Pathogenic diversity of and the emergence of ’exotic’ islands in the gene stream Charles M. Dozois, Roy Curtiss

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Charles M. Dozois, Roy Curtiss. Pathogenic diversity of Escherichia coli and the emergence of ’exotic’ islands in the gene stream. Veterinary Research, BioMed Central, 1999, 30 (2-3), pp.157-179. ￿hal- 00902564￿

HAL Id: hal-00902564 https://hal.archives-ouvertes.fr/hal-00902564 Submitted on 1 Jan 1999

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Pathogenic diversity of Escherichia coli and the emergence of ’exotic’ islands in the gene stream

Charles M. Dozois Roy Curtiss III*

Department of Biology, Washington University, Campus Box 1 137, One Brookings Drive, Saint-Louis, MO 63130, USA

(Received 27 October 1998; accepted 8 December 1998)

Abstract - Escherichia coli is a highly adaptive bacterial species that is both a member of the com- mensal intestinal flora and a versatile pathogen associated with numerous types of intestinal and systemic infections in humans and other animals. The spectrum of diseases caused by E. coli is due to the acquisition of specific virulence genes harbored on plasmids, bacteriophages, or within distinct DNA segments termed pathogenicity islands (PAls) that are absent from the genomes of commen- sal E. coli strains. PAls are likely to have been transferred horizontally and may have integrated into the E. coli chromosome through bacteriophage or plasmid integration or transposition. The con- tribution of intergenic inheritance to the adaptation and evolution of E. coli, types of PAls associated with different groups of pathogenic E. coli and approaches to identify unique sequence islands (USis), some of which might confer pathogenicity, in E. coli and other bacteria are presented. © Inra/Elsevier, Paris.

Escherichia coli / pathogenicity island / horizontal gene transfer / adaptation

Résumé - Diversité du pouvoir pathogène d’Escherichia coli et fluidité du génome bactérien : acquisition d’ADN « exotiques » organisés en îlots au sein du chromosome. E.ccherichia coli est une espèce bactérienne ayant une grande faculté d’adaptation. Elle fait partie de la flore intesti- nale commensale mais c’est également un pathogène versatile responsable d’infections systémiques et intestinales chez l’homme et les animaux. La grande diversité des maladies provoquées par E. coli est due à l’acquisition de gènes de virulence spécifiques se trouvant sur des plasmides et des bactériophages ou à l’intérieur de régions chromosomiques appelées îlots de pathogénicité (PAIs). Ceux-ci sont absents du génome des souches d’E. coli commensales. L’acquisition des PAIs est probablement la conséquence d’un transfert horizontal d’ADN étranger au génome des E. coli lors de l’intégration dans le chromosome de bactériophages, de plasmides ou de transposons. Nous pré- sentcrons dans cette revue la contribution de ces transferts d’ADN à l’adaptation et à l’évolution de E. coli, les types de PAIs associés aux différents groupes de E. coli pathogènes ainsi que les approches permettant l’identification des îlots constitués de séquences uniques (USIs) et dont une partie pour- rait conférer le pouvoir pathogène chez E. cnli et chez d’autres bactéries. © Inra/Elsevier, Paris. Escherichia coli / îlot de pathogénicité / transfert horizontal de gène / adaptation

* Correspondence and reprints Tel.: ( I ) 314 935 6819; Fax: ( 1) 314 935 7246; e-mail: dozoisC!biodec.wustl.edu 1. INTRODUCTION 111, 176]. Other E. coli pathogens are asso- ciated with host-specific diseases such as air- sacculitis and cellulitis in caused In addition to a common member poultry by being avian E. coli diarrhea of the intestinal microflora of humans and pathogenic (APEC), in rabbits caused rabbit other animals, subsets of by enteropathogenic particular E. coli and E. coli that cause Escherichia coli strains have the (REPEC) [2, 37] adapted edema disease in pigs [l76]. capacity to cause a variety of intestinal and systemic infections. Different types of The schism that separates commensal E. pathogenic E. coli exhibit host and tissue coli strains from pathogenic E. coli and the specificity as well as age specificity for par- diversity of diseases they cause has largely ticular hosts [48, 66, 162]. The diarrheagenic been attributed to the acquisition of different E. coli pathogens include: enterotoxinogenic sets of virulence genes that permit E. coli E. coli (ETEC) associated with travellers’ pathogens to gain entry to, colonize, and diarrhea and porcine post-weaning diarrhea; promote the development of specific patho- enteropathogenic E. coli (EPEC) that cause logical changes in host niches that are inac- watery diarrhea in children and animals, cessible or inhospitable to commensal E. enterohemorrhagic E. coli (EHEC) associ- coli [52]. Genes contributing to the viru- ated with hemorrhagic colitis and hemolytic lence of pathogenic E. coli, as well as other uremic syndrome, enteroaggregative E. coli bacterial pathogens, have been acquired by (EAEC) associated with persistent diarrhea, inheritance of specific plasmids or phages and enteroinvasive E. coli (EIEC) and and through the integration of large blocks Shigella that cause invasive intestinal infec- of DNA, termed pathogenicity islands tions, watery diarrhea and in (PAls), into their chromosomes [52, 63, 70, humans and other primates [75, 120, 122]. 101 J. In this review, we summarize some E. coli associated with extra-intestinal dis- of the aspects of PAIs and discuss the mech- eases include uropathogenic E. coli (UPEC) anisms by which PAIs may have been that cause urinary tract infections in humans, acquired. PAls that have been identified and dogs and cats [13, 41, 61, 175], E. coli asso- characterized in pathogenic E. coli are ciated with neonatal [174], and reviewed, and finally some of the E. coli associated with septicemia in humans approaches that may be useful for identify- [24, 153, 174] and other animals [4, 46, 71, ing and characterizing PAIs are presented. 2. EXOTIC ISLANDS IN THE GENE [16] has allowed Lawrence and Ochman STREAM CONTRIBUTE TO [100] to estimate that at least 17.6 % of the BACTERIAL EVOLUTION open reading frames (ORFs), 755 of 4 288 ORFs totalling 547.8 kb, in the E. coli K- PAIs have been described as large and 12 genome were acquired through horizon- sometimes unstable DNA regions that are tal gene transfer. The distribution of these present in pathogenic variants of a bacterial horizontally acquired segments in E. coli species, but are absent from strains of the K-12 [100], as well as PAIs in pathogenic E. same species that exhibit low or no viru- coli (table o and other bacteria [63, 70, 101 ] lence [63, 70, 101]. These DNA islands are are often associated with tRNA gene loci. ’exotic’ in the sense that genes encoded by In addition, horizontally acquired regions these sequences often possess a nucleotide of E. coli K-12 are also frequently associated composition and codon usage that is atypi- with the presence of insertional sequence cal to that of the bacterial genome within (IS) elements. Similarly, PAIs from differ- which they presently reside. Differences in ent bacterial species are frequently associ- G+C content and codon usage suggest that ated with IS-related, or plasmid-encoded these DNA regions were most likely sequences. These findings suggest that the acquired at some time in the past from for- mechanisms for horizontal transfer of PAIs eign species that may now be extinct, as well as the acquisition of metabolic or through lateral transmission. This rationale niche adaptive genes by commensal E. coli is based on the idea that, when first acquired, strains are shared. DNA from sources with differ- intergenic The association of tRNA ing G+C contents will demonstrate the great- genes impli- cates temperate as the medi- est level of sequence divergence from the bacteriophages ators inte- recipient into which it has integrated. Over- of horizontal gene transfers. The sites of a number of time random genetic drift should amelio- gration bacteriophages, such as P4 and related are within rate the third codon to a base composi- coliphages, (P3) or to tRNA Hori- tion that is more like that of the adjacent genes [31, 142]. surrounding zontal tRNA hom- average G+C content of the host [6, 99]. acquisition through gene ing bacteriophages is a plausible means of Incorporation of foreign DNA into bac- facilitating intergenic recombination since terial is not restricted to and genomes PAls, tRNA sites are highly conserved across it is likely that horizontal gene transfer is a species. The association of different PAIs force in the evolution of bacterial major with tRNA genes and the presence of phage- such as E. coli or Salmonella enter- genomes or plasmid-related sequences bordering or ica Bacterial of E. [98, 100]. populations within PAIs supports the likelihood that coli or Salmonella have been shown to be these virulence-associated sequences may clonal based on multilocus elec- enzyme have been acquired from bacteriophages or trophoresis (MEE) [154, 17!]. Sequencing integrative plasmids that carried bacterio- of common housekeeping genes encoding phage integrase or attachment sites. IS functions shared all strains required by sequences may also mediate acquisition or within a group demonstrate limited DNA loss of PAls, either directly by transposi- transfer In among clones [25, 121, 164!. tion events in the case of functional mobile involved in clone contrast, genes adaptation elements such as the IS]OO flanked unstable and diversification show a of higher degree pgm locus of Yersinia pe.rtis [54, 55,134]. and include intergenic acquisition rfb gene The presence of non-functional remnants of clusters 141, 159, as well as PAI- [9, 168] IS sequences or repetitive sequences, how- encoded genes [63, 70, 101].]. ever, may also contribute to integration, sub- Analysis of the complete genome sequent recombination or deletion of hori- sequence of the E. coli K-12 strain MG1655 zontally acquired DNA. The genomes of natural E. coli isolates and adults. Uropathogenic E. coli (UPEC) based on physical mapping vary by as much comprise a distinct subset of E. coli strains as 1 Mb, with chromosome sizes ranging that belong to certain 0 serogroups and pos- from 4.5 to 5.5 Mb in length [12]. Clinical E. sess virulence determinants which are less coli isolates possess the largest genome frequently associated with E. coli isolated sizes, whereas E. coli K-12 is situated at the from the fecal flora. These virulence deter- lower end of the genome sizes with 4.63 minants include P and S/F 1 C fimbriae, aer- Mb. This further supports the likelihood that obactin, hemolysin, cytotoxic necrotizing the continued success and adaptation of E. factor 1 (CNFI), and certain capsular (K) coli to exploit specialized niches involves antigens [41, 86]. UPEC-associated viru- gene capture from external sources. There lence genes are generally encoded on the are therefore numerous and diverse addi- chromosome and are often clustered together tions to pathogenic E. coli strains compared on discrete blocks of DNA [51, 79, 103]. to the adaptive changes that E. coli K-122 These distinct virulence gene blocks that has acquired. could spontaneously delete from the chro- mosome of UPEC strains 536 and J96 were first termed DNA islands’ 3. E. COLI PATHOGENICITY ’pathogenicity by Hacker et al. and later shortened to ISLANDS [69], ’pathogenicity islands’ [68]. The PAls of three different UPEC strains J96 and Various virulence-associated genes have (536, CFT073) have been characterized. There are integrated into the genome of E. coli, and diverse differences in the five PAls charac- have endowed this organism with a capacity terized from these three strains to successfully adapt to a pathogenic lifestyle including sizes of the PAls, different direct in different host tissues and species and cause flanking association with different a spectrum of different disease syndromes. In repeat sequences, its broadest definition horizontally acquired tRNA genes and location on the E. coli K-122 none of the PAIs segments of DNA that contribute to the map (table I). Interestingly, pathogenicity of E. coli and closely related from strains J96 and CFT703 share any com- mon for the and Shigella spp. could also include virulence sequences except hly pap [65, 163]. The fact that and pap and temperate as well as genes hly plasmids phages, in large blocks of DNA containing virulence sequences occur independently the of some E. coli [44, genes that have integrated into the chromo- genomes pathogenic 58, 86, 109] also the likelihood that some. The acquisition of plasmids and tem- supports various PAIs associated with UPEC strains perate phages harboring genes that encode have and have toxins, adhesins, or other virulence factors emerged independently different addi- most definitely contributes to the virulence of undergone significantly gene tions or deletions over time. certain pathogenic E. coli and Shigella spp. [7, 8, 120, 127]. Strictly speaking, these genetic elements are not, however, consid- 3.1.1. PAI- and PAI-2 of UPEC ered to be PAIs and are not included in the strain 536 following sections. A summary of some of the characteristics of the PAls described Uropathogenic E. coli strain 536 below are in table I. presented (06:K15:H31) contains two chromosomal copies of hly gene clusters encoding 3.1. Pathogenicity islands associated hemolysin, produces S fimbriae, P-related with uropathogenic E. coli fimbriae (Prf) and type 1 fimbriae and is resistant to serum [69, 92]. Spontaneous E. coli strains are the most frequent cause hemolysin-negative deletion mutations from of urinary tract infections (UTIS) in children strain 536 occur at a high rate (10-3 to 10-4) in vitro [67] and were also observed in iso- located on two distinct PAls that could lates from the urinary tract of infected rats excise from the chromosome of strain 536 [68]. Loss of the hemolysin phenotype was [93]. Loss of these PAIs resulted in aviru- associated with the loss of large DNA lence of the strain, sensitivity to serum, and regions flanking the hly clusters [67, 93]. also affected the expression of S and type Genomic analysis and sequencing demon- I fimbrial adhesin production, although strated that these hly gene clusters are these fimbrial gene clusters were not asso- ciated with the PAIs and were not excised Inhibition of type 1 fimbrial production in from the chromosome of strain 536 PAI strain 536 PAI-2 mutants is clearly depen- deletion mutants [69, 93]. dent on leuX [146]. The gene encoding the FimB recombinase that mediates a switch The two PAIs of E. coli 536 are termed PAI-1 and PAI-2. PAI-1 is 70 kb in size, to phase-on expression for type 1 fimbriae in strain 536 contains five contains a hly gene cluster (hly I) and is TRNAeu-specific 5 L TTG codons and its is located at 82.6 min on the E. coli K-12 map expression suppressed [19, 93], whereas PAI-2 is 190 kb in size, entirely in a leuX-negative background, however, the five TTG codons to contains a second hly gene cluster (hly II), a altering P-related fimbriae encoding the prf gene CTG in fimB renders fim expression inde- of leuX is that other cluster, and is located at 96.9 min on the E. pendent [ 146].1t likely characteristics such as coli K-12 map [19]. PAI- and PAI-2 are leuX-dependent flanked, respectively, by 16- and 18-bp enterobactin and flagellar production and are also due to a for a direct repeats that share the motif ‘TTCGA’, motility dependence substantial amount of TTG codons for and these repeats are part of tRNA encoding of these of genes. PAI- is flanked by the .selC seleno- expression products. Expression leuX also contributes to the survival of strain cysteinyl tRNA (tRNASe°) gene [21 ], whereas PAI-2 is flanked by leuX, the 536 in mouse bladder mucus 140] and colo- leucinyl tRNA (tRNAsLeu) [95] encoding nization and nephropathogenicity in rats gene. Excision of either PAI leaves only [161!.] . one direct repeat and results in disruption and abolition of of se/C or leuX transcription In addition to the effects of tRNA gene 191. excision and the loss of virulence factors on Loss of the PAls from strain 536 does the PAIs themselves, another PAI excision- indeed result in a loss of virulence; how- associated phenomenon has been observed. ever, it appears that much of the reduction in Loss of PAI-2 by strain 536 greatly reduces virulence is not directly associated with loss the transcription of sfa genes that encode of the hly, prf or other as yet unknown genes the S fimbriae [69, 93]. However, decreased located on these unstable DNA regions. .s/ii expression is not dependent on leuX, but Rather, decreased virulence of strain 536 is attributed to the absence of the prf PAI deletion mutants is primarily due to the encoded regulatory genes pr/7 and prj8 disruption of the tRNA gene leuX upon exci- [ I 1 8 PrtT and PrfB are highly similar to sion of PAI-2, resulting in alterations in the .sfaB and .sfoC gene products that medi- global gene transcription [145]. Absence of ate regulation of expression of S fimbriae, tRNAse! due to disruption of .selC affected and Prfl and PrfB enhance expression of S the metabolism of a strain 536 PAI-1 dele- fimbriae in strain 536 by cross-talk in trans tion mutant and rendered the strain inca- [ 1 18 Overall, the spontaneous loss of PAls pable of anaerobic growth owing to an inca- from strain 536, the excision of tRNA genes, pacity to produce formate dehydrogenases and alterations in the metabolism and global (which contain selenocysteine) and an regulation of virulence genes are inextrica- inability to utilize selenium, but did not bly linked. Although Hacker et al. have also affect the virulence of the strain 11451. In demonstrated spontaneous loss of PAIs in contrast, absence of tRNA5 caused by other UPEC, namely PAI-5 of strain J96 excision of PAI-2 had no effect on anaero- [68!, it it remains to be demonstrated if spon- bic growth, but rendered the strain serum taneous loss of other PAIs associated with sensitive, dramatically affected production E. coli from UTI also occur and whether of enterobactin-mediated iron acquisition, such events are also mediated by tRNA gene type I and S fimbriae, and flagella, and associated direct repeats that disrupt a tRNA greatly reduced the virulence of this strain. gene upon excision. 3.1.2. PAI-4 and PAI-5 also inserted near pheV in certain pathogenic of UPEC strain J96 E. coli strains [34, 150] (see below). Random sequencing of cosmid subclones UPEC strain J96 (04:K6) is a prototype containing sequences unique to PAI-4 or E. coli strain from which various extra- PAI-5 demonstrated similarities to genes intestinal E. coli virulence factors have been encoding adhesins and other virulence deter- cloned and characterized [77, 125, 170]. minants from diverse pathogens including Strain J96 possesses two copies of gene clus- Streptococcus parasanguis, Proteus ters encoding P (pap-encoded) and P-related mirabilis, Serratia mareescens, and entero- (prs-encoded) fimbriae, produces F1C and toxinogenic E. coli as well as similarities to type I fimbriae, contains two hly gene clus- IS elements (IS]OO, IS630, IS971 ), R1 plas- ters encoding hemolysin and produces cyto- mid genes, and P4 and P2 phage genes. toxic necrotizing factor type 1 (CNF1). All Aside from the hly and paplprs gene clusters of these virulence-associated genes are few common sequences internal to PAI-4 located on the chromosome. Some of the and PAI-5 were observed, except for virulence factors on strain J96 and other sequences related to phage P4 located at the UPEC strains are linked to each other [51, borders of the two islands. Although the 68, 74, 78, 79, 103]. Blum et al. [20] demon- PAI-4 and PAI-5 insertion sites are associ- strated that the virulence-associated genes ated with phenylalanyl tRNA-encoding encoding Prs fimbriae, hemolysin and CNFI genes, differences in the sequences present on strain J96 were contained within a 610-kb within and flanking these PAIs suggest that Sfil fragment and are part of a PAI that has they were acquired and have evolved inde- the capacity to spontaneously delete from pendently. Although Hacker et al. have iso- the chromosome [68]. This PAI is now lated spontaneous PAI-5 deletion mutants termed E. coli PAI-5, whereas another of strain J96 [20, 68], other groups have region containing the pap and hly gene clus- observed that the PAls of strain J96 are sta- ters is termed PAI-4 [70, 163].] , ble (E. Oswald, pers. comm.). PAI-4 and PAI-5 have been further char- Aside from the pap- and hly-related gene acterized by analysis of overlapping cos- clusters, it is currently unknown whether mid clones, hybridization to the E. coli K- I 2 the PAI-4 and PAI-5 sequences related to Kohara ordered phage library [94], and virulence genes from diverse sources play a sequencing of internal and flanking regions role in UTI pathogenesis or whether they [ 163]. PAI-5 spans I 10 kb and is inserted are associated with other UPEC or at 94 min on the E. coli K-12 map. The pre- pathogenic E. coli strains associated with cise junction points of PAI-5 contained human or animal infections. Certain E. coli nearly perfect 135-bp direct repeats with an associated with diarrhea and septicemia of intact PheU (P/K’!) gene at the right junction cattle and pigs produce CNF1 and also pos- and a 25-bp segment of the 3’ end of PheU sess hlv and pap gene clusters [46, 108J. at the left junction. The phe U gene encodes The genes encoding CNF1, hemolysin and phenylalanyl tRNAPheu and is located at 94 P-related adhesins are clustered together on min on the E. coli K-12 map [ 16]. The size PAls in these porcine and bovine E. coli of PAI-4 was only partially determined and strains and fall into two distinct groups: i) spans over 170 kb. The right junction of PAls that are inserted near PheU as is the PAI-4 was within 250 bp of another pheny- case for PAI-5 of strain J96; and ii) strains lalanyl tRNA gene, pheV, located at 67 min that contain PAI-5-type islands that are on the E. coli K-12 map and also exhibited inserted elsewhere in the chromosome (Her- sequences similar to the k-l!s (group II and III ault et al., unpublished results). Whether capsular polysaccharide) genes. Group lI these PAls contribute to intestinal or extra- and group III capsule-encoding genes are intestinal infections in pigs is currently being investigated (Oswald and Fairbrother, pers. contains 44 ORFs [65]. In addition to the comm.). pap and hly gene cluster, there are two notable regions showing homologies to other 3.1.3. The PAI of UPEC strain sequences. The left-hand region is highly CFT073 (PAI-6) similar to a 6.3-kb segment of DNA from enteropathogenic E. coli (EPEC) strain The UPEC strain CFT073 was isolated E2348/69 (accession no. U85771) (pers. from the blood and urine of a woman with obs.). This PAI-6 region homologous to that of EPEC E. coli K-12- acute pyelonephritis. This strain is Hly+, the strain contains related located at 26.8 min to Pap+, Foc+ and Fim+ and is known to con- genes adjacent dadX as well as three ORFs absent from E. tain two functional pap gene clusters [88]. coli K-12 that be involved in iron trans- One of these pap gene clusters lies within a may The second notable is an 58-kb PAI (that we have named PAI-6) and port. homology ORF at the end of the PAI that was initially identified and characterized by (R4) right ordered overlapping cosmid clones and is homologous to an iron siderophore recep- tor from Bordetella In addi- sequencing of flanking DNA that was also bronchiseptica. there are ORFs similar to common to E. coli K- 12 188]. The location tion, tranposase IS or of PAI-6 within the genome of CFT073 is genes, sequences plasmid-encoded less clear compared to most other described genes located before the pap gene cluster PAls. Cosmid sequencing determined that and in between the pap and laly gene clus- the left flanking region starts 7 bp down- ters. There is also a stretch of 8 kb within stream of dadX (26.7 min), whereas the right PAI-6 that is similar to six ORFs from E. end terminates within an uncharacterized coli K-12 that are located at 59.8 min. The G+C content of E. coli gene (f447) (GenBank accession no. PAI-6, excluding sequences with to E. cnli K-12 is ECAE000365) at a site that is 75 bp down- homology DNA, 42.9 % and is different from stream of the metv tRNA gene (63.5 min). significantly The authors confirmed that these E. coli K- that of the E. coli K-12 genome (50.8 %) the G+C mol % of the PAIs 12 sequences flank PAI-6 and suggested [ 1 6]. Similarly, from strains 536 and J96 have also been that CFT073 may have undergone chromo- somal rearrangements relative to the K-122 estimated to be 41 % )70j supporting the likelihood that within these UPEC linkage map. However, it remains to be sequences determined if strain CFT073 has incurred PAIs have been acquired by lateral gene major chromosomal inversions centered transfer from organisms with a lower aver- around PAI-6 or whether additional DNA age G+C content. sequences homologous to those located at 26.7 or 63.5 min of E. coli K-12 have inserted adjacent to either side of PAI-6. 3.2. Locus of enterocyte effacement The junctions on either side of the PAI are (LEE) (PAI-3) bordered by 9-bp direct repeats and may have mediated the of PAI-6 into integration Certain diarrheagenic E. coli and other the PAI-6 to chromosome; however, appears pathogenic enterobacteria adhere intimately be stable [88]. of PAI-6 were quite Regions to and remodel the actin cytoskeleton of used as and to 80 % of probes hybridized host cells, to the formation of what the DNA from strains associated with leading are called attaching and effacing (A/E) or whereas 22 °lo pylonephrtis cystitis, only lesions [117, l58] (for recent reviews see of the DNA from fecal strains on average this volume of (30 with these Veterinary Research hybridized probes [65, 88[. (1999)) and [42, 56, 89, 90, 1201). Entero- Much of a 61-kb region encompassing bacterial pathogens known to cause these the 586-kb PAI-6 has been sequenced and lesions are widespread among different ani- mal species and include enteropathogenic than selC in bovine EPEC and EHEC strains E. coli (EPEC) from humans [120], swine [62] and is not the site of insertion in rabbit [73, 180], cattle [33, 57], dogs [5, 47] and enteropathogenic E. coli (REPEC) [102] rabbits [l, 2, 37], enterohemorrhagic E. coli and EHEC strains of serogroup 0103 (E. (EHEC) in humans and cattle [22, 120]; cer- Oswald, pers. comm.). tain strains alvei and the of Hafnia [144] Weiler et al. [ 173] found that the insertion murine Citrobacter rodentium pathogen site of the LEE is dependent on the evolu- C. (formerly freundii biotype 4280) [60, tionary lineage of the strains. Two groups 151].] . of related strains called EPEC1 and EHEC1, Initial studies demonstrated that a gene belonging to serotypes 0127:H6, 055:H6, 055:H7 and were inserted at termed eae (for E. coli attaching and effac- 0157:H7, selC, whereas a second cluster of EPEC and ing) [84], was required for the development of A/E lesions by EPEC strains, but that EHEC strains (EPEC2 and EHEC2) belong- alone did not induce A/E lesions when trans- ing to serogroups 0111, 026, 045 and 0128 had LEE that were inserted elsewhere ferred to E. coli K-12. The eae gene was on the chromosome. A second found to be located on the chromosome [85]] integration site for the LEE has been determined for and similar sequences were present in other A/E-producing E. coli and enterobacteria some strains belonging to the EHEC2 lin- [11, 59, 85, 151, 178]. eage [157]. Certain EHEC2 strains of serogroups 0111 or 026 were shown to McDaniel et al. [115] demonstrated that contain LEE insertions within the tRNA the A/E of EPEC strain E2348/69 capacity gene pheU (pheR), located at 94 min on the was encoded a !35-kb (0127:H6) by large E. coli K-12 map, which is also the inser- region termed the LEE (for locus of ente- tion site of PAI-5 of UPEC strain J96 [163].]. rocyte effacement). Hybridization of seg- It is likely that the LEE has inserted within ments of the LEE demonstrated that similar more than just these two sites as E. coli and genetic loci were present in other A/E other enterobacteria that induce A/E lesions but were absent from non-A/E pathogens, comprise a diverse evolutionary group. As E. E. coli K-12 and other pathogenic coli, an example, a strain of serotype bacterial The when cloned pathogens. LEE, O 111 ab:H25 that belongs to a lineage other into E. coli K-12 endowed these recipients, than the EPEC/EHEC groups 1 and 2 [172] strains with a capacity to induce A/E lesions contains a LEE that is inserted elsewhere on cultured cells demon- epithelial [114], than the selC or pheU sites [157]. Despite that this DNA can strating unique region the fact that the LEE appears to have autonomously mediate the characteristic inserted at multiple times and within multi- trait of attaching and effacing E. coli. ple sites during the evolution of enterobac- The LEE of strain E2348/69 is inserted teria that induce A/E lesions, and in con- trast to some other E. coli PAls, the LEE 16 bp downstream of the TRNAS&dquo; gene SeIC seems to be stable. located at 82.6 min on the E. coli chromo- some [1 15], the exact site of insertion of The complete sequence of the LEE from PAI-1 from uropathogenic E. coli strain 536 strain E2348/69 encompasses 35 624 bp [19]. Interestingly, selC is also the site of [50] and has an average G+C content of insertion of the E. coli P4-like retronphage 38.4 %, well below the E. coli K-12 chro- <&R73 [80, 160J. Although the LEE inser- mosome value of 50.8 % [16], supporting tion site was first characterized as selC in a the likelihood that the LEE, as with many few EPEC and EHEC strains [ I 15], in many other PAIs, originated from a bacterial cases the LEE has inserted at locations other species with a sufficiently different G+C than .relC [62, 157, 173]. The LEE was content. Analysis of the sequence predicts 411 shown to be generally inserted at sites other ORFs within the LEE island that comprise at least three major functional regions from locus is 43 359 bp in size. This increased left to right: i) a large region encoding a size as compared to the E2348/69 LEE is type III secretion apparatus; ii) a central due to a 7.5-kb putative P4 family prophage region containing the intimin adhesin encod- designated 933L present at the left end of ing eae [59], and tir (translocated intimin the EDL933 LEE. The integration site for receptor) genes [91]; and iii) a region encod- bacteriophage P4 is known to be a tRNA ing secreted Esp (E. coli secreted proteins) gene [132], and the P4-like retronphage gene products required for signal transduc- OR73 inserts within the selC tRNA gene. tion in host cells. Numerous gene names [80, 160!. The LEE of three related EHEC1 have also been changed to follow a more strains (one 055:H7 and two 0157:H7 standardized nomenclature of type III secre- serovars) described by Wieler et al. [173] tion-encoding genes [23, 177]. Uncharac- also contained prophage sequences, whereas terized ORFs include a H-NS related regu- these sequences were absent from E2348/69 latory protein (Orfl), situated at the start of and two clonally related 055:H6 EPEC1I the first operon encoding type III secretion strains. Aside from the presence of the genes. HN-S proteins are global regulators prophage 933L, the LEEs of E2348/69 and that bind to curved DNA regions that are EDL933 are quite homologous. Both LEE often AT-rich, and it is compelling to spec- sequences contain 41 conserved ORFs and ulate that Orfl may regulate expression of an average nucleotide identity of 93.9 %. functional genes within the AT-rich LEE. Interestingly, individual genes exhibiting Three ORFs encode proteins that bear sim- lower identity scores and the most non- ilarities to proteins on virulence plasmids synonymous base changes included the of Shigella and Salmonella spp. Sixteen genes encoding proteins that interact directly remaining ORFs exhibit no relevant homolo- with the host (eae, tir, espA, espB, espD), gies to current database entries. Orfd4 has whereas most of the other genes were highly recently been characterized and named espF homologous. Differences between proteins [116]. EspF is a proline-rich secreted protein in the EPEC and EHEC LEEs may reflect that is exported by the type III secretion some of the discrepancies that have been apparatus. Non-coding sequences on the observed in the mechanisms underlying strain E2348/69 LEE include previously pathogenic adaptation of EPEC and EHEC described remnants of an IS600 transposase- strains and host specificity [89, 120]. related sequence at the extreme right end [43], a large enterobacterial repeat inter- consensus at the left genic (ERIC) sequence 3.3. Sugar islands: group II and III end of the LEE, as well as 1 570 bp of polysaccharide capsule-encoding deleted DNA to selC that is adjacent pre- gene clusters sent in E. coli K-12 but absent from sequences flanking the LEE in strain E2348/69 [115]. The absence of these K- E. coli that cause extra-intestinal disease in humans and animals are often 12 flanking sequences suggests that the LEE encapsu- lated [82, 83]. All known E. coli may have inserted in the past through a capsules are encoded on the chromo- mechanism involving IS sequences, but that (K antigens) some and are divided into three such mobile insertion elements were per- major based on differences in chemical haps excised and truncated owing to recom- groups, Genes I bination or deletion events. composition [128]. encoding group capsules are located near the rfb and his loci The LEE from EHEC strain EDL933 at 45 min on the E. coli map and may be (0157:H7) has recently been published and produced in varying amounts by most E. compared to the E2348/69 LEE [130]. The coli strains [ 148]. In contrast, groups II and EDL933 LEE which is inserted at the selc III capsules are encoded by gene clusters of about 20 kb in size situated near serA [ 126, E coli, they can in certain cases also be con- 167], and are absent from E. coli K-12. Cer- sidered as pathogenicity islands. tain group II (e.g. Kl, K4, K5) and group III (e.g. K54, K10) K antigens are associ- ated with invasive E. coli strains causing 3.4. Unique DNA regions specific to meningitis, or urinary tract infections E. coli causing meningitis or sepsis in humans or animals. Some E. coli group II or III capsules are also structurally related to Pathogenic E. coli that cause meningitis capsules of other invasive pathogens such and sepsis in humans belong to a limited as and group of strains that frequently possess cer- influenzae [83, 119]. Furthermore, group II tain virulence traits. In particular, the K11 or III capsules have been shown to be capsule and certain 0 serogroups as well as required for enhanced virulence and inva- other virulence factors including S/F I C and siveness of extra-intestinal E. coli strains P fimbrial adhesins, hemolysin, the aer- [ 17, 149]. obactin siderophore system and IbelO (inva- Sequencing of regions flanking the K5 sion of brain endothelium) protein are asso- and Kl (group II)- and K54 (group III)- ciated with E. coli causing neonatal sepsis or encoding gene clusters has pinpointed the meningitis [14, 15, 24, 96, 156]. insertion sites to I50 bp 3’ of tRNA pheV Bloch et al. have devised a means of [34, 150] at 67.0 min of the E. coli chro- determining chromosome physical distance mosome. The insertion region of PAI-4 from discrepancies between E. coli strains, and uropathogenic E. coli strain J96 is also used this approach to chart an extensive located at 67.0 in the region of pheV (see physical map of the genome of the E. coli above) [163]. Recently, it has been shown strain RS218 (018:K1:H7), isolated from that the genes specifying synthesis of the neonatal meningitis [18, 147]. This com- group III K54 capsule are inserted within a parative E. coli genome analysis system group 11-type gene cluster and were proba- involves the use of engineered Tn70-based bly acquired through IS//0-mediated hori- mobile markers containing rare-cutting zontal gene transfer [150]. Furthermore, restriction endonuclease (RCP) sites 106, group II and III capsular antigen genes have 107J and parallel analysis of E. coli RS2188 a low G+C content (33-42 %) compared to and E. coli K-12 restriction fragment sizes the E. coli K-12 chromosomc, again sug- by pulsed field gel electrophoresis. 1S70 gesting interspecific gene transfer. markers in E. coli K-12 can be transferred to E. coli or other strains of interest Group II and III capsule-encoding gene pathogenic P1-mediated clusters produce over 80 different polysac- by bacteriophage transduction, charide surface structural antigens [ 83 It and the sizes of restriction fragments between E. coli K-12 and E. coli is likely that ancestral capsule gene clusters wild-type the RCP cassettes can be determined were acquired by horizontal gene transfer bearing and near the /7/K’! locus and have since evolved compared. by recombination and additional horizontal By comparative genome analysis of strain gene transfer events that have conserved the RS218 and E. coli K-12 MG 1655, it is esti- flanking genes required for proper transport mated that the genome of RS218 is 11 % and assembly of these bacterial surface greater than MG 1655 and contains 12 addi- structures. This has resulted in an array of tional regions of greater than 20 kb that are antigenically diverse capsular types among absent from E. coli MG 1655 [ 147]. The kps different E. coli strains. As these chromo- gene cluster encoding the K I capsule, the somally encoded group II and III capsular ifb genes involved in 0 antigen synthesis, polysaccharide-encoding gene clusters may theyfa . gene cluster encoding the S fimbriae, also contribute to the virulence of pathogenic the ibe]O gene involved in invasion of the brain endothelial cell barrier [76], and the of the APEC strain x7122 (078:H7:K80) temperate À phage-encoded bor gene [7, 8]] that are not present in E. coli K-12. By using are now known to be encoded within some this approach, 12 unique sequence islands of the additional ’loops’ or ’bulges’ in strain (USIs) from strain x7122 were identified RS218 [17]. The importance of the unique and were mapped by hybridization to the E. region containing the Kl capsule has been coli K-12 ordered Kohara phage library [94]. clearly demonstrated in strain RS218 [17]. Four of these unique regions in proximity P1 transduction-mediated construction of a to 0, 6.1, 45 and 66.6 min were replaced by RS218-K-12 chimera bearing a replacement using sequential P phage transduction with of the unique region encompassing the KIl lysates from E. coli K-12 strains bearing capsule resulted in lack of invasiveness in different antibiotic resistance markers flank- newborn rats as compared to the wild-type ing either side of the unique regions. Two of RS218 strain. In addition, both the chimera these regions, the 45.0-min region, encoding and RS218 strain grew equally well in rich the 078 rfb gene cluster, and the 0.0-min or minimal medium indicating that lack of region, encoding as yet uncharacterized invasiveness was not due to non-specific genes were necessary for virulence of strain alterations to the physiology of the replace- x7122 in an intra-air sac infection model. ment mutant. Whether some of the other Replacement of other x7122 USIs with E. regions present in strain RS218 and other coli K-12 DNA, determination of whether invasive E. coli strains should be termed any of these regions are required for viru- pathogenicity islands is a question that lence, and determination of the sizes and requires further elucidation. It is probable further characterization of these unique that some of these greater than 20-kb regions regions are currently underway in our labo- were acquired by horizontal gene transfer; ratory. however, further studies will be needed to determine whether these regions contribute to the pathogenesis of meningitis and sep- 3.6. Pathogenicity islands present in ticemia. Yersinia spp. and certain E. coli pathotypes 3.5. of avian Unique regions pathogenic strains of Yersinia E. coli Highly pathogenic spp. comprise Y. pestis (the bacil- lus), Y. h.seudotuberculosis and Y. entero- Avian E. coli (APEC) com- pathogenic coliticcr biotype I B. In addition to the a limited set of E. coli strains, prise partic- presence of virulence of 02 and which plasmids, high- ularly serogroups 078, O1, pathogenicity strains contain chromosomal are associated with respiratory disease (air- determinants on ’high-pathogenicity islands’ sacclulitis), and other septicemia patholo- (HPI) that are absent from low- or non- gies in poultry [39, 64]. Virulence factors pathogenic Yersinia spp. Yersinia HPis con- known to be associated with APEC include tain genes involved in iron uptake and stor- aerobactin resistance to serum production, age and are for virulence in mice and the of required production adhesins 138, 44, 45, [28, 55]. The HPI from Y. hestis, also known 97, 113, 135J. The mechanisms which by as the pigmentation (pgm) locus [54J, spans APEC cause disease are poultry largely 102 kb and contains three known virulence- unknown. associated factors: i) a hemin storage (hms) To determine the genetic basis for the locus that gives a pigmented phenotype to virulence of APEC, Brown and Curtiss [26] colonies grown on agar plates containing utilized a subtractive DNA hybridization Congo red [ 13) ii) irpl and irp2 [105! and technique to identify regions in the genome ybtT and ybtE [ 10[ genes associated with yersiniabactin production; and iii) the fyuA irp-fyua HPI of Yersinia spp.[152]. HPI (psn) gene that encodes the yersiniabactin sequences were frequent in enteroaggrega- receptor and also acts as a receptor for the tive E. coli (EA/EC) (93 %) and blood cul- bacteriocin pesticin [72]. The Y. pestis HPI ture isolates (80 %), were less frequent in is flanked by ISJ00 sequences [134] and enteroinvasive E. coli (EIEC) (27 %), can frequently delete from the chromosome enteropathogenic E. coli (EPEC) (5 %) and [54, 55]. Loss of the HPI results in loss of enterotoxinogenic E. coli (ETEC) (5 %), pigmentation and reduced virulence in sub- and were absent from enterohemorrhagic cutaneously infected mice [81]. E. coli (EHEC), Shigella and Salmonella Highly pathogenic Y. Jzseudotuberculo- strains. Production of irp2- and,fyuA-encoded was also demonstrated in HPI con- sis and Y. enterocolitica 1B are pigmenta- proteins tion-negative and contain truncated HPIs. taining E. coli. The hm.s locus of Y. pe.stis was absent from all E. coli, These smaller HPIs span 45 kb and contain HPI-positive the yersiniabactin production locus - irpl, although the irp2 and fyuA sequences from irp2, ybt homologues (irp4 and irp5) [1291[ E. coli were closer to the Y. hestis lineage of the HPI and were also associated with the and’M/t /B genes - but lack the funs gene and a.s;! tRNA However, insertion loca- IS]OO t7anking sequences [29, 140]. HPis gene. in Y. pseudotuberculosis and Y. cn!/’6’<;’!/- tion of HPI sequences within the E. coli has not been determined. As with itica are more stable than the larger Y. pesti.s genome the Yer.sinia some E. coli strains HPI, although spontaneous deletion of either spp. (3 %) dele- fyuA alone or bothM/t /y and irp2 can occur containing irp2 genes exhibitedJYuA [29, 134J. The HPI of Y. enterolitica I B tions. The,fyuA-irp genes have a G+C con- strain Ye 8081 has been further character- tent of 56-59 % which is higher than that ized and compared with other Yersinia HPI of the overall G+C content of Yer.sinia mol or E. coli [29]. In addition to the irp-fyuA gene cluster, (46-50 °!o) (50.8 %). Hence, it is that the HPI island a gene encoding tRNA-Asn, almost identi- likely pathogenicity associated with an a.sn tRNA has dis- cal to the asnT gene of E. coli, and single gene seminated from an with a copies of four other repeat sequence (RS) organism higher elements that include IS7328 and IS]400 G+C content to Yersinia spp. and to certain E. cnli strains either or were identified. The HPI was conserved for pathogenic directly the HPI-encoded the most part among other Y. enterolitica sequentially. Clearly contribute to the virulence of strains except for the 15-kb stretch that con- sequences tains three of the RS elements located down- Yer.sinia species. Whether HPI-encoded pro- teins contribute to the virulence of stream of fyuA. It is hypothesized that the E. coli, EA/EC or asn tRNA gene and/or RS sequences on the pathogenic particularly E. coli from humans outskirts of the HPI may play some role in septicemia-causing remains to be determined. At the the instability of the island leading to exci- present, of HPI-associated clusters in sion of part or all of the HPI in certain strains presence gene [29, 139J] pathogenic E. coli or commensal E. coli strains from animals has yet to be investi- Two lineages of the Yersinia HPI have gated. been established based on DNA sequence comparisons: a Y. enterolitica I B group and a Y. Pe.stislY. pseudotuberculosis group [140!. Interestingly, four pesticin-sensitivc 3.7. Pathogenicity islands in Slzigella E. coli strains were shown to contain func- tional Yersinia irh2 andfyuA genes related Shigella spp. are associated with inva- to the Y. pestis lineage. A more extensive sive intestinal disease, watery diarrhea and investigation has demonstrated that certain dysentery in humans and other primates and pathogenic E. coli from humans contain thc comprise four different groups - S. dy.se n- teriae, S. flexneri, S. boydii and S. sonnei - intron-like sequence. Flanking regions from all of which can be considered as clones of this strain were specific to she+ Shigella E. coli [123, 136]. Shigella spp. invade the strains, indicating that the she PAI is spe- colonic epithelium, and elaborate one or cific to a limited group of Shigella strains. more secretory enterotoxins, phenotypes These findings correlate with the fact that that are encoded by both plasmid and chro- ShETI is almost exclusively associated with mosomal loci [127]. In addition, to viru- S. flexneri 2a strains [122].] . lence plasmid-encoded genes, PAIs have been identified on the chromosome of 2a [137, 138]. A 99-kb 3.8. Sometimes less is more: deletion from the chromosome of Shigella ’black holes’ in Shigella and flexneri 2a strain YSH6000 was identified enteroinvasive E. coli and localized between the Shigella ompA and situated on the pyrC gene homologues Unlike most pathogenic or commensal NotI fragment D of the Shigella physical E. coli strains, enteroinvasive E. coli (EIEC) min on E. coli map [124] (22-24 K-12) and Shigella spp. are typically unable to fer- Loss of this 99-kb resulted [137]. fragment ment lactose and also lack the cadA gene in a reduction in contact hemolysin activ- that encodes lysine decarboxylase (LDC). ity and rendered the strain sensitive to ampi- Recently, Maurelli et al. [112] identified and cillin, , streptomycin large genomic gaps they termed ’black tetracycline. holes’ in the region containing cada, in and EIEC. To The 99-kb S. flexneri 2a deletion mutant Shigelln spp. investigate whether lack of LDC contributes was used to identify a second Shigella production to the pathogenicity of Shigella.flexneri 2a, flexneri PAI with a capacity to sponta- neously delete from the chromosome by Maurelli et al. [ 112] initially attempted to introduce cadA into S. jlexneri P1 using an ’island probing’ counterselection by phage transduction with an E. coli No method [138]. A tetAR cassette, encoding K-12 lysate. LDC+ transductants were obtained, how- tetracycline resistance was inserted within ever, that may have the she gene of the Tets 99-kb deletion suggesting S. flexneri a deletion to E. coli K-122 mutant by homologous recombination. The large compared that recombination in she gene encodes the ShMu protein, a mem- precludes legitimate the cadA. Alterna- ber of the IgA protease-like autotransporters, region encompassing a a copy of cadA and also encompasses the setla and setlbb tively, plasmid bearing was transformed into S. it genes encoding the Shigella enterotoxin 1 flexneri rendering LDC+. LDC had no effect on the (ShETl) [53, 1381. Tets revertants were activity of S. flexneri to invade and selected for by growing cells on plates con- capacity lyse cells in culture; however, cadA+ S. flexneri taining fusaric acid [110], and were obtained exhibited little enterotoxin and was at a of 10-5 to 10-6. Restriction activity frequency attenuated in virulence. The digests migrated on pulsed-field demon- specific gels inhibitor of ShET 1 and ShET2 enterotoxin strated that these Tets revertants had lost a was shown to be cadaverine, a of -51 kb from their genome that activity region metabolic of cadA-mediated was located on Not! fragment D of the product lysine Shigella flexneri physical map 124]. A total decarboxylation. of 25 kb flanking either side of she was Physical enzyme restriction mapping sequenced and found to contain a second demonstrated that the S. flexneri 2a strain IgA-protease autotransporter-related gene has a large deletion of -190 kb compared designated .sigA, complete copies of IS2 and to E. coli K-12 in the region surrounding IS600 and an IS629 sequence that was dis- cadA (93.9 min on E. coli K-12), and large rupted by an E. coli chromosomal group II gaps in Shigella spp. and EIEC were deter- mined using hybridization probes spanningg equivalent E. coli K-12 DNA sequences the cadA region from lexA (91.7 min) to bordering them are, however, difficult to vacB (95.0 min.). These findings illustrate assess. Spontaneous deletions in strains, as that genomic deletions or ’black holes’ and demonstrated by the studies of Ritter et al. loss of a metabolic gene present in non- [145, 146], may involve the disruption of pathogenic E. coli as well as the acquisition tRNA genes that affect global regulation. of virulence genes encoded on plasmids, For this reason, the replacement of USIs by phages or PAls can contribute to the adap- the corresponding flanking E. coli K-122 tation of E. coli (Shigella spp.) to a specific DNA by PI phage-mediated transduction pathogenic lifestyle. The search for other [l7, 26] or other methods to delete large and ’black hole’ genomic deletions by compar- defined chromosomal regions [87, 165] may ative physical mapping and analysis of bio- be more suitable for determining whether chemical or physiological differences USIs contribute to the pathogenicity of par- between closely related pathogenic and non- ticular E. coli strains. pathogenic counterparts or related pathogenss that exhibit tropisms for different host The availability of the complete E. coli species merits further investigation. K-12 genome [16] as well as refined meth- ods for genetic manipulation of E. coli and analysis of genomes by physical mapping and 4. CONCLUSION - DISCOVERING pulsed-field electrophoresis are a tremendous for AND EXPLORING UNCHARTED advantage characterizing and the location of PAIs or ISLANDS determining black holes in E. coli and Shigella [18, 112, 138, 147 Other recent methods that select Throughout the descriptions of PAls pre- for specifically unique sequences or genes sented above various approaches were taken required for survival or virulence in the host to identify and characterize different E. coli may also lead to the discovery of new USIs PAls. Methods to detect the loss of viru- or black holes that may contribute to the lence-associated and to first genes identify of E. coli or other bacterial PAIs included the detection of the pathogenicity sponta- pathogens. Examples of such technologies neous loss of the in hemolysin phenotype include subtractive DNA [26, UPEC strains 536 and J96 hybridization phenotypes [67, 49] and subtractive RNA hybridization [133, as well as the method 68], island-probing 1661, signature tagging [32, 155], in vivo that uses counterselection for tetracycline- expression technology [35, 36, 104, 169], sensitive revertants insertion of following representational difference analysis [27, 30],] , a TetAR cassette into a on the specific gene and mRNA differential [3, 179]. chromosome A number of other display [1381. Furthermore, DNA of PAls [50, counterselectable markers that be sequencing may 65, 163] and for unstable computer-assisted sequence adapted screening potentially comparisons will provide insights into the PAIs has recently been presented by Reyrat mechanisms through which PAIs may have et al. [ 143]. been acquired as well as the possible func- Screening methods such as island probing tions of PAI-encoded proteins. The use of combined with physical genome analysis infection studies using animal models or by pulsed-field electrophoresis may be very cell and tissue cultures should provide an beneficial for initially detecting and sizing indication of the roles such PAI-encoded unstable regions that are potentially PAIs genes may play in pathogenesis. However, and in analyzing their mechanisms of spon- as there may be a number of homologous taneous deletion. The determination of the or heterologous loci encoding for common role that newly identified USIs may con- virulence attributes within the genome of tribute to virulence without knowing the particular pathogenic E. coli strains, dele- tion of individual USIs that may contribute !7] Barondess J.J., Beckwith J., A bacterial viru- lence determinant encoded col- to the of by lysogenic cumulatively pathogenic capacity iphage lambda, Nature 346 (1990) 871-874. a strain not alter the viru- may dramatically [8![ Barondess J.J., Beckwith J., borgene of phage lence of a strain. Determining whether lambda, involved in serum resistance, encodes sequences encoded by USIs are common to a widely conserved outer membrane lipopro- particular pathogenic strains should also tein, J. Bacteriol. 177 (1995) 1247-1253. [9! Bastin D.A., Reeves P.R., and anal- provide an indication as to whether these Sequence ysis of the 0 antigen gene (rfb) cluster of DNA sequence regions may promote the Escherichia coli 0 III, l, Gene 164 (1995) 17-23. pathogenicity of these strains, and will pro- ! 10! Bearden S.W., Fetherston J.D., Perry R.D., vide insight into what gene products may Genetic organization of the yersiniabactin be potentially useful for the development biosynthetic region and construction of avirulent mutants in Yer.sinia pestis, Infect. Immun. 65 of protective vaccines against these (1997)1659-1668. pathogens. [ I[ Beebakhee G., Louie M., De Azavedo J., Brun- ton J., Cloning and nucleotide sequence of the eae gene homologue from enterohemorrhagic Escherichia coli serotype 0157:H7, FEMS ACKNOWLEDGMENTS Microbiol. Lett. 70 ( 1992) 63-68. [ 12! Bergthorsson U., Ochman H., Distribution of This work was supported by US Department chromosome length variation in natural isolates of Agriculture National Research Initiative Com- of Escherichia coli, Mol. Biol. Evol. 15 (1998) petitive Grant 97-35204-4512. C.M.D. is the 6-16. recipient of a fellowship from the Canadian Nat- ! 131[ Beutin L., Escherichia coli as a pathogen in ural Sciences and Engineering Research Council. dogs and cats, Vet. Res. 30 (1999) 285-298. 1141 Bingen E., Bonacorsi S., Brahimi N., Denamur E., Elion J., Virulence patterns of Escherichia coli K strains associated with neonatal menin- REFERENCES gitis, J. Clin. Microbiol. 35 ( 1997) 2981-2982. ! 15! 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