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author:("kmeth, Jens")
1.  A Biochemical Analysis of the Interaction of Porphyromonas gingivalis HU PG0121 Protein with DNA 
PLoS ONE  2014;9(3):e93266.
K-antigen capsule, a key virulence determinant of the oral pathogen Porphyromonas gingivalis, is synthesized by proteins encoded in a series of genes transcribed as a large polycistronic message. Previously, we identified a 77-base pair inverted repeat region with the potential to form a large stem-loop structure at the 5′ end of this locus. PG0121, one of two genes flanking the capsule operon, was found to be co-transcribed with the operon and to share high similarity to the DNA binding protein HU from Escherichia coli. A null mutation in PG0121 results in down-regulation of transcription of the capsule synthesis genes and production of capsule. Furthermore, we have also shown that PG0121 gene can complement multiple deficiencies in a strain of E. coli that is deficient for both the alpha and beta subunits of HU. Here, we examined the biochemical properties of the interaction of PG0121 to DNA with the emphasis on the kinds of nucleic acid architectures that may be encountered at the 77-bp inverted repeat. We have concluded that although some DNA binding characteristics are shared with E. coli HU, HU PG0121 also shows some distinct characteristics that set it apart from other HU-like proteins tested to date. We discuss our results in the context of how PG0121 may affect the regulation of the K-antigen capsule expression.
doi:10.1371/journal.pone.0093266
PMCID: PMC3969353  PMID: 24681691
2.  Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis 
PLoS ONE  2014;9(3):e89334.
Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition.
doi:10.1371/journal.pone.0089334
PMCID: PMC3958366  PMID: 24642967
3.  Plaque Bacterial Microbiome Diversity in Children Younger than 30 Months with or without Caries Prior to Eruption of Second Primary Molars 
PLoS ONE  2014;9(2):e89269.
Objective
Our primary objective is to phylogenetically characterize the supragingival plaque bacterial microbiome of children prior to eruption of second primary molars by pyrosequencing method for studying etiology of early childhood caries.
Methods
Supragingival plaque samples were collected from 10 caries children and 9 caries-free children. Plaque DNA was extracted, used to generate DNA amplicons of the V1–V3 hypervariable region of the bacterial 16S rRNA gene, and subjected to 454-pyrosequencing.
Results
On average, over 22,000 sequences per sample were generated. High bacterial diversity was noted in the plaque of children with caries [170 operational taxonomical units (OTU) at 3% divergence] and caries-free children (201 OTU at 3% divergence) with no significant difference. A total of 8 phyla, 15 classes, 21 orders, 30 families, 41 genera and 99 species were represented. In addition, five predominant phyla (Firmicute, Fusobacteria, Proteobacteria, Bacteroidetes and Actinobacteria) and seven genera (Leptotrichia, Streptococcus, Actinomyces, Prevotella, Porphyromonas, Neisseria, and Veillonella) constituted a majority of contents of the total microbiota, independent of the presence or absence of caries. Principal Component Analysis (PCA) presented that caries-related genera included Streptococcus and Veillonella; while Leptotrichia, Selenomonas, Fusobacterium, Capnocytophaga and Porphyromonas were more related to the caries-free samples. Neisseria and Prevotella presented approximately in between. In both groups, the degree of shared organism lineages (as defined by species-level OTUs) among individual supragingival plaque microbiomes was minimal.
Conclusion
Our study represented for the first time using pyrosequencing to elucidate and monitor supragingival plaque bacterial diversity at such young age with second primary molar unerrupted. Distinctions were revealed between caries and caries-free microbiomes in terms of microbial community structure. We observed differences in abundance for several microbial groups between the caries and caries-free host populations, which were consistent with the ecological plaque hypothesis. Our approach and findings could be extended to correlating microbiomic changes after occlusion establishment and caries treatment.
doi:10.1371/journal.pone.0089269
PMCID: PMC3938432  PMID: 24586647
4.  Major Membrane Protein TDE2508 Regulates Adhesive Potency in Treponema denticola 
PLoS ONE  2014;9(2):e89051.
The cultivation and genetic manipulation of Treponema denticola, a Gram-negative oral spirochaeta associated with periodontal diseases, is still challenging. In this study, we formulated a simple medium based on a commercially available one, and established a transformation method with high efficiency. We then analyzed proteins in a membrane fraction in T. denticola and identified 16 major membrane-associated proteins, and characterized one of them, TDE2508, whose biological function was not yet known. Although this protein, which exhibited a complex conformation, was presumably localized in the outer membrane, we did not find conclusive evidence that it was exposed on the cell surface. Intriguingly, a TDE2508-deficient mutant exhibited significantly increased biofilm formation and adherent activity on human gingival epithelial cells. However, the protein deficiency did not alter autoaggregation, coaggregation with Porphyromonas gingivalis, hemagglutination, cell surface hydrophobicity, motility, or expression of Msp which was reported to be an adherent molecule in this bacteria. In conclusion, the major membrane protein TDE2508 regulates biofilm formation and the adhesive potency of T. denticola, although the underlying mechanism remains unclear.
doi:10.1371/journal.pone.0089051
PMCID: PMC3931704  PMID: 24586498
5.  Subinhibitory Concentrations of Triclosan Promote Streptococcus mutans Biofilm Formation and Adherence to Oral Epithelial Cells 
PLoS ONE  2014;9(2):e89059.
Triclosan is a general membrane-active agent with a broad-spectrum antimicrobial activity that is commonly used in oral care products. In this study, we investigated the effect of sub-minimum inhibitory concentrations (MICs) of triclosan on the capacity of the cariogenic bacterium Streptococcus mutans to form biofilm and adhere to oral epithelial cells. As quantified by crystal violet staining, biofilm formation by two reference strains of S. mutans was dose-dependently promoted, in the range of 2.2- to 6.2-fold, by 1/2 and 1/4 MIC of triclosan. Observations by scanning electron microscopy revealed the presence of a dense biofilm attached to the polystyrene surface. Growth of S. mutans in the presence of triclosan at sub-MICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. The expression of several genes involved in adherence and biofilm formation in S. mutans was investigated by quantitative RT-PCR. It was found that sub-MICs of triclosan significantly increased the expression of comD, gtfC, and luxS, and to a lesser extent of gtfB and atlA genes. These findings stress the importance of maintaining effective bactericidal concentrations of therapeutic triclosan since sub-MICs may promote colonization of the oral cavity by S. mutans.
doi:10.1371/journal.pone.0089059
PMCID: PMC3923858  PMID: 24551218
6.  Probing Oral Microbial Functionality – Expression of spxB in Plaque Samples 
PLoS ONE  2014;9(1):e86685.
The Human Oral Microbiome Database (HOMD) provides an extensive collection of genome sequences from oral bacteria. The sequence information is a static snapshot of the microbial potential of the so far sequenced species. A major challenge is to connect the microbial potential encoded in the metagenome to an actual function in the in vivo oral biofilm. In the present study we took a reductionist approach and identified a considerably conserved metabolic gene, spxB to be encoded by a majority of oral streptococci using the HOMD metagenome information. spxB encodes the pyruvate oxidase responsible for the production of growth inhibiting amounts of hydrogen peroxide (H2O2) and has previously been shown as important in the interspecies competition in the oral biofilm. Here we demonstrate a strong correlation of H2O2 production and the presence of the spxB gene in dental plaque. Using Real-Time RT PCR we show that spxB is expressed in freshly isolated human plaque samples from several donors and that the expression is relative constant when followed over time in one individual. This is the first demonstration of an oral community encoded gene expressed in vivo suggesting a functional role of spxB in oral biofilm physiology. This also demonstrates a possible strategy to connect the microbial potential of the metagenome to its functionality in future studies by identifying similar highly conserved genes in the oral microbial community.
doi:10.1371/journal.pone.0086685
PMCID: PMC3906080  PMID: 24489768
7.  Multiple Roles of RNase Y in Streptococcus pyogenes mRNA Processing and Degradation 
Journal of Bacteriology  2013;195(11):2585-2594.
Control over mRNA stability is an essential part of gene regulation that involves both endo- and exoribonucleases. RNase Y is a recently identified endoribonuclease in Gram-positive bacteria, and an RNase Y ortholog has been identified in Streptococcus pyogenes (group A streptococcus [GAS]). In this study, we used microarray and Northern blot analyses to determine the S. pyogenes mRNA half-life of the transcriptome and to understand the role of RNase Y in global mRNA degradation and processing. We demonstrated that S. pyogenes has an unusually high mRNA turnover rate, with median and mean half-lives of 0.88 min and 1.26 min, respectively. A mutation of the RNase Y-encoding gene (rny) led to a 2-fold increase in overall mRNA stability. RNase Y was also found to play a significant role in the mRNA processing of virulence-associated genes as well as in the rapid degradation of rnpB read-through transcripts. From these results, we conclude that RNase Y is a pleiotropic regulator required for mRNA stability, mRNA processing, and removal of read-through transcripts in S. pyogenes.
doi:10.1128/JB.00097-13
PMCID: PMC3676074  PMID: 23543715
8.  The Extra-Cytoplasmic Function Sigma Factor SigX Modulates Biofilm and Virulence-Related Properties in Pseudomonas aeruginosa  
PLoS ONE  2013;8(11):e80407.
SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the “chaperones and heat shock proteins”, “antibiotic resistance and susceptibility”, “energy metabolism”, “protein secretion/export apparatus”, and “secreted factors”, and “motility and attachment” classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes.
doi:10.1371/journal.pone.0080407
PMCID: PMC3832394  PMID: 24260387
9.  Stress Responses of the Industrial Workhorse Bacillus licheniformis to Osmotic Challenges 
PLoS ONE  2013;8(11):e80956.
The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress.
doi:10.1371/journal.pone.0080956
PMCID: PMC3858371  PMID: 24348917
10.  Metabolic Proximity in the Order of Colonization of a Microbial Community 
PLoS ONE  2013;8(10):e77617.
Microbial biofilms are often composed of multiple bacterial species that accumulate by adhering to a surface and to each other. Biofilms can be resistant to antibiotics and physical stresses, posing unresolved challenges in the fight against infectious diseases. It has been suggested that early colonizers of certain biofilms could cause local environmental changes, favoring the aggregation of subsequent organisms. Here we ask whether the enzyme content of different microbes in a well-characterized dental biofilm can be used to predict their order of colonization. We define a metabolic distance between different species, based on the overlap in their enzyme content. We next use this metric to quantify the average metabolic distance between neighboring organisms in the biofilm. We find that this distance is significantly smaller than the one observed for a random choice of prokaryotes, probably reflecting the environmental constraints on metabolic function of the community. More surprisingly, this metabolic metric is able to discriminate between observed and randomized orders of colonization of the biofilm, with the observed orders displaying smaller metabolic distance than randomized ones. By complementing these results with the analysis of individual vs. joint metabolic networks, we find that the tendency towards minimal metabolic distance may be counter-balanced by a propensity to pair organisms with maximal joint potential for synergistic interactions. The trade-off between these two tendencies may create a “sweet spot” of optimal inter-organism distance, with possible broad implications for our understanding of microbial community organization.
doi:10.1371/journal.pone.0077617
PMCID: PMC3813667  PMID: 24204896
11.  The Effect of pstS and phoB on Quorum Sensing and Swarming Motility in Pseudomonas aeruginosa 
PLoS ONE  2013;8(9):e74444.
Pseudomonas aeruginosa is an opportunistic pathogen that can cause a wide range of infections and inflammations in a variety of hosts, such as chronic biofilm associated lung infections in Cystic Fibrosis patients. Phosphate, an essential nutrient, has been recognized as an important signal that affects virulence in P. aeruginosa. In the current study we examined the connection between phosphate regulation and surface motility in P. aeruginosa. We focused on two important genes, pstS, which is involved in phosphate uptake, and phoB, a central regulator that responds to phosphate starvation. We found that a mutant lacking pstS is constantly starved for phosphate and has a hyper swarming phenotype. Phosphate starvation also induced swarming in the wild type. The phoB mutant, on the other hand, did not express phosphate starvation even when phosphate was limited and showed no swarming. A double mutant lacking both genes (pstS and phoB) showed a similar phenotype to the phoB mutant (i.e. no swarming). This highlights the role of phoB in controlling swarming motility under phosphate-depleted conditions. Finally, we were able to demonstrate that PhoB controls swarming by up-regulating the Rhl quorum sensing system in P. aeruginosa, which resulted in hyper production of rhamonlipids: biosurfactants that are known to induce swarming motility.
doi:10.1371/journal.pone.0074444
PMCID: PMC3762822  PMID: 24023943
12.  Characterization of Pyruvate Uptake in Escherichia coli K-12 
PLoS ONE  2013;8(6):e67125.
The monocarboxylate pyruvate is an important metabolite and can serve as sole carbon source for Escherichia coli. Although specific pyruvate transporters have been identified in two bacterial species, pyruvate transport is not well understood in E. coli. In the present study, pyruvate transport was investigated under different growth conditions. The transport of pyruvate shows specific activities depending on the growth substrate used as sole carbon source, suggesting the existence of at least two systems for pyruvate uptake: i) one inducible system and probably highly specific for pyruvate and ii) one system active under non-induced conditions. Using the toxic pyruvate analog 3-fluoropyruvate, a mutant was isolated unable to grow on and transport pyruvate. Further investigation revealed that a revertant selected for growth on pyruvate regained the inducible pyruvate transport activity. Characterization of pyruvate excretion showed that the pyruvate transport negative mutant accumulated pyruvate in the growth medium suggesting an additional transport system for pyruvate excretion. The here presented data give valuable insight into the pyruvate metabolism and transport of E. coli suggesting the presence of at least two uptake systems and one excretion system to balance the intracellular level of pyruvate.
doi:10.1371/journal.pone.0067125
PMCID: PMC3688616  PMID: 23818977
13.  Role of LytF and AtlS in eDNA Release by Streptococcus gordonii 
PLoS ONE  2013;8(4):e62339.
Extracellular DNA (eDNA) is an important component of the biofilm matrix produced by many bacteria. In general, the release of eDNA is associated with the activity of muralytic enzymes leading to obvious cell lysis. In the Gram-positive oral commensal Streptococcus gordonii, eDNA release is dependent on pyruvate oxidase generated hydrogen peroxide (H2O2). Addition of H2O2 to cells grown under conditions non-permissive for H2O2 production causes eDNA release. Furthermore, eDNA release is maximal under aerobic growth conditions known to induce pyruvate oxidase gene expression and H2O2 production. Obvious cell lysis, however, does not occur. Two enzymes have been recently associated with eDNA release in S. gordonii. The autolysin AtlS and the competence regulated murein hydrolase LytF. In the present report, we investigated the role of both proteins in the H2O2 dependent eDNA release process. Single and double mutants in the respective genes for LytF and AtlS released less eDNA under normal growth conditions, but the AtlS mutant was still inducible for eDNA release by external H2O2. Moreover, we showed that the AtlS mutation interfered with the ability of S. gordonii to produce eDNA release inducing amounts of H2O2. Our data support a role of LytF in the H2O2 eDNA dependent release of S. gordonii as part of the competence stress pathway responding to oxidative stress.
doi:10.1371/journal.pone.0062339
PMCID: PMC3634736  PMID: 23638042
14.  CcpA Regulates Biofilm Formation and Competence in Streptococcus gordonii 
Molecular Oral Microbiology  2011;27(2):83-94.
Summary
Streptococcus gordonii is an important member of the oral biofilm community. As oral commensal streptococci, S. gordonii is considered beneficial in promoting biofilm homeostasis. CcpA is known as central regulator of carbon catabolite repression in Gram-positive bacteria and is also involved in the control of virulence gene expression. To further establish the role of CcpA as central regulator in S. gordonii, the effect of CcpA on biofilm formation and natural competence of S. gordonii was investigated. These phenotypic traits have been suggested to be important to oral streptococci in coping with environmental stress. Here we demonstrate that a CcpA mutant was severely impaired in its biofilm forming ability, showed a defect in extracellular polysaccharide production and reduced competence. The data suggest that CcpA is involved in the regulation of biofilm formation and competence development in S. gordonii.
doi:10.1111/j.2041-1014.2011.00633.x
PMCID: PMC3296961  PMID: 22394467
15.  Evaluation of bacteria-induced enamel demineralization using optical profilometry 
Objectives
Streptococcus mutans is considered a major causative of tooth decay due to it’s ability to rapidly metabolize carbohydrates such as sucrose. One prominent excreted end product of sucrose metabolism is lactic acid. Lactic acid causes a decrease in the pH of the oral environment with subsequent demineralization of the tooth enamel. Biologically relevant bacteria-induced enamel demineralization was studied.
Methods
Optical profiling was used to measure tooth enamel decay with vertical resolution under one nanometer and lateral features with optical resolution as a result of S. mutans biofilm exposure. Comparison measurements were made using AFM.
Results
After 72 hr of biofilm exposure the enamel displayed an 8-fold increase in the observed roughness average, (Ra), as calculated over the entire measured array. Similarly, the average root mean square (RMS) roughness, RRMS, of the enamel before and after biofilm exposure for 3 days displayed a 7-fold increase. Further, the direct effect of chemically induced enamel demineralization using biologically relevant organic acids was shown. Optical profiles of the enamel surface after addition of a 30% lactic acid solution showed a significant alteration in the surface topography with a corresponding increase in respective surface roughness statistics. Similar measurements with 10% citric acid over seconds and minutes give insight into the demineralization process by providing quantitative measures for erosion rates: comparing surface height and roughness as metrics.
Significance
The strengths of optical profilometry as an analytical tool for understanding and analyzing biologically relevant processes such as biofilm induced tooth enamel demineralization were demonstrated.
doi:10.1016/j.dental.2009.07.012
PMCID: PMC3454478  PMID: 19732947
enamel erosion; optical profilometry; biofilm; Streptococcus mutans; enamel demineralization; citric acid; lactic acid; AFM
16.  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
17.  Dynamics of speB mRNA Transcripts in Streptococcus pyogenes 
Journal of Bacteriology  2012;194(6):1417-1426.
Streptococcus pyogenes (group A streptococcus [GAS]) is a human-specific pathogen that causes a variety of diseases ranging from superficial infections to life-threatening diseases. SpeB, a potent extracellular cysteine proteinase, plays an important role in the pathogenesis of GAS infections. Previous studies show that SpeB expression and activity are controlled at the transcriptional and posttranslational levels, though it had been unclear whether speB was also regulated at the posttranscriptional level. In this study, we examined the growth phase-dependent speB mRNA level and decay using quantitative reverse transcription-PCR (qRT-PCR) and Northern blot analyses. We observed that speB mRNA accumulated rapidly during exponential growth, which occurred concomitantly with an increase in speB mRNA stability. A closer observation revealed that the increased speB mRNA stability was mainly due to progressive acidification. Inactivation of RNase Y, a recently identified endoribonuclease, revealed a role in processing and degradation of speB mRNA. We conclude that the increased speB mRNA stability contributes to the rapid accumulation of speB transcript during growth.
doi:10.1128/JB.06612-11
PMCID: PMC3294869  PMID: 22267517
18.  The Role of Hydrogen Peroxide in Environmental Adaptation of Oral Microbial Communities 
Oral streptococci are able to produce growth-inhibiting amounts of hydrogen peroxide (H2O2) as byproduct of aerobic metabolism. Several recent studies showed that the produced H2O2 is not a simple byproduct of metabolism but functions in several aspects of oral bacterial biofilm ecology. First, the release of DNA from cells is closely associated to the production of H2O2 in Streptococcus sanguinis and Streptococcus gordonii. Extracellular DNA is crucial for biofilm development and stabilization and can also serve as source for horizontal gene transfer between oral streptococci. Second, due to the growth inhibiting nature of H2O2, H2O2 compatible species associate with the producers. H2O2 production therefore might help in structuring the initial biofilm development. On the other hand, the oral environment harbors salivary peroxidases that are potent in H2O2 scavenging. Therefore, the effects of biofilm intrinsic H2O2 production might be locally confined. However, taking into account that 80% of initial oral biofilm constituents are streptococci, the influence of H2O2 on biofilm development and environmental adaptation might be under appreciated in current research.
doi:10.1155/2012/717843
PMCID: PMC3405655  PMID: 22848782
19.  Hydrogen Peroxide-Dependent DNA Release and Transfer of Antibiotic Resistance Genes in Streptococcus gordonii ▿  
Journal of Bacteriology  2011;193(24):6912-6922.
Certain oral streptococci produce H2O2 under aerobic growth conditions to inhibit competing species like Streptococcus mutans. Additionally, H2O2 production causes the release of extracellular DNA (eDNA). eDNA can participate in several important functions: biofilm formation and cell-cell aggregation are supported by eDNA, while eDNA can serve as a nutrient and as an antimicrobial agent by chelating essential cations. eDNA contains DNA fragments of a size that has the potential to transfer genomic information. By using Streptococcus gordonii as a model organism for streptococcal H2O2 production, H2O2-dependent eDNA release was further investigated. Under defined growth conditions, the eDNA release process was shown to be entirely dependent on H2O2. Chromosomal DNA damage seems to be the intrinsic signal for the release, although only actively growing cells were proficient eDNA donors. Interestingly, the process of eDNA production was found to be coupled with the induction of the S. gordonii natural competence system. Consequently, the production of H2O2 triggered the transfer of antibiotic resistance genes. These results suggest that H2O2 is potentially much more than a simple toxic metabolic by-product; rather, its production could serve as an important environmental signal that facilitates species evolution by transfer of genetic information and an increase in the mutation rate.
doi:10.1128/JB.05791-11
PMCID: PMC3232836  PMID: 21984796
21.  Characterization of competence and biofilm development of a Streptocccus sanguinis endocarditis isolate 
Molecular oral microbiology  2011;26(2):117-126.
Streptococcus sanguinis is an oral commensal bacterium and endogenous pathogen in the blood, which generally is naturally competent to take up extracellular DNA. Regarded as a stress response, competence development enables S. sanguinis to acquire new genetic material. The sequenced reference strain SK36 encodes and expresses the genes required for competence (com) and uptake of DNA. Isolated from blood cultures of a confirmed case of infective endocarditis, strain 133–79 encodes all necessary com genes but is not transformable under conditions permissive for competence development in SK36. Using synthetic competence-stimulating peptides (sCSP) based on sequences of SK36 and 133–79 comC, both strains developed competence at similar frequencies in cross-transformation experiments. Furthermore, downstream response pathways are similar in strains SK36 and 133–79 since platelet aggregation and biofilm formation appeared unaffected by CSP. Collectively, the data indicate that strains SK36 and 133–79 respond to CSP similarly, strongly suggesting that endogenous production or release of CSP from 133–79 is impaired.
doi:10.1111/j.2041-1014.2010.00602.x
PMCID: PMC3075536  PMID: 21375702
22.  Counteractive Balancing of Transcriptome Expression Involving CodY and CovRS in Streptococcus pyogenes▿† 
Journal of Bacteriology  2011;193(16):4153-4165.
Streptococcus pyogenes (group A streptococcus [GAS]) responds to environmental changes in a manner that results in an adaptive regulation of the transcriptome. The objective of the present study was to understand how two global transcriptional regulators, CodY and CovRS, coordinate the transcriptional network in S. pyogenes. Results from expression microarray data and quantitative reverse transcription-PCR (qRT-PCR) showed that the global regulator CodY controls the expression of about 250 genes, or about 17% of the genome of strain NZ131. Additionally, the codY gene was shown to be negatively autoregulated, with its protein binding directly to the promoter region with a CodY binding site. In further studies, the influence of codY, covRS, and codY-covRS mutations on gene expression was analyzed in growth phase-dependent conditions using C medium, reported to mimic nutritional abundance and famine conditions similar to those found during host GAS infection. Additional biological experiments of several virulence phenotypes, including pilin production, biofilm formation, and NAD glycohydrolase activity, demonstrated the role that both CodY and CovRS play in their regulation. Correlation analysis of the overall data revealed that, in exponentially growing cells, CodY and CovRS act in opposite directions, with CodY stimulating and CovRS repressing a substantial fraction of the core genome, including many virulence factors. This is the first report of counteractive balancing of transcriptome expression by global transcription regulators and provides important insight into how GAS modulates gene expression by integrating important extracellular and intracellular information.
doi:10.1128/JB.00061-11
PMCID: PMC3147680  PMID: 21705595
23.  Environmental Influences on Competitive Hydrogen Peroxide Production in Streptococcus gordonii ▿  
Applied and Environmental Microbiology  2011;77(13):4318-4328.
Streptococcus gordonii is an important member of the oral biofilm. One of its phenotypic traits is the production of hydrogen peroxide (H2O2). H2O2 is an antimicrobial component produced by S. gordonii that is able to antagonize the growth of cariogenic Streptococcus mutans. Strategies that modulate H2O2 production in the oral cavity may be useful as a simple therapeutic mechanism to improve oral health, but little is known about the regulation of H2O2 production. The enzyme responsible for H2O2 production is pyruvate oxidase, encoded by spxB. The functional studies of spxB expression and SpxB abundance presented in this report demonstrate a strong dependence on environmental oxygen tension and carbohydrate availability. Carbon catabolite repression (CCR) modulates spxB expression carbohydrate dependently. Catabolite control protein A (CcpA) represses spxB expression by direct binding to the spxB promoter, as shown by electrophoretic mobility shift assays (EMSA). Promoter mutation studies revealed the requirement of two catabolite-responsive elements (CRE) for CcpA-dependent spxB regulation, as evaluated by spxB expression and phenotypic H2O2 production assays. Thus, molecular mechanisms for the control of S. gordonii spxB expression are presented for the first time, demonstrating the possibility of manipulating H2O2 production for increased competitive fitness.
doi:10.1128/AEM.00309-11
PMCID: PMC3127700  PMID: 21571883
24.  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
25.  Catabolite Control Protein A Controls Hydrogen Peroxide Production and Cell Death in Streptococcus sanguinis▿  
Journal of Bacteriology  2010;193(2):516-526.
Streptococcus sanguinis is a commensal oral bacterium producing hydrogen peroxide (H2O2) that is dependent on pyruvate oxidase (Spx) activity. In addition to its well-known role in bacterial antagonism during interspecies competition, H2O2 causes cell death in about 10% of the S. sanguinis population. As a consequence of H2O2-induced cell death, largely intact chromosomal DNA is released into the environment. This extracellular DNA (eDNA) contributes to the self-aggregation phenotype under aerobic conditions. To further investigate the regulation of spx gene expression, we assessed the role of catabolite control protein A (CcpA) in spx expression control. We report here that CcpA represses spx expression. An isogenic ΔccpA mutant showed elevated spx expression, increased Spx abundance, and H2O2 production, whereas the wild type did not respond with altered spx expression in the presence of glucose and other carbohydrates. Since H2O2 is directly involved in the release of eDNA and bacterial cell death, the presented data suggest that CcpA is a central control element in this important developmental process in S. sanguinis.
doi:10.1128/JB.01131-10
PMCID: PMC3019840  PMID: 21036992

Results 1-25 (37)