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Chlamydia psittaci is an avian pathogen and zoonotic agent of atypical pneumonia. The most pathogenic C. psittaci strains cluster into the 6BC clade, predicted to have recently emerged globally. Exposure to infected parrots is a risk factor with limited evidence also of an indirect exposure risk. Genome sequencing was performed on six Australian human and a single avian C. psittaci strain isolated over a 9 year period. Only one of the five human patients had explicit psittacine contact. Genomics analyses revealed that the Australian C. psittaci strains are remarkably similar, clustering tightly within the C. psittaci 6BC clade suggested to have been disseminated by South America parrot importation. Molecular clock analysis using the newly sequenced C. psittaci genomes predicted the emergence of the 6BC clade occurring approximately 2,000 years ago. These findings reveal the potential for an Australian natural reservoir of C. psittaci 6BC strains. These strains can also be isolated from seriously ill patients without explicit psittacine contact. The apparent recent and global spread of C. psittaci 6BC strains raises important questions over how this happened. Further studies may reveal whether the dissemination of this important zoonotic pathogen is linked to Australian parrot importation rather than parrots from elsewhere.

Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species., (© 2016 S. Karger AG, Basel.)

Chlamydia pneumoniae is an obligate intracellular bacterium implicated in a wide range of human diseases including atherosclerosis and Alzheimer's disease. Efforts to understand the relationships between C. pneumoniae detected in these diseases have been hindered by the availability of sequence data for non-respiratory strains. In this study, we sequenced the whole genomes for C. pneumoniae isolates from atherosclerosis and Alzheimer's disease, and compared these to previously published C. pneumoniae genomes. Phylogenetic analyses of these new C. pneumoniae strains indicate two sub-groups within human C. pneumoniae, and suggest that both recombination and mutation events have driven the evolution of human C. pneumoniae. Further fine-detailed analyses of these new C. pneumoniae sequences show several genetically variable loci. This suggests that similar strains of C. pneumoniae are found in the brain, lungs and cardiovascular system and that only minor genetic differences may contribute to the adaptation of particular strains in human disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Chlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts., Methods: Whole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts., Results: Three porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock. Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec., Conclusions: This study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.

In humans, the vaginal microbiota is thought to be the first line of defense again pathogens including Chlamydia trachomatis. The guinea pig has been extensively used as a model to study chlamydial infection because it shares anatomical and physiological similarities with humans, such as a squamous vaginal epithelium as well as some of the long-term outcomes caused by chlamydial infection. In this study, we aimed to evaluate the guinea pig-C. caviae model of genital infection as a surrogate for studying the role of the vaginal microbiota in the early steps of C. trachomatis infection in humans. We used culture-independent molecular methods to characterize the relative and absolute abundance of bacterial phylotypes in the guinea pig vaginal microbiota in animals non-infected, mock-infected or infected by C. caviae. We showed that the guinea pig and human vaginal microbiotas are of different bacterial composition and abundance. Chlamydia caviae infection had a profound effect on the absolute abundance of bacterial phylotypes but not on the composition of the guinea pig vaginal microbiota. Our findings compromise the validity of the guinea pig-C. caviae model to study the role of the vaginal microbiota during the early steps of sexually transmitted infection., (© FEMS 2015.)

Chlamydia pecorum is an important global pathogen of livestock, and it is also a significant threat to the long-term survival of Australia's koala populations. This study employed a culture-independent DNA capture approach to sequence C. pecorum genomes directly from clinical swab samples collected from koalas with chlamydial disease as well as from sheep with arthritis and conjunctivitis. Investigations into single-nucleotide polymorphisms within each of the swab samples revealed that a portion of the reads in each sample belonged to separate C. pecorum strains, suggesting that all of the clinical samples analyzed contained mixed populations of genetically distinct C. pecorum isolates. This observation was independent of the anatomical site sampled and the host species. Using the genomes of strains identified in each of these samples, whole-genome phylogenetic analysis revealed that a clade containing a bovine and a koala isolate is distinct from other clades comprised of livestock or koala C. pecorum strains. Providing additional evidence to support exposure of koalas to Australian livestock strains, two minor strains assembled from the koala swab samples clustered with livestock strains rather than koala strains. Culture-independent probe-based genome capture and sequencing of clinical samples provides the strongest evidence yet to suggest that naturally occurring chlamydial infections are comprised of multiple genetically distinct strains., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Summary: Circleator is a Perl application that generates circular figures of genome-associated data. It leverages BioPerl to support standard annotation and sequence file formats and produces publication-quality SVG output. It is designed to be both flexible and easy to use. It includes a library of circular track types and predefined configuration files for common use-cases, including. (i) visualizing gene annotation and DNA sequence data from a GenBank flat file, (ii) displaying patterns of gene conservation in related microbial strains, (iii) showing Single Nucleotide Polymorphisms (SNPs) and indels relative to a reference genome and gene set and (iv) viewing RNA-Seq plots., Availability and Implementation: Circleator is freely available under the Artistic License 2.0 from http://jonathancrabtree.github.io/Circleator/ and is integrated with the CloVR cloud-based sequence analysis Virtual Machine (VM), which can be downloaded from http://clovr.org or run on Amazon EC2., (© The Author 2014. Published by Oxford University Press.)

Background: Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced., Results: Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations., Conclusions: The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen.

Unlabelled: It is not currently possible to predict the probability of whether a woman with a chlamydial genital infection will develop pelvic inflammatory disease (PID). To determine if specific biomarkers may be associated with distinct chlamydial pathotypes, we utilized two Chlamydia muridarum variants (C. muridarum Var001 [CmVar001] and CmVar004) that differ in their abilities to elicit upper genital tract pathology in a mouse model. CmVar004 has a lower growth rate in vitro and induces pathology in only 20% of C57BL/6 mouse oviducts versus 83.3% of oviducts in CmVar001-infected mice. To determine if chemokine and cytokine production within 24 h of infection is associated with the outcome of pathology, levels of 15 chemokines and cytokines were measured. CmVar004 infection induced significantly lower levels of CXCL1, CXCL2, tumor necrosis factor alpha (TNF-α), and CCL2 in comparison to CmVar001 infection with similar rRNA (rs16) levels for Chlamydiae. A combination of microRNA (miRNA) sequencing and quantitative real-time PCR (qRT-PCR) analysis of 134 inflammation-related miRNAs was performed 24 h postinfection to determine if the chemokine/cytokine responses would also be reflected in miRNA expression profiles. Interestingly, 12 miRNAs (miR-135a-5p, miR298-5p, miR142-3p, miR223-3p, miR299a-3p, miR147-3p, miR105, miR325-3p, miR132-3p, miR142-5p, miR155-5p, and miR-410-3p) were overexpressed during CmVar004 infection compared to CmVar001 infection, inversely correlating with the respective chemokine/cytokine responses. To our knowledge, this is the first report demonstrating that early biomarkers elicited in the host can differentiate between two pathological variants of chlamydiae and be predictive of upper tract disease., Importance: It is apparent that an infecting chlamydial population consists of multiple genetic variants with differing capabilities of eliciting a pathological response; thus, it may be possible to identify biomarkers specific for a given virulence pathotype. miRNAs are known to regulate genes that in turn regulate signaling pathways involved in disease pathogenesis. Importantly, miRNAs are stable and can reflect a tissue response and therefore have the potential to be biomarkers of disease severity. Currently, with respect to chlamydial infections, there is no way to predict whether an infected patient is more or less likely to develop PID. However, data presented in this study indicate that the expression of a specific miRNA profile associated with a virulent variant early in the infection course may be predictive of an increased risk of pelvic inflammatory disease, allowing more aggressive treatment before significant pathology develops., (Copyright © 2014 Yeruva et al.)

Chlamydia suis is a natural pathogen of pigs (Sus scrofa) and causes conjunctivitis, pneumonia, enteritis, and various reproductive disorders that adversely impact this economically important animal. Here, we report the first C. suis genome, that of C. suis MD56, isolated from a conjunctival swab of a weaned piglet.

Background: Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing., Results: Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection., Conclusions: Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.

We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness). Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

Among chlamydial virulence factors are the type III secretion (T3S) system and its effectors. T3S effectors target host proteins to benefit the infecting chlamydiae. The assortment of effectors, each with a unique function, varies between species. This variation likely contributes to differences in host specificity and disease severity. A dozen effectors of Chlamydia trachomatis have been identified; however, estimates suggest that more exist. A T3S prediction algorithm, SVM-based Identification and Evaluation of Virulence Effectors (SIEVE), along with a Yersinia surrogate secretion system helped to identify a new T3S substrate, CT082, which rather than functioning as an effector associates with the chlamydial envelope after secretion. SIEVE was modified to improve/expand effector predictions to include all sequenced genomes. Additional adjustments were made to the existing surrogate system whereby the N terminus of putative effectors was fused to a known effector lacking its own N terminus and was tested for secretion. Expansion of effector predictions by cSIEVE and modification of the surrogate system have also assisted in identifying a new T3S substrate from C. psittaci. The expanded predictions along with modifications to improve the surrogate secretion system have enhanced our ability to identify novel species-specific effectors, which upon characterization should provide insight into the unique pathogenic properties of each species., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

Chlamydia psittaci is a highly prevalent avian pathogen and the cause of a potentially lethal zoonosis, causing life-threatening pneumonia in humans. We report the genome sequences of C. psittaci 6BC, the prototype strain of the species, and C. psittaci Cal10, a widely used laboratory strain.

Chlamydia pecorum is an obligate intracellular bacterial pathogen that causes diverse disease in a wide variety of economically important mammals. We report the finished complete genome sequence of C. pecorum E58, the type strain for the species.

Background: Chlamydia pneumoniae is a widespread pathogen causing upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. Previous whole genome analyses have focused on four essentially clonal (> 99% identity) C. pneumoniae human genomes (AR39, CWL029, J138 and TW183), providing relatively little insight into strain diversity and evolution of this species., Results: We performed individual gene-by-gene comparisons of the recently sequenced C. pneumoniae koala genome and four C. pneumoniae human genomes to identify species-specific genes, and more importantly, to gain an insight into the genetic diversity and evolution of the species. We selected genes dispersed throughout the chromosome, representing genes that were specific to C. pneumoniae, genes with a demonstrated role in chlamydial biology and/or pathogenicity (n = 49), genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6), and extrachromosomal elements (9 plasmid and 2 bacteriophage genes)., Conclusions: We have identified strain-specific differences and targets for detection of C. pneumoniae isolates from both human and animal origin. Such characterisation is necessary for an improved understanding of disease transmission and intervention.

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non-Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials.

Zoonotic infections are a growing threat to global health. Chlamydia pneumoniae is a major human pathogen that is widespread in human populations, causing acute respiratory disease, and has been associated with chronic disease. C. pneumoniae was first identified solely in human populations; however, its host range now includes other mammals, marsupials, amphibians, and reptiles. Australian koalas (Phascolarctos cinereus) are widely infected with two species of Chlamydia, C. pecorum and C. pneumoniae. Transmission of C. pneumoniae between animals and humans has not been reported; however, two other chlamydial species, C. psittaci and C. abortus, are known zoonotic pathogens. We have sequenced the 1,241,024-bp chromosome and a 7.5-kb cryptic chlamydial plasmid of the koala strain of C. pneumoniae (LPCoLN) using the whole-genome shotgun method. Comparative genomic analysis, including pseudogene and single-nucleotide polymorphism (SNP) distribution, and phylogenetic analysis of conserved genes and SNPs against the human isolates of C. pneumoniae show that the LPCoLN isolate is basal to human isolates. Thus, we propose based on compelling genomic and phylogenetic evidence that humans were originally infected zoonotically by an animal isolate(s) of C. pneumoniae which adapted to humans primarily through the processes of gene decay and plasmid loss, to the point where the animal reservoir is no longer required for transmission.

Comparative genomic analysis of a wild-type strain of the ovine pathogen Chlamydia abortus and its nitrosoguanidine-induced, temperature-sensitive, virulence-attenuated live vaccine derivative identified 22 single nucleotide polymorphisms unique to the mutant, including nine nonsynonymous mutations, one leading to a truncation of pmpG, which encodes a polymorphic membrane protein, and two intergenic mutations potentially affecting promoter sequences. Other nonsynonymous mutations mapped to a pmpG pseudogene and to predicted coding sequences encoding a putative lipoprotein, a sigma-54-dependent response regulator, a PhoH-like protein, a putative export protein, two tRNA synthetases, and a putative serine hydroxymethyltransferase. One of the intergenic mutations putatively affects transcription of two divergent genes encoding pyruvate kinase and a putative SOS response nuclease, respectively. These observations suggest that the temperature-sensitive phenotype and associated virulence attenuation of the vaccine strain result from disrupted metabolic activity due to altered pyruvate kinase expression and/or alteration in the function of one or more membrane proteins, most notably PmpG and a putative lipoprotein.

Whole-genome sequencing has been skewed toward bacterial pathogens as a consequence of the prioritization of medical and veterinary diseases. However, it is becoming clear that in order to accurately measure genetic variation within and between pathogenic groups, multiple isolates, as well as commensal species, must be sequenced. This study examined the pangenomic content of Escherichia coli. Six distinct E. coli pathovars can be distinguished using molecular or phenotypic markers, but only two of the six pathovars have been subjected to any genome sequencing previously. Thus, this report provides a seminal description of the genomic contents and unique features of three unsequenced pathovars, enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. We also determined the first genome sequence of a human commensal E. coli isolate, E. coli HS, which will undoubtedly provide a new baseline from which workers can examine the evolution of pathogenic E. coli. Comparison of 17 E. coli genomes, 8 of which are new, resulted in identification of approximately 2,200 genes conserved in all isolates. We were also able to identify genes that were isolate and pathovar specific. Fewer pathovar-specific genes were identified than anticipated, suggesting that each isolate may have independently developed virulence capabilities. Pangenome calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors. This comparative study of the species E. coli, while descriptive, should provide the basis for future functional work on this important group of pathogens.

A multilocus VNTR analysis (MLVA) system for detection of tandem repeats across the whole genome of Chlamydophila psittaci has been developed. Twenty selected genetic loci were initially tested on 9 avian reference strains including representatives of all major serotypes (A to F). Thereafter, 8 loci were retained for a more complete study performed on over 150 C. psittaci isolates from different bird species and geographical origins. Comparative analysis of the MLVA results and those obtained from currently available methods including serotyping and/or ompA sequencing indicate that the MLVA system provides an additional level of discrimination, with 20 distinct patterns identified to date. The newly developed MLVA system therefore provides a highly sensitive, high resolution test for the differentiation of C. psittaci isolates from different origins that is suitable for molecular epidemiological studies.

Dichelobacter nodosus causes ovine footrot, a disease that leads to severe economic losses in the wool and meat industries. We sequenced its 1.4-Mb genome, the smallest known genome of an anaerobe. It differs markedly from small genomes of intracellular bacteria, retaining greater biosynthetic capabilities and lacking any evidence of extensive ongoing genome reduction. Comparative genomic microarray studies and bioinformatic analysis suggested that, despite its small size, almost 20% of the genome is derived from lateral gene transfer. Most of these regions seem to be associated with virulence. Metabolic reconstruction indicated unsuspected capabilities, including carbohydrate utilization, electron transfer and several aerobic pathways. Global transcriptional profiling and bioinformatic analysis enabled the prediction of virulence factors and cell surface proteins. Screening of these proteins against ovine antisera identified eight immunogenic proteins that are candidate antigens for a cross-protective vaccine.

Coastal aquatic environments are typically more highly productive and dynamic than open ocean ones. Despite these differences, cyanobacteria from the genus Synechococcus are important primary producers in both types of ecosystems. We have found that the genome of a coastal cyanobacterium, Synechococcus sp. strain CC9311, has significant differences from an open ocean strain, Synechococcus sp. strain WH8102, and these are consistent with the differences between their respective environments. CC9311 has a greater capacity to sense and respond to changes in its (coastal) environment. It has a much larger capacity to transport, store, use, or export metals, especially iron and copper. In contrast, phosphate acquisition seems less important, consistent with the higher concentration of phosphate in coastal environments. CC9311 is predicted to have differences in its outer membrane lipopolysaccharide, and this may be characteristic of the speciation of some cyanobacterial groups. In addition, the types of potentially horizontally transferred genes are markedly different between the coastal and open ocean genomes and suggest a more prominent role for phages in horizontal gene transfer in oligotrophic environments.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique "genomic islands" identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

Type IV fimbriae are expressed by several bacterial pathogens and are essential for virulence in Dichelobacter nodosus, which causes ovine footrot. We have identified a two-component signal transduction system (PilR/S) and an alternative sigma factor (sigma 54) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in D. nodosus. Western blots showed that in both pilR and rpoN mutants, fimbrial subunit production was significantly reduced by a process that was shown to occur at a PilR- and sigma 54-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and sigma 54, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and sigma 54 regulated genes other than fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a sigma 54-dependent PilR/S system that also indirectly controls protease secretion.

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.

Background: The first microbial genome sequence, Haemophilus influenzae, was published in 1995. Since then, more than 400 microbial genome sequences have been completed or commenced. This massive influx of data provides the opportunity to obtain biological insights through comparative genomics. However few tools are available for this scale of comparative analysis., Results: The BLAST Score Ratio (BSR) approach, implemented in a Perl script, classifies all putative peptides within three genomes using a measure of similarity based on the ratio of BLAST scores. The output of the BSR analysis enables global visualization of the degree of proteome similarity between all three genomes. Additional output enables the genomic synteny (conserved gene order) between each genome pair to be assessed. Furthermore, we extend this synteny analysis by overlaying BSR data as a color dimension, enabling visualization of the degree of similarity of the peptides being compared., Conclusions: Combining the degree of similarity, synteny and annotation will allow rapid identification of conserved genomic regions as well as a number of common genomic rearrangements such as insertions, deletions and inversions. The script and example visualizations are available at: http://www.microbialgenomics.org/BSR/.

The expression of iron regulated genes in bacteria is typically controlled by the ferric uptake regulator (Fur) protein, a global transcriptional repressor that regulates functions as diverse as iron acquisition, oxidative stress, and virulence. We have identified a fur homologue in Dichelobacter nodosus, the causative agent of ovine footrot, and shown that it complements an Escherichia coli fur mutant. Homology modeling of the D. nodosus Fur protein with the recently solved crystal structure of Fur from Pseudomonas aeruginosa indicated extensive structural conservation. As Southern hybridization analysis of different clinical isolates of D. nodosus indicated that the fur gene was present in all of these strains, the fur gene was insertionally inactivated to determine its functional role. Analysis of these mutants by various techniques did not indicate any significant differences in the expression of known virulence genes or in iron-dependent growth. However, we determined several Fur regulatory targets by two-dimensional gel electrophoresis coupled with mass spectrometry. Analysis of proteins from cytoplasmic, membrane, and extracellular fractions revealed numerous differentially expressed proteins. The transcriptional basis of these differences was analyzed by using quantitative reverse transcriptase PCR. Proteins with increased expression in the fur mutant were homologues of the periplasmic iron binding protein YfeA and a cobalt chelatase, CbiK. Down-regulated proteins included a putative manganese superoxide dismutase and ornithine decarboxylase. Based on these data, it is suggested that in D. nodosus the Fur protein functions as a regulator of iron and oxidative metabolism.

We present the complete 2,843,201-bp genome sequence of Treponema denticola (ATCC 35405) an oral spirochete associated with periodontal disease. Analysis of the T. denticola genome reveals factors mediating coaggregation, cell signaling, stress protection, and other competitive and cooperative measures, consistent with its pathogenic nature and lifestyle within the mixed-species environment of subgingival dental plaque. Comparisons with previously sequenced spirochete genomes revealed specific factors contributing to differences and similarities in spirochete physiology as well as pathogenic potential. The T. denticola genome is considerably larger in size than the genome of the related syphilis-causing spirochete Treponema pallidum. The differences in gene content appear to be attributable to a combination of three phenomena: genome reduction, lineage-specific expansions, and horizontal gene transfer. Genes lost due to reductive evolution appear to be largely involved in metabolism and transport, whereas some of the genes that have arisen due to lineage-specific expansions are implicated in various pathogenic interactions, and genes acquired via horizontal gene transfer are largely phage-related or of unknown function.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

Genes for a putative membrane associated protein (mvi -homologue) and a 48 kDa protein (ctr48) in Chlamydia trachomatis were characterized. The mvi -homologue has 12 transmembrane domains and shows considerable homology to the members of this gene family in various organisms. The ctr48 has a leader sequence and the C-proximal half is tryptophan-rich. The latter region shares 65% identity with the N-proxima third of C. pneumoniae 76 kDa protein over an overlap of 231 amino acid residues. The genes for the mvi -homologue and the ctr48 are present in the B, Ba, D, E, J and L2 serotypes of C. trachomatis. Immediately downstream from the ctr48 gene are multiple stop codons which are followed by a functional rho-independent terminator. The mvi -homologue and ctr48 genes are independently transcribed, albeit poorly in serotype B. However, protein products corresponding to these genes could not be detected by western blotting in HEp2 cells infected with C. trachomatis. Nevertheless, antibodies to peptides corresponding to these proteins were detected in sera with high micro-immunofluorescence titre against C. trachoImatic, collected from a Chlamydia -endemic population. These results suggest that the mvi -homologue and ctr48 are expressed by C. trachomatis during natural infection., (Copyright 2000 Academic Press.)

To meet the demands for improved carbohydrate analysis techniques, a method of sugar extraction and analysis has been developed using ion chromatography. The sample preparation method, which uses filtration and extraction steps, has been shown to adequately isolate glucose, fructose, and sucrose from other food components in raw and processed vegetables. Resulting sample preparations were successfully analyzed by ion chromatography using pulsed amperometric detection, which is a sensitive, rapid, and accurate technique. Sugar concentrations in a number of different vegetable products were successfully determined in this way.

High-quality echocardiograms were performed on 146 normal individuals whose ages ranged from 3 to 73 years (mean 27 years). Normal values for mitral diastolic excursion and E-F slope, the chamber dimensions of the right ventricle, left atrium, and left ventricle, the aortic root dimension, and thickness of the interventricular septum and left ventricular posterior wall were determined. Each tracing was then read independently by two experienced echocardiographers. The extent of interobserver variability was calculated and expressed as a percent of the mean. The 95 per cent confidence limits for these estimates were calculated. Small but significant interobserver variability was found for all nine of these commonly measured echocardiographic parameters. Observer variability is a small but potentially important consideration in investigative echocardiography.

Quantitative spectral (frequency) analyses were performed on 60 carotid bruits in 48 patients with suspected carotid stenosis. The technic was totally noninvasive. Bruits were recorded at the skin surface, analyzed by a minicomputer, and the degree of arterial stenosis estimated using a recently derived theory of sound production by turbulent blood flow. This method has been named phonoangiography. Fifty of the 60 bruits (83 per cent) had spectrums characteristic of turbulent blood flow, and a residual lumen diameter could be calculated. Results from the noninvasvie technic compared well with data obtained from radiographic carotid arteriograms in the 50 bruits with internal carotid artery stenosis. The residual lumen diameter estimated from phonoangiography differed from the radiographic value by less than 1 mm in 83 per cent and less than 1.5 mm in 92 per cent of the studies. Phonoangiography appears to be a useful method for assessment of carotid artery stenosis.

Clinical data and echocardiographic findings were correlated in 20 patients with pericardial effusion. Moderate to large effusions were associated with increased motion of the entire heart within the pericardial sac. A correlation was found between the estimated volume of fluid and the diastolic excursion and velocity of the right ventricular and left ventricular walls (P less than 0.01). For any given volume of fluid as estimated from the echocardiogram neoplastic effusions resulted in greater increments in wall motion (P less than 0.02). Patterns of "pseudo" mitral valve prolapse occurred and were correlated with the extent and timing of cardiac swinging and heart rate. A diminished E-F slope of the mitral valve echo and notch on the right ventricular epicardial echo during early systole were found in all four patients with pericardial tamponade. These preliminary observations suggest that echocardiographic examination of patients with pericardial effusion may provide clues to the presence or absence of tamponade in addition to providing an estimate of the quantity of fluid in the pericardial sac.

A high-resolution method of spectral analysis, of the class generally called "maximum entropy method," was used in a study of aortic porcine valve closing sounds in 37 patients (ages 19 to 76). Spectra from 27 normal xenografts, implanted from 2 weeks to 61 months previously, were characterized by a dominant frequency peak, F1, at 89 +/- 15 Hz (mean +/- SD), with a lower amplitude peak, F2, at 154 +/- 25 Hz. Eight of nine patients with aortic porcine valve dysfunction were proved surgically to have leaflet degeneration or infection and had either F1 (139 +/- 54 Hz) and/or F2 (195 +/- 74 Hz) significantly higher than normal (p less than .001). In two patients with paravalvar leak but no leaflet abnormality, F1 and F2 were in the normal range. Estimation of F1 and F2 was highly reproducible and was unaffected by duration of implant up to 5 years. Spectral analysis of aortic porcine valve closing sounds by the maximum entropy method may be useful for detection of intrinsic xenograft dysfunction.

A relation between the peak transaortic pressure gradient and the frequency content of the murmur (r = 0.79) was demonstrated in a prospective "test" set of 50 patients with the clinical diagnosis of aortic stenosis. After heart sounds were recorded and digitized, three segments of the systolic murmur were isolated and analyzed by fast Fourier transform technique. An average frequency spectrum was quantitated by a previously described empiric spectral estimator. Clinical data and spectral ratio were correlated with the transaortic pressure gradient and aortic valve area was calculated from cardiac catheterization data. The best prediction of the transaortic pressure gradient was obtained when a 170 ms murmur segment was analyzed and when the predictive algorithm also included the aortic dimension (r = 0.87). The aortic valve area was poorly predicted (r = -0.48) unless estimates of blood flow and valvular calcification were included in the algorithm (r = 0.84). Further refinement of this technique may provide a non-invasive and clinically useful method for the estimation of aortic valve stenosis.

The prevalence of mitral valve prolapse has been established in selected groups of patients but not in the general population. The present study was designed to define the echocardiographic spectrum of mitral valve motion in a population of young individuals without clinical evidence of significant cardiac disease or hypertension. Echocardiograms were performed on 136 normal volunteers. Six subjects (4.4 per cent) had mitral valve prolapse. Eighteen subjects (13.2 per cent) had a lesser degree of posterior systolic motion of the mitral valve leaflets which was suggestive but not diagnostic of prolapse. Minor degrees of posterior systolic mitral valve motion may represent a variant of normal. Caution should be exercised in making the echocardiographic diagnosis of mitral prolapse until this question is settled.

During protein-energy malnutrition appositional bone growth in the seventh caudal vertebra of the rat slows and finally ceases. During rehabilitation appositional growth begins agains and attains a rate in excess of that of the controls. This may account for alterations in skeletal proportions resulting from malnutrition.

Left ventricular performance was studied in 15 patients with severe, primary hypothyroidism (mean serum total thyroxine of 0.8 mug per 100 ml and serum thyrotropin of 160 muU per milliliter). Pretreatment systolic-time intervals were characterized by prolongation of the pre-ejection period (delta PEP = +30) and reduction of the left ventricular ejection period (delta LVET = -23) with a resultant increase in the PEP/LVET ratio (0.47). Nine of 14 patients demonstrated pericardial effusions. These abnormalities were reversed with physiologic thyroxine replacement. Further reductions of the delta PEP and PEP/LVET ratio occurred with supraphysiologic doses (200 to 300 mug per day). During therapy, delta PEP was inversely correlated with serum thyroxine (P less than 0.001) and directly correlated with serum thyrotropin (P less than 0.001). Thus physiologic thyroid hormone replacement, appropriately adjusted to need, appears necessary in hypothyroidism for optimal left ventricular function.