Guy Plunkett

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.

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.

Emergent properties of reduced-genome Escherichia coli

With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.

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.

Comparative Genomics of Salmonella enterica Serovar Typhi Strains Ty2 and CT18

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.

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 DNAStars 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.

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.

Genome of Bacteriophage P1

P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from sigma(70) promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli

Sequences of four new heat-shock (HS) genes of Escherichia coli organized into two operons were determined. The operon at 83 min specifies two proteins of 15.8 kDa (HslT) and 16.1 kDa (HslS), which are identical to IbpA and IbpB, respectively. Expression of mRNA from a sigma 32-dependent promoter of the hslTS/ibpAB operon is stimulated 30-75-fold upon temperature upshift. The transcription start point (tsp) is located at a G, 96 bp upstream from the AUG start codon of hslT/ibpA. The deduced amino acid sequences of HslT/IbpA and HslS/IbpB are 48% identical to each other and were found to be remotely related to the chloroplast low-molecular-weight HS protein, which is highly conserved among plants. The second hs operon is much less actively stimulated by temperature upshift, although it has a hs promoter that perfectly matches the consensus of promoters recognized by sigma 32. Located at 88.9 min, the hslVU operon specifies proteins of 19.1 kDa (HslV) and 49.6 kDa (HslU). Multiple tsp were found in this operon. HslV is remotely related to the eukaryotic proteasome proteins, and HslU is very similar to a Pasteurella haemolytica protein of unknown function. Both HslU and the P. haemolytica protein share a ATP/GTP-binding motif near their N-termini. The two operons described here are transcribed counterclockwise on the standard genetic map.

Novel phosphotransferase-encoding genes revealed by analysis of the Escherichia coli genome: a chimeric gene encoding an Enzyme I homologue that possesses a putative sensory transduction domain.

Two genes (ptsI and ptsA) that encode homologues of the energy coupling Enzyme I of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system (PTS) have previously been identified on the Escherichia coli chromosome. We here report the presence of a third E. coli gene, designated ptsP, that encodes an Enzyme I homologue, here designated Enzyme INtr. Enzyme INtr possesses an N-terminal domain homologous to the N-terminal domains of NifA proteins [(127 amino acids (aa)] joined via two tandem flexible linkers to the C-terminal Enzyme I-like domain (578 aa). Structural features of the putative ptsP operon, including transcriptional regulatory signals, are characterized. We suggest that Enzyme INtr functions in transcriptional regulation of nitrogen-related operons together with previously described PTS proteins encoded within the rpoN operon. It may thereby provide a link between carbon and nitrogen assimilatory pathways.