© 2005 Nature Publishing Group

100 | FEBRUARY 2005 | VOLUME 3 www.nature.com/reviews/micro

This month we discuss the insights thatgenomics can provide into the evolution andspeciation of virulent human pathogens, usingtwo pertinent examples from the Yersinia andBurkholderia genera.

Yersinia pestis is renowned for being the agent ofthe Black Death and is one of the most potentbacterial pathogens that can infect man (FIG. 1).Fully virulent strains of this pathogen havebeen sequenced in the past, but the publicationof the whole genome sequence of an avirulentisolate, Y. pestis strain 91001, by Song and col-leagues1 provides an intriguing comparison.Biochemically, Y. pestis strain 91001 is a mem-ber of biovar Mediaevalis, in common with thepreviously sequenced KIM 10+ strain (KIM)2.Taken with Y. pestis strain CO92 (biovarOrientalis)3, the genomes of three Y. pestisstrains have now been sequenced. Y. pestis strain91001 was isolated from Brandt’s vole

(Microtus brandti), which is a small rodentspecies found in China. This strain is lethal inmice — the LD

50in mice is just 23 cells — but

infection of rabbits, or even human volunteers,with challenge doses in excess of 107 cells failedto cause plague.

Song et al. identify differences between thehuman virulent and avirulent strains and goon to discuss whether Y. pestis strain 91001could indeed be a member of a new Y. pestisbiovar. Y. pestis strain 91001 contains the threeessential virulence plasmids pCD1, pMT1 andpPCP1, which the authors note containsequence modifications in genes such as yopMand pla that might explain the avirulent phe-notype of this strain in humans and rabbits.Whole-genome comparisons of Y. pestis strain91001 with Y. pestis strains CO92 and KIMrevealed considerable intrachromosomalrearrangements, largely mediated by insertionsequence (IS) elements, and the presence of a

large number of pseudogenes (141), some ofwhich are intact in the virulent strains.

The chromosome of Y. pestis strain 91001 isslightly smaller than the previous two Y. pestisgenomes (4.6 Mb), but all three genomes share90% of their coding sequences, with between3–56 unique genes in pairwise comparisons.Y. pestis strain 91001 also has several largeunique regions including DFR4, which is onlyfound in the Y. pestis strains isolated fromMicrotus species and in Yersinia pseudotubercu-losis. Moreover Song et al. note that eventhough Y. pestis strains KIM and 91001 belongto the same biovar, there is more sequence sim-ilarity between the KIM and CO92 strains thanbetween KIM and 91001, which is further evi-dence that strain 91001 belongs to a different,perhaps earlier, lineage of Y. pestis. Other dis-tinguishing features include a mutation in thenapA gene that differs from that found in othermembers of biovar Mediaevalis as well as several biochemical traits, which indicate thatstrain 91001 represents a novel clade, whichthe authors name biovar Microtus.

Y. pseudotuberculosis is a pathogen thatcauses diarrhoea, has a broad host range and iswidely distributed in the environment. As theclosest relative of Y. pestis — ~1, 500–20, 000years since the divergence of these species —the genome sequence of Y. pseudotuberculosisstrain IP32953 (REF. 4) is a useful aid to explain-ing how a potent pathogen seemingly evolvedfrom a relatively benign species (reviewed inREF. 5). Chain et al. present a detailed compari-son of the Y. pseudotuberculosis and Y. pestischromosomes and plasmids. The genome ofY. pseudotuberculosis strain IP32953 comprises a4.7-Mb chromosome, the well-characterizedYersinia general virulence plasmid pYV (68 kb)and a novel cryptic plasmid pYptb32953 (27 kb).

Brothers in armsNicholas Thomson, Matthew Holden and Julian Parkhill

GENOME WATCH

NEWS & ANALYSIS

Countries that reported plague, 1970–1998

Regions where plague occurs in animals

Justinian plague(5th–7th century)

32

1

Black Death(13th–15th century)

Modern plague(1870s onwards)

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Figure 1 | World distribution of plague, 1998. Reproduced with permission from REF. 5 © (2004) Nature Reviews Microbiology,Macmillan Magazines Ltd.

© 2005 Nature Publishing Group

N E W S & A N A L Y S I S

NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | FEBRUARY 2005 | 101

Of the predicted 3,974 genes on the chromo-some, 75% have highly conserved (>97%amino acid identity) orthologues in Y. pestis(CO92 and KIM10). Like Y. pestis strains KIM,CO92 and 91001, pseudogenes are also presentin the Y. pseudotuberculosis strain IP32953genome — 62 have been identified, of which 43are present in all the Y. pestis strains. With thebenefit of a three-way comparison between Y. pseudotuberculosis strain IP32953 and Y. pestisstrains KIM and CO92, Chain et al. identified149 extra pseudogenes in Y. pestis, which, iffunctional, would encode surface-exposed,regulatory and virulence proteins, possiblysignificant in the pathogenesis of this bacterium.

Chain et al. used PCR to screen the Y. pseudotuberculosis strain IP32953-specificgenes against a selection of Y. pestis and Y. pseudotuberculosis isolates and found 11genes that are unique to Y. pseudotuberculosisspecies. This gene-set included genes that areinvolved in general metabolism, which mighthave been lost from Y. pestis, and glucan (osmo-protectant) biosynthetic genes. Using the sameapproach 32 Y. pestis-unique genes were foundthat could represent important targets forfuture study.

IS element expansion was one of the moststriking observations made from the analysisof Yersinia genomes. Y. pseudotuberculosis con-tains 20 IS elements compared with 117, 138and 109 in Y. pestis strains KIM, CO92 and91001, respectively. It is evident that IS elementshave driven genome fluidity in the yersiniae,which is responsible for some of the Y. pseudo-tuberculosis-unique regions, owing to IS-medi-ated deletion in Y. pestis, and the considerableamount of rearrangements that have occurredsince Y. pseudotuberculosis and Y. pestisdiverged, and more recently after the split of theY. pestis biovars. Chain et al. were not able toinclude Y. pestis strain 91001 in their study, so itis with great anticipation that we await a newcomparison of the non-plague and plagueyersiniae genomes.

Further insight into the evolution and

speciation of virulent human pathogens hasbeen provided by the genomes of Burkholderiapseudomallei 6 and Burkholderia mallei 7 (FIG. 2).Both organisms are category B biothreatagents and cause clinically and pathologicallysimilar illnesses. However, the natural reser-voirs of the two organisms are markedly differ-ent; B. pseudomallei is a soil saprophyte and B. mallei survives only in equine hosts. Despitethe differences in their ecology, the two organ-isms are closely related. Phylogenetic analysisusing multilocus sequence typing has indicatedthat B. mallei is a clone of B. pseudomallei8,thereby making these two genomes ideal for acomparative analysis to unravel the short-termevolutionary events that have shaped theirniche adaptation.

B. pseudomallei is the causative agent ofmelioidosis, which is a complex disease thatcan involve many organs and tissues in thebody and has high fatality rates. The disease isendemic in south-east Asia and northernAustralia, and occurs after contamination ofskin lesions or by inhalation after contact withwater or soil. A striking feature of the disease isthe incubation period, which can extend fromtwo days to several years — the longestrecorded period being 26 years in a VietnamWar veteran.

The versatility of B. pseudomallei isreflected in the 7.25-Mb bipartite genome,which encodes a large metabolic repertoireand many putative survival and virulencefunctions. The diversity and apparent redun-dancy of some of these functions indicatesniche-specific functionality. For example,there are three complete type III secretionsystems in B. pseudomallei. One of the sys-tems is similar in sequence and organizationto the Inv/Mxi-Spa type III systems of theanimal and human pathogens Salmonellaand Shigella, and the other two are similar tothe type III (hrp) systems of the plantpathogens Ralstonia solanacearum andXanthomonas species. The presence of threedistinct systems raises interesting questionsabout diversity of the cell–cell interactions ofwhich B. pseudomallei is capable as part of itsversatile existence.

An insight into some of the short-term evo-lution events that have shaped the B. pseudo-mallei genome was gained by the identificationof sixteen genomic islands (which comprise6.1% of the genome) that encode a diversearray of functions. The differential distributionof these horizontally transferred regions in thewider population was confirmed by probingphylogenetically diverse clinical and environ-mental isolates of B. pseudomallei fromThailand using multiplex PCR.

Although acquisition of DNA seems to

have been intrinsic to the evolution ofB. pseudomallei, quite the opposite is true of B. mallei. B. mallei is the aetiological agentof glanders, which is a disease of horses,mules and donkeys and which can be trans-mitted to humans to cause a life-threateningillness. Unlike B. pseudomallei, it is not able tosurvive in the soil and is host-restricted. Thebipartite genome of B. mallei is 1.41 Mbsmaller than the B. pseudomallei genome.Pairwise comparisons revealed that a largeproportion of the ‘extra DNA’ in B. pseudo-mallei is actually DNA that has been deletedfrom the B. mallei genome since it divergedfrom a common ancestor. Genomic islandsalso seem to be absent from B. mallei, withthe exception of a single region that seems tocontain the remnants of a genomic island.

In common with the Y. pestis genome, theB. mallei genome contains an increased num-ber of IS elements, IS-mediated intrachro-mosomal rearrangements, and detectablepseudogenes in comparison with its moreversatile relation. For both of these organ-isms the recent evolutionary transition fromversatile to specialized pathogens has resultedin marked genomic changes that belie theirrelationships to their brothers in arms.

Nicholas Thomson, Matthew Holden and JulianParkhill are at the Sanger Institute, WellcomeTrust Genome Campus, Hinxton, CambridgeCB10 1SA, UK.e-mail: microbes@sanger.ac.ukdoi:10.1038/nrmicro1092Published online 10th January 2005

1. Song, Y. et al. Complete genome sequence of Yersiniapestis strain 91001, an isolate avirulent to humans. DNARes. 11, 179–197 (2004).

2. Deng, W. et al. Genome sequence of Yersinia pestis KIM. J. Bacteriol. 184, 4601–4611 (2002).

3. Parkhill, J. et al. Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413, 523–527 (2001).

4. Chain, P. S. et al. Insights into the evolution of Yersinia pestisthrough whole-genome comparison with Yersiniapseudotuberculosis. Proc. Natl Acad. Sci. USA 101,13826–13831 (2004).

5. Wren, B. W. The yersiniae — a model genus to study therapid evolution of bacterial pathogens. Nature Rev.Microbiol. 1, 55–64 (2003).

6. Holden, M. T. G. et al. Genomic plasticity of the causativeagent of melioidosis, Burkholderia pseudomallei. Proc. NatlAcad. Sci. USA 101, 14244–14255 (2004).

7. Nierman, W. C. et al. Structural flexibility in the Burkholderiamallei genome. Proc. Natl Acad. Sci. USA 101, 14246–14251(2004).

8. Godoy, D. et al. Multilocus sequence typing and evolutionaryrelationships among the causative agents of melioidosis andglanders, Burkholderia pseudomallei and Burkholderiamallei. J. Clin. Microbiol. 41, 2068-2079 (2003).

Online links

DATABASESThe following terms in this article are linked online to:Entrez: http://www.ncbi.nlm.nih.gov/Entrez/Burkholderia mallei | Burkholderia pseudomallei | Y. pestis strainCO92 | Y. pestis strain 91001 | Y. pestis KIM 10 strain | Y. pseudotuberculosis strain IP32953

FURTHER INFORMATIONThe Pathogen Sequencing Unit:http://www.sanger.ac.uk/Projects/Pathogens/Access to this links box is available online.

Figure 2 | Actin tail formation by Burkholderia pseudomallei. Macrophage filamentous actin was stained with FITC-conjugatedphalloidin (green) and bacteria were stained with an antibodyconjugate that detects B. pseudomallei LPS (red). Image courtesyof Mark P. Stevens, Institute for Animal Health, Compton, UK.