (AI)-2, the proposed signal for bacterial interspecies communication. AI-2 is synthesized by many bacterial species in three enzymatic steps. The last step, a LuxS-catalyzed reaction, leads to the formation of DPD, which spontaneously cyclizes into AI-2. AI-2-like activity of the synthesized molecule was ascertained by the Vibrio harveyi bioassay. To further validate the biological activity of synthetic DPD and to explore its potential in studying DPD (AI-2)-mediated signaling, a Salmonella typhimurium luxS mutant was constructed. Expression of the AI-2 regulated lsr operon can be rescued in this luxS mutant by addition of synthetic DPD or genetic complementation. Biofilm formation by S. typhimurium has been reported to be defective in a luxS mutant, and this was confirmed in this study to test DPD for chemical complementation. However, biofilm formation of the luxS mutant cannot be restored by addition of DPD. In contrast, introduction of luxS under control of its own promoter complemented biofilm formation. Further results demonstrated that biofilm formation of the luxS mutant cannot be restored with luxS under control of the strong nptII promoter. This indicates that altering the intrinsic promoter activity of luxS affects Salmonella biofilm formation. Conclusively, we synthesized biologically active DPD. Using this chemical compound in combination with genetic approaches opens new avenues in studying AI-2-mediated signaling.     

A Mutation in the luxS gene influences Listeria monocytogenes biofilm formation

Using a Vibrio harveyi reporter strain, we demonstrated that Listeria monocytogenes secretes a functional autoinducer 2 (AI-2)-like signal. A luxS-deficient mutant produced a denser biofilm and attached to a glass surface 19-fold better than the parent strain. Exogenous AI-2 failed to restore the wild-type phenotype to the mutant. It seems that an intact luxS gene is associated with repression of components required for attachment and biofilm formation.     

Possible quorum sensing in the rumen microbial community: detection of quorum-sensing signal molecules from rumen bacteria

The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria. The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules. Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P. ruminicola subsp. ruminicola and R. flavefaciens. These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen.     

The LuxS-dependent autoinducer AI-2 controls the expression of an ABC transporter that functions in AI-2 uptake in Salmonella typhimurium

In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI-2, originally discovered in the quorum-sensing bacterium Vibrio harveyi, is made by many species of Gram-negative and Gram-positive bacteria. In every case, production of AI-2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI-2 in most of these luxS-containing species of bacteria are not known. Here, we describe the identification and characterization of AI-2-regulated genes in Salmonella typhimurium. We find that LuxS and AI-2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)-type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium. A gene encoding a DNA-binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI-2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI-2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI-2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI-2 into the cells. It is intriguing that an operon regulated by AI-2 encodes functions resembling the ribose transporter, given recent findings that AI-2 is derived from the ribosyl moiety of S-ribosylhomocysteine.     

Tobacco smoke augments Porphyromonas gingivalis-Streptococcus gordonii biofilm formation

Smoking is responsible for the majority of periodontitis cases in the US and smokers are more susceptible than non-smokers to infection by the periodontal pathogen Porphyromonas gingivalis. P. gingivalis colonization of the oral cavity is dependent upon its interaction with other plaque bacteria, including Streptococcus gordonii. Microarray analysis suggested that exposure of P. gingivalis to cigarette smoke extract (CSE) increased the expression of the major fimbrial antigen (FimA), but not the minor fimbrial antigen (Mfa1). Therefore, we hypothesized that CSE promotes P. gingivalis-S. gordonii biofilm formation in a FimA-dependent manner. FimA total protein and cell surface expression were increased upon exposure to CSE whereas Mfa1 was unaffected. CSE exposure did not induce P. gingivalis auto-aggregation but did promote dual species biofilm formation, monitored by microcolony numbers and depth (both, p<0.05). Interestingly, P. gingivalis biofilms grown in the presence of CSE exhibited a lower pro-inflammatory capacity (TNF-α, IL-6) than control biofilms (both, p<0.01). CSE-exposed P. gingivalis bound more strongly to immobilized rGAPDH, the cognate FimA ligand on S. gordonii, than control biofilms (p<0.001) and did so in a dose-dependent manner. Nevertheless, a peptide representing the Mfa1 binding site on S. gordonii, SspB, completely inhibited dual species biofilm formation. Thus, CSE likely augments P. gingivalis biofilm formation by increasing FimA avidity which, in turn, supports initial interspecies interactions and promotes subsequent high affinity Mfa1-SspB interactions driving biofilm growth. CSE induction of P. gingivalis biofilms of limited pro-inflammatory potential may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced infectious diseases and conditions.     

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans

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 (luxS(Sm)) 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.     

Assessment of the roles of LuxS, S-ribosyl homocysteine, and autoinducer 2 in cell attachment during biofilm formation by Listeria monocytogenes EGD-e

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5alpha, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10 degrees C, 15 degrees C, 25 degrees C, and 42 degrees C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.     

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

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

Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2

Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence. This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers. While most autoinducers are species specific, autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi, is produced and detected by many Gram-negative and Gram-positive bacteria. The crystal structure of the V. harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester. Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium. We find that LsrB binds a chemically distinct form of the AI-2 signal, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), that lacks boron. Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling.     

A LuxS-dependent cell-to-cell language regulates social behavior and development in Bacillus subtilis

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.