this diversity is located in discrete gene clusters that are spread throughout the different genomes. In contrast to this diversity, these enteric microorganisms exhibit marked synteny in their largescale genomic organization, bearing in mind that E. coli and S. enterica diverged about 100 Myr ago 28. The conserved genes may be a re¯ection of the basic lifestyle of the bacteria, requiring intestine colonization, environmental survival and transmission. The unique gene clusters probably contribute to adaptation to environmental niches and to pathogenicity. The pseudogene complement of S. typhi has implications for our understanding of the tight host restriction of this organism, and raises the question of whether it may be possible to eradicate S. typhi and typhoid fever altogether. M Methods Salmonella typhi CT18 was isolated in December 1993, at the Mekong Delta region of Vietnam, from a 9-year-old girl who was suffering from typhoid. The strain was isolated from blood using routine culture methods 23 , and after serological and metabolic con-®rmation of the strain as S. typhi it was immediately frozen in glycerol at-70 8C. The genome sequence was obtained from 97,000 end sequences (giving 7.9´coverage) derived from several pUC18 genomic shotgun libraries (with insert sizes ranging from 1.4 to 4.0 kb) using dye terminator chemistry on ABI377 automated sequencers. This was supplemented with 0.7´sequence coverage from M13mp18 libraries with similar insert sizes. End sequences from a larger insert plasmid (pSP64; 1.9´clone coverage, 10±14-kb insert size) and lambda (lambda-FIX-II; 0.4´clone coverage, 20±22-kb insert size) libraries were used as a scaffold, and the ®nal assembly was veri®ed by comparison with restriction-enzyme digest patterns using pulsed-®eld gel electrophoresis (data not shown). Total sequence coverage was 9.1´. The sequence was assembled, ®nished and annotated as described 29 , using Artemis 30 to collate data and facilitate annotation. In addition we used a gene®nder that was trained speci®cally for S. typhi, which uses a hidden Markov model with modules for the coding region, start and stop codons, and the ribosome-binding site (T.S.L. and A.K., unpublished data). The genome and proteome sequences of S. typhi and S. typhimurium or E. coli were compared in parallel to identify deletions and insertions using the Artemis Comparison Tool (ACT) (K. Rutherford, unpublished data; see also http://www.sanger.ac.uk/Software/ ACT/). Pseudogenes had one or more mutations that would ablate expression, and were identi®ed by direct comparison with S. typhimurium; each of the inactivating mutations was subsequently checked against the original sequencing data.

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their approximately 4,400 protein coding sequences: 173 in Paratyphi A and approximately 210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).

Construction of physical maps of genomes by pulsed-field gel electrophoresis requires enzymes which cut the genome into an analyzable number of fragments; most produce too many fragments. The enzyme I-Ceu I, encoded by a mobile intron in the chloroplast 23S ribosomal RNA (rrl) gene of Chlamydomonas eugametos, cuts a 26-bp site in the rrl gene. This enzyme digests DNA of Salmonella typhimurium at seven sites, each corresponding to one of the rrl genes of the rrn operons, but at no other site. These seven fragments were located on the previously determined Xba I physical map, and the I-Ceu I sites, and thus the rrn genes of S. typhimurium, were mapped on the 4800-kb chromosome. Eschenchia coli K-12 also yields seven fragments of sizes similar to those of S.

Our recent studies have shown that the dendritic cell-specific ICAM nonintegrin CD209 (DC-SIGN) specifically binds to the core LPS of Escherichia coli K12 (E. coli), promoting bacterial adherence and phagocytosis. In this current study, we attempted to map the sites within the core LPS that are directly involved in LPS-DC-SIGN interaction. We took advantage of four sets of well-defined core LPS mutants, which are derived from E. coli, Salmonella enterica serovar Typhimurium, Neisseria gonorrhoeae, and Haemophilus ducreyi and determined interaction of each of these four sets with DC-SIGN. Our results demonstrated that N-acetylglucosamine (GlcNAc) sugar residues within the core LPS in these bacteria play an essential role in targeting the DC-SIGN receptor. Our results also imply that DC-SIGN is an innate immune receptor and the interaction of bacterial core LPS and DC-SIGN may represent a primeval interaction between Gram-negative bacteria and host phagocytic cells.

Endonuclease digestion of the 4,800-kb chromosome of SabloneUa lyphimurium LT2 yielded 24 XbaI fragments, 12 BIn! fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis.The 90-kb plasmid pSLT has oneXbaI site and one BinI site. The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography. Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome. One hundred nine independent TnlO insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and Bin1 sites in TnlO. The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn1O insertions were located by centisome around the map. There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes. All seven rRNA genes were located on the map; all have the Ceu! digestion site, all four with the tRNA gene for glutamate have theXbaI and the BinI sites, but only four of the seven have the Bin! site in the 16S rRNA (rrs) gene. Their inferred orientation of transcription is the same as in Escherichia coli. A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E. coli, observed earlier by others, has been confirmed. The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E. coli, but the XbaI and BinI sites outside the mrn genes show very little conservation.