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Since our chapter on genome sequencing using the GS-FLX pyrosequencer in the First Edition of this book, significant advances have been made in next-generation DNA sequencing (NGS) technology. Not only has the GS-FLX become extinct, but the more recent introduction and establishment of the so-called third-generation DNA sequencers by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) has revolutionized genomics yet again by generating ultra-long (100,000 basepair) sequence reads concomitant with an incredible reduction in cost per sequenced basepair. Unfortunately, the ultra-high sequence yields of third-generation sequencers are compromised by their inherent sequencing error rates, prompting an alternative sequencing strategy, i.e., a hybrid sequencing strategy, which combines PacBio/ONT primary datasets with complementary datasets generated by mainstream short-read NGS platforms, e.g., Illumina or Ion Torrent. Although the concept of a hybrid sequencing strategy is not new, existing yields and accuracy of ultra-long and short-read sequencing technologies makes such a strategy achievable, resulting in complete genome sequences in one hit. In this chapter, we describe our updated laboratory and bioinformatic protocols that will allow the average research group to obtain complete oral microbial genome sequences assembled from a combination of DNA sequence data generated by NGS and third-generation platforms.

Genes encoding the subunits of the membrane-bound F1F0-ATPase (responsible for exporting protons from the cytoplasm and contributing to acid tolerance) were sequenced for 24 non-mutans streptococci isolated from carious lesions. Isolates, mostly Streptococcus salivarius, displayed a continuum of acid tolerance thresholds ranging from pH 4.55 to 3.39, but amino acid alignments of F1F0-ATPase subunits revealed few non-synonymous substitutions and these were unrelated to acid tolerance. Thus, the F1F0-ATPase is highly-conserved among S. salivarius isolates despite varying acid tolerance thresholds, supporting the contention that acid tolerance is determined by the level of gene/protein expression rather than variation in molecular structure.

The human oral cavity is home to one of the most diverse, dynamic and complex collections of microbes (the ‘oral microbiota’) with an estimated 700–1,000 different microbial species inhabiting a va...

The oral microbiome is ecologically diverse, complex, dynamic, and little understood. We describe the microbiota of four oral habitats in a birth cohort at age 32 and examine differences by sex, oral hygiene, and current smoking status, dental caries, and periodontal health. Oral biofilm samples collected from anterior labial supragingival, posterior lingual supragingival, subgingival, and tongue sites of 841 Dunedin Multidisciplinary Health and Development Study members were analysed using checkerboard DNA-DNA hybridization; focusing on 30 ecologically important bacterial species. The four habitats exhibited distinct microbial profiles that differed by sex. Streptococcus gordonii was more dominant in supragingival and tongue biofilms of males; Porphyromonas gingivalis exhibited higher relative abundance in subgingival biofilm of females. Males had higher scores than females for periodontal pathogens at supragingival sites. The relative abundance of several putative caries and periodontal pathogens differed in smokers and non-smokers. With poor oral hygiene significantly higher proportions of Gram-negative facultative anaerobes were present in subgingival biofilm and there were higher scores for the principal components characterised by putative cariogenic and periodontal pathogens at each site. Distinctive microenvironments shape oral biofilms and systematic differences exist by sex, oral hygiene, and smoking status.

Genes encoding the subunits of the membrane-bound F1F0-ATPase (responsible for exporting protons from the cytoplasm and contributing to acid tolerance) were sequenced for 24 non-mutans streptococci isolated from carious lesions. Isolates, mostly Streptococcus salivarius, displayed a continuum of acid tolerance thresholds ranging from pH 4.55 to 3.39, but amino acid alignments of F1F0-ATPase subunits revealed few non-synonymous substitutions and these were unrelated to acid tolerance. Thus, the F1F0-ATPase is highly-conserved among S. salivarius isolates despite varying acid tolerance thresholds, supporting the contention that acid tolerance is determined by the level of gene/protein expression rather than variation in molecular structure.

In order to assess the colonization efficacy of the oral probiotic Streptococcus salivarius K12, a rapid method for specific detection and enumeration of the strain was developed. Here, we describe a two-step TaqMan™ quantitative PCR assay using primer-probe combinations targeting genes of the locus encoding the lantibiotic bacteriocin salivaricin B.

Objective To characterize the bacteriocin-like inhibitory-substances (BLIS) activity of Streptococcus macedonicus MBF10-2 [named BLIS(MBF10-2)], a bacteriocinogenic strain isolated from an Indonesian tofu byproduct. Methods BLIS(MBF10-2) was obtained by culturing the bacterium, and standard deferred antagonism assays were used to demonstrate its activity. The antibacterial testing of fractions collected by filtration using 3–30 kDa cut-off membrane sizes were carried out by performing well diffusion method. Results The growth of Micrococcus luteus , Streptococcus pyogenes , Lactococcus lactis , Leuconostoc mesenteroides and Weissella confusa were inhibited by BLIS(MBF10-2). Interestingly, BLIS-containing fractions obtained from sequential application on ultrafiltration membranes indicated that this bacterium Streptococcus macedonicus MBF10-2 could produces at least two antimicrobial peptides activities, one of which is likely to be a lantibiotic peptide. Potential synergistic activity against certain Gram-positive (but not Gram-negative) species when partnered with antibiotics (ampicillin, tetracycline or kanamycin) were observed. Conclusion Combination of some BLIS(MBF10-2) active fractions with antibiotics (ampicillin, tetracycline or kanamycin) could demonstrate synergistic activities against certain Gram-positive species.

Orthodontic braces can impede oral hygiene and promote halitosis. The aim of the study was to investigate the effect of the oral probiotic Streptococcus salivarius M18 on oral hygiene indices and halitosis in patients wearing orthodontic braces. The study was a prospective, randomized, triple-blind, placebo-controlled trial. Patients undergoing fixed orthodontic treatment were randomly allocated to a probiotic group (n = 32) and a placebo group (n = 32). Patients consumed 2 lozenges d-1 for one month. Assessments were taken at baseline, at the end of the intervention, and at a 3 month follow-up. The outcome measures were plaque index (PI), gingival index (GI) and halitosis-causing volatile sulfur compound (VSC) levels. The dental biofilms before and after the intervention were analyzed utilizing next-generation sequencing of bacterial 16S rRNA genes. PI and GI scores were not significantly influenced by the probiotic intervention (intervention × time: p > 0.05). The level of VSCs decreased significantly in both the probiotic group (VSC reduction = -8.5%, 95%CI = -7.4% to -9.1%, p = 0.015) and the placebo group (-6.5%, 95%CI = -6.0% to -7.4%, p = 0.039) after 1 month intervention. However, at the 3 month follow-up, the VSC levels in the placebo group returned to baseline levels whereas those in the probiotic group decreased further (-10.8%, 95%CI = -10.5% to -12.9%, p = 0.005). Time, but not treatment, was associated with the decrease in microbial community alpha diversity and a modest effect on beta diversity. Oral probiotic S. salivarius M18 reduced the level of halitosis in patients with orthodontic braces, but had minimal effects on PI, GI and dental biofilm microflora.

Dental caries is an infectious disease that is continuing to increase in prevalence, reducing the quality of life for millions worldwide as well as causing considerable expense, with an estimated US$108 billion spent on dental care in the USA each year. Oral probiotics are now being investigated to determine whether they could play a role in the prevention and treatment of this disease. Streptococcus salivarius strain JH is a potential probiotic candidate that produces multiple proteinaceous antimicrobials (bacteriocins), the inhibitory spectrum of which includes Streptococcus mutans, one of the principal causative agents of dental caries. The genome of strain JH has previously been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3, streptin and streptococcin SA-FF22. Here we show that strain JH also produces salivaricin E, a 32 aa lantibiotic with a mass of 3565.9 Da, which is responsible for the inhibition of S. mutans growth. In addition, strain JH was shown to produce dextranase, an enzyme that hydrolyses (1 → 6)-α-D-glucosidic linkages, at levels higher than any other S. salivarius tested. In vitro testing showed that partial hydrolysis of the exopolymeric substances of S. mutans, using strain JH dextranase, improved the anti-S. mutans inhibitory activity of the lytic bacteriocin, zoocin A. The multiple bacteriocin and dextranase activities of strain JH support its candidature for development as an oral probiotic.

Background:The development of antibiotic resistance amongst bacterial pathogens and a population explosion, e.g. in countries such as Indonesia, are two issues the world is facing today. These issues have stimulated interest in the development of new antimicrobial therapeutic agents and contraceptive strategies, such as novel spermicides. Bacteriocins, which are bacterially-derived antimicrobial peptides, may fulfill some of the criteria for these new agents.Methods:Weissella confusa MBF8-1, originally isolated from a homemade soy product, exhibits antibacterial activity that was subsequently found to be plasmid-encoded, presumably by three peptides Bac1, Bac2 and Bac3. In the present study, we tested cell-free MBF8-1 bacteriocin preparations and chemically-synthesized versions of Bac1, Bac2 and Bac3 peptides for (i) its antibacterial activity against the indicator bacterium Leuconostoc mesenteroides and (ii) its ability to affect the motility of spermatozoa. Nisin, a known lantibiotic bacteriocin, was used as the control.Results:Here, we demonstrate that synthetic Bac1, in combination with synthetic Bac2, was sufficient to inhibit the growth of L. mesenteroides and affect sperm motility. However, the presence of all three synthetic peptides, s-Bac1, s-Bac2 and s-Bac3, was required for full potency.Conclusion:In summary, the bacteriocin-like peptides of W. confusa MBF8-1 have the potential to be developed as a narrow-spectrum antimicrobial agent and a novel spermicidal agent.

Since the pioneering work of van Leeuwenhoek in 1684, subsequently built upon by other renowned microbiologists Robert Koch, Willoughby Miller and GV Black, oral microbiology has developed innovative techniques to study the oral microflora (now termed the 'oral microbiome'). The advent of molecular techniques such as DNA-DNA hybridization, polymerase chain reaction and DNA sequencing has created an array of opportunities to construct a comprehensive picture of the diversity and composition of the oral microbiome. Approximately 700 oral bacterial species have been identified, of which 50% have yet to be cultivated, and some of these are known only by their signature DNA sequences. The synergism of ever-evolving culture-based and state-of-the-art culture-independent molecular techniques has facilitated in-depth understanding of the dynamics, acquisition and transfer of oral bacteria, along with their role in oral and general health and disease. Further research is needed to not only analyse but also to make sense of the ever-increasing volumes of data which these molecular techniques (especially high-throughput DNA sequencing) are generating, as well as why particular bacteria are present and what they are 'actually doing' there. This review presents a comprehensive literature search of oral microbiology-related methods currently used to study the oral microbiome.

We report here the draft genome sequence of Weissella confusa MBF8-1, an isolate from a homemade fermented soybean product that produces sucrases and exhibits antibacterial (bacteriocin) activity. The draft genome of W. confusa MBF8-1 comprises a 2.2-Mbp chromosome and a 17.8-kbp bacteriocin-encoding plasmid. Two putative glucansucrase genes were also identified.

Streptococcus salivarius has an exclusive and intimate association with humans. We are its sole natural host, and its contribution to the relationship appears overwhelmingly benevolent. Beautifully adapted to its preferred habitat, the human tongue, it only rarely ventures far from this location in the healthy host and indeed appears ill-equipped to become invasive due to a scarcity of virulence attributes. We consider that its strategically advantageous lingual location and numerical predominance allow S. salivarius to carry out a population surveillance and modulation role within the oral microbiota. Some strains are armed with complex arrays of targeted antibiotic weaponry, much of which belongs to the lantibiotic class of bacteriocins and a key to their ability to assemble and utilize this armament is their possession of transmissible multi-bacteriocin-encoding megaplasmid DNA. This review traces the origins of research into S. salivarius bacteriocins and bacteriocin-like inhibitory substances, showcases some of the inhibitory activities that we currently have knowledge of, and speculates about potential directions for ongoing investigation and probiotic application of this previously under-rated human commensal.

Background: Previous studies have demonstrated variable effects on systemic inflammatory and immune responses following improved periodontal health. This study examined changes in serum levels of the inflammatory mediators IL-1β, IL-6, TNF-α and sICAM-1, and antibodies to Porphyromonas gingivalis, human heat shock protein (hHSP) 60 and P. gingivalis GroEL following improvement in periodontal health in high cardiovascular (CV) risk and low CV-risk patients. Methods: Patients retrospectively selected from a longitudinal study, had undergone yearly periodontal examinations and peripheral blood collections. They had demonstrated a quantifiable improvement in periodontal health (>60% reduction in number of sites with probing depth ≥4 mm from the baseline visit) and could be classified as either high CV-risk (≥6 classical risk factors, n = 13) or low CV-risk (≤1 classical risk factor, n = 14). Serum levels of the cytokines and antibodies were measured using ELISA. Results: For sICAM-1 and anti-P. gingivalis GroEL and anti-hHSP60 antibodies, most patients recorded decreased levels. Reductions in serum sICAM-1 levels were more notable in low CV-risk patients (p = 0.006); and reductions in levels of anti-P. gingivalis GroEL and anti-hHSP60 antibodies (p = 0.001 and 0.009 respectively) were more notable in high CV-risk patients. Conclusions: This study found that subsequent to improved periodontal health, the anti-HSP (HSP60 and GroEL) antibody response was reduced, particularly for high CV-risk patients. sICAM-1 levels were also lowered, more so for low CV-risk patients.

Dysgalacticin is a novel bacteriocin produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 that has a narrow spectrum of antimicrobial activity directed primarily against the principal human streptococcal pathogen Streptococcus pyogenes. Unlike many previously described bacteriocins of Gram-positive bacteria, dysgalacticin is a heat-labile 21.5 kDa anionic protein that kills its target without inducing lysis. The N-terminal amino acid sequence of dysgalacticin [Asn-Glu-Thr-Asn-Asn-Phe-Ala-Glu-Thr-Gln-Lys-Glu-Ile-Thr-Thr-Asn-(Asn)-Glu-Ala] has no known homologue in publicly available sequence databases. The dysgalacticin structural gene, dysA, is located on the indigenous plasmid pW2580 of strain W2580 and encodes a 220 aa preprotein which is probably exported via a Sec-dependent transport system. Natural dysA variants containing conservative amino acid substitutions were also detected by sequence analyses of dysA elements from S. dysgalactiae strains displaying W2580-like inhibitory profiles. Production of recombinant dysgalacticin by Escherichia coli confirmed that this protein is solely responsible for the inhibitory activity exhibited by strain W2580. A combination of in silico secondary structure prediction and reductive alkylation was employed to demonstrate that dysgalacticin has a novel structure containing a disulphide bond essential for its biological activity. Moreover, dysgalacticin displays similarity in predicted secondary structure (but not primary amino acid sequence or inhibitory spectrum) with another plasmid-encoded streptococcal bacteriocin, streptococcin A-M57 from S. pyogenes, indicating that dysgalacticin represents a prototype of a new class of antimicrobial proteins.

In naturally-competent streptococci such as Streptococcus pneumoniae, expression of the late competence operons is regulated by ComX (sigma(X)), the competence-specific alternative sigma factor. In this study, duplicate genes (comR1 and comR2) encoding the putative ComX homologue of the oral bacterium Streptococcus gordonii were identified. Like the identical twin comX loci of S. pneumoniae, both comR determinants are independently functional as well as responsive to the ComDE signal transduction system activated by competence-stimulating peptide. However, in contrast to the comX system, nucleotide sequence analyses in combination with in trans complementation studies with a comR null mutant demonstrate that the identical 83 bp tracts (Region I) located immediately upstream of the comR structural genes are insufficient to confer wild-type competence levels. Wild-type transformation levels required additional distal nonhomologous DNA segments (Region II). Our findings suggest that alternative regulatory elements, under overall control of the ComDE pathway, may influence expression of the comR loci.

Bacteriocins are ribosomally synthesised antimicrobial proteins produced by bacteria that generally kill or inhibit species or strains closely related to the producer. Members of the genus Streptococcus produce a myriad of bacteriocins, most of which are small ( 10 kDa) streptococcal bacteriocins have also been identified. In this paper, we describe the biochemical and genetic characteristics of the large bacteriocins currently studied in our laboratory, including the identification of stellalysin, a new lytic bacteriocin produced by Streptococcus constellatus subsp. constellatus .

M. RAHIMI, N.C.K. HENG, J.A. KIESER AND G.R. TOMPKINS. 2005. Aims: This study assessed, for forensic purposes, the feasibility of genotypically matching oral streptococci recovered from recent human bite marks with those from the teeth of the biter. Methods and Results: Streptococci were isolated from the incisors of eight volunteers. Arbitrarily primed PCR (AP-PCR) distinguished 106 streptococcal genotypes among the participants, each harbouring at least eight distinct strains. In a crime simulation, a sample from an experimental bite mark was analysed by an experimenter unaware of its origin. The bacteria were unambiguously matched to the biter by comparing the amplicon profiles with those from the eight participants. In contrast, bacteria from an additional bite mark (not generated by one of the original participants) could not be matched to any of the eight participants. Between 20 and 78% of catalogued bacterial genotypes were recovered 12 months later from each participant. Throughout the study period, none of the bacterial genotypes were shared between participants. Conclusions: Streptococci isolated from recent bite marks can be catalogued by AP-PCR and matched to the teeth responsible for the bite. Significance and Impact of the Study: The study provides ! proof of concept" that genotypic analysis of streptococci from bite marks may provide valuable forensic evidence in situations where the perpetrator’s DNA cannot be recovered.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).

Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing ′ ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, ′ bglM (β-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase ( xylA ) and peptide methionine sulfoxide reductase ( msrB ), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.

Background and Objective There is a paucity of data in relation to the possible emergence of triclosan (TCS)-resistant bacteria following long-term exposure to TCS toothpaste. Therefore, this study investigated whether long-term continuous exposure to TCS in toothpaste selects for TCS-resistant bacteria within the oral biofilm. Material and Methods Dental plaque samples were collected from 40 individuals during year 5 of a randomised controlled trial. Participants had been randomly assigned to use TCS (3000 μg/mL TCS) (n = 18) or placebo toothpaste (n = 22). Diluted plaque samples were plated on to Wilkins–Chalgren agar plates containing 5% (v/v) laked sheep red blood cells and TCS (concentrations ranging from 25 to 150 μg/mL) and incubated at 37°C under microaerophilic and anaerobic conditions for 2–10 d. Selected bacterial isolates were identified by partial 16S rDNA sequencing and TCS minimum inhibitory concentration (MIC) determined for each isolate. Results At 3000 μg/mL TCS no growth was observed under microaerophilic or anaerobic conditions in either group. The MICs of TCS for all isolates ranged from 125 to 1000 μg/mL in both groups. Species common to both groups had similar MICs. Veillonella parvula and Campylobacter gracilis were the most frequent isolates from both groups, with similar MICs in both groups. Conclusion The use of TCS-containing toothpaste did not appear to lead to an increase in MIC of TCS of oral bacterial isolates.

Members of the Gram-positive lactic acid bacteria (LAB) are well-known for their beneficial properties as starter cultures and probiotics. Many LAB species produce ribosomally synthesized proteinaceous antibiotics (bacteriocins). Weissella confusa MBF8-1 is a strain isolated from a fermented soybean product that not only produces useful exopolysaccharides but also exhibits bacteriocin activity, which we call weissellicin MBF. Here, we show that bacteriocin production by W. confusa MBF8-1 is specified by a large plasmid, pWcMBF8-1. Plasmid pWcMBF8-1 (GenBank accession number [KR350502][1]), which was identified from the W. confusa MBF8-1 draft genome sequence, is 17 643 bp in length with a G + C content of 34.8% and contains 25 open reading frames (ORFs). Six ORFs constitute the weissellicin MBF locus, encoding three putative double-glycine-motif peptides (Bac1, Bac2, Bac3), an ABC transporter complex (BacTE) and a putative immunity protein (BacI). Two ORFs encode plasmid partitioning and mobilization proteins, suggesting that pWcMBF8-1 is transferable to other hosts. To the best of our knowledge, plasmid pWcMBF8-1 not only represents the first large Weissella plasmid to be sequenced but also the first to be associated with bacteriocin production in W. confusa . [1]: /lookup/external-ref?link_type=GEN&access_num=KR350502&atom=%2Ffemsle%2F363%2F8%2Ffnw059.atom

Aim To determine the relationship between periodontal pathogen load and anti-human heat shock protein 60 (hHSP60) antibodies in patients with established cardiovascular disease (CVD). Materials and Methods Participants were cardiovascular patients (n similar to=similar to 74) with a previous hospital admission for myocardial infarction. Concurrent periodontal pathogen load of Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia and Aggregatibacter actinomycetemcomitans was determined using quantitative real-time PCR. Serum antibodies to these pathogens, GroEL and hHSP60 were determined using an ELISA. Results There was a trend for increasing anti-hHSP60 antibody as the number of bacterial species increased. The strongest positive correlations were found between anti-hHSP60 levels and numbers of T. forsythia (r similar to=similar to 0.43; p similar to

Streptococcus salivarius is a Gram-positive bacterial commensal and pioneer colonizer of the human oral cavity. Many strains produce ribosomally synthesized proteinaceous antibiotics (bacteriocins), and some strains have been developed for use as oral probiotics. Here, we present the draft genome sequence of the bacteriocin-producing oral probiotic S. salivarius strain M18.

Recent research has provided mechanistic insight into the important contributions of the gut microbiota to vertebrate biology, but questions remain about the evolutionary processes that have shaped this symbiosis. In the present study, we showed in experiments with gnotobiotic mice that the evolution of Lactobacillus reuteri with rodents resulted in the emergence of host specialization. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and we identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and it identified genomic events that have shaped this process., Author Summary The gastrointestinal microbiota of vertebrates is important for nutrient utilization, resistance against pathogens, and immune maturation of its host, but little is known about the evolutionary relationships between vertebrates and individual bacterial members of these communities. Here we provide robust evidence that the evolution of the gut symbiont Lactobacillus reuteri with vertebrates resulted in the emergence of host specialization. Genomic approaches using a combination of genome sequence comparisons and microarray analysis were used to identify the host-specific genome content in rodent and human strains and the evolutionary events that resulted in host adaptation. The study revealed divergent patterns of genome evolution in rodent and human lineages and a distinct genome inventory in host-restricted sub-populations of L. reuteri that reflected the niche characteristics in the gut of their particular vertebrate hosts. The ecological significance of representative rodent-specific genes was demonstrated in gnotobiotic mice. In conclusion, this work provided evidence that the vertebrate gut symbiont Lactobacillus reuteri, despite the likelihood of horizontal transmission, has remained stably associated with related groups of vertebrate hosts over evolutionary time and has evolved a lifestyle specialized to these host animals.

DysI is identified as the protein that confers specific immunity to dysgalacticin, a plasmid-encoded streptococcal bacteriocin. dysI is transcribed as part of the copG - repB - dysI replication-associated operon. DysI appears to function at the membrane level to prevent the inhibitory effects of dysgalacticin on glucose transport, membrane integrity, and intracellular ATP content.

For over 30 years, the chain termination method of DNA sequencing (commonly known as Sanger sequencing) has been the mainstay of any DNA sequencing project. In the past, whole-genome sequencing employing exclusively Sanger chemistry has been a labor-intensive and costly exercise and an option unfeasible for the average research group. However, within the last 4 years, the introduction of three high-throughput sequencing technologies (454, SOLiD, and Illumina) has revolutionized genomics by facilitating unprecedented levels (up to gigabasepairs) of reliable DNA sequence output in a relatively short time frame and at a much lower cost per sequenced basepair. Here, we provide laboratory and bioinformatic protocols that will allow the average research group to undertake high-throughput sequencing of oral bacterial genomes using the Roche Genome Sequencer FLX System which employs 454 pyrosequencing technology.

For over 30 years, the chain termination method of DNA sequencing (commonly known as Sanger sequencing) has been the mainstay of any DNA sequencing project. In the past, whole-genome sequencing employing exclusively Sanger chemistry has been a labor-intensive and costly exercise and an option unfeasible for the average research group. However, within the last 4 years, the introduction of three high-throughput sequencing technologies (454, SOLiD, and Illumina) has revolutionized genomics by facilitating unprecedented levels (up to gigabasepairs) of reliable DNA sequence output in a relatively short time frame and at a much lower cost per sequenced basepair. Here, we provide laboratory and bioinformatic protocols that will allow the average research group to undertake high-throughput sequencing of oral bacterial genomes using the Roche Genome Sequencer FLX System which employs 454 pyrosequencing technology.

Members of the Gram-positive bacterial genus Streptococcus are a diverse collection of species inhabiting many body sites and range from benign, nonpathogenic species to those causing life-threatening infections. The streptococci are also prolific producers of bacteriocins, which are ribosomally synthesized proteinaceous antibiotics that kill or inhibit species closely related to the producer bacterium. With the emergence of bacterial resistance to conventional antibiotics, there is an impetus to discover, and implement, new and preferably ‘natural’ antibiotics to treat or prevent bacterial infections, a niche that bacterial interference therapy mediated by bacteriocins could easily fill. This review focuses on describing the diversity of bacteriocins produced by streptococci and also puts forth a case for Streptococcus salivarius, a nonpathogenic and numerically predominant oral species, as an ideal candidate for development as the model probiotic for the oral cavity. S. salivarius is a safe species that not only produces broad-spectrum bacteriocins but harbors bacteriocin-encoding (and bacteriocin-inducing) transmissible DNA entities (megaplasmids).

Streptococcus uberis , a causal agent of bovine mastitis, produces ubericin A, a 5.3-kDa class IIa (pediocin-like) bacteriocin, which was purified and characterized. The uba locus comprises two overlapping genes: ubaA (ubericin A precursor peptide) and ubaI (putative immunity protein). Ubericin A is the first streptococcal class IIa bacteriocin to be characterized.

The production of streptocins STH 1 and STH 2 by Streptococcus gordonii DL1 (Challis) is directly controlled by the competence regulon, which requires intact comR and comAB loci. The streptocin ( sth ) locus comprises two functional genes, sthA and sthB . Whereas STH 1 activity requires sthA alone, STH 2 activity depends on both genes.

Aims: To determine the degree of overlap in strain types of Campylobacter jejuni isolated from clinical cases and water samples from the Taieri catchment in the South Island of New Zealand. Methods and Results: Thermophilic Campylobacter were collected from human cases of infection, the main stem of the Taieri River and streams within distinct land-use types over a 1-year period. Campylobacter jejuni (187 isolates) and Campylobacter lari (four isolates) were identified using a multiplex polymerase chain reaction protocol. Isolates were typed by the Penner method and pulsed-field gel electrophoresis (PFGE) utilizing two restriction endonucleases. Several serotypes and PFGE types occurred in both water samples and clinical cases when the restriction profiles for each enzyme were considered separately. However, when PFGE profiles and serotyping were combined, there was no overlap between Camp. jejuni types from water and clinical cases. Conclusions: The results of this study indicate that recreational water in the Taieri catchment is not a major source of campylobacteriosis in the Dunedin area. Significance and Impact of the Study: This study suggests the risk of acquiring campylobacteriosis from surface waters in the Taieri catchment is considerably lower than previously predicted and highlights the necessity of using two endonucleases in PFGE typing.

Microcins are peptide antibiotics (

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic bacteriocin (mutacin) activity. In this study, we have combined bioinformatic and mutational analyses to identify the ABC transporter designated NlmTE, which is required for mutacin biogenesis in strain UA159 as well as in another mutacin producer, S. mutans N.

Production of the novel bacteriocin streptococcin A-M57 (SA-M57) by Streptococcus pyogenes strains of M-protein type 57 is plasmid-associated. Plasmid pDN571 (3351 bp) harbored by S. pyogenes 71–724, the prototype M-type 57 strain, has been completely sequenced and contains three putative open reading frames (repA, scnM57 and ORF3). In addition, the double-strand and single-strand (SSO) origins of replication were identified. Analysis of the replication-associated genetic elements places pDN571 in the ubiquitous pC194/pUB110 family of rolling-circle plasmids. The SSO of pDN571 is of the ssoA type. SA-M57 (encoded by scnM57) is synthesized as a secreted 179-amino acid polypeptide with a 27-residue secretion signal peptide and has no homology to proteins of known function.

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA . An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.

Strains of the gastrointestinal species Lactobacillus reuteri were electrotransformed with plasmid constructs containing the endo-1,3-1,4-beta-glucanase gene (bglM) of Bacillus macerans. The enzyme was expressed and secreted by the lactobacilli. A plasmid construct containing the bglM gene lacking its promoter was derived and was demonstrated to be useful as a promoter probe vector.