Complete DNA Sequence Analysis of Enterohemorrhagic Escherichiacoli Plasmid pO157_2 in �-Glucuronidase-Positive E. coli O157:H7Reveals a Novel Evolutionary Path
L. V. Rump,a J. Meng,a,b E. A. Strain,c G. Cao,a,d M. W. Allard,d and N. Gonzalez-Escalonad
Department of Nutrition and Food Sciencea and Joint Institute for Food Safety and Applied Nutrition (JIFSAN),b University of Maryland, and Biostatistics Branchc andDivision of Microbiology,d Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland, USA
Strains of enterohemorragic Escherichia coli (EHEC) O157:H7 that are non-sorbitol fermenting (NSF) and �-glucuronidase neg-ative (GUD�) carry a large virulence plasmid, pO157 (>90,000 bp), whereas closely related sorbitol-fermenting (SF) E. coliO157:H� strains carry plasmid pSFO157 (>120,000 bp). GUD� NSF O157:H7 strains are presumed to be precursors of GUD�
NSF O157:H7 strains that also carry pO157. In this study, we report the complete sequence of a novel virulence plasmid,pO157-2 (89,762 bp), isolated from GUD� NSF O157:H7 strain G5101. PCR analysis confirmed the presence of pO157-2 in sixother strains of GUD� NSF O157:H7. pO157-2 carries genes associated with virulence (e.g., hemolysin genes) and conjugation(tra and trb genes) but lacks katP and espP present in pO157. Comparative analysis of the three EHEC plasmids shows thatpO157-2 is highly related to pO157 and pSFO157 but not ancestral to pO157. These results indicated that GUD� NSF O157:H7strains might not be direct precursors to GUD� NSF O157:H7 as previously proposed but rather have evolved independentlyfrom a common ancestor.
large outbreaks of severe enteric infections including bloody diar-rhea, hemorrhagic colitis, and hemolytic uremic syndrome world-wide (7, 10). It has been proposed that highly pathogenic E. coliO157:H7 arose from an ancestral enteropathogenic E. coli (EPEC)O55:H7 through sequential acquisition of virulence factors, phe-notypic traits, and serotypic change (8, 9, 28). In this evolutionarymodel for O157:H7, six clonal complexes (A1 to A6) carryingcharacteristic traits were proposed. Ancestral O55:H7 EPECstrains belong to clonal complexes (CCs) A1 and A2. The somatic(O) antigen change from O55 to O157 gave rise to a hypotheticalintermediary (CC A3) (29). This was followed by a divergence intwo separate O157 clonal lineages of nonmotile sorbitol-ferment-ing (SF) O157:H� strains (A4) and non-sorbitol-fermenting(NSF) O157:H7 strains (CC A5). The most typical O157:H7 phe-notype at present (CC A6) is believed to have emerged from CCA5 strains by the loss of �-glucuronidase activity (GUD�), causedby a mutational inactivation of the uidA (9). These CC A6 strainshave spread widely into disparate locales and now account formost food-borne illness caused by EHEC (29).
The pathogenicity of O157:H7 is among other factors associ-ated with the possession of a large EHEC virulence plasmid. CC A6strains carry pO157 (�90 kb), and CC A4 strains carry a largerplasmid (�120 kb), namely, pSFO157 (2). Different genes andgene clusters including the EHEC hemolysin (hly) operon locatedon pO157 have been linked to virulence (21, 23). pO157 further-more carries katP, encoding a periplasmic catalase-peroxidase (3);a type II secretion system (etp operon) (22), toxB, involved inadherence (26); and espP, encoding a secreted serine protease (4).Although NSF O157 and SF O157 strains are closely related andshare virulence factors such as the attaching and effacing (eae)locus and Shiga toxin (stx) genes, pSFO157-carrying strains donot show hemolytic activity despite carrying the hyl operon andlacking katP and espP. Ancestral O55:H7 strains carry pO55,
which does not possess most virulence genes present on pO157 orpSFO157; however, pO55 does contain the etp operon (31). Allthree plasmids contain incomplete tra gene clusters and thus arepresumably incapable of conjugative transfer, as many of themissing genes are required for conjugation (16). Thereby, it ap-pears that the plasmids were inherited from a common ancestor ofO157:H7 and O55:H7 but have undergone major structuralchanges (31).
Brunder at al. (1) suggested that pO157 and pSFO157 evolvedfrom a common ancestor plasmid probably present in CC A3 bythe acquisition and loss of various genes due to insertion/transpo-sition events and possibly plasmid fusion. Results by Rump et al.(20) showed that the virulence plasmid in CC A5 strains lack someof the pO157 regions containing insertion sequence 629 (IS629),suggesting the presence of a different plasmid. Due to these dis-crepancies with the proposed stepwise emergence of E. coli O157:H7, we determined the complete sequence of the plasmids presentin CC A5 strain (G5101) and two CC A4 strains (493/89 andH2687) in order to gain an insight into the emergence of pO157and pSFO157.
MATERIALS AND METHODSBacterial strains. Strains used in this study are listed in Table 1. The SFO157 strains (493/89 and H2687) were originally isolated in Germany(12) and Scotland (27) and carry stx2, representing strains of CC A4 (9).
GUD� NSF O157:H7 strain G5101 harbors both stx1 and stx2 and belongsto CC A5. All strains examined were from the culture collection of theDivision of Microbiology at the U.S. Food and Drug Administration(FDA), Center for Food Safety and Applied Nutrition (CFSAN). Strainswere grown overnight in Luria-Bertani (LB) medium at 37°C with shaking(250 rpm).
Plasmid extraction, sequencing, and contig assembly. PlasmidDNAs were extracted from overnight cultures of strains G5101, 493/89,and H2687 using the PureYield plasmid maxiprep system kit (Promega,Madison, WI) following the manufacturer’s instructions. The plasmidgenomes were sequenced using 454 Titanium pyrosequencing (Roche,Branford, CT) according to the manufacturer’s instructions at 20� to100� coverage. Plasmid sequence contigs were assembled using CLCGenomics Workbench software version 4.7.1 (CLC bio, Germantown,MD) with the following reference sequences: pSFO157 of E. coli O157:H�
strain 3072/96 (GenBank accession no. AF401292) (1) and pO157 of E.coli O157:H7 strain Sakai (GenBank accession no. NC_002128) (17).Contigs were manually arranged according to their contiguous sequencehomologies using BioEdit version 7.0.9.0 (11). Contig organization forthe novel G5101 plasmid (pO157-2) was confirmed by PCR bridging ad-jacent fragments. Sequences were annotated using the NCBI ProkaryoticGenomes Automatic Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html) (14). Comparison of the sequenced plas-mids with pO157 and pSFO157 was performed with Mauve version 2.3.1(6) and visualized with CGView (http://wishart.biology.ualberta.ca/cgview/index.html) (25). The overall similarities of the considered plas-mid core genes (excluding mobile elements) of pO157-2 with pO157 andpSFO157 were 99.8 and 99.4%, respectively.
Plasmid confirmation by PCR. Specific PCRs were conducted to es-tablish the presence of each individual plasmid in the strains analyzed.Primers employed are listed in Table S1 in the supplemental material. Thefollowing genes or regions were used as target indicators for the presenceof each plasmid: espP (pO157), traC (pSFO157), and repA3-repE
(pO157-2, region 4); yihA (each plasmid produced a different PCR prod-uct size); and letA-etpH (pO157/pSFO157; negative for pO157-2). PCRamplifications were performed using 5 ng of plasmid DNA in a final vol-ume of 30 �l. The PCR mixture contained 1 U of Taq polymerase (Qiagen,Valencia, CA), 1� Taq polymerase buffer, 3.5 mM MgCl2, 125 �M eachdeoxynucleoside triphosphate (dNTP), 150 nM each primer pair. PCRconditions were as follows: 1 cycle of 95°C for 2 min, followed by 30 cyclesof 95°C for 1 min, 58°C for 1 min, and 72°C for 1.5 min, and a finalextension at 72°C for 10 min. Amplicons were visualized on a 1% agarosegel in Tris-borate-EDTA (TBE) buffer containing 0.3 �g/ml ethidiumbromide.
Nucleotide sequence accession numbers. The plasmid sequences forpO157-2 (G5101) and pSFO157 (493/89 and H2687) are available inGenBank under accession numbers AETX01000217, AETY01000205, andAETZ01000210, respectively.
RESULTSPlasmid content in E. coli strains 493/89, H2687, and G5101.The assembly of plasmid contigs confirmed the presence of a plas-mid highly similar to pSFO157 from strain 3072/96 in twoO157:H� strains (493/89 and H2687) belonging to CC A4 (1),showing only minor differences (single nucleotide polymor-phisms [SNPs]). pSF493/89 had 3 SNPs (positions 32471, 87701,and 115470) and pSF2687 had 7 SNPs (positions 6860, 47558,55997, 87698, 87701, 90799, and 115470) compared to pSFO157of strain 3072/96. PCR analysis confirmed the presence ofpSFO157 in four other O157:H� CC A4 strains. Strain G5101contains a novel plasmid (pO157-2) resembling both pO157 andpSFO157 (Fig. 1A and B).
Assembly and characterization of pO157-2 from E. coliO157:H7 strain G5101. Twelve contigs were obtained for
TABLE 1 Serotype, sequence type, characteristics, and isolation information of E. coli strains used in this study
Strain Serotype stxa
Special characteristics
STd CCe Sourcef Countryf Yearf
Accession no. orreferenceGUDb SORc Plasmid
Sakai O157:H7 1,2 � � pO157 66 A6 Human Japan 1996 NC_002695EDL933 O157:H7 1,2 � � pO157 66 Food USA 1982 AE005174EC4115 O157:H7 1,2 � � pO157 66 Food USA 2006 NC_011353TW14359 O157:H7 1,2 � � pO157 66 ? USA 2006 CP001368EDL931 O157:H7 1,2 � � pO157 66 Human USA 1983 24MA6 O157:H7 2 � � pO157 66 Food Malaysia 1998 11550654 O157:H7 2 � � pO157 66 ? USA 2009FDA 413 O157:H7 2 � � pO157 66 ? ? ? 11G5101 O157:H7 1,2 � � pO157_2 65 A5 Human USA 1995 12TW06289 O157:H7 1,2 � � pO157_2 65 ? USA 1997 25TW06290 O157:H7 1,2 � � pO157_2 65 ? USA 1997 12EC97144 O157:H7 1,2 � � pO157_2 65 ? Japan 1997 8EC96038 O157:H7 1,2 � � pO157_2 65 ? Japan 1996 12EC96012 O157:H7 1,2 � � pO157_2 65 ? Japan 1996 12493-89 O157:H� 2 � � pSFO157 75 Human Germany 1989 125412-89 O157:H� 2 � � pSFO157 75 A4 Human Germany 1989 24H56929 O157:H� 2 � � pSFO157 76 ? Finland 1999 12H56909 O157:H� 2 � � pSFO157 76 ? Finland 1999 12H1085c O157:H� 2 � � pSFO157 76 Cat Scotland 2003 12H2687 O157:H� 2 � � pSFO157 76 Human Scotland 2003 12a stx, Shiga toxin gene.b GUD, �-glucuronidase activity.c SOR, sorbitol fermentation.d ST, sequence type as determined by a combination of seven genes (http://www.shigatox.net/ecmlst/cgi-bin/dst).e CC, clonal complex (12).f ?, Unknown.
pO157-2. The assembly of contigs, closing gaps using sequencedPCR products, revealed a circular plasmid of 89,762 bp (GC con-tent of 47.71%) that contained 107 annotated open readingframes (ORFs), including multiple transposons and insertion el-ements (Fig. 1A; also see Table S2 in the supplemental material).pO157-2 carries virulence-associated regions like the hly operon,the etp operon, and toxB but lacks the espP and katP genes. Al-though pO157-2 shares more sequence similarities with pO157(Fig. 1), its sequence analysis is based on pSFO157, since pSFO157is thought to be evolutionarily older than pO157 (1). In accor-dance with the annotations in pSFO157 (5), pO157-2 was dividedinto five discrete regions with the origins of replication definingthe regions (1) and the finO gene as the first gene of the analysis(Fig. 2).
Region 1 (finO to repA1) (position 1 to 4353) carries eightpotential ORFs of an IncFII minimal replication origin (1). Sharedgenes of pO157 and pO157-2 are 100% similar in sequence, com-pared to 93 to 97% similarity to genes in pSFO157 (see Table S2 inthe supplemental material). Major differences between the threeplasmids are in yihA and copB, with pSFO157 carrying a completeyihA (590 bp), which is truncated in both pO157 and pO157-2(339 bp; internal deletion). A truncated IS629 interrupts yihA up-stream (position 88 and 89) in pO157-2 (Fig. 2A). In addition,both pO157-2 and pO157 carry a copB gene that shared 65% se-quence identity with its homologous gene in pSFO157, located atthe end of this region (Fig. 2A).
Region 2 differs significantly between plasmids (Fig. 2B).pO157-2 shares none of the genes present in pSFO157 (sfp cluster
FIG 1 EHEC plasmid pO157-2 map and comparison with two other EHEC plasmids. (A) Genetic map of the novel virulence EHEC plasmid pO157-2 generatedwith CGView (25). Blue block arrows in outer circle denote coding regions in plasmid indicating ORF transcription direction. G�C content is shown in themiddle circle, and deviation from the average G�C content (47.71%) is shown in the innermost circle. BLAST comparisons with other two EHEC plasmids areshown in red (pO157) and green (pSFO157). (B) Comparison of whole sequences of plasmid pO157-2 and plasmids pO157 and pSFO157 with MAUVE. Eachplasmid genome is laid out in a horizontal track, and homologous segments are indicated in the same color and connected across genomes. Respective scales showthe sequence coordinates in base pairs. A colored similarity plot is shown for each genome, the height of which is proportional to the level of sequence identityin that region.
encoding fimbriae and mannose-resistant hemagglutinationgenes) and pO157 (katP and espP) and containing solely completeand incomplete ISs and transposons (IS1294, truncated Tn2501,ISEc8). pO157-2 shares most of the ISs flanking the sfp cluster with
pSFO157 with the addition of ISEc8, which is the second mostprevalent IS found in O157:H7 strains (after IS629). ISEc8 isunique to pO157-2 but is also present in pO55 of E. coli O55:H7and in the O157:H7 genome in multiple copies (19).
FIG 2 Schematic comparison of plasmids pO157-2, pO157, and pSFO157. Genes are shown in blocks; shaded blocks share less similarity with other genes. (Ato E) Comparative analysis of gene contents for all three plasmids (pO157, pO157-2, and pSFO157) divided by regions according to pSFO157 (1). Block coloringindicates type of region: insertion sequences are represented by red blocks, transposons are dark orange, hly genes are dark green, etp genes are blue, tra genes arelight green, and toxB is light orange. Areas not present in the plasmids are shown with dotted lines. tr, truncated; hp, genes coding for a hypothetical protein.
Region 3 in pSFO157 carries virulence genes (msbB, etpoperon, hly operon) and the eae-positive conserved fragment (ecf).Both pO157 and pO157-2 lack the replication origin and ISs withthe exception of iso-IS1 remnants. However, all of the followinggenes and operons in this region are present and �99% identicalto those found in pSFO157 (see Table S2 in the supplementalmaterial), showing that this region is more conserved in all threeplasmids than other regions. One exception is an additional IS30alocated between a truncated IS911 and the e-hlyC in pSFO157.
Region 4 in pSFO157 starts at the third origin of replication inthe plasmid (repA3) and comprises seven ORFs (1). This region ispresent in pO157 with the exception of a hypothetical transposon
(42 to 44 partial) but entirely absent from pO157-2. pO157 carriesan additional complete IS629 at the end of this region truncatingrepE.
Region 5 in pSFO157 comprises genes related to F-like plas-mids. It was shown previously that pO157 and pSFO157 differednotably in length in this particular region, which is mainly due tothe lack of a significant amount of transfer genes (tra and trb) inpO157 (Fig. 2F; also see Table S2 in the supplemental material)(1), while repE is truncated in both pO157-2 and pO157 and thelatter contains an IS629. sopA, sopB, and several other genes foundon the F-plasmid (AP001918) until parB are �99% identical insequence in all three plasmids analyzed. The exception is yccB,
which is truncated at the 3= end in pO157 by an unknown element(a possible RNA-directed DNA polymerase or transposon of�2,400 bp). Major differences between plasmids are present in theregion containing transfer genes (Fig. 2E and F). The whole trans-fer gene region between parB and traH observed in pSFO157 ismissing from pO157. Instead, this region in pO157 contains toxBflanked by several complete and incomplete ISs elements (IS3,IS21, IS629). pO157-2 also carries toxB including its surroundingISs and shares the upstream transfer genes (traM to traB) presentin pSFO157. pSFO157, contrary to both pO157 and pO157-2,lacks toxB and carries several more transfer genes. traB is eithertruncated in or absent from all three plasmids: truncated (41%) inboth pSFO157 and pO157-2 and entirely absent from pO157.
Distribution of pO157-2 plasmids in E coli strains belongingto the stepwise emergence of E. coli O157:H7. PCR results showthat pO157, pO157-2, and pSFO157 in the strains analyzed aredistributed according to CCs (see Table S3 in the supplementalmaterial), whereas all CC A6 strains, CC A5 strains, and CC A4strains carry pO157, pO157-2, and pSFO157, respectively.
DISCUSSION
In this study, we described a novel, potentially nonconjugativeplasmid present in GUD� E. coli O157:H7 strains. According tothe stepwise evolutionary model for O157:H7 (8), strain G5101was thought to be a precursor of SOR�, GUD� O157:H7 strains(9). It was previously assumed that clinical O157:H7 isolates carrya virulence plasmid resembling pO157 (24, 30). However, in thisstudy we showed that O157:H7 strains could carry virulence plas-mids other than pO157.
The three closely related plasmids pO157, pSFO157, andpO157-2 found in O157 strains shared several genes and regionsincluding highly conserved virulence factors (etp and hly operons,mspB, sopA, sopB) and the F-plasmid-like leading region (yccB-parB). Sequences within the leading region are also conservedamong other F-like plasmids belonging to the Inc9, B, I, K, and Yincompatibility groups (18). Since none of the plasmids appears tobe conjugative, a recent introduction into E. coli O157 is unlikely.DNA sequence analysis indicated that they have undergone majorstructural changes. The plasmids lack a significant number oftransfer genes: 38 for pO157 (traM-traI), 30 for pO157-2 (traP-traI), and 6 for pSFO157 (traB-traG, artA, trbJ, and trbF) in theF-plasmid-like region. However not all transfer genes are essentialfor plasmid conjugation. traB, however, which is either missing ortruncated in all three plasmids, is essential for conjugative transfer(13), indicating that none of the plasmids is likely conjugative.Comparative analysis showed numerous evidences for a separateemergence of the three plasmids and most likely an early split ofpSFO157. Besides a significantly greater number of transfer genespresent in pSFO157, the plasmid also carries a complete yihA anda distinct copB in region 1. The hypothetical ancestral plasmidpO157A thereby would have likely carried more transfer genes, afunctional yihA, and a different copB. Presumably after the splitfrom pSFO157, transfer genes were lost and the two genes weretruncated in hypothetical common ancestor plasmid pO157B(Fig. 3). Other evidences of this early split and the ongoingchanges are also present; for example, pSFO157 carries the sfpgene cluster, a functional repE, several differences in IS content,and multiple nucleotide differences in shared genes (see Table S1in the supplemental material).
pO157 and pO157-2 are more closely related to each other
than to pSFO157, which is confirmed by the presence of toxB andcopB and the absence of the sfp gene cluster. However, there areseveral additional features indicating that pO157-2, althoughhighly related to pO157, evolved independently from hypotheticalpO157B and unlikely gave rise to plasmid pO157 (Fig. 3). pO157shares genes with pSFO157 in region 4 (ydiA, etpH, kfrA, redD),but these are absent from pO157-2. Since pO157 and pSFO157share genes of this region, genes should be present in pO157-2 inorder to be a possible progenitor to pO157. The deletion of thosegenes in pO157-2 therefore indicates a divergence of pO157 andpO157-2 from pO157B and excludes the possibility of A5 CCstrains being precursor strains to CC A6 strains. Furthermore,after this split, pO157 likely incorporated katP and espP, addi-tional transfer genes (psiB-traK) were lost, and different ISs werealso lost and/or acquired in diverse regions. It is thereby apparentthat especially since it misses elements shared by pSFO157 andpO157, the newly sequenced plasmid pO157-2 is unlikely to be theprecursor of pO157.
The three plasmids contain several ISs, with the majority be-longing to the IS3 and IS21 family located in regions differingbetween plasmids (see Table S2 in the supplemental material).Additionally, pO157 carries IS91 and pO157-2 carries IS66 familymembers. pSFO157 is almost identical in CC A4 strains, butpO157 differs greatly between closely related strains. The recentlydescribed IS excision enhancer (IEE) protein, exclusively presentin O157:H7 strains, significantly increases IS3 family transposi-tion (15). It would therefore be interesting to speculate that thepresence of IS3 family members and IEE lead to rearrangements,insertions, and deletions in pO157B from which pO157 andpO157-2 evolved (Fig. 3). This hypothesis suggests that virulenceplasmids in O157:H7 strains are still changing rapidly, thereby
FIG 3 Proposed evolution of plasmid pO157-2 based on the stepwise evolu-tion model for EHEC O157:H7. Possible acquisitions and/or losses of genes areindicated in branches. Clonal complexes (CCs) A3 to A7 are shown; changespredicted to have occurred: blue, gain of genes or regions; red, loss of genes orregions; gray, particular gene was truncated; green arrow, introduction of in-sertion sequence excision enhancer (IEE) in strains. Red circle, newly intro-duced CC A7 with plasmid pO157-2. Ancestral strain and plasmids A3 and A5(shaded circles) have not been reported.
contributing to pathogenic potential. They could be used for ad-ditional characterization of strains and also serve as markers forrelatedness analysis among strains.
In the present study, we have shown that O157:H7 strains,although displaying characteristics for pO157, could carry distinctplasmids. Plasmid pO157-2 shares high similarity with pO157;nevertheless, structural evidences showed that it evolved indepen-dently from a common ancestor plasmid and pO157-2 carryingstrains belong to a separate clonal lineage (designated CC A7).Zhou et al. (31) also suggested an early split of GUD� NSFO157:H7 strains using a core genome alignment of several O55:H7, O157:H� and O157:H7 genomes. In this model, GUD� strainG5101 (containing pO157-2) split from the O157:H7 evolution-ary path and did not appear to be the ancestral strain for GUD�
NSF CC A6, as also suggested by our results. Therefore, GUD�
O157:H7 strains which either are SOR� (“American clones”) orSOR� (“Japanese clones”) belong to CC A7 and are not precursorsto CC A6 strains as initially proposed (8). This analysis resulted ina novel evolutionary path that may offer insight into the evolu-tionary history of O157 populations.
ACKNOWLEDGMENTS
We thank Ruth E. Timme for her assistance with the annotation of theplasmids.
This project was supported by an appointment to L.V.R. through theResearch Fellowship Program for the Center for Food Safety and AppliedNutrition administered by the Oak Ridge Associated Universities througha contract with the FDA. This project was supported by the FDA FoodsProgram Intramural Funds.
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