[show abstract][hide abstract] ABSTRACT: Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.
Journal of Bacteriology 12/2004; 186(22):7773-82. · 3.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Bacteriology 05/2003; 185(7):2330-7. · 3.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps (XhoI and PvuII) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.
Applied and Environmental Microbiology 01/2003; 68(12):6321-31. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. Salmonella enterica serovar Typhi is a human-specific patho- gen causing enteric typhoid fever, a severe infection of the reticuloendothelial system (8, 10, 14). Although difficult to estimate, it is thought that at least 16 million cases and 500,000 deaths occur each year around the world (8). The early admin- istration of antibiotic treatment has proven to be highly effec- tive in eliminating infections, but indiscriminate use of antibi- otics has led to the emergence of multidrug-resistant strains of S. enterica serovar Typhi (13, 31). Since typhoid is becoming difficult to treat with conventional drugs, information about the whole genome sequence and genes of S. enterica serovar Typhi will help to reveal more specific targets for drugs aimed at disease treatment and vaccines for prevention. Although S. enterica serovar Typhi can grow in laboratory media and sur- vive in other hosts, such as experimental mice, humans are the only known natural hosts. The mouse model is a poor repre- sentive of the human disease. Experimental infections of chim- panzees or human volunteers have been the only way to relate bacterial genetic characteristics with pathogenic effects. Con- sequently, what is known about S. enterica serovar Typhi pathogenicity has been largely extrapolated from studies of S. enterica serovar Typhimurium infections in mice. In this work, we present the genome sequence of the well- studied pathogenic strain Ty2. This strain was the foundation for vaccine development and was the parent of mutant strains Ty21a and CVD908 and their derivatives, used in trials of live attenuated vaccines (16). Isolated before the emergence of drug resisitance in the 1970s, it contains no plasmids. In con- trast, recently isolated S. enterica serovar Typhi strain CT18 (25) carries multiple drug resistance cassettes on a large plas- mid and contains a second large plasmid closely related to pMT1 of Yersinia pestis. The genome sequence of CT18 was recently determined at the Sanger Centre, Hinxton, United Kingdom (GenBank accession number AL513382), and we have used this sequence to perform a detailed comparison of the two genomes.
Journal of Bacteriology - J BACTERIOL. 01/2003; 185(7):2330-2337.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Bacteriology 09/2002; 184(16):4601-11. · 3.19 Impact Factor