Nicole T. Perna

Genome sequence of enterohaemorrhagic Escherichia coli O157:H7

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions—all of which could be targets for surveillance.

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.

Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli

We present the complete genome sequence of uropathogenic Escherichia coli, strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli. The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.

PATTERNS OF NUCLEOTIDE COMPOSITION AT FOURFOLD DEGENERATE SITES OF ANIMAL MITOCHONDRIAL GENOMES

Three statistics (%GC, GC-skew, and AT-skew) can be used to describe the overall patterns of nucleotide composition in DNA sequences. Fourfold degenerate third codon positions from 16 animal mitochondrial genomes were analyzed. The overall composition, as measured by %GC, varies from 3.6 %GC in the honeybee to 47.2 %GC in human mtDNA. Compositional differences between strands of the mitochondrial genome were quantified using the two skew statistics presented in this paper. Strand-specific distribution of bases varies among animal taxa independently of overall %GC. Compositional patterns reflect the substitution process. Description of these patterns may aid in the formation of hypotheses about substitutional mechanisms.

Escherichia coli K-12: a cooperatively developed annotation snapshot—2005

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.

Complete Genome Sequence and Comparative Genomics of Shigella flexneri Serotype 2a Strain 2457T

ABSTRACT We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organisms distinctive lifestyle that have yet to be explained.

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. Contact: rissman@wisc.edu Supplementary information: Supplementary data are available at Bioinformatics online and http://gel.ahabs.wisc.edu

Host Range and Molecular Phylogenies of the Soft Rot Enterobacterial Genera Pectobacterium and Dickeya

ABSTRACT Pectobacterium and Dickeya spp. are related broad-host-range entero-bacterial pathogens of angiosperms. A review of the literature shows that these genera each cause disease in species from at least 35% of angiosperm plant orders. The known host ranges of these pathogens partially overlap and, together, these two genera are pathogens of species from 50% of angiosperm plant orders. Notably, there are no reported hosts for either genus in the eudicots clade and no reported Dickeya hosts in the magnoliids or eurosids II clades, although Pectobacterium spp. are pathogens of at least one plant species in the magnoliids and at least one in each of the three eurosids II plant orders. In addition, Dickeya but not Pectobacterium spp. have been reported on a host in the rosids clade and, unlike Pectobacterium spp., have been reported on many Poales species. Natural disease among nonangiosperms has not been reported for either genus. Phylogenetic analyses of sequences concatenated from regions of seven housekeeping genes (acnA, gapA, icdA, mdh, mtlD, pgi, and proA) from representatives of these genera demonstrated that Dickeya spp. and the related tree pathogens, the genus Brenneria, are more diverse than Pectobacterium spp. and that the Pectobacterium strains can be divided into at least five distinct clades, three of which contain strains from multiple host plants.

Identification and Characterization of lpfABCC′DE, a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7

ABSTRACT The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC′DE) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (lpf) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC′DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim−) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation.

ASAP, a systematic annotation package for community analysis of genomes

ASAP (a systematic annotation package for community analysis of genomes) is a relational database and web interface developed to store, update and distribute genome sequence data and functional characterization (https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm). ASAP facilitates ongoing community annotation of genomes and tracking of information as genome projects move from preliminary data collection through post-sequencing functional analysis. The ASAP database includes multiple genome sequences at various stages of analysis, corresponding experimental data and access to collections of related genome resources. ASAP supports three levels of users: public viewers, annotators and curators. Public viewers can currently browse updated annotation information for Escherichia coli K-12 strain MG1655, genome-wide transcript profiles from more than 50 microarray experiments and an extensive collection of mutant strains and associated phenotypic data. Annotators worldwide are currently using ASAP to participate in a community annotation project for the Erwinia chrysanthemi strain 3937 genome. Curation of the E. chrysanthemi genome annotation as well as those of additional published enterobacterial genomes is underway and will be publicly accessible in the near future.