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Intraspecies variation in bacterial genomes: the need for a species genome concept Ruiting Lan and Peter R. Reeves

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acteria are characterized Bacterial populations are clonal. Their absence of the relevant genes, by extensive intraspecies evolution involves not only divergence and this is perhaps generally variation. There is not between orthologous genes but also gain the case. only the sequence variation of genes from other clones or species, One could of course sefound in all species, but also which has only recently been widely quence a large number of isothe presence or absence of appreciated through macrorestriction lates for each species to obtain whole genes or clusters of mapping, genomic subtraction and this all-important sequence, genes. In most eukaryotes, an complete genome sequencing. Genes can but that is expensive and has individual genome sequence also be lost in response to selection or by only been completed for two will provide us with the vast random mutation after becoming isolates of two species: Helicomajority of the genes for that redundant. The bacterial genome is a bacter pylori2,3 and Neisseria species. This is not the case for meningitidis4,5. Work is in dynamic structure and intraspecies bacteria. Only if we have the progress on more than one variation needs to be included in genome sequence for all the DNA imisolate of a number of other analysis if we are to gain insight into the portant for a species do we refull species genome. species [see TIGR’s website ally have the ‘species’ genome, (http://www.tigr.org) for R. Lan and P.R. Reeves* are in the Dept of as distinct from an individual a list of genomes currently Microbiology, Bldg G08, University of Sydney, genome. It would probably being sequenced] but this will NSW 2006, Sydney, Australia. require the sequence of many still give only a small sample of *tel: 161 2 9351 6045, individual genomes to even the variation, and other apfax: 161 2 9351 4571, e-mail: [email protected] approximate the full species proaches are being developed. genome, and alternative approaches are therefore required. This review will Genome-comparison techniques focus on the genome differences between clones of Macrorestriction mapping or pulse-field gel eleca species and the forces involved in generating this trophoresis, which separates large DNA fragments variation. after digestion of chromosomal DNA by rare-cutting enzymes, has greatly advanced our understanding of Assessing genome variation within species species genome structure. Macrorestriction mapping It has long been known that bacteria can carry plas- detects genome rearrangements as well as substantial mids or lysogenic bacteriophages and that these gene additions; this technique has now been carried elements are, in general, present in only some strains. out for many species and has demonstrated the ubiqThere have been no extensive studies of the number uity of intraspecies genome size variation6,7. Bloch of forms that can exist in a species, but it is certainly and colleagues8 developed a novel macrorestriction very large. For example, there are nine pro- mapping method using extremely rare enzyme sites phages/cryptic prophages in Escherichia coli K-12 that are introduced into the chromosome on trans(Ref. 1). Some are simply parasitic and not an integral posons and then moved between strains. The part of the genome, but others carry genes that make chromosome can then be cut into large-sized fragthem important for cell survival. There are also ments with fixed-reference terminal loci, allowing groups of genes found in the chromosome of some comparison of fragments with the same end points strains but not others. These are clearly part of the from different isolates. Genomic subtraction is based on the hybridization genome and it is this variation that will most concern us. The best known of these groups are the of DNA from two genomes and removal of any compathogenicity islands (PAIs), which carry genes that mon sequences9, allowing direct analysis of strainconfer specific aspects of pathogenicity. As another specific DNA. A simplification of the procedure has example, strains of many bacterial species vary in been reported recently10. Genomic subtraction is metabolic capability, in areas such as which sugars or probably the best method to explore genome differother substrates can be utilized; in some cases, this ences for a large number of isolates. One can use the has been shown to be the result of the presence or subtracted DNA to screen plasmid/cosmid libraries, 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(00)01791-1 TRENDS

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or it can be cloned directly, allowing access to the unique DNA regions11. Another powerful tool for comparative genome analysis is DNA microarray or DNA chip technology. Behr et al.12 exploited sv. Typhimurium LT2 – – this technology to compare the genomes of 13 variants of the sv. Typhimurium (#2) 98% 2% tuberculosis Bacille Calmette– SPI-2 Guérin (BCG) vaccine strain, sv. Muenchen 92% 5% 39 fragments which was derived from a viruSPI-1 lent isolate of Mycobacterum 12 Metabolic sv. Typhi 88% 9% bovis early this century, with 6 Phages that of Mycobacterium tubercu2 O-antigens Subspecies V N/A 20% losis H37Rv. Of the 3924 open 3 Virulence reading frames (ORFs) of M. tu14 Not defined E. coli K-12 45% 30% berculosis H37Rv, 3902 were spotted on a microarray and hytrends in Microbiology bridized with labeled BCG total Fig. 1. Genomic variation in Salmonella enterica. The dendrogram shows the relationships of three genomic DNA. One region was S. enterica subspecies I serovars (including two strains of sv. Typhimurium), S. enterica subabsent from all BCG strains, perspecies V and Escherichia coli K-12. S. enterica subspecies II, IIIa, IIIb, IV, VI and VII were omitted haps representing loss during as no subtractive hybridization data were available for these subspecies. The branching order was the initial process of attenuation. inferred from multilocus enzyme electrophoresis (MLEE) and sequence data14,39. Two significant events in the evolution of virulence occurred through the gain of pathogenicity islands: Salmonella Four regions were apparently pathogenicity island 1 (SPI-1; found in all subspecies) and SPI-2 (found in all subspecies except deleted in various BCG strains V)22 are marked on the nodes. DNA–DNA hybridization data with reference to S. enterica LT2 are during subsequent passages. from Crosa40. The percentage of non-homologous DNA for S. enterica strains was determined with Nine regions, of a total of 50 kb reference to S. enterica LT2 by analysis of residual DNA after subtractive hybridization11, and for E. coli K-12 with reference to S. enterica LT2 by comparison of genome data41 (incomplete for LT2). containing 61 ORFs, were found 39 random fragments sampled from the 20% of DNA present in LT2 but absent in subspecies V to be absent in BCG strains and were sequenced11 and the sequences have been used to identify 35 of the corresponding genes virulent M. bovis strains. Alusing databases for genomes being sequenced for S. enterica sv. Typhimurium and Typhi (see webthough they were regarded as sites http://genome.wustl.edu/gsc/bacterial/salmonella.shtml and http://www. M. bovis deletions12,13, it is surely sanger.ac.uk/Projects/S_typhi/). The boxed text shows the general categories for the 23 of these genes for which such information was obtained in BLAST searches in Genbank. The majority equally likely that many of these are metabolic pathway genes, possibly gained to expand ecological niches during divergence of genes were gained by M. tuberS. enterica. culosis when it became a humanspecific pathogen. Behr et al.12 reported that their current microarray detects de- to probe LT2 DNA cosmids. The Typhimurium, letions as small as 2 kb (i.e. 1/2000th of a genome), Muenchen, Typhi and subspecies V strains were estiillustrating the power of this technology. However, a mated to differ from LT2 by 2%, 5%, 9% and 20%, serious drawback of this approach is that it is unidi- respectively. This correlates with the divergence of rectional, that is, we can only use it to screen for genes the four strains found by multilocus enzyme electrowe have already identified. phoresis (MLEE), sequence variation of housekeeping genes and DNA–DNA hybridization data (Fig. 1). Genome variation in Salmonella enterica and E. coli Further insight was gained by sequencing 39 fragGenome size in S. enterica and E. coli has been shown ments from the subspecies-V-strain-subtracted LT2 to vary by as much as 20% between isolates. DNA11. Sixteen had a G1C content below the species average, indicating derivation from distantly related S. enterica species, but over half had a G1C content within the There are .2000 serovars of S. enterica. Initially, full normal range for the species. Of six for which a dataspecies names were given but all serovars are now in base search indicated a function, the majority related one species, S. enterica, with subspecies V having ar- to the utilization of specific substrates. We have now guable status as a separate species, Salmonella bon- used the partial genome sequences of S. enterica sv. gori. S. enterica has a well-defined subspecies struc- Typhimurium LT2 and Typhi (http://genome. ture with deep branch lengths14. We have analysed wustl.edu/gsc/bacterial/salmonella.shtml the genome differences of four strains by genomic and http://www.sanger.ac.uk/Projects/S_ subtraction11. Strain LT2 (sv. Typhimurium) was typhi/) to obtain the full sequence of the genes used as the target and separately subtracted by sub- sampled by 35 of the 39 fragments. BLAST searches species I strains of serovars Typhimurium, Muenchen suggest that 12 are related to metabolism, six to and Typhi, and a subspecies V strain. The amount of phage, two to O-antigen synthesis and three to viruLT2 DNA not present in each of the other strains was lence (Fig. 1). Of course, some of the variation in, for estimated by using the LT2 DNA left after subtraction example, metabolic pathway genes could relate to

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virulence but, if so, the relevance is not immediately obvious, and it appears that much of the variation relates to aspects of S. enterica ecology other than virulence. E. coli E. coli is a diverse species with commensal isolates as well as many pathogenic clones. There is a great variation in genome size. By genomic subtraction against the reference K-12 strain, an avian enteropathogenic E. coli strain was found to carry a total of 12 unique regions with an estimated 350-kb unique DNA15. Using their modified macrorestriction mapping, Bloch’s group16 identified the differences from the K-12 genome in RS218, a newborn-sepsis-associated strain, and J96, a urinary tract infection (UTI) isolate. Strain RS218 has 10 additions totalling 537 kb and one 20-kb deletion. Strain J96 has 493 kb of additional DNA at four locations and two deletions of 53 and 35 kb. Two of the J96 segments are known PAIs17. Our final example is the well-known clone O157:H7, which is being sequenced and is reported to have a genome 20% larger than that of K-12 (Ref. 18). A more comprehensive but less detailed picture of genome differences in this species was obtained by Bergthorsson and Ochman19, who measured the genome sizes of 35 isolates of the ECOR set (a widely used reference collection of E. coli strains) by I-ceuI macrorestriction mapping. The chromosome lengths ranged from 4.5 to 5.5 Mb, with the variation being dispersed throughout the genome. The major E. coli groups differ in average genome size, with groups B2 and D having the largest genomes. PAIs have contributed to this variably present DNA. Boyd and Hartl20 showed that of four virulence genes (hly, kps, pap and sfa) from UTI-associated PAIs17, at least two are present in the majority of B2 and D strains but only sporadically elsewhere. This provides just a hint of the patterns we can expect to see as we explore the species genome. Genome variation in H. pylori and N. meningitidis There are complete genome sequences available for two isolates each of H. pylori and N. meningitidis, allowing gene-by-gene comparison of genome variation. H. pylori H. pylori occupies a single niche, the gastric mucosa, but causes a range of diseases and was the first bacterial species for which the complete genome sequence of two isolates was determined2,3. The two isolates of H. pylori essentially provide a random sampling of the species variation. One strain, J99, was from a patient with a duodenal ulcer and the other, 26695, was from a gastritis patient. Eighty nine of the 1495 genes (6%) in J99 and 117 of the 1552 genes (7.5%) in 26695 are specific to their respective strain, with almost half of these genes being clustered in a single hypervariable region. Sequence divergence in other genes ranges from 0–30%. As yet, little is known of the features of the strain-specific

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genes, with only 51 of the total of 206 assigned to a functional category, including DNA restriction or modification (31 genes), cell-envelope synthesis (6), cellular processes (6), DNA replication (4), energy metabolism (3) and phospholipid metabolism (1). N. meningitidis N. meningitidis is generally a commensal that lives on the mucosa of the nasopharynx. However, some strains are invasive and can enter first the bloodstream and then the cerebrospinal fluid, causing meningitis. Most of the pathogenic strains fall into three serogroups and representatives of the group A (Strain MC58) and group B (strain Z2491) serogroups have been sequenced4,5. We downloaded sequences for all ORFs of MC58 and Z2491 (2155 and 2121 ORFs, respectively) and performed BLASTN searches for homologous genes. Two hundred and thirty nine (11.1%) of the MC58 ORFs and 208 (9.8%) of the Z2491 ORFs are not present in the other strain. We used a P value of 10220 for the BLASTN non-homologous cut-off. However, a proportion of the ORFs of MC58 and Z2491 with a BLASTN P value of 10220–10250 (42 and 59 ORFs, respectively) are only partially homologous but not included. Of the strainspecific ORFs, 87% are of unknown function. Only 31 of the 239 MC58 ORFs and 28 of the 207 Z2491 ORFs have been assigned (putative) functions including, for example, capsule biosynthesis, as expected, and membrane proteins. In summary, studies based on subtractive hybridization or genome comparisons show that a substantial amount of DNA present in one strain can be absent in another. Horizontal gene transfer and niche adaptation Bacterial clones are often adapted to specific niches21 (see Box 1). In E. coli and S. enterica, both well-studied organisms at the population level, there are many clones that are specialized in the disease they cause and the hosts they colonize. It has been proposed that such clones are maintained by niche adaptation21, and that new clones arise by horizontal transfer of beneficial genes when clones are adapting to new niches. It is important to note that the transfer of a gene or genes beneficial to the recipient will be followed by selection of the recombinant, which gives a high probability of fixation in the clone and allows the expansion of niches. This can occur even in highly clonal species with very low levels of recombination21. The variation in genes present in different clones provides the major genetic diversity within a species, with the extent of the diversity depending on the range of niches occupied. PAIs PAIs have been a major focus of the study of pathogenicity. The PAIs of S. enterica are particularly interesting in terms of the evolution of virulence and adaptation. There are five PAIs known in this species. The distribution of the known PAIs in different subspecies of S. enterica suggests that the acquisition of PAIs by

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horizontal transfer was the essential step in becoming a pathogen22. SPI-1 contains genes required for the invasion of epithelial cells23 and is present in all subspecies but absent in E. coli24. This suggests that, after acquisition of SPI-1, S. enterica became capable of invading intestinal epithelium and multiplying within gut-associated lymphoid tissue and effectively established a niche different from the niche in the gut lumen occupied by its close relative E. coli. The acquisition of additional pathogenicity islands, after the divergence of subspecies V, probably allowed S. enterica to spread from the intestinal tissue into the bloodstream and multiply within macrophages, thus expanding the niche to intracellular locations. SPI-2 is required for systemic infections and is present in all subspecies apart from subspecies V (Refs 24,25). SPI-3 (Ref. 26) and possibly SPI-4 (Ref. 27) are required for survival within macrophages, whereas SPI-5 (Ref. 28) carries genes required for enteropathogenicity. SPI-3 is variably present in all subspecies and must have a complex history26, but we have no distribution information for SPI-4 or SPI-5. O-antigens The variation within a species can be looked at either as polymorphism or as a result of lateral transfer between clones. In the case of S. enterica, as a highly clonal species, O-antigen variation can be viewed as a stable polymorphism within a species, maintained by niche selection. Antigenic diversity is common and is probably a widely used mechanism of evading the immune system. The O-antigen is an extremely variable and antigenic surface polysaccharide, with 46 known forms in S. enterica. Twenty five forms are present in at least three subspecies and only five rare forms are limited to a single subspecies. The distribution of O-antigens provides evidence of extensive movement of the O-antigen-encoding genes in the seven subspecies29. Evidence for such transfer can be seen in the gnd gene, which is adjacent to the O-antigen locus. As for other genes, subspecies-specific forms of gnd can be recognized, but frequently a part of the gene is cotransferred with the O-antigen genes, carrying its subspecies signature30. Gene loss in bacterial evolution Genes that for some reason become deleterious or are no longer beneficial to a clone will be lost by the accumulation of mutations and deletions; this presumably balances gains by lateral transfer. Lysine decarboxylase (LDC) is widely present in E. coli but is usually absent in Shigella and enteroinvasive (EIEC) strains of E. coli. When the LCD-encoding gene was cloned into a Shigella strain there was a significant reduction in virulence (fitness)31. LCD catalyses a reaction to produce cadaverine, which inhibits the enterotoxin and hence attenuates virulence, and the region containing the LDC-encoding gene was found to be deleted in Shigella and EIEC clones. This is an excellent example of a gene becoming deleterious in a new niche, thereby generating strong selection pressure for gene loss. The selection pressure for loss of activity

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Box 1. Definition of species in higher organisms and bacteria The species concept is generally applied to bacteria, yet it has long been recognized that there are difficulties. In sexually reproducing higher organisms, species are, in general, readily recognized in any well-worked groups. There are many definitions of a species but that of Mayr a ‘groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups’ is widely accepted and appropriate for our purpose. The important aspects of such species that differentiate them from bacteria are: (1) that each individual acquires its genome almost equally from two parents, with the corollary that genetic polymorphisms are reassorted each generation such that one can treat each individual as having a near-random sample, at least for the local gene pool; and (2) that as species diverge hybrids become less viable, as they will at best combine an assortment of all characters that differentiate the two species, and so be adapted to neither niche, and at worst will suffer genetic incompatibility, leading to imperfect development. Bacteria propagate by binary fission; gene exchange is therefore relatively rare, occuring by transfer of one or few genes from one individual to another. This enables extreme diversity to develop by niche adaptation of clonesb and also enables genes to transfer between species. Perhaps surprisingly, despite occasional suggestions that the latter aspect renders the traditional species concept inapplicable, bacterial species are also readily recognized in well-studied cases. When housekeeping genes (part of the core set of genes discussed in the text) are sequenced, each species has a well-defined cluster of related sequences, with only occasional evidence of interspecific transfer. It seems clear that, despite the opportunity for gene transfer between species, many and perhaps most genes in a species can be recognized by their sequence as being that particular species. That is not to say that at some time in the past the gene did not arrive from outside. Thus, although there are differences between species concept in bacteria and sexually reproducing higher organisms, in both cases species can be recognized by phenotypes and by reference to a gene pool. References a Mayr, E. (1940) Speciation phenomena in birds. Am. Nat. 74, 249–278 b Reeves, P.R. (1992) Variation in O antigens, niche specific selection and bacterial populations. FEMS Microbiol. Lett. 100, 509–516

will be high, as these strains live mainly within gut epithelial cells, and the expression of LDC effectively removes its ability to live in this niche. Another Shigella example involves Shigella sonnei, which has an O-antigen identical to that of serotype 17 of Plesiomonas shigelloides. The S. sonnei O-antigen gene cluster is on a plasmid and appears to have been acquired by lateral transfer from P. shigelloides32. The original chromosomal O-antigen genes have been inactivated by a deletion that fused the O-antigen cluster and the upstream colanic acid cluster33. One can speculate that the new O-antigen offered a selective advantage in adapting to a new environment, whereas the old O-antigen might have exerted a severe burden on the cell, leading to its eventual inactivation by deletion. There are other examples of genes in O-antigen gene clusters that have clearly undergone substantial mutational changes after becoming redundant, such as wbaE in the

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Questions for future research • How can strain-specific DNA be collected systematically? Can novel methods be developed? • What is the best way to define a bacterial species (i.e. a good species concept) and the best way to define strains for coverage of a species genome? • How many genes are present in the core set of genes and how extensive are auxiliary genes in a species? • What is the extent of gene polymorphism present within a species? • How many decaying genes are present in a genome?

O-antigen IIA gene cluster of Yersinia pseudotuberculosis34 and wzy genes in the B and D1 antigen gene clusters of S. enterica35. The wzy gene in group B strains, for example, has many regions deleted, which total 73% of the gene, but there are sufficient regions remaining to identify it as a remnant of a wzy gene that is very similar to the functional wzy gene of D3. This loss of DNA will presumably continue until there is no remnant of the gene left. We suggest that this whole process be called gene decay. The species genome concept Even the limited data available on pairwise comparisons show that up to 20% of the DNA in one strain can be absent in another. The total amount of such DNA is, at present, unknown, but it is part of the genome of that particular species. Clearly, comparison with different strains will add to the DNA in this class. If we accept the concept of the species genome, comprising all genes found in the species, then the genes of any individual will include two components: the core set of genes and the auxiliary genes. Genes found in most individuals, which we can call the core set of genes for that species, are the genes that determine those properties characteristic of all members of the species. Additionally, each strain will have some auxiliary genes, which determine properties found in some but not all members of the species. The distinction will not be absolute but we believe it provides a useful framework. Suitable boundaries might become obvious as our knowledge of intraspecies variation grows, but we suggest as a starting point that genes found in 95% or more of isolates form the core set and genes found in 1–95% of isolates form part of the auxiliary set of genes; those present in ,1% are provisionally treated as foreign genes or genes being lost from the species. The cutoffs are arbitrary, and with better knowledge one would define the lower cutoff in terms of genes that persisted in the species for long enough to show that their presence was maintained by selection. The tools of subtractive hybridization and microarray technology will enable these components of the genome to be identified. For the core genome, one can use microarrays to determine how many genes of a reference strain are present in 95% or more of a set of isolates. The auxiliary set of genes comprises those in the species genome but not in the core set of genes. To determine the latter is not straightforward but, for

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example, if a number of strains are compared with a reference strain by subtractive hybridization, for each strain one can collect the DNA present in it but not the reference strain. From comparison of the individual pools of subtracted DNA one can determine how much of it is present in one, two, three and so on, of the strains, and then determine statistically how many isolates need to be studied to achieve a reasonable approximation of the species genome. The application of these methods will give us an overview of the genome of a bacterial species, and of course provide the DNA to enable characterization of the two species genome components. The core set of genes as we define it is not equivalent to the ‘minimal’ set of genes described by Hutchison et al.36 Our core set includes all genes generally found in any individual of the species and will include genes not required for growth in the specific experimental growth conditions used to define the ‘minimal’ gene set. Conclusions and prospects Bacterial taxa at all levels seem to be defined in large part by the presence or absence of genes relative to other taxa. This is apparent from the description of, for example, species and genera, which are often defined by a combination of biochemical functions, and confirmed by the genome sequences now available. The phenomenon also applies to clones of a species, as they also vary in the genes present. It is important that in the new genome era this intraspecies variation be recognized by exploring the gene content of the whole species, the ‘species genome’; only rarely will the genome of a single isolate represent the genetic potential of a bacterial species. DNA chip technology provides a powerful means to scan for the presence or absence of the genes already sequenced12. However, the major task will be to find the genes not present in the sequenced isolates. A systematic approach is needed, as it is this which will provide the ultimate gateway to an understanding of the evolution and biology of the species. We have reviewed the currently used methods. Subtractive hybridization remains the method of choice for extending the species genome as discussed above for an avian pathogen of E. coli15. Microarray technology seems best suited for studying the distribution of known genes, which, until now has been done mainly by subtractive hybridization for S. enterica11 as discussed, and more recently for Neisseria species37. It is also interesting to speculate on why horizontal transfer is so common in bacteria. It probably relates to the very nature of single-celled organisms. A newly transferred gene is of immediate benefit to the whole organism and, furthermore, will be easily passed on to the next generation. In a multicellular organism it could benefit only one cell and its descendants within the organism, and in most animals only if it can be passed on in the germline to the next generation. Also, in animals and plants the major adaptations involve changes in developmental processes, which are more likely to require fine-tuning than added

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functions. Even desirable new functions will generally only be useful if expression is regulated to occur at the appropriate time and place, whereas bacteria can often gain benefit without regulation, which can be added later. In the circumstances, it is hardly surprising that horizontal gene transfer is so important in prokaryote evolution and indeed seems to go back to the very origins of the three major domains38. Acknowledgements Research in the authors’ laboratory is supported by the Australian Research Council and National Health and Medical Research Council. We thank the reviewers for helpful suggestions. References 1 Blattner, F.R. et al. (1997) The complete genome sequence of Escherichia coli K-12. Science 277, 1453–1474 2 Tomb, J.F. et al. (1997) The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539–547 3 Alm, R.A. et al. (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397, 176–180 4 Tettelin, H. et al. (2000) Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287, 1809–1815 5 Parkhill, J. et al. (2000) Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404, 502–506 6 Liu, S-L. and Sanderson, K.E. (1996) Highly plastic chromosomal organization in Salmonella typhi. Proc. Natl. Acad. Sci. U. S. A. 93, 10303–10308 7 Leblond, P. and Decaris, B. (1998) Chromosome geometry and intraspecific genetic polymorphism in Gram-positive bacteria revealed by pulsed-field gel electrophoresis. Electrophoresis 19, 582–588 8 Rode, C.K. et al. (1995) New tools for integrated genetic and physical analysis of the Escherichia coli chromosome. Gene 166, 1–9 9 Straus, D. and Ausubel, F.M. (1990) Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc. Natl. Acad. Sci. U. S. A. 87, 1889–1893 10 Akopyants, N.S. et al. (1998) PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc. Natl. Acad. Sci. U. S. A. 95, 13108–13113 11 Lan, R. and Reeves, P.R. (1996) Gene transfer is a major factor in bacterial evolution. Mol. Biol. Evol. 13, 47–55 12 Behr, M.A. et al. (1999) Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520–1523 13 Young, D.B. and Robertson, B.D. (1999) TB vaccines: global solutions for global problems. Science 284, 1479–1480 14 Selander, R.K. et al. (1996) Evolutionary genetics of Salmonella enterica. In Escherichia coli and Salmonella: Cellular and Molecular Biology (2nd edn) (Vol. 2) (Neidhardt, F.C. et al., eds), pp. 2691–2707, ASM Press 15 Brown, P. and Curtiss, R. (1996) Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain. Proc. Natl. Acad. Sci. U. S. A. 93, 11149–11154 16 Rode, C.K. et al. (1999) Type-specific contribution to chromosome size differences in Escherichia coli. Infect. Immun. 67, 230–236 17 Hacker, J. et al. (1997) Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol. Microbiol. 23, 1089–1097 18 Perna, N.T. et al. (1998) Comparative genomics of E. coli K–12, O157:H7 and related enterobacterial pathogens. In ASM Conference on Small Genomes, p. 7, ASM Press 19 Bergthorsson, U. and Ochman, H. (1998) Distribution of chromosome length variation in natural isolates of Escherichia coli. Mol. Biol. Evol. 15, 6–16

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20 Boyd, E.F. and Hartl, D.L. (1998) Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution. J. Bacteriol. 180, 1159–1165 21 Reeves, P.R. (1992) Variation in O antigens, niche-specific selection and bacterial populations. FEMS Microbiol. Lett. 100, 509–516 22 Baumler, A.J. (1997) The record of horizontal gene transfer in Salmonella. Trends Microbiol. 5, 318–322 23 Mills, D.M. et al. (1995) A 40-kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome. Mol. Microbiol. 15, 749–759 24 Ochman, H. and Groisman, E.A. (1996) Distribution of pathogenicity islands in Salmonella spp. Infect. Immun. 64, 5410–5412 25 Hensel, M. et al. (1997) Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12. J. Bacteriol. 179, 1105–1111 26 Blanc-Potard, A.B. et al. (1999) The SPI-3 pathogenicity island of Salmonella enterica. J. Bacteriol. 181, 998–1004 27 Wong, K.K. et al. (1998) Identification and sequence analysis of a 27-kilobase chromosomal fragment containing a Salmonella pathogenicity island located at 92 minutes on the chromosome map of Salmonella enterica serovar Typhimurium LT2. Infect. Immun. 66, 3365–3371 28 Wood, M.W. et al. (1998) Identification of a pathogenicity island required for Salmonella enteropathogenicity. Mol. Microbiol. 29, 883–891 29 Reeves, P.R. (1995) Role of O-antigen variation in the immune response. Trends Microbiol. 3, 381–386 30 Thampapillai, G. et al. (1994) Molecular evolution in the gnd locus of Salmonella enterica. Mol. Biol. Evol. 11, 813–828 31 Maurelli, A.T. et al. (1998) ‘Black holes’ and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 95, 3943–3948 32 Houng, H.H. et al. (1997) The roles of IS630 sequence in the expression of the form I antigen of Shigella sonnei: molecular and evolutionary aspects. In Ecology of Pathogenic Bacteria: Molecular and Evolutionary Aspects (van der Zeijst, B.A.M. et al., eds), pp. 282–283, Elsevier Science 33 Lai, V. et al. (1998) Escherichia coli clone Sonnei (Shigella sonnei) had a chromosomal O-antigen gene cluster prior to gaining its current plasmid-borne O-antigen genes. J. Bacteriol. 180, 2983–2986 34 Hobbs, M. and Reeves, P.R. (1995) Genetic organisation and evolution of Yersinia pseudotuberculosis 3,6-dideoxyhexose biosynthetic genes. Biochim. Biophys. Acta 1245, 273–277 35 Curd, H. et al. (1998) Relationships among the O-antigen gene clusters of Salmonella enterica groups B, D1, D2 and D3. J. Bacteriol. 180, 1002–1007 36 Hutchison, C.A. et al. (1999) Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286, 2165–2169 37 Perrin, A. et al. (1999) Identification of regions of the chromosome of Neisseria meningitidis and Neisseria gonorrhoeae which are specific to the pathogenic Neisseria species. Infect. Immun. 67, 6119–6129 38 Nelson, K.E. et al. (1999) Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399, 323–329 39 Beltran, P. et al. (1991) Reference collection of strains of the Salmonella typhimurium complex from natural populations. J. Gen. Microbiol. 137, 601–606 40 Crosa, J.H. et al. (1973) Molecular relationships among the Salmonellae. J. Bacteriol. 115, 307–315 41 Wong, R.M-Y. et al. (1999) Sample sequencing of a Salmonella typhimurium LT2 lambda library: comparison to the Escherichia coli K12 genome. FEMS Microbiol. Lett. 173, 411–423

VOL. 8

NO. 9

SEPTEMBER 2000