Methods to Study Antagonistic Activities Among Oral Bacteria.

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.

Compartmentalized biosynthesis of mycophenolic acid.

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. 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.

Genome-wide screening of potential RNase Y-processed mRNAs in the M49 serotype Streptococcus pyogenes NZ131.

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.

Veillonella Catalase Protects the Growth of Fusobacterium nucleatum in Microaerophilic and Streptococcus gordonii-Resident Environments.

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 as , produce HO to inhibit the growth of competitors, like the mutans streptococci, as well as strict anaerobic middle and later colonizers of the dental biofilm. Interestingly, species, as early colonizers, physically interact (coaggregate) with A putative catalase gene () is found in most sequenced species; however, the function of this gene is unknown.

Complete Genome Sequence of OK5, the First Transformable Strain in the Species.

The strain OK5 was isolated from a human saliva sample and was the first strain shown to be genetically transformable in the 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 OK5.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

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.

Natural Competence Is Common among Clinical Isolates of and Is Useful for Genetic Manipulation of This Key Member of the Oral Microbiome.

The six 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 biology, largely due to the difficulties associated with their genetic manipulation.

Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleT.

The cytochrome P450 enzyme OleT 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 HO 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.

Methods to Study Antagonistic Activities Among Oral Bacteria.

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.

In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins.

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.

Identification and characterization of a haem biosynthesis locus in Veillonella.

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.

Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite.

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).

The Sialic Acid Binding Protein, Hsa, in Streptococcus gordonii DL1 also Mediates Intergeneric Coaggregation with Veillonella Species.

Dental biofilm development involves initial colonization of the tooth's surface by pioneer colonizers, followed by cell-cell coaggregation between the pioneer and later colonizers. Streptococcus gordonii is one of the pioneer colonizers. In addition to its role in oral biofilm development, S.

Establishment of a counter-selectable markerless mutagenesis system in Veillonella atypica.

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.

RNases J1 and J2 are critical pleiotropic regulators in Streptococcus mutans.

In recent years, it has become increasingly evident that post-transcriptional control mechanisms are the principal source of gene regulation for a large number of prokaryotic genetic pathways, particularly those involved in virulence and environmental adaptation. Post-transcriptional regulation is largely governed by RNA stability, which itself is determined by target accessibility to RNase degradation. In most Firmicutes species, mRNA stability is strongly impacted by the activity of two recently discovered RNases referred to as RNase J1 and RNase J2.

Functional characterization of MpaG', the O-methyltransferase involved in the biosynthesis of mycophenolic acid.

Mycophenolic acid (MPA, 1) is a clinically important immunosuppressant. In this report, a gene cluster mpa' responsible for the biosynthesis of 1 was identified from Penicillium brevicompactum NRRL 864. The S-adenosyl-L-methionine-dependent (SAM-dependent) O-methyltransferase encoded by the mpaG' gene was functionally and kinetically characterized in vitro.

The Streptococcus mutans irvA gene encodes a trans-acting riboregulatory mRNA.

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.

A YadA-like autotransporter, Hag1 in Veillonella atypica is a multivalent hemagglutinin involved in adherence to oral streptococci, Porphyromonas gingivalis, and human oral buccal cells.

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.

Improved production of fatty alcohols in cyanobacteria by metabolic engineering.

Background: Fatty alcohols are widely used in industrial chemicals. The biosynthetic pathways for fatty alcohols are diverse and widely existing in nature. They display a high capacity to produce fatty alcohols by the metabolic engineering of different microbe hosts.

Ubiquitous sialometabolism present among oral fusobacteria.

Fusobacterium nucleatum is a ubiquitous member of the human oral flora and is associated with the development of periodontitis and a variety of other types of polymicrobial infections of the mucosa. In the oral cavity, this species is one of the few that is prevalent in both healthy and diseased subgingival plaque. Using microarray analysis, we examined the transcriptional response of F.

[Construction and evaluation of efficient gene expression platforms in Synechocystis sp. strain PCC6803].

For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803.

Development of a tunable wide-range gene induction system useful for the study of streptococcal toxin-antitoxin systems.

Despite the plethora of genetic tools that have been developed for use in Streptococcus mutans, the S. mutans genetic system still lacks an effective gene induction system exhibiting low basal expression and strong inducibility. Consequently, we created two hybrid gene induction cassettes referred to as Xyl-S1 and Xyl-S2.

Construction, characterization and application of molecular tools for metabolic engineering of Synechocystis sp.

An integrative gene expression system has been constructed for the directional assembly of biological components in Synechocystis PCC6803. We have characterized 11 promoter parts with various expression efficiencies for genetic engineering of Synechocystis for the production of fatty alcohols. This was achieved by integrating several genetic modifications including the expression of multiple-copies of fatty acyl-CoA reductase (FAR) under the control of strong promoters, disruption of the competing pathways for poly-β-hydroxybutyrate and glycogen synthesis, and for peptide truncation of the FAR.

Cloning-independent plasmid construction for genetic studies in streptococci.

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.