In common with other reported Shigella strains, the genome of Sf8401 contains a circular chromosome. Since the complete sequences of pWR501 and pWR100 are known 1516, we present here only the chromosome sequence, which is 4,574,284 bp in length with an average GC content of 50.92% and encodes 97 tRNA genes (Table 1, Additional file 1). Its size is a little smaller than that of Sf301, mainly because SHI-1 is absent (see below). Sf8401 has 7 rRNA operons, with 4 copies in one replichore and 3 in the other, while Sf301 has 5 copies in one replichore and 2 in the other. These result from one intra-replichore inversion near the replication origin (Fig. 1). Comparison of Sf8401 with Sf301 reveals that more than 97% of the genome sequence is shared between the two strains. The architecture of the Sf8401 genome is similar to Sf301 but the overall colinearity is broken by 7 translocations and inversions involving DNA segments >5 kb (Fig. 1). Among the 4194 proteomes of MG1655, 3098 proteins (74%) are shared by Sf8401 and Sf301 and may be regarded as the "backbone" of S. flexneri (Additional file 2), while 114 are pseudogenes in both genomes. Two hundred and sixty-six of the 1096 non-backbone proteins were predicted to be metabolism-related by the Clusters of Orthologous Groups (COGs) database. This reflects the evolution of S. flexneri from a non-pathogenic E. coli ancestor to a facultative intracellular pathogen. Among the 393 Sf8401 specific proteins (compared with MG1655) only 28 are specific to all known Shigella genomes; most of them are bacteriophage-related proteins or hypothetical proteins (Additional file 3).

IS elements are ubiquitous in bacterial genomes and important factors in evolution 17. The IS insertion can cause gene inactivation, activate cryptic genes or alter the expression of adjacent genes 18. The numbers and species of IS elements in the Sf8401 genome are similar to previously-determined Shigella genomes. In total, the IS elements encode 485 ORFs and make up 6.37% (291.3 kb) of the chromosome, and the predominant species is IS1. A distinct difference is that Sf301 has 13 copies of iso-IS10R, while Sf8401 and other sequenced Shigella chromosomes have not. This might be used as a marker for epidemiological studies. Furthermore, IS elements are capable of causing various genetic rearrangements such as deletions, inversions and translocations 1920. Unlike the inversion reported in Yersinia pestis 21, S. enterica serovar Typhi 22 and E. coli K-12 strain W3110 1323, which are associated with rRNA homologies, inversions in Sf8401 are probably mediated by IS elements. Inversions 1 and 5 (Fig. 1) occur around SHI-O and SHI-2, which are mediated by IS629 and IS1. Besides these major inversions, there are other four inverted regions, inversions 2, 3, 4 and 6 (Fig. 1), which are probably mediated by IS4, ISSfl2, IS600 and IS1 respectively.

LPS is an important virulence factor in Shigella 24. Since the immune response to Shigella spp. is O-antigen specific, an immune response to a specific O antigen does not protect against infection with other serotypes. Therefore, the capacity to alter serotypes may be advantageous for Shigella spp. in the infectious process 25. In Sf8401, the serotype conversion region, i.e. SHI-O 26, is located around 300 kb (Fig. 1). In SHI-O the 3 genes termed gtrA, gtrB and gtrV (a serotype-specific glycosyltransferase) are putatively involved in glucosylation reactions 27. The mean GC content of gtrA and gtrB is 42.71%, and the GC content of gtrV is 32.99%. All three are lower than the whole genome GC content (50.92%). Compared with Sf301, gtrA and gtrB are highly conserved and interchangeable among serotypes, whereas gtr (serotype) appears to be unique to each bacteriophage 2829, which indicates that these genes have been acquired from lysogeny caused by bacteriophages.

The SHI-O component differs between Sf301 and Sf8401 (Fig. 2a). Although type II antigen is encoded by an inducible bacteriophage, SfII 30, little genomic sequence of bacteriophage SfII remains in the SHI-O of 2457T and Sf301 except the key genes related to antigenic variation 13. However, the Sf8401 SHI-O includes not only the type V antigen gene, which comes from bacteriophage SfV, but also another 15 kb segment of bacteriophage SfV 2731.

Temperate bacteriophages of S. flexneri play an important role in serotype conversion. Bacteriophages SfV and SfII encode the factors involved in glucosylation of the O-antigen, and lysogenization results in conversion of serotype Y strains to serotypes 5 and 2 2730. The defining point for evolution of S. flexneri 2 and 5 from the S. flexneri ancestor is probably the acquisition of the precursor of the current-day O-antigen modification genes. It can be supposed that the S. flexneri ancestor acquired diverse O-antigen modification genes along with related bacteriophage sequences when the bacteriophages were integrated into the bacterial genomes. The genome rearrangements caused by IS elements in far-flung evolutional processes have interrupted these bacteriophage sequences in many Shigella genomes. However, there are ~15 kb of bacteriophage SfV sequence remnants in Sf8401, though far less bacteriophage SfII sequence has remained in Sf301. This might be because the Sf8401 genome is less dynamic than that of Sf301, or alternatively, it may suggest that Sf8401 arose later than Sf301. This hypothesis requires further investigation.

Shigella pathogenicity island SHI-1 encodes three characterized proteins: SigA, Pic and the enterotoxin ShET1. Functional analysis shows that SigA is cytopathic for HEp-2 cells and at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops 6. The Pic protein, a serine protease, is involved in mucinase activity, serum resistance and hemagglutination 9. Furthermore, ShET1 encoded by set1A and set1B could increase fluid accumulation in the rabbit loop model.

SHI-1 is located directly downstream of the pheV tRNA gene and includes an imperfect repeat of the 3'-end 22 bp of the pheV gene at the right boundary in Sf301 (Fig. 2b). Studies of SHI-1 distribution (she PAI) show that intact SHI-1 is present in all tested serotype 2a strains of S. flexneri but absent from some S. flexneri serotype strains such as 1a, 1b, 3b, 4 and 5 32. It is therefore not surprising that SHI-1 is wholly absent from Sf8401. However, the homolog of SF3004, a hypothetical protein located downstream of SHI-1 in Sf301, is situated next to the pheV gene in Sf8401 (SFV3027). Further investigation shows that the homolog of SF3004 is only present adjacent to SHI-1 in the Sf301 and 2457T genomes and the S. flexneri 2a she pathogenicity island 6. So the presence of SFV3027 suggests that Sf8401 might have contained SHI-1 during its evolutionary history but lost it for unknown reasons. Sakellaris et al. 33 demonstrated that the spontaneous and precise excision of SHI-1 can occur via recombination between a 22 bp sequence at the 3' terminus of pheV and an imperfect direct repeat at the pheV-distal boundary of the SHI-1. Thus, we have probably witnessed the case in Sf8401. Undoubtedly, the mechanism by which SHI-1 is stabilized in S. flexneri 2a strains will be an interesting focus for further studies.

SHI-2 encodes the synthesis and transport of aerobactin, a hydroxamate siderophore associated with increased virulence in enteric bacteria 1034, and is located downstream of the selC tRNA gene in Sf8401. The conservation of the component, organization and integration site of SHI-2 in Sf301 and Sf8401 implies that it was acquired by the S. flexneri ancestor before the serotypes diverged. However, the iut/iuc operon is located on the leading strand and on the counter-clockwise site of the replication origin in Sf301, but on the lagging strand and on the clockwise site in Sf8401 (Fig. 2c). Our previous genome studies revealed that SHI-2 in S. sonnei Ss046 was unlinked with the selC gene by an inversion, and SHI-3 of S. boydii Sb227 that carries a similar iut/iuc operon is linked with the pheU tRNA locus 14. So in view of this information, the observation that the iut/iuc operon can inserted into a variety of different loci suggests that it is highly mobile and may be acquired by additional human or animal pathogens 10.

In Sf8401, SHI-2 is involved in inversion 5 that spans oriC (Fig. 1). All sequenced Shigella chromosomes have inversions at oriC and terC, which is suggested to be a common evolutionary feature of bacterial genomes 35. In contrast to Sf301, inversion 5 is found from 3597 to 4100 kb in Sf8401, which appears to be mediated by the boundary IS1 copies in Sf8401 (Fig. 2c). However, the ~22 kb center region of inversion 5 that covers oriC retains colinearity with that of Sf301 (Fig. 2c; the block colored green). Since this region is sandwiched between two copies of IS4, it implies that two or more inversions have occurred: an inversion was followed by a re-inversion to restore colinearity.

Inversions can produce an "X" shape, which changes the positions of these sequences from their natural locations 35, and the distance from the iut/iuc operon to oriC in Sf8401 is different from that in Sf301 due to inversion 5 and the internal re-inversion mentioned above. Owing to bidirectional replication, there are extra copies of genes close to oriC, resulting in increased gene expression 36, and since dosage differences may cause the strengths of promoters to be evolutionarily optimized for their specific positions, cells in which genes are a different distance from oriC are at a selective disadvantage 37. Hence, whether the change in position of the iut/iuc operon in Sf8401 has any influence on expression needs to be determined.

The E. coli bet gene cluster (betABIT) contributes to the pathway for glycine-betaine biosynthesis from choline 38 and is located close to SHI-O in Sf8401 but absent from Sf301 and 2457T. Among eubacteria-compatible solutes, the most widespread is glycine-betaine, and this is the only osmoprotectant synthesized by E. coli 3940. The choline-glycine-betaine pathway confers a high level of osmotic tolerance on E. coli 38. Whether this system offers an advantage to S. flexneri 5b for environmental survival over S. flexneri 2a or plays a role during infection requires further investigation. However, the inversion around the SHI-O may contribute to the deletion of the bet operon from S. flexneri 2a (Fig. 2a).

There are some differences in metabolic and physiological pathways between Sf8401 and Sf301. In both strains, some key metabolic pathways were inactivated by the creation of independent pseudogenes. For instance, the loss of ability to utilize D-sorbitol is due to the inactive states of srlE and srlA in Sf8401 and Sf301, respectively. It seems that Shigella strains have a general tendency to lose some pathways and functions, and this tendency has given rise to convergent evolution 41. The microorganisms need to adapt to new niches by adopting a strictly pathogenic life-style. There must be some genes that provide little overall selective benefit in a new situation. These genes will be eliminated through mutational bias favoring deletions for the lack of selective force to maintain them 42. Such functions, no longer active in one serotype but expressed in another, may lead to a better understanding of the diversity of the two serotypes and the evolution of S. flexneri.

Shigella flexneri 5b, strain 8401, was isolated and sequenced from epidemic in China, kindly provided by the National Institute for Communicable Disease Control and Prevention, Chinese Centre for Disease Control and Prevention.

The whole genome sequence shotgun libraries for Sf8401 were established as described previously 1214, and ABI3730 automated sequencers were used for sequence collection. 48,000 clones were sequenced from both ends, giving rise to 8 times coverage of the genome. Sequences were assembled initially using the phred/phrap program with the Q20 criteria 43 when the sequence coverage was ~4-fold over the estimated size of the genome. The Consed program was used for sequence finishing 43. Gaps among contigs were closed either by primer walking on selected clones, which were identified by analysis on the forward and the reversed links between contigs using a perl/Tk script, or by sequencing the DNA amplicons generated by polymerase chain reaction (PCR). Glimmer 2.0, a program that searches for protein coding regions, was used to identify those ORFs possessing more than 30 consecutive codons 44. Overlapping and closely clustered ORFs were manually inspected. Predicted polypeptide sequences were used to search the non-redundant protein database with BLASTP, and the clusters of orthologous groups of proteins (COGs) database was used to identify families to which predicted proteins are related 45. Those cases in which a stop condon or deletion has resulted in an encoded protein that is less than 80% of the length of its counterpart in K-12 genome and those cases in which a frameshift of insertion has altered more than 20% of the amino acid sequence were classed as pseudogenes. Mobile elements and repetitive sequences were identified using the IS FINDER database 46. GenomeComp was used for genomic comparison with default parameters 47. The comparison figures used in Figure 1 and 2 were exported from GenomeComp with a 1500 bp filter setting along with the scale setting of 2000 for chromosomes. The KEGG database was used for the metabolic pathways analysis 48.

Complete genome sequence of Sf8401 has been deposited in the Genbank. The accession number for chromosome is: CP000266.