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author:("Qi, fengxiao")
1.  Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems 
Applied and Environmental Microbiology  2013;79(20):6375-6384.
Despite the plethora of genetic tools that have been developed for use in Streptococcus mutans, the S. mutans genetic system still lacks an effective gene induction system exhibiting low basal expression and strong inducibility. Consequently, we created two hybrid gene induction cassettes referred to as Xyl-S1 and Xyl-S2. Both Xyl-S cassettes are xylose inducible and controlled by the Bacillus megaterium xylose repressor. The Xyl-S cassettes each demonstrated >600-fold-increased reporter activity in the presence of 1.2% (wt/vol) xylose. However, the Xyl-S1 cassette yielded a much higher maximum level of gene expression, whereas the Xyl-S2 cassette exhibited much lower uninduced basal expression. The cassettes also performed similarly in Streptococcus sanguinis and Streptococcus gordonii, which suggests that they are likely to be useful in a variety of streptococci. We demonstrate how both Xyl-S cassettes are particularly useful for the study of toxin-antitoxin (TA) modules using both the previously characterized S. mutans mazEF TA module and a previously uncharacterized HicAB TA module in S. mutans. HicAB TA modules are widely distributed among bacteria and archaea, but little is known about their function. We show that HicA serves as the toxin component of the module, while HicB serves as the antitoxin. Our results suggest that, in contrast to that of typical TA modules, HicA toxicity in S. mutans is modest at best. The implications of these results for HicAB function are discussed.
doi:10.1128/AEM.02320-13
PMCID: PMC3811191  PMID: 23934493
2.  The Mutacins of Streptococcus mutans: Regulation and Ecology 
Molecular Oral Microbiology  2011;27(2):57-69.
SUMMARY
Streptococcus mutans is generally recognized as a causative agent of human dental caries. The production of mutacins (bacteriocins) by S. mutans is considered to be an important factor in the colonization and establishment of S. mutans in the dental biofilm. Two types of mutacins have been characterized: the lantibiotics and the non-lantibiotics. The lantibiotics generally have a wider spectrum of activity than the non-lantibiotics, which make them attractive targets for development into new antimicrobial modalities. The non-lantibiotics are much more prevalent among strains of S. mutans and play a significant role in both community and population level interactions in the dental biofilm. These interactions are directly mediated through the ComCDE two-component system and the newly characterized LytTR Regulation Systems HdrRM and BrsRM. These systems coordinate natural competence development and mutacin production as a means to acquire transforming DNA either by killing closely related streptococcal species in the vicinity of S. mutans, or through an altruistic suicide mechanism among a subpopulation of competent cells within the S. mutans community. As more S. mutans strains are sequenced, it is anticipated that additional mutacins with novel functions will be discovered, which may yield further insights into the ecological role of mutacins within the oral biofilm.
doi:10.1111/j.2041-1014.2011.00634.x
PMCID: PMC3296966  PMID: 22394465
mutacin; Streptococcus mutans; bacteriocin
3.  CcpA-Dependent Carbohydrate Catabolite Repression Regulates Galactose Metabolism in Streptococcus oligofermentans 
Journal of Bacteriology  2012;194(15):3824-3832.
Streptococcus oligofermentans is an oral commensal that inhibits the growth of the caries pathogen Streptococcus mutans by producing copious amounts of H2O2 and that grows faster than S. mutans on galactose. In this study, we identified a novel eight-gene galactose (gal) operon in S. oligofermentans that was comprised of lacABCD, lacX, and three genes encoding a galactose-specific transporter. Disruption of lacA caused more growth reduction on galactose than mutation of galK, a gene in the Leloir pathway, indicating that the principal role of this operon is in galactose metabolism. Diauxic growth was observed in cultures containing glucose and galactose, and a luciferase reporter fusion to the putative gal promoter demonstrated 12-fold repression of the operon expression by glucose but was induced by galactose, suggesting a carbon catabolite repression (CCR) control in galactose utilization. Interestingly, none of the single-gene mutations in the well-known CCR regulators ccpA and manL affected diauxic growth, although the operon expression was upregulated in these mutants in glucose. A double mutation of ccpA and manL eliminated glucose repression of galactose utilization, suggesting that these genes have parallel functions in regulating gal operon expression and mediating CCR. Electrophoretic mobility shift assays demonstrated binding of CcpA to the putative catabolite response element motif in the promoter regions of the gal operon and manL, suggesting that CcpA regulates CCR through direct regulation of the transcription of the gal operon and manL. This provides the first example of oral streptococci using two parallel CcpA-dependent CCR pathways in controlling carbohydrate metabolism.
doi:10.1128/JB.00156-12
PMCID: PMC3416513  PMID: 22609925
4.  Establishment of a Tractable Genetic Transformation System in Veillonella spp. 
We have constructed the first Escherichia coli-Veillonella shuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinical Veillonella strain. A highly transformable Veillonella strain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for future Veillonella genetic studies.
doi:10.1128/AEM.00196-12
PMCID: PMC3346448  PMID: 22344660
5.  Oligomerization of the Response Regulator ComE from Streptococcus mutans Is Affected by Phosphorylation 
Journal of Bacteriology  2012;194(5):1127-1135.
We have previously characterized the interactions of the response regulator ComE from Streptococcus mutans and DNA binding sites through DNase I footprinting and electrophoretic mobility shift assay analysis. Since response regulator functions are often affected by their phosphorylation state, we investigated how phosphorylation affects the biochemical function of ComE. Unlike many response regulators, we found that the phosphorylation state of ComE does not likely play a role in DNA binding affinity but rather seems to induce the formation of an oligomeric form of the protein. The role of this oligomerization state for ComE function is discussed.
doi:10.1128/JB.06565-11
PMCID: PMC3294772  PMID: 22210762
6.  Differential response of Streptococcus mutans towards friend and foe in mixed-species cultures 
Microbiology  2011;157(Pt 9):2433-2444.
In the oral biofilm, the ‘mitis’ streptococci are among the first group of organisms to colonize the tooth surface. Their proliferation is thought to be an important factor required for antagonizing the growth of cariogenic species such as Streptococcus mutans. In this study, we used a three-species mixed culture to demonstrate that another ubiquitous early colonizing species, Veillonella parvula, can greatly affect the outcome of the competition between a pair of antagonists such as S. mutans and Streptococcus gordonii. Transcriptome analysis further revealed that S. mutans responds differentially to its friend (V. parvula) and foe (S. gordonii). In the mixed culture with S. gordonii, all but one of the S. mutans sugar uptake and metabolic genes were downregulated, while genes for alternative energy source utilization and H2O2 tolerance were upregulated, resulting in a slower but persistent growth. In contrast, when cultured with V. parvula, S. mutans grew equally well or better than in monoculture and exhibited relatively few changes within its transcriptome. When V. parvula was introduced into the mixed culture of S. mutans and S. gordonii, it rescued the growth inhibition of S. mutans. In this three-species environment, S. mutans increased the expression of genes required for the uptake and metabolism of minor sugars, while genes required for oxidative stress tolerance were downregulated. We conclude that the major factors that affect the competition between S. mutans and S. gordonii are carbohydrate utilization and H2O2 resistance. The presence of V. parvula in the tri-species culture mitigates these two major factors and allows S. mutans to proliferate, despite the presence of S. gordonii.
doi:10.1099/mic.0.048314-0
PMCID: PMC3352174  PMID: 21565931
7.  Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿ 
Applied and Environmental Microbiology  2011;77(22):8025-8033.
Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.
doi:10.1128/AEM.06362-11
PMCID: PMC3208986  PMID: 21948849
8.  Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans▿† 
Journal of Bacteriology  2011;193(14):3642-3652.
In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.
doi:10.1128/JB.00155-11
PMCID: PMC3133340  PMID: 21602345
9.  Mutanobactin A from the human oral pathogen Streptococcus mutans is a cross-kingdom regulator of the yeast-mycelium transition† 
Organic & biomolecular chemistry  2010;8(24):5486-5489.
The recent investigation of a gene cluster encoding for a hybrid PKS-NRPS metabolite in the oral pathogen Streptococcus mutans UA159 yielded evidence that this natural product might play an important role regulating a range of stress tolerance factors. We have now characterized the major compound generated from this gene cluster, mutanobactin A, and demonstrated that this secondary metabolite is also capable of influencing the yeast-mycelium transition of Candida albicans.
doi:10.1039/c0ob00579g
PMCID: PMC2992086  PMID: 20852771
10.  Identification of a Novel Bacteriocin Regulatory System in Streptococcus mutans 
Molecular microbiology  2010;78(6):1431-1447.
Summary
Recently, we described the function of an uncharacterized two-gene regulatory system consisting of a LytTR family transcription regulator and a putative membrane protein, which we referred to as the hdrRM operon. In this study, we determined that the HdrRM system controls the expression of an analogous uncharacterized regulatory system annotated as SMU.2080 and SMU.2081. Like hdrRM, the SMU.2080–2081 operon encodes a LytTR family transcription regulator and putative membrane protein, which we now refer to as BrsR and BrsM, respectively. Examination of the regulatory mechanism of the BrsRM system suggests that BrsM serves to antagonize the function of the transcription regulator BrsR. Further analyses of the regulatory role of BrsR determined that it functions as a transcription activator for a variety of bacteriocins and bacteriocin-related genes. In vitro electromobility shift assays confirmed that BrsR binds to the promoter regions of several bacteriocin genes and requires the presence of a LytTR family consensus direct repeat in order to stably bind DNA. In addition, we identified a novel regulatory scheme in which both the HdrRM and BrsRM systems coregulate each other and ultimately determine whether bacteriocin production will inhibit competitor organisms or result in lethality to the producer.
doi:10.1111/j.1365-2958.2010.07417.x
PMCID: PMC3059113  PMID: 21143316
bacteriocin; LytTR; oral bacteria; Streptococcus; cell death
11.  Rapid Probing of Biological Surfaces with a Sparse-Matrix Peptide Library 
PLoS ONE  2011;6(8):e23551.
Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained “hits” against all biological surfaces probed. Sequence refinement of the “hits” led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue.
doi:10.1371/journal.pone.0023551
PMCID: PMC3156232  PMID: 21858167
12.  Genomic Island TnSmu2 of Streptococcus mutans Harbors a Nonribosomal Peptide Synthetase-Polyketide Synthase Gene Cluster Responsible for the Biosynthesis of Pigments Involved in Oxygen and H2O2 Tolerance ▿ †  
Applied and Environmental Microbiology  2010;76(17):5815-5826.
The oral biofilm community consists of >800 microbial species, among which Streptococcus mutans is considered a primary pathogen for dental caries. The genomic island TnSmu2 of S. mutans comprises >2% of the genome. In this study, we demonstrate that TnSmu2 harbors a gene cluster encoding nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and accessory proteins and regulators involved in nonribosomal peptide (NRP) and polyketide (PK) biosynthesis. Interestingly, the sequences of these genes and their genomic organizations and locations are highly divergent among different S. mutans strains, yet each TnSmu2 region encodes NRPS/PKS and accessory proteins. Mutagenesis of the structural genes and putative regulatory genes in strains UA159, UA140, and MT4653 resulted in colonies that were devoid of their yellow pigmentation (for strains UA140 and MT4653). In addition, these mutant strains also displayed retarded growth under aerobic conditions and in the presence of H2O2. High-performance liquid chromatography profiling of cell surface extracts identified unique peaks that were missing in the mutant strains, and partial characterization of the purified product from UA159 demonstrated that it is indeed a hybrid NRP/PK, as predicted. A genomic survey of 94 clinical S. mutans isolates suggests that the TnSmu2 gene cluster may be more prevalent than previously recognized.
doi:10.1128/AEM.03079-09
PMCID: PMC2935078  PMID: 20639370
13.  Regulation of ciaXRH Operon Expression and Identification of the CiaR Regulon in Streptococcus mutans▿  
Journal of Bacteriology  2010;192(18):4669-4679.
The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the −10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes.
doi:10.1128/JB.00556-10
PMCID: PMC2937423  PMID: 20639331
14.  The cia Operon of Streptococcus mutans Encodes a Unique Component Required for Calcium-Mediated Autoregulation 
Molecular microbiology  2008;70(1):112-126.
Streptococcus mutans is a primary pathogen for dental caries in humans. CiaR and CiaH of S. mutans comprise a two-component signal transduction system (TCS) involved in regulating various virulent factors. However, the signal that triggers the CiaRH response remains unknown. In this study, we show that calcium is a signal for regulation of the ciaRH operon, and that a double-glycine-containing small peptide encoded within the ciaRH operon (renamed ciaX) mediates this regulation. CiaX contains a serine-aspartate (SD) domain that is shared by calcium-binding proteins. A markerless in-frame deletion of ciaX reduced ciaRH operon expression and diminished the calcium repression of operon transcription. Point mutations of the SD-domain resulted in the same phenotype as the in-frame deletion, indicating that the SD-domain is required for CiaX function. Further characterization of ciaX demonstrated that it is involved in calcium mediated biofilm formation. Furthermore, inactivation of ciaR or ciaH led to the same phenotype as the in-frame deletion of ciaX, suggesting that all three genes are involved in the same regulatory pathway. Sequence analysis and real-time RT-PCR identified a putative CiaR binding site upstream of ciaX. We conclude that the ciaXRH operon is a three-component, self-regulatory system modulating cellular functions in response to calcium.
doi:10.1111/j.1365-2958.2008.06390.x
PMCID: PMC2955730  PMID: 18681938
15.  The hdrRM Operon of Streptococcus mutans Encodes a Novel Regulatory System for Coordinated Competence Development and Bacteriocin Production▿  
Journal of Bacteriology  2010;192(7):1844-1852.
The Streptococcus mutans hdrRM operon encodes a novel two-gene regulatory system induced by high cell density. Previous studies identified hdrM as the only known negative regulator of competence development in S. mutans. In the present study, we demonstrated that the HdrRM system bypasses the prototypical competence gene regulators ComC and ComDE in the transcriptional regulation of the competence-specific sigma factor comX and the late competence genes. Similarly, the HdrRM system can abrogate the requirement for ComE to produce the bacteriocin mutacin IV. To further probe the regulatory mechanism of hdrRM, we created an hdrR overexpression strain and showed that it could reproduce each of the hdrM competence and mutacin phenotypes, indicating that HdrM acts as a negative regulator of HdrR activity. Using a mutacin IV-luciferase reporter, we also demonstrated that the hdrRM system utilizes the same promoter elements recognized by ComE and thus appears to comprise a novel regulatory pathway parallel to ComCDE.
doi:10.1128/JB.01667-09
PMCID: PMC2838059  PMID: 20118256
16.  Genes Involved in the Repression of Mutacin I Production in Streptococcus mutans 
Microbiology (Reading, England)  2009;155(Pt 2):551-556.
Streptococcus mutans is considered a primary pathogen for human dental caries. Its ability to produce a variety of peptide antibiotics called mutacins may play an important role in its invasion and establishment in the dental biofilm. S. mutans strain UA140 produces two types of mutacins, the lantibiotic mutacin I and the non-lantibitoc mutacin IV. In a previous study, we constructed a random insertional-mutation library to screen for genes involved in regulating mutacin I production, and found 25 genes/operons that have a positive effect on mutacin I production. In this study, we continued our previous work to identify genes that are negatively involved in mutacin I production. By using a high phosphate BHI plate that inhibited mutacin I production of the wild-type, we isolated 77 clones that consistently produced mutacin I under repressive conditions. From the 34 clones that we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups including the central metabolism, surface binding, sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when it is usually not expressed in the wild-type. These results further demonstrate an intimate and intricate connection between mutacin production and the overall cellular homeostasis.
doi:10.1099/mic.0.021303-0
PMCID: PMC2946218  PMID: 19202103
17.  The Streptococcus mutans IrvR Repressor Is a CI-Like Regulator That Functions through Autocleavage and Clp-Dependent Proteolysis▿  
Journal of Bacteriology  2009;192(6):1586-1595.
Previous work has shown that irvR is required for the proper regulation of genetic competence and dextran-dependent aggregation due to its ability to repress the transcription regulator irvA. In this study, we determined the mechanism used to relieve the repression of irvA. We demonstrate that IrvR is a “LexA-like” protein with four conserved amino acid residues likely required for IrvR autocleavage activity. Furthermore, recombinant IrvR protein purified from Escherichia coli was competent to undergo autocleavage in vitro. Using several truncated IrvR constructs, we show that the amino acids adjacent to the autocleavage site are essential for relieving irvA repression and engaging the irvA-dependent regulatory pathway primarily through the ClpXP and ClpCP proteases. By extending the IrvR C terminus with an epitope derived from the autocleavage site, we were also able to create a constitutive Clp-dependent degradation of the full-length IrvR protein. This suggests that the derepression of irvA occurs through a two-step mechanism involving the initial autocleavage of IrvR and exposure of a proteolytic degradation sequence followed by Clp-dependent degradation of the IrvR DNA binding domain. Thus, irvA derepression is highly analogous to the genetic switch mechanism used to regulate lysogeny in bacteriophages.
doi:10.1128/JB.01261-09
PMCID: PMC2832535  PMID: 20038591
18.  Bacterial and Host Interactions of Oral Streptococci 
DNA and Cell Biology  2009;28(8):397-403.
The oral microbial flora comprises one of the most diverse human-associated biofilms. Its development is heavily influenced by oral streptococci, which are considered the main group of early colonizers. Their initial attachment determines the composition of later colonizers in the oral biofilm and impacts the health or disease status of the host. Thus, the role of streptococci in the development of oral diseases is best described in the context of bacterial ecology, which itself is further influenced by interactions with host epithelial cells, the immune system, and salivary components. The tractability of the oral biofilm makes it an excellent model system for studies of complex, biofilm-associated polymicrobial diseases. Using this system, numerous cooperative and antagonistic bacterial interactions have been demonstrated to occur within the community and with the host. In this review, several recent identified interactions are presented.
doi:10.1089/dna.2009.0868
PMCID: PMC2903342  PMID: 19435424
19.  Specific Binding and Mineralization of Calcified Surfaces by Small Peptides 
Calcified Tissue International  2009;86(1):58-66.
Several small (<25aa) peptides have been designed based on the sequence of the dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)n repeats, and though similar repeated sequences—(NTT)n, (DTT)n, (ETT)n, (NSS)n, (ESS)n, (DAA)n, (ASS)n, and (NAA)n—also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)n peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed.
doi:10.1007/s00223-009-9312-0
PMCID: PMC2798077  PMID: 19949943
Dentin phosphoprotein; Peptide; Mineralization
20.  SO-LAAO, a Novel l-Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H2O2 from Peptone▿ † 
Journal of Bacteriology  2008;190(13):4716-4721.
We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H2O2) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H2O2 produced on peptone by S. oligofermentans was mainly derived from seven l-amino acids, i.e., l-aspartic acid, l-tryptophan, l-lysine, l-isoleucine, l-arginine, l-asparagine, and l-glutamine, indicating the possible existence of l-amino acid oxidase (LAAO) that can produce H2O2 from l-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aaoSo, was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H2O2 formation from the seven l-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aaoSo mutant of S. oligofermentans did lose H2O2 formation from the seven l-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aaoSo mutant, indicating the gene's importance in interspecies competition.
doi:10.1128/JB.00363-08
PMCID: PMC2446784  PMID: 18469105
21.  The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis 
Microbiology (Reading, England)  2007;153(Pt 6):1799-1807.
In Streptococcus pneumoniae, competence and bacteriocin genes are controlled by two two-component systems, ComED and BlpRH, respectively. In Streptococcus mutans, both functions are controlled by the ComED system. Recent studies in S. mutans revealed a potential ComE binding site characterized by two 11 bp direct repeats shared by each of the bacteriocin genes responsive to the competence-stimulating peptide (CSP). Interestingly, this sequence was not found in the upstream region of the CSP structural gene comC. Since comC is suggested to be part of a CSP-responsive and ComE-dependent autoregulatory loop, it was of interest to determine how it was possible that the ComED system could simultaneously regulate bacteriocin expression and natural competence. Using the intergenic region IGS1499, shared by the CSP-responsive bacteriocin nlmC and comC, it was demonstrated that both genes are likely to be regulated by a bifunctional ComE. In a comE null mutant, comC gene expression was increased similarly to a fully induced wild-type. In contrast, nlmC gene expression was nearly abolished. Deletion of ComD exerted a similar effect on both genes to that observed with the comE null mutation. Electrophoretic mobility shift assays (EMSAs) with purified ComE revealed specific shift patterns dependent on the presence of one or both direct repeats in the nlmC–comC promoter region. The two direct repeats were also required for the promoter activity of both nlmC and comC. These results suggest that gene regulation of comC in S. mutans is fundamentally different from that reported for S. pneumoniae, which implicates a unique regulatory mechanism that allows the coordination of bacteriocin production with competence development.
doi:10.1099/mic.0.2007/005975-0
PMCID: PMC2062498  PMID: 17526837
22.  Coordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species 
Molecular microbiology  2005;57(2):392-404.
It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced coordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral bio-film, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.
doi:10.1111/j.1365-2958.2005.04695.x
PMCID: PMC1262684  PMID: 15978073
23.  Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿  
Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents.
doi:10.1128/AAC.01270-06
PMCID: PMC1855471  PMID: 17296741
24.  Enhancement of Antimicrobial Activity against Pseudomonas aeruginosa by Coadministration of G10KHc and Tobramycin▿  
Antimicrobial Agents and Chemotherapy  2006;50(11):3833-3838.
Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic fibrosis. Previously, we converted the wide-spectrum antimicrobial peptide novispirin G10 into a selectively-targeted antimicrobial peptide (STAMP), G10KHc. Compared to novispirin G10, the STAMP had an enhanced ability to kill Pseudomonas mendocina. In this study, we explored the activity of G10KHc against P. aeruginosa. G10KHc was found to be highly active (as active as tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were cotreated with G10KHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to G10KHc-mediated cell membrane disruption. These results suggest that G10KHc may be useful against P. aeruginosa during acute and chronic infection states, especially when it is coadministered with tobramycin.
doi:10.1128/AAC.00509-06
PMCID: PMC1635211  PMID: 16940063
25.  Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide 
Antimicrobial Agents and Chemotherapy  2006;50(11):3651-3657.
Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.
doi:10.1128/AAC.00622-06
PMCID: PMC1635210  PMID: 17060534

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