Search

Most bacteria in nature exist in multispecies communities known as biofilms. In the natural habitat where resources (nutrient, space, etc.) are usually limited, individual species must compete or collaborate with other neighboring species in order to perpetuate in the multispecies community. The human oral cavity is colonized by700 microbial species known as the indigenous microbiota. This indigenous flora normally maintains an ecological balance through antagonistic as well as mutualistic interspecies interactions. However, environmental perturbation may disrupt this balance, leading to overgrowth of pathogenic species which could in turn initiate diseases such as dental caries (tooth decay) and periodontitis (gum disease). Understanding the mechanisms of diversity maintenance may help developing novel approaches to manage these "polymicrobial diseases". In this chapter, we will focus on a well-characterized form of biochemical warfare: bacteriocins produced by Streptococcus mutans, a primary dental caries pathogen, and hydrogen peroxide (H

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic in human history and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, its biosynthetic mechanisms have remained a long-standing mystery. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and β-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB’ as the long-sought key enzyme responsible for the oxidative cleavage of sesquiterpene side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC’ and O-methyltransferase MpaG’, the Golgi apparatus-associated prenyltransferase MpaA’, the endoplasmic reticulum-bound oxygenase MpaB’ and P450-hydrolase fusion enzyme MpaDE’, and the peroxisomal acyl-CoA hydrolase MpaH’. The whole pathway is elegantly co-mediated by these compartmentalized enzymes, together with the peroxisomal β-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic pathway, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.Significance StatementHere we elucidate the full biosynthetic pathway of the fungal natural product mycophenolic acid (MPA), which represents an unsolved mystery for decades. Besides the intriguing enzymatic mechanisms, we reveal that the MPA biosynthetic enzymes are elegantly compartmentalized; and the subcellular localization of the acyl-CoA hydrolase MpaH’ in peroxisomes is required for the unique cooperation between biosynthetic and β-oxidation catabolism machineries. This work highlights the importance of a cell biology perspective for understanding the unexplored organelle-associated essential catalytic mechanisms in natural product biosynthesis of fungi and other higher organisms. The insights provided by our work will also benefit future efforts for both industrial strain improvement and novel drug development.

Cytochrome P450 enzymes are highly diversified biocatalysts associated with steroid biosynthesis, xenobiotic metabolism, biosynthesis of natural products, and industrial oxidation reactions. A typical P450 catalytic cycle requires sequential transfer of two electrons from NAD(P)H to the heme-iron reactive center for O2 activation. For the most abundant bacterial Class I P450 systems, this important process is usually mediated by two redox partner proteins including an FAD-containing ferredoxin reductase (FdR) and a small iron–sulfur protein, ferredoxin (Fdx). However, it is often unclear which pair of Fdx and FdR among multiple redox partners is the optimal one for a specific Class I P450 enzyme. To address this important but underexplored question, herein, a reaction matrix network with 16 Fdxs, 8 FdRs, and 6 P450s (against 7 substrates) was constituted. By analyzing the reactivity profiles of 896 P450 reactions, together with phylogenetic analysis, redox potential measurements, structural simulations, a...

The irreversibly diminishing reserves and the fluctuating price of crude oil, together with the serious concerns on the global environment and energy security, all suggests an unpleasant future of fossil fuel. Within this context, it is imperative to find alternative eco-friendly fuel with properties that highly mimic fossil fuel. Aliphatic alka(e)nes generated from renewable resources via biosynthetic pathways perfectly suits the criteria and represents the ideal ″drop-in″ biofuels. α-Alkenes are special microbial natural products. These biohydrocarbons have attracted much attention due to their potential application as biofuels and biomaterials. By coupling the activity of lipase and P450 decarboxylase, lipids (i.e. triglycerides) can be transformed into α-alkenes via free fatty acid intermediates. To improve the conversion efficiency of lipids to α-alkenes, in this study, four different versions of fusion proteins of the lipase TlL from Thermomyces lanuginosus and the P450 decarboxylase OleTJE from Jeotgalicoccus sp. ATCC 8456, including FusA (His6-TlL-linker-OleTJE), FusB (TlL-Linker-OleTJE-His6), FusC (His6-OleTJE-linker-TlL) and FusD (OleTJE- linker-TlL-His6), were constructed and comparatively evaluated. The optimal fusion enzyme FusC was able to convert a number of triacylglycerols including trilaurin (C12), trimyristin (C14) and tripalmitin (C16) into corresponding α-alkenes via the coupled hydrolysis and decarboxylation. The 31.7% yield of 1-tridecene from trimyristin demonstrates good substrate channeling effect. Due to the higher catalytic efficiency compared to the mixed TlL and OleTJE, as well as the simplified enzyme purification and hence the lower cost, this fusion strategy may hold significant potential of application.

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an ‘all-or-none’ pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry.

Objective The release of unpolymerized monomers and by-products of resin composites influences biofilm growth and confounds the measurement of metabolic activity. Current assays to measure biofilm viability have critical limitations and are typically not performed on relevant substrates. The objective of the present study was to determine the utility of firefly luciferase assay for quantification of the viability of intact biofilms on a resin composite substrate, and correlate the results with a standard method (viable colony counts). Methods Disk-shaped specimens of a dental resin composite were fabricated, wet-polished, UV-sterilized, and stored in water. Biofilms of Streptococcus mutans (strain UA159 modified by insertion of constitutively expressed firefly luc gene) were grown (1:500 dilution; anaerobic conditions, 24 h, 37 °C) in two media concentrations (0.35x and 0.65x THY medium supplemented with 0.1% sucrose; n = 15/group). An additional group of specimens with biofilms grown in 0.65x + sucrose media was treated with chlorhexidine gluconate solution to serve as the control group. Bioluminescence measurements of non-disrupted biofilms were obtained after addition of d -Luciferin substrate. The adherent biofilms were removed by sonication, and bioluminescence of sonicated bacteria was then measured. Viable colony counts were performed after plating sonicated bacteria on THY agar plates supplemented with spectinomycin. Bioluminescence values and cell counts were correlated using Spearman correlation tests ( α = 0.05). Results Strong positive correlations between viable colony counts and bioluminescence values, both before- and after-sonication, validated the utility of this assay. Significance A novel non-disruptive, real-time bioluminescence assay is presented for quantification of intact S. mutans biofilms grown on a resin composite, and potentially on antibacterial materials and other types of dental biomaterials.

Summary Dental biofilm development is a sequential process, and adherence between microbes and the salivary pellicle (adhesion) as well as among different microbes (co-adhesion or coaggregation) plays a critical role in building a biofilm community. The Veillonella species are among the most predominant species in the oral cavity and coaggregate with many initial, early, middle, and late colonizers. Similar to oral fusobacteria, they are also considered bridging species in biofilm development. However, the mechanism of this ability has yet to be reported, due to the previous lack of a genetic transformation system in the entire genus. In this study, we used our recently discovered transformable Veillonella strain, Veillonella atypica OK5, to probe the mechanism of coaggregation between Veillonella species and other oral bacteria. By insertional inactivation of all eight putative hemagglutinin genes, we identified one gene, hag1, which is involved in V. atypica coaggregation with the initial colonizers Streptococcus gordonii, Streptococcus oralis and Streptococcus cristatus, and the periodontal pathogen Porphyromonas gingivalis. The hag1 mutant also abolished adherence to human buccal cells. Inhibition assays using various chemical or physiological treatments suggest different mechanisms being involved in coaggregation with different partners. The entire hag1 gene was sequenced and shown to be the largest known bacterial hemagglutinin gene.

In both prokaryotes and eukaryotes, insight into gene function is typically obtained by in silico homology searches and/or phenotypic analyses of strains bearing mutations within open reading frames. However, the studies herein illustrate how mRNA function is not limited to the expression of a cognate protein. We demonstrate that a stress-induced protein-encoding mRNA (irvA) from the dental caries pathogen Streptococcus mutans directly modulates target mRNA (gbpC) stability through seed pairing interactions. The 5' untranslated region of irvA mRNA is a trans riboregulator of gbpC and a critical activator of the DDAG stress response, whereas IrvA functions independently in the regulation of natural competence. The irvA riboregulatory domain controls GbpC production by forming irvA-gbpC hybrid mRNA duplexes that prevent gbpC degradation by an RNase J2-mediated pathway. These studies implicate a potentially ubiquitous role for typical protein-encoding mRNAs as riboregulators, which could alter current concepts in gene regulation.

The oral biofilm is a multispecies community in which antagonism and mutualism coexist among friends and foes to keep an ecological balance of community members. The pioneer colonizers, such asStreptococcus gordonii, produce H2O2to inhibit the growth of competitors, like the mutans streptococci, as well as strict anaerobic middle and later colonizers of the dental biofilm. Interestingly,Veillonellaspecies, as early colonizers, physically interact (coaggregate) withS. gordonii. A putative catalase gene (catA) is found in most sequencedVeillonellaspecies; however, the function of this gene is unknown. In this study, we characterized the ecological function ofcatAfromVeillonella parvulaPK1910 by integrating it into the only transformable strain,Veillonella atypicaOK5, which iscatAnegative. The strain (OK5-catA) became more resistant to H2O2. Further studies demonstrated that thecatAgene expression is induced by the addition of H2O2or coculture withS. gordonii. Mixed-culture experiments further revealed that the transgenic OK5-catAstrain not only enhanced the growth ofFusobacterium nucleatum, a strict anaerobic periodontopathogen, under microaerophilic conditions, but it also rescuedF. nucleatumfrom killing byS. gordonii. A potential role of catalase in veillonellae in biofilm ecology and pathogenesis is discussed here.IMPORTANCEVeillonellaspecies, as early colonizers, can coaggregate with many bacteria, including the initial colonizerStreptococcus gordoniiand periodontal pathogenFusobacterium nucleatum, during various stages of oral biofilm formation. In addition to providing binding sites for many microbes, our previous study also showed thatVeillonellaproduces nutrients for the survival and growth of periodontal pathogens. These findings indicate thatVeillonellaplays an important “bridging” role in the development of oral biofilms and the ecology of the human oral cavity. In this study, we demonstrated that the reducing activity ofVeillonellacan rescue the growth ofFusobacterium nucleatumnot only under microaerophilic conditions, but also in an environment in whichStreptococcus gordoniiis present. Thus, this study will provide a new insight for future studies on the mechanisms of human oral biofilm formation and the control of periodontal diseases.

The Veillonella atypica strain OK5 was isolated from a human saliva sample and was the first strain shown to be genetically transformable in the Veillonella genus. Genetic studies using this strain have helped us gain much insight into the ecology of human oral biofilms. Here, we report the complete genome sequence of V. atypica OK5.

The cytochrome P450 enzyme OleTJE from Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H2O2 as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleTJE through extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleTJE. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleTJE towards myristic acid were determined.

The six Veillonella species found in the human oral cavity are among the most abundant members of the oral flora, occurring in both supra- and subgingival dental plaque as well as on the oral mucosa. Epidemiological data have also implicated these species in the development of the most common oral diseases. Despite their ubiquity, abundance, and ecological significance, surprisingly little is known about Veillonella biology, largely due to the difficulties associated with their genetic manipulation. In an effort to improve genetic analyses of Veillonella species, we isolated a collection of veillonellae from clinical plaque samples and screened for natural competence using a newly developed transformation protocol. Numerous strains of V. parvula were found to exhibit a natural competence ability that was highly influenced by growth medium composition. By exploiting this ability, we were able to utilize cloning-independent allelic exchange mutagenesis to identify the likely source of DNA uptake machinery within a locus homologous to type II secretion systems (T2SS). Interestingly, V. parvula natural competence was found to exhibit a clear hierarchy of preference for different sources of DNA (plasmid < PCR product < genomic DNA), which is unlike most naturally competent species. Genomic comparisons with other members of the Veillonellaceae family suggest that natural competence is likely to be widely distributed within this group. To the best of our knowledge, this study is the first demonstration of natural competence and targeted allelic exchange mutagenesis within the entire Veillonellaceae family and demonstrates a simple and rapid method to study Veillonella genetics.

Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in Escherichia coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5 × 103 to 2 × 105 CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli–Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts.

Most bacteria in nature exist in multispecies communities known as biofilms. In the natural habitat where resources (nutrient, space, etc.) are usually limited, individual species must compete or collaborate with other neighboring species in order to perpetuate in the multispecies community. The human oral cavity is colonized by >700 microbial species known as the indigenous microbiota. This indigenous flora normally maintains an ecological balance through antagonistic as well as mutualistic interspecies interactions. However, environmental perturbation may disrupt this balance, leading to overgrowth of pathogenic species which could in turn initiate diseases such as dental caries (tooth decay) and periodontitis (gum disease). Understanding the mechanisms of diversity maintenance may help developing novel approaches to manage these "polymicrobial diseases." In this chapter, we focus on a well-characterized form of biochemical warfare: bacteriocins produced by Streptococcus mutans, a primary dental caries pathogen, and hydrogen peroxide (H2O2) produced by several oral commensal streptococci. We will describe detailed methodologies on the competition assay, isolation, purification, and characterization of bacteriocins.

Haemin/haem is one of the essential nutrients required by periodontopathogens such as Porphyromonas gingivalis to grow in vitro. In the oral cavity, this nutrient is believed to be provided by the crevicular fluid, a serum-like exudate produced during gum inflammation. However, P. gingivalis is also present in the healthy dental biofilm where inflammation is absent. This study was designed to answer the question: what organism(s) in the healthy dental biofilm provides haemin/haem to those periodontal pathogens? We report here that veillonellae, a group of bridging species in dental biofilm development, harbour a complete gene cluster for haem biosynthesis. Haemin production was detected from cell lysate, suggesting that the haem biosynthesis pathway is functional in veillonellae. Using the only transformable strain Veillonella atypica OK5, we inactivated specific key genes in the haem biosynthesis pathway. Inactivation of hemE, encoding the enzyme uroporphyrinogen decarboxylase, not only abolished haemin production but also significantly decreased OK5-supported growth of P. gingivalis. A luciferase gene reporter to the hemEHG operon demonstrated up-regulation of operon expression by P. gingivalis. Analysis of all sequenced genomes of oral bacteria in the HOMD database identified three genera (Veillonella, Propionibacterium and Aggregatibacter) that have a complete haem biosynthesis gene cluster, suggesting that they all could be potential haemin/haem providers in the dental biofilm.

The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-l-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.

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.

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.

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.

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.

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.

Interactions between oral streptococci have developed because of ecological pressures within the oral environment. The high abundance of oral streptococci makes it very likely that they have evolved close relationships manifested in diverse interspecies interactions. Interspecies interactions in the oral biofilm can be defined by the purpose and nature of the interactions. Although there are many potential interactions between oral streptococci and other members of the oral biofilm community as well as the host, this chapter specifically focuses on streptococcus-streptococcus interactions. By promoting genetic exchange, the interspecies interaction, although not beneficial for the lysed bacterial species, is important in the context of evolution. A counteroffensive strategy was recently identified for Streptococcus oligofermentans, another oral streptococcal species and competitor of S. mutans. Surprisingly, S. oligofermentans is able to utilize the lactic acid produced by cariogenic species, such as S. mutans, to generate hydrogen peroxide, leading to growth inhibition of the lactic acid producer. Expanding research into multispecies models is desirable and would most likely reveal interesting synergistic behavior of oral streptococci. The close relationship of the oral streptococci enables the reconstruction of biochemical networks from already-existing metabolic functions identified in other streptococci. The spatial-temporal developmental patterns of oral streptococci in the biofilm, including the molecular mechanisms, are characterized. The oral biofilm is easily accessible, and the variety of genetic manipulation options for oral streptococci allows for experimental verification of in silico predictions.

Veillonellae are one of the most prevalent and predominant microorganisms in both the supra- and subgingival plaques of the human oral cavity. Veillonellae's mutualistic relationships with the early, middle, and late colonizers of the oral cavity make them an important component of oral biofilm ecology. Unlike other ubiquitous early colonizers in the oral cavity, surprisingly little is known about Veillonella biology due to our lack of ability to genetically transform this group of bacteria. The objective of this study was to test the transformability of veillonellae. Using Veillonella parvula strain PK1910, we first obtained spontaneous mutations conferring streptomycin resistance. These mutations all carry a K43N substitution in the RpsL protein. Using the mutated rpsL gene as a selection marker, a variety of conditions were tested and optimized for electroporation. With the optimized protocol, we were able to introduce the first targeted mutation into the chromosome of V. parvula PK1910. Although more studies are needed to develop a robust genetic manipulation system in veillonellae, our results demonstrated, for the first time, that V. parvula is transformable, at least for strain PK1910.

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.

Human dental plaque is a complex microbial community containing an estimated 700 to 19,000 species/phylotypes. Despite numerous studies analysing species richness in healthy and diseased human subjects, the true genomic composition of the human dental plaque microbiota remains unknown. Here we report a metagenomic analysis of a healthy human plaque sample using a combination of second-generation sequencing platforms. A total of 860 million base pairs of non-human sequences were generated. Various analysis tools revealed the presence of 12 well-characterized phyla, members of the TM-7 and BRC1 clade, and sequences that could not be classified. Both pathogens and opportunistic pathogens were identified, supporting the ecological plaque hypothesis for oral diseases. Mapping the metagenomic reads to sequenced reference genomes demonstrated that 4% of the reads could be assigned to the sequenced species. Preliminary annotation identified genes belonging to all known functional categories. Interestingly, although 73% of the total assembled contig sequences were predicted to code for proteins, only 51% of them could be assigned a functional role. Furthermore, ~2.8% of the total predicted genes coded for proteins involved in resistance to antibiotics and toxic compounds, suggesting that the oral cavity is an important reservoir for antimicrobial resistance.

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.

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 H 2 O 2 . 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.

RNase Y is a major endoribonuclease in Group A streptococcus (GAS) and other Gram‐positive bacteria. Our previous study showed that RNase Y was involved in mRNA degradation and processing in GAS. We hypothesized that mRNA processing regulated the expression of important GAS virulence factors via altering their mRNA stabilities and that RNase Y mediated at least some of the mRNA‐processing events. The aims of this study were to (1) identify mRNAs that were processed by RNase Y and (2) confirm the mRNA‐processing events. The transcriptomes of Streptococcus pyogenes NZ131 wild type and its RNase Y mutant (Δrny) were examined with RNA‐seq. The data were further analyzed to define GAS operons. The mRNA stabilities of the wild type and Δrny at subgene level were determined with tiling array analysis. Operons displaying segmental stability in the wild type but not in the Δrny were predicted to be RNase Y processed. Overall 865 operons were defined and their boundaries predicted. Further analysis narrowed down 15 mRNAs potentially processed by RNase Y. A selection of four candidates including folC1 (folylpolyglutamate synthetase), prtF (fibronectin‐binding protein), speG (streptococcal exotoxin G), ropB (transcriptional regulator of speB), and ypaA (riboflavin transporter) mRNAs was examined with Northern blot analysis. However, only folC1 was confirmed to be processed, but it is unlikely that RNase Y is responsible. We conclude that GAS use RNase Y to selectively process mRNA, but the overall impact is confined to selected virulence factors.

Previous studies have identified the hdrRM operon as a novel regulatory system induced by conditions of high cell density. Little is known about the genes under the control of this system, but a variety of important phenotypes are associated with either hdrR overexpression or mutation of hdrM. To characterize the regulatory function of the HdrRM system in Streptococcus mutans we used a microarray approach to compare the transcriptional profiles of an hdrR overexpression strain with an hdrM mutant. Both strains exhibited almost identical profiles, which included all of the known late competence genes as well as a variety of competence-induced bacteriocins. Through a combination of real-time reverse transcription-polymerase chain reaction (PCR), reporter gene analysis and random amplification of complementary DNA ends PCR, we confirmed the role of comX as a central intermediate regulator of numerous genes in the hdrRM regulon. Through these studies, we also identified novel comX-regulated genes required for natural competence. Taken together, our results suggest that the primary function of the HdrRM system is to regulate the late competence genes together with various bacteriocins. This occurs independently of the ComCDE system, even though both systems regulate nearly identical genes. This suggests that S. mutans has multiple parallel input sensory systems that control the same output response: the induction of natural competence and concurrent production of bacteriocins.

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.

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-lantibiotic 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 brain heart infusion agar medium 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 for which we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups, including central metabolism, surface binding and sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when the gene 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.

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.

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans , the primary cariogenic pathogen, by producing hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 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 H 2 O 2 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 aao S o , 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 H 2 O 2 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 aao S o mutant of S. oligofermentans did lose H 2 O 2 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 aao S o mutant, indicating the gene's importance in interspecies competition.

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.

Genetic studies of Streptococcus mutans have benefited greatly from the numerous techniques that have been successfully adapted for use in this organism. One notable exception is the lack of a negative selection system that can be employed for the easy isolation of markerless in-frame deletions. In this study, we report the development of a galK/galactose-based negative selection system in S. mutans for this purpose. This system consists of a recipient strain (IFD140) that contains a deletion in the galKTE operon and a suicide vector (pIFD-Sm) that carries the S. mutans galK open reading frame fused to the constitutive lactate dehydrogenase (ldh) promoter. Using this system we created a markerless in-frame deletion in the beta-galactosidase (lacG) gene within the S. mutans lactose operon. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of the galactose-resistant colonies and resulted in in-frame deletions in 50% of the screened isolates. Based on the ratio of galactose-resistant cells to total cells, we determined that plasmid excision occurred at a frequency of approximately 1/3000 cells. Furthermore, the simplicity of this system should make it adaptable for use in numerous other gram-positive and gram-negative organisms.

Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella.

Streptococcus mutans is a major cariogenic inhabitant of the high cell density oral biofilm (dental plaque). In previous studies, we showed that production of one of its virulence factors, the bacteriocin mutacin IV, was regulated by high cell density as well as the competence regulatory system ComED. In this study, we utilized luciferase fusions and real-time reverse transcriptase polymerase chain reaction (RT-PCR), to demonstrate that high cell density and ComED also regulate an uncharacterized group of mutacin and mutacin-like genes. Under high cell density or in the presence of externally added competence-stimulating peptide (CSP), gene expression increased 10- to 30-fold. Interestingly, high cell density was able to bypass the requirement for CSP addition. However, both cell density and CSP-dependent gene expression had a strict requirement for the ComE response regulator.

Streptococcus oligofermentans is a newly characterized species belonging to the mitis group of oral streptococci. So far no correlation has been demonstrated between S. oligofermentans and dental caries. Furthermore, a reverse correlation has been observed between the number of S. oligofermentans and the number of Streptococcus mutans, a major cariogenic pathogen, in the oral cavity. These properties suggest that S. oligofermentans may have a potential to be used as a 'probiotics' for caries prevention. In this study, we aim to establish a genetic system in S. oligofermentans to further study the biology of this new species. Using homologous regions of the comCDE locus in other streptococci, the comC gene was isolated and sequenced. A synthetic competence-stimulating peptide (CSP) was synthesized and shown to be able to effectively induce competence in S. oligofermentans. This CSP-induced transformation system in S. oligofermentans was used to construct green fluorescent protein (gfp) and luciferase (luc) reporter systems, both of which are driven by the lactate dehydrogenase (ldh) promoter. These reporter systems were further shown to be highly expressed in planktonic and biofilm cells, suggesting that these reporter systems can be used in future ecological studies of S. oligofermentans.

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 s pecifically (or s electively) t argeted a nti m icrobial p eptides (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 c ompetence s timulating p eptide (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.

Streptococcus mutansis known as a primary pathogen responsible for dental caries. One of the virulence factors ofS. mutansin cariogenicity is its ability to attach to the tooth surface and form a biofilm. Several surface proteins have been shown to be involved in this process. A 29 kDa surface protein named wall-associated protein A (WapA, antigen A or antigen III), was previously used as a vaccine in animal studies for immunization against dental caries. However, the function of WapA inS. mutansis still not clear. This study characterized the function of WapA in cell surface structure and biofilm formation. Compared to the wild-type, thewapAmutant had much-reduced cell chain length, diminished cell–cell aggregation, altered cell surface ultrastructure, and unstructured biofilm architecture. Furthermore,in vivoforce spectroscopy revealed that the cell surface of thewapAmutant was less sticky than that of the wild-type cells. More interestingly, these phenotypic differences diminished as sucrose concentration in the medium was increased to 0.5 %. Real-time RT-PCR analysis demonstrated that sucrose strongly repressedwapAgene expression in both planktonic and biofilm cells. These results suggest that the WapA protein plays an important structural role on the cell surface, which ultimately affects sucrose-independent cell–cell aggregation and biofilm architecture.

The microbiota plays a crucial role in experimental models of inflammatory bowel disease, but the exact mechanisms of its effects are unknown. These studies took two molecular approaches to this question. The first used amplification of the 16s ribosomal DNA to define microbial diversity in the colon. Although there were differences in colitic and non-colitic mice, we could not determine whether this was primary or secondary to the disease. The second approach used serologic expression cloning to identify the microbial proteins stimulating the pathogenic immune response. Previously unknown microbial flagellins were the dominant cluster of antigens identified. About half of the sera from patients with Crohn's disease have IgG antibodies to these flagellins.

Streptococcus mutans is a primary pathogen associated with dental caries. Its bacteriocin (mutacin) production ability is thought to play an important role in maintaining competitiveness in the multispecies oral biofilm. Previous studies have demonstrated that the production of the lantibiotic, mutacin I, is responsive to multiple input signals and that a putative inducible repressor, irvA, seems to be involved in the luxS-mediated mutacin I gene regulation pathway. In this study, we demonstrate that these multiple inputs can be divided into two pathways: irvA-dependent and irvA-independent. Similar to luxS, signals mediated through vicK, pttB and hk03 exert their effect possibly through modulating irvA transcription, whereas signals mediated through ciaH, hrcA, adhE, and Smu1281 exert their effect through an unknown mechanism independent of irvA.

Currently available antimicrobials exhibit broad killing with regard to bacterial genera and species. Indiscriminate killing of microbes by these conventional antibiotics can disrupt the ecological balance of the indigenous microbial flora, often resulting in negative clinical consequences. Species-specific antimicrobials capable of precisely targeting pathogenic bacteria without damaging benign microorganisms provide a means of avoiding this problem. In this communication, we report the successful creation of the first synthetic, target-specific antimicrobial peptide, G10KHc, via addition of a rationally designed Pseudomonas -specific targeting moiety (KH) to a generally killing peptide (novispirin G10). The resulting chimeric peptide showed enhanced bactericidal activity and faster killing kinetics against Pseudomonas spp. than G10 alone. The enhanced killing activities are due to increased binding and penetration of the outer membrane of Pseudomonas sp. cells. These properties were not observed in tests of untargeted bacterial species, and this specificity allowed G10KHc to selectively eliminate Pseudomonas spp. from mixed cultures. This work lays a foundation for generating target-specific “smart” antimicrobials to complement currently available conventional antibiotics.