Prevalence of Bacteria of Division TM7 in Human Subgingival Plaque and Their Association with Disease

Members of the uncultivated bacterial division TM7 have been detected in the human mouth, but little information is available regarding their prevalence and diversity at this site. Human subgingival plaque samples from healthy sites and sites exhibiting various stages of periodontal disease were analyzed for the presence of TM7 bacteria. TM7 ribosomal DNA (rDNA) was found in 96% of the samples, and it accounted for approximately 0.3%, on average, of all bacterial rDNA in the samples as determined by real-time quantitative PCR. Two new phylotypes of this division were identified, and members of the division were found to exhibit filamentous morphology by fluorescence in situ hybridization. The abundance of TM7 rDNA relative to total bacterial rDNA was higher in sites with mild periodontitis (0.54% ± 0.1%) than in either healthy sites (0.21% ± 0.05%, P \u3c 0.01) or sites with severe periodontitis (0.29% ± 0.06%, P \u3c 0.05). One division subgroup, the I025 phylotype, was detected in 1 of 18 healthy samples and 38 of 58 disease samples. These data suggest that this phylotype, and the TM7 bacterial division in general, may play a role in the multifactorial process leading to periodontitis

Methanogenic \u3cem\u3eArchaea\u3c/em\u3e and human periodontal disease

Archaea have been isolated from the human colon, vagina, and oral cavity, but have not been established as causes of human disease. In this study, we reveal a relationship between the severity of periodontal disease and the relative abundance of archaeal small subunit ribosomal RNA genes (SSU rDNA) in the subgingival crevice by using quantitative PCR. Furthermore, the relative abundance of archaeal small subunit rDNA decreased at treated sites in association with clinical improvement. Archaea were harbored by 36% of periodontitis patients and were restricted to subgingival sites with periodontal disease. The presence of archaeal cells at these sites was confirmed by fluorescent in situ hybridization. The archaeal community at diseased sites was dominated by a Methanobrevibacter oralis-like phylotype and a distinct Methanobrevibacter subpopulation related to archaea that inhabit the gut of numerous animals. We hypothesize that methanogens participate in syntrophic relationships in the subgingival crevice that promote colonization by secondary fermenters during periodontitis. Because they are potential alternative syntrophic partners, our finding of larger Treponema populations sites without archaea provides further support for this hypothesis

Bordetella pertussis, the Causative Agent of Whooping Cough, Evolved from a Distinct, Human-Associated Lineage of B. bronchiseptica

Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu), and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica–like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu), and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed

Uncovering cis Regulatory Codes Using Synthetic Promoter Shuffling

Revealing the spectrum of combinatorial regulation of transcription at individual promoters is essential for understanding the complex structure of biological networks. However, the computations represented by the integration of various molecular signals at complex promoters are difficult to decipher in the absence of simple cis regulatory codes. Here we synthetically shuffle the regulatory architecture — operator sequences binding activators and repressors — of a canonical bacterial promoter. The resulting library of complex promoters allows for rapid exploration of promoter encoded logic regulation. Among all possible logic functions, NOR and ANDN promoter encoded logics predominate. A simple transcriptional cis regulatory code determines both logics, establishing a straightforward map between promoter structure and logic phenotype. The regulatory code is determined solely by the type of transcriptional regulation combinations: two repressors generate a NOR: NOT (a OR b) whereas a repressor and an activator generate an ANDN: a AND NOT b. Three-input versions of both logics, having an additional repressor as an input, are also present in the library. The resulting complex promoters cover a wide dynamic range of transcriptional strengths. Synthetic promoter shuffling represents a fast and efficient method for exploring the spectrum of complex regulatory functions that can be encoded by complex promoters. From an engineering point of view, synthetic promoter shuffling enables the experimental testing of the functional properties of complex promoters that cannot necessarily be inferred ab initio from the known properties of the individual genetic components. Synthetic promoter shuffling may provide a useful experimental tool for studying naturally occurring promoter shuffling

Comparative Genomics of Bordetella pertussis Reveals Progressive Gene Loss in Finnish Strains

BACKGROUND: Bordetella pertussis is a gram-negative bacterium that infects the human respiratory tract and causes pertussis or whooping cough. The disease has resurged in many countries including Finland where the whole-cell pertussis vaccine has been used for more than 50 years. Antigenic divergence has been observed between vaccine strains and clinical isolates in Finland. To better understand genome evolution in B. pertussis circulating in the immunized population, we developed an oligonucleotide-based microarray for comparative genomic analysis of Finnish strains isolated during the period of 50 years. METHODOLOGY/PRINCIPAL FINDINGS: The microarray consisted of 3,582 oligonucleotides (70-mer) and covered 94% of 3,816 ORFs of Tohama I, the strain of which the genome has been sequenced. Twenty isolates from 1953 to 2004 were studied together with two Finnish vaccine strains and two international reference strains. The isolates were selected according to their characteristics, e.g. the year and place of isolation and pulsed-field gel electrophoresis profiles. Genomic DNA of the tested strains, along with reference DNA of Tohama I strain, was labelled and hybridized. The absence of genes as established with microarrays, was confirmed by PCR. Compared with the Tohama I strain, Finnish isolates lost 7 (8.6 kb) to 49 (55.3 kb) genes, clustered in one to four distinct loci. The number of lost genes increased with time, and one third of lost genes had functions related to inorganic ion transport and metabolism, or energy production and conversion. All four loci of lost genes were flanked by the insertion sequence element IS481. CONCLUSION/SIGNIFICANCE: Our results showed that the progressive gene loss occurred in Finnish B. pertussis strains isolated during a period of 50 years and confirmed that B. pertussis is dynamic and is continuously evolving, suggesting that the bacterium may use gene loss as one strategy to adapt to highly immunized populations

In Search of an Uncultured Human-Associated TM7 Bacterium in the Environment

We have identified an environmental bacterium in the Candidate Division TM7 with ≥98.5% 16S rDNA gene homology to a group of TM7 bacteria associated with the human oral cavity and skin. The environmental TM7 bacterium (referred to as TM7a-like) was readily detectable in wastewater with molecular techniques over two years of sampling. We present the first images of TM7a-like cells through FISH technique and the first images of any TM7 as viable cells through the STARFISH technique. In situ quantification showed TM7 concentration in wastewater up to five times greater than in human oral sites. We speculate that upon further characterization of the physiology and genetics of the TM7a-like bacterium from environmental sources and confirmation of its genomic identity to human-associated counterparts it will serve as model organisms to better understand its role in human health. The approach proposed circumvents difficulties imposed by sampling humans, provides an alternative strategy to characterizing some diseases of unknown etiology, and renders a much needed understanding of the ecophysiological role hundreds of unique Bacteria and Archaea strains play in mixed microbial communities

Development of the Human Infant Intestinal Microbiota

Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract

Comparative genomics of prevaccination and modern Bordetella pertussis strains

Contains fulltext : 89571.pdf (publisher's version ) (Open Access)BACKGROUND: Despite vaccination since the 1950s, pertussis has persisted and resurged. It remains a major cause of infant death worldwide and is the most prevalent vaccine-preventable disease in developed countries. The resurgence of pertussis has been associated with the expansion of Bordetella pertussis strains with a novel allele for the pertussis toxin (Ptx) promoter, ptxP3, which have replaced resident ptxP1 strains. Compared to ptxP1 strains, ptxP3 produce more Ptx resulting in increased virulence and immune suppression. To elucidate how B. pertussis has adapted to vaccination, we compared genome sequences of two ptxP3 strains with four strains isolated before and after the introduction vaccination. RESULTS: The distribution of SNPs in regions involved in transcription and translation suggested that changes in gene regulation play an important role in adaptation. No evidence was found for acquisition of novel genes. Modern strains differed significantly from prevaccination strains, both phylogenetically and with respect to particular alleles. The ptxP3 strains were found to have diverged recently from modern ptxP1 strains. Differences between ptxP3 and modern ptxP1 strains included SNPs in a number of pathogenicity-associated genes. Further, both gene inactivation and reactivation was observed in ptxP3 strains relative to modern ptxP1 strains. CONCLUSIONS: Our work suggests that B. pertussis adapted by successive accumulation of SNPs and by gene (in)activation. In particular changes in gene regulation may have played a role in adaptation