Escherichia coli genes and pathways involved in surviving extreme exposure to ionizing radiation.

To further an improved understanding of the mechanisms used by bacterial cells to survive extreme exposure to ionizing radiation (IR), we broadly screened nonessential Escherichia coli genes for those involved in IR resistance by using transposon-directed insertion sequencing (TraDIS). Forty-six genes were identified, most of which become essential upon heavy IR exposure. Most of these were subjected to direct validation. The results reinforced the notion that survival after high doses of ionizing radiation does not depend on a single mechanism or process, but instead is multifaceted. Many identified genes affect either DNA repair or the cellular response to oxidative damage. However, contributions by genes involved in cell wall structure/function, cell division, and intermediary metabolism were also evident. About half of the identified genes have not previously been associated with IR resistance or recovery from IR exposure, including eight genes of unknown function.

Evolution of extreme resistance to ionizing radiation via genetic adaptation of DNA repair.

By directed evolution in the laboratory, we previously generated populations of Escherichia coli that exhibit a complex new phenotype, extreme resistance to ionizing radiation (IR). The molecular basis of this extremophile phenotype, involving strain isolates with a 3-4 order of magnitude increase in IR resistance at 3000 Gy, is now addressed. Of 69 mutations identified in one of our most highly adapted isolates, functional experiments demonstrate that the IR resistance phenotype is almost entirely accounted for by only three of these nucleotide changes, in the DNA metabolism genes recA, dnaB, and yfjK. Four additional genetic changes make small but measurable contributions. Whereas multiple contributions to IR resistance are evident in this study, our results highlight a particular adaptation mechanism not adequately considered in studies to date: Genetic innovations involving pre-existing DNA repair functions can play a predominant role in the acquisition of an IR resistance phenotype. DOI: http://dx.doi.org/10.7554/eLife.01322.001.

Using comparative genomics for inquiry-based learning to dissect virulence of Escherichia coli O157:H7 and Yersinia pestis.

Genomics and bioinformatics are topics of increasing interest in undergraduate biological science curricula. Many existing exercises focus on gene annotation and analysis of a single genome. In this paper, we present two educational modules designed to enable students to learn and apply fundamental concepts in comparative genomics using examples related to bacterial pathogenesis. Students first examine alignments of genomes of Escherichia coli O157:H7 strains isolated from three food-poisoning outbreaks using the multiple-genome alignment tool Mauve. Students investigate conservation of virulence factors using the Mauve viewer and by browsing annotations available at the A Systematic Annotation Package for Community Analysis of Genomes database. In the second module, students use an alignment of five Yersinia pestis genomes to analyze single-nucleotide polymorphisms of three genes to classify strains into biovar groups. Students are then given sequences of bacterial DNA amplified from the teeth of corpses from the first and second pandemics of the bubonic plague and asked to classify these new samples. Learning-assessment results reveal student improvement in self-efficacy and content knowledge, as well as students' ability to use BLAST to identify genomic islands and conduct analyses of virulence factors from E. coli O157:H7 or Y. pestis. Each of these educational modules offers educators new ready-to-implement resources for integrating comparative genomic topics into their curricula.

Multiplex sequencing of seven ocular herpes simplex virus type-1 genomes: phylogeny, sequence variability, and SNP distribution.

PURPOSE: Little is known about the role of sequence variation in the pathology of HSV-1 keratitis virus. The goal was to show that a multiplex, high-throughput genome-sequencing approach is feasible for simultaneously sequencing seven HSV-1 ocular strains. METHODS: A genome sequencer was used to sequence the HSV-1 ocular isolates TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4, in a single lane. Reads were mapped to the HSV-1 strain 17 reference genome by high-speed sequencing. ClustalW was used for alignment, and the Mega 4 package was used for phylogenetic analysis (www.megasoftware.net). Simplot was used to compare genetic variability and high-speed sequencing was used to identify SNPs (developed by Stuart Ray, Johns Hopkins University School of Medicine, Baltimore, MD, http://sray.med.som.jhml.edu/SCRoftware/simplot). RESULTS: Approximately 95% to 99% of the seven genomes were sequenced in a single lane with average coverage ranging from 224 to 1345. Phylogenetic analysis of the sequenced genome regions revealed at least three clades. Each strain had approximately 200 coding SNPs compared to strain 17, and these were evenly spaced along the genomes. Four genes were highly conserved, and six were more variable. Reduced coverage was obtained in the highly GC-rich terminal repeat regions. CONCLUSIONS: Multiplex sequencing is a cost-effective way to obtain the genomic sequences of ocular HSV-1 isolates with sufficient coverage of the unique regions for genomic analysis. The number of SNPs and their distribution will be useful for analyzing the genetics of virulence, and the sequence data will be useful for studying HSV-1 evolution and for the design of structure-function studies.

Annotation and overview of the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 draft genome reveals the virulence gene complement of a tumour-inducing pathogen of woody hosts.

Pseudomonas savastanoi pv. savastanoi is a tumour-inducing pathogen of Olea europaea L. causing olive knot disease. Bioinformatic analysis of the draft genome sequence of strain NCPPB 3335, which encodes 5232 predicted coding genes on a total length of 5856 998 bp and a 57.12% G + C, revealed a large degree of conservation with Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tabaci 11528. However, NCPPB 3335 contains twelve variable genomic regions, which are absent in all previously sequenced P. syringae strains. Various features that could contribute to the ability of this strain to survive in a woody host were identified, including broad catabolic and transport capabilities for degrading plant-derived aromatic compounds, the duplication of sequences related to the biosynthesis of the phytohormone indoleacetic acid (iaaM, iaaH) and its amino acid conjugate indoleacetic acid-lysine (iaaL gene), and the repertoire of strain-specific putative type III secretion system effectors. Access to this seventh genome sequence belonging to the 'P. syringae complex' allowed us to identify 73 predicted coding genes that are NCPPB 3335-specific. Results shown here provide the basis for detailed functional analysis of a tumour-inducing pathogen of woody hosts and for the study of specific adaptations of a P. savastanoi pathovar.

Directed evolution of ionizing radiation resistance in Escherichia coli.

We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D(37) values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

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.

ASAP: a resource for annotating, curating, comparing, and disseminating genomic data.

ASAP is a comprehensive web-based system for community genome annotation and analysis. ASAP is being used for a large-scale effort to augment and curate annotations for genomes of enterobacterial pathogens and for additional genome sequences. New tools, such as the genome alignment program Mauve, have been incorporated into ASAP in order to improve display and analysis of related genomes. Recent improvements to the database and challenges for future development of the system are discussed. ASAP is available on the web at https://asap.ahabs.wisc.edu/asap/logon.php.