Virulence factor landscape of a Staphylococcus aureus sequence type 45 strain, MCRF184.

BACKGROUND: We describe the virulence factors of a methicillin-sensitive Staphylococcus aureus sequence type (ST) 45 strain, MCRF184, (spa type t917), that caused severe necrotizing fasciitis in a 72-year-old diabetic male. The genome of MCRF184 possesses three genomic islands: a relatively large type III νSaα with 42 open reading frames (ORFs) that includes superantigen- and lipoprotein-like genes, a truncated νSaß that consists mostly of the enterotoxin gene cluster (egc), and a νSaγ island with 18 ORFs including α-toxin. Additionally, the genome has two phage-related regions: phage φSa3 with three genes of the immune evasion cluster (IEC), and an incomplete phage that is distinct from other S. aureus phages. Finally, the region between orfX and orfY harbors a putative efflux pump, acetyltransferase, regulators, and mobilization genes instead of genes of SCCmec. RESULTS: Virulence factors included phenol soluble modulins (PSMs) α1 through α4 and PSMs ß1 and ß2. Ten ORFs identified in MCRF184 had not been reported in previously sequenced S. aureus strains. CONCLUSION: The dire clinical outcome in the patient and the described virulence factors all suggest that MCRF184, a ST45 strain is a highly virulent strain of S. aureus.

Complete Genome Sequence of Staphylococcus aureus MCRF184, a Necrotizing Fasciitis-Causing Methicillin-Sensitive Sequence Type 45 Staphylococcus Strain.

We report here the complete genome sequence of a highly virulent methicillin-sensitive Staphylococcus aureus strain, MCRF184, belonging to sequence type 45. This staphylococcal strain was isolated from a surgical biopsy specimen from a patient with necrotizing fasciitis.

The non-protein coding breast cancer susceptibility locus Mcs5a acts in a non-mammary cell-autonomous fashion through the immune system and modulates T-cell homeostasis and functions.

INTRODUCTION: Mechanisms underlying low-penetrance, common, non-protein coding variants in breast cancer risk loci are largely undefined. We showed previously that the non-protein coding mammary carcinoma susceptibility locus Mcs5a/MCS5A modulates breast cancer risk in rats and women. The Mcs5a allele from the Wistar-Kyoto (WKy) rat strain consists of two genetically interacting elements that have to be present on the same chromosome to confer mammary carcinoma resistance. We also found that the two interacting elements of the resistant allele are required for the downregulation of transcript levels of the Fbxo10 gene specifically in T-cells. Here we describe mechanisms through which Mcs5a may reduce mammary carcinoma susceptibility. METHODS: We performed mammary carcinoma multiplicity studies with three mammary carcinoma-inducing treatments, namely 7,12-dimethylbenz(a)anthracene (DMBA) and N-nitroso-N-methylurea (NMU) carcinogenesis, and mammary ductal infusion of retrovirus expressing the activated HER2/neu oncogene. We used mammary gland and bone marrow transplantation assays to assess the target tissue of Mcs5a activity. We used immunophenotyping assays on well-defined congenic rat lines carrying susceptible and resistant Mcs5a alleles to identify changes in T-cell homeostasis and function associated with resistance. RESULTS: We show that Mcs5a acts beyond the initial step of mammary epithelial cell transformation, during early cancer progression. We show that Mcs5a controls susceptibility in a non-mammary cell-autonomous manner through the immune system. The resistant Mcs5a allele was found to be associated with an overabundance of gd T-cell receptor (TCR)+ T-cells as well as a CD62L (L-selectin)-high population of all T-cell classes. In contrast to in mammary carcinoma, gdTCR+ T-cells are the predominant T-cell type in the mammary gland and were found to be overabundant in the mammary epithelium of Mcs5a resistant congenic rats. Most of them simultaneously expressed the CD4, CD8, and CD161α markers. In cultured T-cells of Mcs5a resistant congenic rats we found increased mitogen-induced proliferation and production of Th1 cytokines IFNg, IL-2, and Tumor Necrosis Factor (TNF), but not Th2 cytokines IL-4 and IL-6, or Th17 cytokine IL-17 when compared with susceptible control rats. CONCLUSIONS: These data support a hypothesis that Mcs5a displays a non-mammary cell-autonomous mechanism of action to modulate breast cancer risk through the immune system. The resistant Mcs5a allele is associated with alterations in T-cell homeostasis and functions, and overabundance of γδTCR+ T-cells in carcinogen-exposed mammary epithelium.

Genome sequence of the plant-pathogenic bacterium Dickeya dadantii 3937.

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.

progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement.

BACKGROUND: Multiple genome alignment remains a challenging problem. Effects of recombination including rearrangement, segmental duplication, gain, and loss can create a mosaic pattern of homology even among closely related organisms. METHODOLOGY/PRINCIPAL FINDINGS: We describe a new method to align two or more genomes that have undergone rearrangements due to recombination and substantial amounts of segmental gain and loss (flux). We demonstrate that the new method can accurately align regions conserved in some, but not all, of the genomes, an important case not handled by our previous work. The method uses a novel alignment objective score called a sum-of-pairs breakpoint score, which facilitates accurate detection of rearrangement breakpoints when genomes have unequal gene content. We also apply a probabilistic alignment filtering method to remove erroneous alignments of unrelated sequences, which are commonly observed in other genome alignment methods. We describe new metrics for quantifying genome alignment accuracy which measure the quality of rearrangement breakpoint predictions and indel predictions. The new genome alignment algorithm demonstrates high accuracy in situations where genomes have undergone biologically feasible amounts of genome rearrangement, segmental gain and loss. We apply the new algorithm to a set of 23 genomes from the genera Escherichia, Shigella, and Salmonella. Analysis of whole-genome multiple alignments allows us to extend the previously defined concepts of core- and pan-genomes to include not only annotated genes, but also non-coding regions with potential regulatory roles. The 23 enterobacteria have an estimated core-genome of 2.46Mbp conserved among all taxa and a pan-genome of 15.2Mbp. We document substantial population-level variability among these organisms driven by segmental gain and loss. Interestingly, much variability lies in intergenic regions, suggesting that the Enterobacteriacae may exhibit regulatory divergence. CONCLUSIONS: The multiple genome alignments generated by our software provide a platform for comparative genomic and population genomic studies. Free, open-source software implementing the described genome alignment approach is available from http://gel.ahabs.wisc.edu/mauve.

Reordering contigs of draft genomes using the Mauve aligner.

SUMMARY: Mauve Contig Mover provides a new method for proposing the relative order of contigs that make up a draft genome based on comparison to a complete or draft reference genome. A novel application of the Mauve aligner and viewer provides an automated reordering algorithm coupled with a powerful drill-down display allowing detailed exploration of results. AVAILABILITY: The software is available for download at http://gel.ahabs.wisc.edu/mauve.

The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse.

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.

Chemopreventive effects of celecoxib are limited to hormonally responsive mammary carcinomas in the neu-induced retroviral rat model.

INTRODUCTION: While current breast cancer chemoprevention strategies using selective estrogen response modulators and aromatase inhibitors are quite successful, their effects are limited to hormonally responsive breast cancer. Hormonally nonresponsive breast cancer (including estrogen receptor-negative cancer) is associated with poor prognosis for patients, and few chemoprevention agents exist for this type of cancer. The cyclooxygenase-2 inhibitor celecoxib (Celebrex) is a nonsteroidal anti-inflammatory drug and as such is a potential candidate for the prevention of hormonally nonresponsive breast cancer. METHODS: The chemopreventive effects of celecoxib were evaluated in the neu-induced retroviral rat mammary carcinogenesis model, to assess the efficacy of celecoxib on hormonally responsive and hormonally nonresponsive mammary carcinomas. RESULTS: Dietary celecoxib at 1,200 mg/kg diet was highly efficacious in the prevention of hormonally responsive mammary carcinomas in intact rats, decreasing tumor multiplicity by 56% (P < 0.0001) and by 74% (P = 0.0002) in two independent experiments. No significant effect was found, however, on hormonally nonresponsive mammary carcinomas of ovariectomized rats. Treatment with a combination diet, consisting of tamoxifen at 2 mg/kg diet and celecoxib at 1,200 mg/kg diet, reduced tumor multiplicity by 72% (P = 0.0002) in intact rats. This reduction was not statistically different from that observed with celecoxib alone. Furthermore, long-term treatment with celecoxib was not associated with reductions in tumor volume in either intact rats or ovariectomized rats. In contrast, tamoxifen treatment and the combination regimen caused significant reductions in tumor volumes in intact rats (P = 0.01 and P = 0.004, respectively). Consistent with these data, decreases in proliferation and increases in apoptosis were detected in tamoxifen-treated and combination diet-treated tumors. No such modulations were observed in celecoxib-treated tumors. CONCLUSION: The chemopreventive effects of celecoxib appear to be limited to modulations in multiplicity of hormonally responsive mammary carcinomas. The fact that no synergistic or additive effects were observed in combination diet-treated rats raises the question of whether celecoxib is suitable for the prevention of hormonally nonresponsive breast cancer or for use in combination therapy with selective estrogen response modulators or aromatase inhibitors.

Enteropathogen Resource Integration Center (ERIC): bioinformatics support for research on biodefense-relevant enterobacteria.

ERIC, the Enteropathogen Resource Integration Center (www.ericbrc.org), is a new web portal serving as a rich source of information about enterobacteria on the NIAID established list of Select Agents related to biodefense-diarrheagenic Escherichia coli, Shigella spp., Salmonella spp., Yersinia enterocolitica and Yersinia pestis. More than 30 genomes have been completely sequenced, many more exist in draft form and additional projects are underway. These organisms are increasingly the focus of studies using high-throughput experimental technologies and computational approaches. This wealth of data provides unprecedented opportunities for understanding the workings of basic biological systems and discovery of novel targets for development of vaccines, diagnostics and therapeutics. ERIC brings information together from disparate sources and supports data comparison across different organisms, analysis of varying data types and visualization of analyses in human and computer-readable formats.

A new asset for pathogen informatics--the Enteropathogen Resource Integration Center (ERIC), an NIAID Bioinformatics Resource Center for Biodefense and Emerging/Re-emerging Infectious Disease.

ERIC (Enteropathogen Resource Information Center) is one of the National Institute of Allergy and Infectious Diseases (NIAID) Bioinformatics Resource Centers for Biodefense and Emerging/Re-emerging Infectious Disease. ERIC serves as a comprehensive information resource for five related pathogens: Yersinia enterocolitica, Yersinia pestis, diarrheagenic E. coli, Shigella spp., and Salmonella spp. ERIC integrates genomics, proteomics, biochemical and microbiological information to facilitate the interpretation and understanding of ERIC pathogens and select related non-pathogens for the advancement of diagnostics, therapeutics, and vaccines. ERIC (www.ericbrc.org) is evolving to provide state-of-the-art analysis tools and data types, such as genome sequencing, comparative genomics, genome polymorphisms, gene expression, proteomics, and pathways as well as expertly curated community genome annotation. Genome sequence and genome annotation data and a variety of analysis and tools for eight strains of Yersinia enterocolitica and Yersinia pestis pathogens (Yersinia pestis biovars Mediaevalis KIM, Mediaevalis 91001, Orientalis CO92, Orientalis IP275, Antiqua Angola, Antiqua Antiqua, Antiqua Nepal516, and Yersinia enterocolitica 8081) and two strains of Yersinia pseudotuberculosis (Yersinia pseudotuberculosis IP32953 and IP31758) are currently available through the ERIC portal. ERIC seeks to maintain a strong collaboration with the scientific community so that we can continue to identify and incorporate the latest research data, tools, and training to best meet the current and future needs of the enteropathogen research community. All tools and data developed under this NIAID contract will be freely available. Please contact info@ericbrc.org for more information.

Rat Mcs5a is a compound quantitative trait locus with orthologous human loci that associate with breast cancer risk.

Breast cancer risk is a polygenic trait. To identify breast cancer modifier alleles that have a high population frequency and low penetrance we used a comparative genomics approach. Quantitative trait loci (QTL) were initially identified by linkage analysis in a rat mammary carcinogenesis model followed by verification in congenic rats carrying the specific QTL allele under study. The Mcs5a locus was identified by fine-mapping Mcs5 in a congenic model. Here we characterize the Mcs5a locus, which when homozygous for the Wky allele, reduces mammary cancer risk by 50%. The Mcs5a locus is a compound QTL with at least two noncoding interacting elements: Mcs5a1 and Mcs5a2. The resistance phenotype is only observed in rats carrying at least one copy of the Wky allele of each element on the same chromosome. Mcs5a1 is located within the ubiquitin ligase Fbxo10, whereas Mcs5a2 includes the 5' portion of Frmpd1. Resistant congenic rats show a down-regulation of Fbxo10 in the thymus and an up-regulation of Frmpd1 in the spleen. The association of the Mcs5a1 and Mcs5a2 human orthologs with breast cancer was tested in two population-based breast cancer case-control studies (approximately 12,000 women). The minor alleles of rs6476643 (MCS5A1) and rs2182317 (MCS5A2) were independently associated with breast cancer risk. The minor allele of rs6476643 increases risk, whereas the rs2182317 minor allele decreases risk. Both alleles have a high population frequency and a low penetrance toward breast cancer risk.

Neu-induced retroviral rat mammary carcinogenesis: a novel chemoprevention model for both hormonally responsive and nonresponsive mammary carcinomas.

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.

Genome-wide detection and analysis of homologous recombination among sequenced strains of Escherichia coli.

BACKGROUND: Comparisons of complete bacterial genomes reveal evidence of lateral transfer of DNA across otherwise clonally diverging lineages. Some lateral transfer events result in acquisition of novel genomic segments and are easily detected through genome comparison. Other more subtle lateral transfers involve homologous recombination events that result in substitution of alleles within conserved genomic regions. This type of event is observed infrequently among distantly related organisms. It is reported to be more common within species, but the frequency has been difficult to quantify since the sequences under comparison tend to have relatively few polymorphic sites. RESULTS: Here we report a genome-wide assessment of homologous recombination among a collection of six complete Escherichia coli and Shigella flexneri genome sequences. We construct a whole-genome multiple alignment and identify clusters of polymorphic sites that exhibit atypical patterns of nucleotide substitution using a random walk-based method. The analysis reveals one large segment (approximately 100 kb) and 186 smaller clusters of single base pair differences that suggest lateral exchange between lineages. These clusters include portions of 10% of the 3,100 genes conserved in six genomes. Statistical analysis of the functional roles of these genes reveals that several classes of genes are over-represented, including those involved in recombination, transport and motility. CONCLUSION: We demonstrate that intraspecific recombination in E. coli is much more common than previously appreciated and may show a bias for certain types of genes. The described method provides high-specificity, conservative inference of past recombination events.

Mauve: multiple alignment of conserved genomic sequence with rearrangements.

As genomes evolve, they undergo large-scale evolutionary processes that present a challenge to sequence comparison not posed by short sequences. Recombination causes frequent genome rearrangements, horizontal transfer introduces new sequences into bacterial chromosomes, and deletions remove segments of the genome. Consequently, each genome is a mosaic of unique lineage-specific segments, regions shared with a subset of other genomes and segments conserved among all the genomes under consideration. Furthermore, the linear order of these segments may be shuffled among genomes. We present methods for identification and alignment of conserved genomic DNA in the presence of rearrangements and horizontal transfer. Our methods have been implemented in a software package called Mauve. Mauve has been applied to align nine enterobacterial genomes and to determine global rearrangement structure in three mammalian genomes. We have evaluated the quality of Mauve alignments and drawn comparison to other methods through extensive simulations of genome evolution.

GRIL: genome rearrangement and inversion locator.

UNLABELLED: GRIL is a tool to automatically identify collinear regions in a set of bacterial-size genome sequences. GRIL uses three basic steps. First, regions of high sequence identity are located. Second, some of these regions are filtered based on user-specified criteria. Finally, the remaining regions of sequence identity are used to define significant collinear regions among the sequences. By locating collinear regions of sequence, GRIL provides a basis for multiple genome alignment using current alignment systems. GRIL also provides a basis for using current inversion distance tools to infer phylogeny. AVAILABILITY: GRIL is implemented in C++ and runs on any x86-based Linux or Windows platform. It is available from http://asap.ahabs.wisc.edu/gril

Genome sequence of Yersinia pestis KIM.

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.