This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Johnson, T. J.
Right arrow Articles by Nolan, L. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, T. J.
Right arrow Articles by Nolan, L. K.

 Previous Article  |  Next Article 

Journal of Bacteriology, January 2006, p. 745-758, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.745-758.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains

Timothy J. Johnson, Kylie E. Siek, Sara J. Johnson, and Lisa K. Nolan*

Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, 1802 Elwood Drive, VMRI #2, Iowa State University, Ames, Iowa 50011

Received 12 August 2005/ Accepted 20 October 2005


arrow
ABSTRACT
 
ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.


arrow
INTRODUCTION
 
Avian pathogenic Escherichia coli (APEC) strains are the etiologic agents of colibacillosis in birds, an important problem in the poultry industry (7). Along with uropathogenic E. coli (UPEC) and the E. coli strain causing neonatal meningitis or septicemias, APEC strains fall under the category of extraintestinal pathogenic E. coli (ExPEC) (39). ExPEC strains are characterized by the possession of virulence factors that enable their extraintestinal lifestyle and make them distinct from commensal and diarrheagenic E. coli strains (39). Among APEC strains, the iroBCDEN locus (11), shown to encode the siderophore salmochelin in Salmonella enterica (16), the aerobactin operon (51), and the yersiniabactin operon (21) are iron acquisition systems thought to contribute to virulence. Other putative APEC virulence factors include those contributing to complement resistance, such as the increased serum survival gene (iss) (31, 33, 37); tsh, the temperature-sensitive hemagglutinin gene (34); and the presence of ColV plasmids (37). In fact, it appears that large virulence plasmids, including ColV plasmids, are a defining feature of the APEC pathotype (37, 44).

ColV and ColV plasmids have interested scientists for many years, with Gratia first describing ColV as "principle V" in 1925 (53). ColV plasmids, which encode ColV production, typically range in size from 80 to 180 kb (53) and encode traits such as aerobactin production (51) and complement resistance (31). Unlike other colicins, ColV itself is a small protein that is exported from the cell and behaves more like a microcin, disrupting the formation of cell membrane potential required for energy production (53). The ColV operon consists of genes for ColV synthesis (cvaC) and ColV immunity (cvi) and two genes for ColV export (cvaA and cvaB) (14). Other traits that have been localized to APEC ColV plasmids include iss (22, 48), the aerobactin operon (19, 23, 49, 51), and tsh (10, 23, 49).

ColV plasmids have been long associated with E. coli virulence (53). However, it was found that the production of the bacteriocin colicin V (ColV), the namesake trait of these plasmids, is not itself directly responsible for this association with virulence (36). Therefore, other traits encoded by these plasmids are likely responsible for their contributions to virulence. To date, the nature of this association has not been fully understood.

Several studies have demonstrated a link between APEC virulence and the possession of ColV plasmids (12, 13, 15, 23, 49, 50). In a previous study, we described a large ColV plasmid, from an APEC isolate, possessing the ColV and aerobactin operons iss, tsh, and traT (23, 24). More recently, Tivendale and colleagues (49) described a similar plasmid occurring in an APEC isolate. Such plasmids appear to be widespread among APEC strains, as gene prevalence studies have shown that many of the genes found on ColV plasmids occur in a large percentage of APEC populations (12, 37). In addition, several studies have directly linked ColV plasmids with the ability to cause disease in production animals (45, 55). Despite the importance of these plasmids with regard to APEC virulence, little sequence data exist for them, hindering further attempts to determine the mechanisms of ColV plasmid-mediated virulence in APEC. In the present study, DNA sequencing was performed on an APEC ColV plasmid to facilitate future studies of similar plasmids and their contributions to APEC virulence. Additionally, populations of APEC and avian commensal E. coli were examined for this plasmid's genes of interest using multiplex PCR.


arrow
MATERIALS AND METHODS
 
Bacterial strains and plasmids. pAPEC-O2-ColV was originally derived from APEC O2 (O2:K2) (23), which was isolated from the joint of a chicken with colibacillosis. In a prior study, APEC O2 (23) was mated with E. coli DH5{alpha}, an avirulent plasmidless strain, and the resulting transconjugant was used as a source of pAPEC-O2-ColV for the present study. Colinearity was previously demonstrated between the donor and transconjugant using Southern hybridizations, PCR, and agarose gel electrophoresis (23). pAPEC-O2-ColV is a large, conjugative plasmid encoding aerobactin production, ColV production, and complement resistance. Additionally, pAPEC-O2-ColV contains sequences homologous to iss, tsh, and traT (23).

Isolates used for the gene prevalence studies were obtained from a variety of sources within the United States, including Georgia, Nebraska, North Dakota, and Minnesota. Of the 794 isolates in this study, 595 originated from sites of infection from birds diagnosed with colibacillosis (APEC), and the remaining 199 isolates were commensal isolates obtained from fecal swabs of apparently healthy chickens and turkeys.

The positive control strain used for multiplex PCR was APEC O2. E. coli DH5{alpha} was used as a negative control for all of the genes studied (40). All bacterial strains and subclones were stored at –70°C in brain heart infusion broth (Difco Laboratories, Detroit, MI) with 10% glycerol until use (41).

DNA isolation and preparation for PCR. pAPEC-O2-ColV DNA was initially obtained from a 1-liter culture grown overnight in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, MI) according to the method described previously by Wang and Rossman (52). Total DNA to be used as a template for PCR was obtained from APEC O2 and each of the 794 E. coli isolates using a boiling lysis procedure (22).

Shotgun library construction and sequencing. Plasmid DNA was sheared, concentrated, and desalted using standard protocols (40). DNA was end repaired (30 min, 15°C; 100-µl reaction mixture consisting of 2 µg sheared DNA, 15 U T4 DNA polymerase, 10 U E. coli DNA polymerase [MBI Fermentas, Vilnius, Lithuania], 500 µM each deoxynucleoside triphosphate, and 10 µl Yellow Tango buffer [MBI Fermentas]), desalted, and tailed with an extra A residue (30 min, 50°C; 100 µl reaction mixture consisting of 2 µg sheared DNA, 50 µM each dCTP, dGTP, and dTTP, 2 mM dATP, 20 U Taq polymerase [MBI Fermentas], and 10 µl Yellow Tango buffer). A-tailed DNA was then size fractionated by electrophoresis, and the 1.5- to 2.5-kb fraction was isolated and purified using standard methods (40) prior to cloning into pGEM-T (Promega, Madison, WI).

Shotgun sequencing was performed by MWG Biotech, Inc. (Hedersberg, Germany). Briefly, plasmid clones were grown for 20 h in 1.8 ml LB broth supplemented with 200 µg ml–1 ampicillin in deep-well boxes. Plasmid DNA was prepared on a RoboPrep2500 DNA-Prep-Robot (MWG Biotech, Ebersberg, Germany) using the NucleoSpin Robot-96 plasmid kit (Macherey & Nagel, Dueren, Germany) and sequenced from both ends with standard primers using BigDye Terminator chemistry (Applied Biosystems, Foster City, CA). The data were collected with ABI 3700 and ABI 3730xl capillary sequencers.

The Universal Genome Walker kit (BD Biosciences Clontech, Palo Alto, CA) was initially used to close remaining gaps by creating inverse primers extending away from known sequences, according to the manufacturer's instructions. Problematic gaps were also subjected to pooled PCR using the technique described previously by Tettelin et al. (48). Amplicons were visualized on a 1% Tris-acetate-EDTA agarose gel run at 9 V/cm for 75 min. Appropriate size markers were also run for comparative purposes. Bands were excised from gels using a clean razor blade, and DNA exposure to ethidium bromide and UV light was kept at a minimum during this procedure. Excised gel fragments were purified using the PCR Clean-up kit (Promega). Purified amplicons were ligated into the pGem-T vector using the T/A Cloning kit (Promega). Ligation products were transformed into competent E. coli JM109 cells (Promega), and transformants were selected on medium containing X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) (0.004%), IPTG (isopropyl-ß-D-thiogalactopyranoside) (0.5 mM), and ampicillin (100 µg/ml). White colonies were picked and screened for insert size with the Colony Fast-Screen kit (Epicenter Technologies, Madison, WI). PCR was used to verify the presence of the desired insert DNA. Several transformants containing appropriate inserts were selected for each primer-walking reaction to ensure at least eightfold sequencing coverage.

Assembly and annotation. Sequencing reads were assembled using SeqMan software from DNASTAR (Madison, WI). Open reading frames (ORFs) in the plasmid sequence were identified using GeneQuest from DNASTAR (Madison, WI), followed by manual inspection. Translated ORFs were then compared to known protein sequences using BLAST (NCBI, August 2005). Those with more than 25% identity, covering more than 60% of the matching protein sequence, were considered matches. Hypothetical proteins with more than 25% identity to one or more previously published proteins were classified as conserved hypothetical proteins, and ORFs with less than 25% identity to any published sequences were classified as hypothetical proteins. The G+C content of individual ORFs was analyzed using GeneQuest (DNASTAR). Insertion sequences (ISs) and repetitive elements were identified using IS FINDER (http://www-is.biotoul.fr/).

Gene prevalence studies. Previously, Rodriguez-Siek et al. (37) examined 451 APEC and 104 avian commensal E. coli isolates for the presence of traits associated with ExPEC virulence. The present study expanded upon that work by adding 144 APEC and 95 commensal E. coli isolates to the isolate set and by screening all 794 isolates for eight additional plasmid-associated genes. Isolates were examined for the presence of pAPEC-O2-ColV-associated genes using several multiplex PCR panels. The genes studied included iss; tsh; cvaA, cvaB, and cvaC of the ColV operon; iutA of the aerobactin operon; iroN of the salmochelin operon; sitA of the sit ABC iron transport operon; hlyF; ompT, a gene encoding an outer membrane protease (37); eitA and eitB (E. coli iron transport), genes of a putative ABC iron transport system; and etsA and etsB (E.coli transport system), genes of a putative ABC transport system contained within pAPEC-O2-ColV.

All primers, annealing temperatures, and expected amplicon sizes are listed in Table 1. Primers were obtained from Integrated DNA Technologies (Coralville, IA). Genes were amplified in three multiplex panels using a modified version of the multiplex PCR technique described previously by Rodriguez-Siek et al. (37, 38). PCR was performed with Amplitaq Polymerase Gold (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Conditions used for PCR were as follows: 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 60°C, and 3 min at 72°C; and a final extension step of 10 min at 72°C. Amplicons were visualized on 2.0% Tris-acetate-EDTA agarose gels alongside a 1-kb ladder (Promega). Reactions were performed three times, and if an amplicon of the predicted size was produced in two of the three reactions, the isolate was considered positive for that gene.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primers used in gene prevalence studies

Statistical analysis. The null hypothesis that the proportion of APEC isolates possessing each gene examined was equal to the proportion of avian commensal E. coli isolates containing the same gene was tested using a Z test on the difference between the proportions (46). Additionally, this test was used to examine codon usage between genes of the putative virulence region of pAPEC-O2-ColV and Escherichia coli K-12 strain MG1655 (3). The {chi}2 test was used for a univariate analysis of the significance of associations between two genes occurring in APEC (46). Gene pairs were classified as associated if they possessed a statistically significant (P ≤ 0.05) {chi}2 value and as highly associated if they possessed a P value of ≤0.0001.


arrow
RESULTS
 
Sequencing of pAPEC-O2-ColV. The focus of this study was pAPEC-O2-ColV, a ColV plasmid occurring in APEC strain O2. In addition to pAPEC-O2-ColV, APEC O2 also possesses pAPEC-O2-R, a 101-kb multidrug resistance plasmid that was sequenced in a previous study (25). Previously, pAPEC-O2-ColV was cotransferred with pAPEC-O2-R into the plasmidless, avirulent strain E. coli DH5{alpha} (Fig. 1), resulting in a transconjugant showing an increase in complement resistance and virulence towards chick embryos compared to the recipient strain (23). The recipient strain that acquired APEC O2's plasmids also became resistant to ampicillin, tetracycline, streptomycin, trimethoprim, a quaternary ammonium compound, sulfamethoxazole, and silver nitrate, all of which are encoded on pAPEC-O2-R (23, 24). It was this multidrug-resistant transconjugant, containing both APEC O2 plasmids, that served as a source of the pAPEC-O2-ColV DNA used in the present study.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 1. Agarose gel electrophoresis of supercoiled plasmid DNA from the donor strain, APEC O2 (lane 1), E. coli DH5{alpha}, the recipient strain (lane 2), and their transconjugant (lane 3). Note that the donor and transconjugant contain pAPEC-O2-R and pAPEC-O2-ColV.

Approximately 2,000 shotgun clones of pAPEC-O2-ColV were arrayed, sequenced, and assembled using the SeqMan program contained within the LaserGene package (DNASTAR).Assembly and subsequent gap closure resulted in the generation of three contiguous sequences: a 93,609-bp region containing numerous virulence-associated genes (Table 2 and Fig. 2), a 48,458-bp region encompassing the full transfer region of pAPEC-O2-ColV (Table 3), and a 37,428-bp region containing genes mostly encoding hypothetical proteins of unknown function (Table 4). The sizes of the three contiguous sequences generated totaled 179,495 bp. Several efforts were made to close remaining gaps between contiguous sequences, including the use of pooled PCR with inverse primers extending away from the ends of the contiguous sequences, long-range PCR in an effort to span gaps and repetitive elements, and genomic walking from the ends of the contiguous sequences. Regardless of the method used, large identical repetitive elements prevented total gap closure. Restriction maps, generated from study of similar ColV plasmids (1, 53), were used to orient the contiguous sequences and close the remaining gaps. Based on all these data, a circular map of pAPEC-O2-ColV was created (Fig. 3), but PCR efforts to close the final three gaps, all of which involved IS1 elements and their flanking sequences, were unsuccessful.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Predicted coding sequences of the putative virulence region of pAPEC-O2-ColV



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Drawing of the putative virulence region of pAPEC-O2-ColV, drawn to scale. The ruler below the image indicates sizes in kilobase pairs. The locations of primers used for PCR are shown immediately below the ruler, and below that are prevalences of the individual genes of the region among APEC strains. The layers of dotted lines are the prevalences of groupings of virulence region genes among APEC strains. For instance, sitA and iutA occur individually in 86% and 80% of the APEC strains tested, respectively; they occur together in 74% of the APEC strains tested. However, sitA, iutA, and hlyF only occur together in 68% of the APEC strains tested. Based on these prevalence data, it appears that this virulence region is composed of two subregions, one that is conserved among the APEC strains used in this study and the other that is more variable in occurrence. The divide between these two subregions appears to occur within the cvaB gene. Genes are color coded as follows: orange, sit operon; red-orange, aerobactin operon; light blue, repA; dark blue, hlyF and ompT; maroon, etsABC; red, iss; purple, salmochelin operon; green, ColV operon; blue, tsh; turquoise, eitABCD; yellow, mobile elements.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Predicted coding sequences of the transfer region of pAPEC-O2-ColV


View this table:
[in this window]
[in a new window]
  		TABLE 4. Predicted coding sequences of the hypothetical region of pAPEC-O2-ColV



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3. Circular genetic map of pAPEC-O2-ColV, drawn to scale. Arrows indicate predicted genes and their directions of transcription. Yellow arrows indicate virulence-associated genes. Blue arrows indicate genes involved in plasmid transfer and maintenance. Red arrows indicate genes involved in plasmid replication. Gray arrows indicate genes of unknown function. Black arrows indicate mobile genetic elements. Orange slashes indicate gaps in contiguous sequence that were unable to be resolved due to IS1 elements.

The 93-kb putative virulence region of pAPEC-O2-ColV was found to contain tsh, a temperature-sensitive hemagglutinin (34); the ColV operon, encoding ColV production (14); iss, the increased serum survival gene involved in complement resistance (18, 23, 33); ompT, an outer membrane protease (37); and hlyF, a putative hemolysin previously identified in an APEC strain (GenBank accession no. AF155222) (Table 2). It also contained several operons associated with iron acquisition including the salmochelin operon, a siderophore iron acquisition system (16); the aerobactin operon, another siderophore system (6); and the sit operon, an ABC transport system (57). Other genes not previously identified as occurring in APEC were also found within this contiguous sequence, including etsA and etsB (E. coli transport system, a novel set of genes identified in this study), genes of a putative ABC transport system; shiF and shiG, genes previously found on a pathogenicity island (PAI) of Shigella flexneri (30); and four genes, eitA to eitD (E. coli iron transport), also novel genes identified in this study, that may encode a putative iron uptake system.

The F-like transfer region of pAPEC-O2-ColV spanned 31,911 bp and contained 30 genes (Table 3). A second replicon of pAPEC-O2-ColV that closely resembles the RepFIIA plasmid replicon (GenBank accession no. M16167) separated the F-like transfer region from the putative virulence region on its 5' end. On the 3' end of the transfer region were approximately 38 kb of genes, encoding hypothetical proteins or conserved hypothetical proteins, for which no functional assignment was available. Overall, the three contiguous sequences of pAPEC-O2-ColV contained 201 predicted ORFs (Tables 2 to 4). Of these coding regions, 47% were found to be of unknown function and 25% were ORFs sharing no significant identity with any available database proteins.

The putative virulence region of pAPEC-O2-ColV was found to begin with the sit ABC transport system, which was followed by the iutABCD and iutA genes of the aerobactin operon and then the RepFIB replicon, containing the repA gene (Fig. 2) (42). Adjacent to the RepFIB region on its 3' end were the insertion sequence IS1, a site-specific integrase, and etsABC, three genes novel to APEC and sharing protein identity with a putative ABC transport system found in Shewanella oneidensis (17) (Table 2). Following etsABC were an assortment of intact and partial IS elements, including IS4 and IS2, followed by iss and the iroBCDEN genes of the salmochelin operon. Adjacent to the salmochelin operon on its 3' end were the cvaABC and cvi genes of the ColV operon and tsh. tsh was surrounded by mobile genetic elements, including a large putative transposase on its 5' end and IS911, IS30, IS91, and an invertase on its 3' end. Following these mobile elements on the 3' end of tsh were the eitABCD genes, novel to APEC and sharing protein identity with a putative ABC iron transport system from the plant pathogen Pseudomonas syringae (4). An intact ColE2 immunity gene, a partial ColE2 structural gene, and remnants of an IS629 element flanked this system on its 3' end.

Overall, this putative virulence region was found to encode two siderophore systems, three putative ABC transport systems, and ColV production and was found to contain iss, hlyF, ompT, tsh, and the RepFIB replicon. Thus, pAPEC-O2-ColV appears to be a member of the IncFIB incompatibility group, based upon BLAST homology and alignment with proteins of the RepFIB replicon. The overall G+C content of the cluster was 48%. Analysis of individual ORFs within this putative virulence region revealed that the 45-kb region from hlyF through cvi possessed a G+C content of 46%, and its 5'- and 3'-flanking regions possessed G+C contents of 52% (Fig. 4).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4. G+C content of individual ORFs within the 93.6-kb virulence cluster of pAPEC-O2-ColV. Dashed lines indicate the average G+C contents of regions of the virulence cluster. Three regions could be discerned based on proximity, gene prevalence, and G+C content (Table 4). The first, running from sitA through int, had an average G+C ratio of 52%. The second region from hlyF through cvi had an average G+C ratio of 46%. The final region, running from ORF67 through IS629, had an average G+C content of 52%. The conserved portion of the virulence cluster contained these first two regions, while the variable portion of the cluster was composed of part of the second region and all of the third region.

Comparative genomics of cluster-related sequences revealed some interesting deviations from previously published patterns. For instance, the aerobactin operon was found to be chromosomally integrated in other pathogens, such as within the SHI-2 and SHI-3 PAIs of Shigella strains (30, 35) and within the chromosome of UPEC strain CFT073 (54). Similarly, the sit iron transport system also appeared to be chromosomally located in other strains, including within a PAI of Salmonella (20) and on the chromosome of UPEC strain CFT073 (54). Comparison of the virulence cluster with previously published sequences from a UPEC transmissible plasmid, p300 (47), and PAI III from UPEC strain 536 (8) revealed that the salmochelin operon was conserved among all three regions. iss was found near the salmochelin operon in a highly conserved arrangement within p300, and tsh and remnants of the ColV operon were also found within portions of PAI III536. Codon usage analysis was performed to test the hypothesis that different patterns of usage occur between genes of the E. coli chromosome andgenes of the putative virulence region of pAPEC-O2-ColV. When frequency distributions for each codonwere examined, 50 out of the 62 codons in pAPEC-O2-ColV's putative virulence region had distributions significantly different from those in E. coli K-12 strain MG1655. A bias was also observed towards rare codons in genes of the putative virulence region, with higher frequencies observed towards AUA (Ile), AGA (Arg), CGA (Arg), CGG (Arg), and CCC (Pro).

Prevalence of plasmid-related genes in avian E. coli. Multiplex PCR was used to examine 595 APEC and 199 avian commensal E. coli strains for the presence of 13 genes found within the putative virulence region of pAPEC-O2-ColV. Results indicated that all of the genes examined were significantly more likely to be found among the APEC isolates than among the commensal isolates (Table 5). Representative genes of the salmochelin, sit, and aerobactin operons, as well as iss and hlyF, occurred in 80% or more of APEC isolates; the putative iron transport genes etsA and etsB occurred in 74.3% of the APEC isolates examined; the putative ABC iron transport system genes eitA and eitB occurred in 38.8% of the APEC isolates examined; cvaA and the 5' end of cvaB occurred in 72.5% and 73.9% of the APEC isolates; and cvaC, tsh, ompT, and the 3' end of cvaB occurred in more than 60% of the APEC isolates. Among the avian commensal E. coli isolates, the least prevalent gene sequences were the 3' end of cvaB as well as cvaC, occurring approximately 19% of the time. iroN, hlyF, iss, etsA, etsB, eitA, eitB, ompT, cvaA, and the 5' end of cvaB all occurred approximately one-quarter of the time among the commensal isolates. iutA, tsh, and sitA occurred 34%, 41%, and 48% of the time, respectively. None of the genes surveyed occurred more than 50% of the time among avian commensal E. coli isolates, and all of the genes surveyed were found in APEC isolates significantly more often than in the commensal isolates.


View this table:
[in this window]
[in a new window]
  		TABLE 5. Comparison of gene prevalence between APEC and avian commensal E. coli isolates

Gene prevalences were also plotted along the map of the putative virulence region (Fig. 2) to determine if a pattern in the occurrence of these genes could be discerned. Based on the resulting plot, it appeared that the putative virulence region could be split into "conserved" and "variable" portions. The "conserved" portion spanned the area from sitA through the 5' end of cvaB. All of the genes of this region screened via PCR occurred individually in more than 67% of the APEC isolates tested and together in 59% of the APEC strains tested. The remainder of the putative virulence region, running from the 3' end of cvaB through eitA, appeared to be more variable among APEC isolates. The genes within this portion of the putative virulence region occurred less often individually than those of the "conserved" portion, and they occurred together in only 26% of the APEC isolates. Additionally, a univariate analysis of the significance of associations between gene pairs was performed for all genes assayed with multiplex PCR. Based on resulting P values obtained using a {chi}2 plot, gene pairs were defined as unassociated (P > 0.05), significantly associated (P≤ 0.05), or highly associated (P ≤ 0.0001) (Table 6). Out of 105 possible gene combinations, 84 were classified as highly associated, 16 were classified as significantly associated, and only 5 were classified as unassociated. All of the gene combinations that were not highly associated involved genes of the "variable" portion of the putative virulence region of pAPEC-O2-ColV.


View this table:
[in this window]
[in a new window]
  		TABLE 6. Correlation of gene pairs among 595 APEC strains isolated from poultry

In an effort to explain the differences in prevalence between the "conserved" and "variable" portions of the putative virulence region, the sequence was examined for mobile elements positioned in such a way that they could render the variable region mobile and subject to loss from the cluster. It was not readily apparent from this examination how insertion sequence-mediated transposition might have produced the observed gene prevalences (Fig. 2 and Table 7).


View this table:
[in this window]
[in a new window]
  		TABLE 7. Regions of the putative virulence region of pAPEC-O2-ColV delineated by proximity, similarity in gene prevalence, and G+C content

So too, it was thought that a G+C analysis of these regions might identify regions of the putative virulence region that share a common origin (Fig. 4). The overall G+C content for the contiguous sequences of pAPEC-O2-ColV was 49.2%. The G+C content of the putative virulence region was 48%. Based on G+C analysis of individual ORFs within the putative virulence region, three distinct regions could be discerned. These regions included one region running from sitA through int, with an average G+C content of 52%; one region running from hlyF through cvi, with an average G+C content of 46%; and a third region running from a putative insertion sequence on the 3' end of cvi through IS629, with an average G+C content of 52%. The first two regions composed the "conserved" portion of pAPEC-O2-ColV's putative virulence region, while a part of the second region and all of the third region comprised the region's "variable" portion. Therefore, it appeared that the conserved portion of the putative virulence region may be composed of two regions of diverse origins.


arrow
DISCUSSION
 
ColV plasmids have long been associated with the virulence of E. coli in general (2, 45, 53) and APEC in particular (9, 10, 13, 15, 23, 24, 49, 55, 56). Interestingly, their association with virulence is not due to their namesake trait of ColV production (36), indicating that genes other than those involved in ColV production must be responsible for this association. Remarkably, despite the long recognition of the association of ColV plasmids and virulence, a ColV plasmid has never been sequenced in its entirety. Here, the first sequence of a ColV plasmid is presented, revealing a 93-kb putative virulence region containing numerous known or putative virulence genes that may account for the association of ColV plasmids with virulence. This region contains several genes or operons previously described as putative APEC virulence factors, including tsh (34), the salmochelin operon (11), and iss (18, 23, 31, 37, 49). This cluster also contains three iron acquisition and transport systems in addition to the salmochelin operon. The sit operon is an ABC transport system, involved in the metabolism of iron and manganese, originally identified in Salmonella enterica serovar Typhimurium (57) and more recently identified in APEC using genomic subtractive hybridization and signature-tagged mutagenesis (28, 43). However, this study is the first report of sit occurring near the aerobactin operon on a ColV plasmid. Two additional putative ABC transport systems are found within the cluster, eitABCD and etsABC. This is also the first report of these systems occurring in E. coli. eitABCD shares low translated protein identity to an iron transport system from the plant pathogen Pseudomonas syringae (4), and etsABC shares identity to an ABC transport system found in Shewanella oneidensis (17). Further work is in progress to determine the functionality of these putative ABC transporters. This putative virulence region also possesses several other genes whose roles have not yet been determined, including shiF, shiG, hlyF, ompT, and several genes which, when translated, encode hypothetical proteins.

Of particular interest is the presence of four sets of genes previously associated with iron acquisition and transport within this 93-kb putative virulence region. Such apparent redundancy suggests that iron acquisition plays an important role in APEC virulence. In addition to the potential iron acquisition and transport systems of APEC O2 presented in this study, this strain also possesses the fyuA and irp2 genes of the yersiniabactin operon and ireA, both of which have been associated with iron acquisition and ExPEC virulence (21, 36, 37). In order to understand APEC's virulence mechanisms, it would seem important to determine if these iron acquisition systems really areredundant or if they have nonoverlapping, specific purposes, such as ensuring that E. coli has an adequate iron supply throughout the different stages of infection. For example, it has been suggested that the sit operon only acts as an iron uptake system during intracellular infection, because this is the only host location in which iron is at a concentration suitable for the ABC transport system to function effectively (5). However, sit, like many of these systems, may be multifunctional, effecting transport of different compounds, such as manganese, at various stages of infection (5). Further studies to assess these iron acquisition and transport genes, their functionality, the conditions of their expression, and their importance to APEC virulence at all stages of infection could prove very helpful in understanding the pathogenesis of avian colibacillosis.

While many individual APEC virulence factors have been identified on large plasmids (10, 19, 49), this is the first report, to our knowledge, of a plasmid-encoded putative virulence region among APEC strains or on a ColV plasmid. Previously, Rodriguez-Siek et al. (37) examined 451 APEC and 104 commensal E. coli isolates for the possession of more than 35 different ExPEC virulence-associated genes. Among the genes examined were iss, cvaC, tsh, sitA, iutA, ompT, and iroN, all found on pAPEC-O2-ColV. The present study expanded that research through the addition of isolates and gene targets to the screening procedures. The genes added to this study included those of the etsABCD cluster, the eitABC cluster, the ColV operon, and hlyF. Many of the genes of this region, including iss, iroN, iutA, sitA, and hlyF, occurred in more than 80% of the APEC isolates and in only about 25% of the avian commensal E. coli isolates examined (Table 5). These results are striking and support the idea that this putative virulence region may be a widespread characteristic of APEC. However, this region does not appear to be intact in all APEC strains, as the prevalence studies show that genes within the "variable" portion of this region (the 3' ends of cvaB, cvaC, tsh, eitA, and eitB) occur less often than genes of the "conserved" portion of the region, including sitA, iroN, iss, iutA, hlyF, etsA, etsB, cvaA, and the 5' end of cvaB. Also, it is possible that some genes of this putative virulence region might be found elsewhere in the APEC genome, such as on non-ColV plasmids or within PAIs on the bacterial chromosome. Indeed, alternative locations for some of these genes have been identified in UPEC strains. For instance, UPEC strain 536 contains PAI III536, which shows some similarity to pAPEC-O2-ColV in both sequence and gene arrangement, leading us to hypothesize that this virulence cluster might be located on the bacterial chromosome in some APEC isolates (8). Interestingly, this UPEC PAI contains the salmochelin operon, tsh, and remnants of the ColV operon, suggesting the possibility that this PAI originated as a ColV plasmid that integrated into the chromosome in a fashion similar to that described previously by Oelschlaeger et al. (32). Also, the iro-iss region of pAPEC-O2-ColV shows 99.9% sequence identity with a UPEC non-ColV plasmid (47), further supporting the idea that the cluster can occur in different locations in the E. coli genome. Indeed, previous studies have demonstrated that ColV plasmids readily integrate into the bacterial chromosome to form Hfr strains and that these cointegrates lose the ability to produce ColV (26). Results of our gene prevalence studies also support this possibility, revealing "conserved" and "variable" portions of the putative virulence region that join within the cvaB gene. Analysis of UPEC PAI III536 showed that it contained remnants of the ColV operon and that it contained a truncated cvaB gene. These results, along with the above-described observations, cause us to speculate that cvaB might be a breakpoint during the integration of ColV-associated sequences into other locations in the bacterial genome. Indeed, our gene prevalence data indicate that cvaA and the 5' end of cvaB occur among APEC isolates at rates similar to that of the "conserved" portion of the putative virulence region, while the 3' end of cvaB and its downstream genes occur among APEC isolates at much lower rates (Fig. 2).

Thus, ColV plasmids might be an evolutionary intermediate for the development of chromosomal PAIs that contain APEC virulence factors (26, 32). Gene prevalence data obtained from this study and that of Rodriguez-Siek et al. (37) support this model of APEC evolution. That is, several isolates can be found that might serve as examples for each stage of development from ColV-encoded virulence traits through PAI-encoded virulence traits. For example, among our collection of APEC isolates, some isolates containing cvaC of the ColV operon and all other virulence genes sought in this study were found, suggesting that these isolates contain plasmids similar to pAPEC-O2-ColV. Also, examples of isolates possessing all of the genes in this study except those of the ColV operon are also found, suggesting that these genes may occur on non-ColV plasmids or within the bacterial chromosome. Isolates can also be found among the APEC strains with PAI III536-like patterns. That is, there are APEC strains containing the salmochelin operon, tsh, and cvaA and the 5' end of cvaB but lacking the 3' end of cvaB and other components of the putative virulence region.

With regard to characterizing the APEC pathotype, of particular interest is the "conserved" portion of the putative virulence region encompassing sitABCD, the aerobactin and salmochelin operons, hlyF, the etsABC transport system, ompT, iss, cvaA, and the 5' portion of cvaB. Selected genes within this span of sequence appear to be highly conserved among APEC isolates, occurring in about 75% or more of the APEC isolates examined. This conserved portion of this putative APEC virulence region may be a defining feature of the APEC pathotype and perhaps a requirement for APEC virulence, regardless of whether or not it occurs on ColV plasmids. Further study will be needed to assess the role of this region in the pathogenesis of avian colibacillosis.

The transfer region of pAPEC-O2-ColV flanks the 3' end of the putative virulence cluster and bears strong similarities to the transfer region of the F plasmid (27). This region is found on the 3' end of an IS1 element following two genes involved in plasmid maintenance and stability, psiA and psiB (29). Downstream of this region, and separating it from the 5' end of pAPEC-O2-ColV's virulence cluster, is a 45-kb stretch of DNA that bears no significant matches within the GenBank databases. This region is noteworthy due to its novel nature, and further work is required to determine the functions of the hypothetical proteins it encodes and their role, if any, in APEC virulence.

In sum, DNA sequencing of pAPEC-O2-ColV, a ColV virulence plasmid occurring in APEC O2, revealed the location of many APEC virulence genes (putative or known), several genes or operons novel to E. coli, and a variety of mobile genetic elements within a putative 93-kb virulence cluster. Portions of this putative virulence region commonly occurred among APEC isolates but not avian commensal E. coli isolates. Genes occurring in the "conserved" portion of this region may occur in the absence of an intact ColV operon in some avian E. coli isolates, which may provide hints as to the evolutionary development of ColV plasmids and chromosomal PAIs. The presence of this virulence cluster appears to discriminate most APEC isolates from commensal E. coli isolates, indicating that this region may prove useful as a target for identification of pathogenic E. coli. Genes within this region likely account for the long association of ColV plasmids with virulence.

The DNA sequence of pAPEC-O2-ColV also contained an intact F-like transfer region and a 45-kb region of novel DNA encoding a number of hypothetical proteins. pAPEC-O2-ColV possesses two plasmid replicons, RepFIB and RepFIIA, as reported elsewhere previously (1). In addition to encoding ColV production, the plasmid also contains an immunity gene towards the bacteriocin ColE2. This plasmid also possesses five copies of the insertion sequence IS1 and two copies of IS2, which likely play an important role in the plasmid's evolution. Overall, this 180-kb ColV plasmid is a mosaic of virulence genes, novel genes, transfer genes, and mobile genetic elements. Further work is needed to determine the roles that certain components of this plasmid have in APEC virulence.


arrow
ACKNOWLEDGMENTS
 
We thank Soren Schubert from the Max von Pettenkofer Institut and Shelley Payne from the University of Texas for providing control strains for these studies.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, 1802 Elwood Drive, VMRI #2, Iowa State University, Ames, IA 50011. Phone: (515) 294-3470. Fax: (515) 294-3839. E-mail: lknolan{at}iastate.edu. Back


arrow
REFERENCES
 
    1
  1. Ambrozic, J., A. Ostroversnik, M. Starcic, I. Kuhar, M. Grabnar, and D. Zgur-Bertok. 1998. Escherichia coli ColV plasmid pRK100: genetic organization, stability and conjugal transfer. Microbiology 144:343-352.[Abstract/Free Full Text]
    2. 2
  2. Binns, M. M., D. L. Davies, and K. G. Hardy. 1979. Cloned fragments of the plasmid ColV,I-K94 specifying virulence and serum resistance. Nature 279:778-781.[CrossRef][Medline]
    3. 3
  3. Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-1474.[Abstract/Free Full Text]
    4. 4
  4. Buell, C. R., V. Joardar, M. Lindeberg, J. Selengut, I. T. Paulsen, M. L. Gwinn, R. J. Dodson, R. T. Deboy, A. S. Durkin, J. F. Kolonay, R. Madupu, S. Daugherty, L. Brinkac, M. J. Beanan, D. H. Haft, W. C. Nelson, T. Davidsen, N. Zafar, L. Zhou, J. Liu, Q. Yuan, H. Khouri, N. Fedorova, B. Tran, D. Russell, K. Berry, T. Utterback, S. E. Van Aken, T. V. Feldblyum, M. D'Ascenzo, W. L. Deng, A. R. Ramos, J. R. Alfano, S. Cartinhour, A. K. Chatterjee, T. P. Delaney, S. G. Lazarowitz, G. B. Martin, D. J. Schneider, X. Tang, C. L. Bender, O. White, C. M. Fraser, and A. Collmer. 2003. The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. USA 100:10181-10186.[Abstract/Free Full Text]
    5. 5
  5. Crosa, J. H., A. R. Mey, and S. M. Payne (ed.). 2004. Iron transport in bacteria. ASM Press, Washington, D.C.
    6. 6
  6. de Lorenzo, V., and J. B. Neilands. 1986. Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli. J. Bacteriol. 167:350-355.[Abstract/Free Full Text]
    7. 7
  7. Dho-Moulin, M., and J. M. Fairbrother. 1999. Avian pathogenic Escherichia coli (APEC). Vet. Res. 30:299-316.[Medline]
    8. 8
  8. Dobrindt, U., G. Blum-Oehler, G. Nagy, G. Schneider, A. Johann, G. Gottschalk, and J. Hacker. 2002. Genetic structure and distribution of four pathogenicity islands (PAI I536 to PAI IV536) of uropathogenic Escherichia coli strain 536. Infect. Immun. 70:6365-6372.[Abstract/Free Full Text]
    9. 9
  9. Doetkott, D. M., L. K. Nolan, C. W. Giddings, and D. L. Berryhill. 1996. Large plasmids of avian Escherichia coli isolates. Avian Dis. 40:927-930.[CrossRef][Medline]
    10. 10
  10. Dozois, C. M., M. Dho-Moulin, A. Bree, J. M. Fairbrother, C. Desautels, and R. Curtiss III. 2000. Relationship between the tsh autotransporter and pathogenicity of avian Escherichia coli and localization and analysis of the tsh genetic region. Infect. Immun. 68:4145-4154.[Abstract/Free Full Text]
    11. 11
  11. Dozois, C. M., F. Daigle, and R. Curtiss III. 2003. Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc. Natl. Acad. Sci. USA 100:247-252.[Abstract/Free Full Text]
    12. 12
  12. Ewers, C., T. Janssen, S. Kiessling, H. C. Philipp, and L. H. Wieler. 2004. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet. Microbiol. 104:91-101.[CrossRef][Medline]
    13. 13
  13. Gibbs, P. E., J. J. Maurer, L. K. Nolan, and R. E. Wooley. 2003. Prediction of chicken embryo lethality with the avian Escherichia coli traits complement resistance, colicin V production, and presence of the increased serum survival gene cluster (iss). Avian Dis. 47:370-379.[CrossRef][Medline]
    14. 14
  14. Gilson, L., H. K. Mahanty, and R. Kolter. 1987. Four plasmid genes are required for colicin V synthesis, export, and immunity. J. Bacteriol. 169:2466-2470.[Abstract/Free Full Text]
    15. 15
  15. Ginns, C. A., M. L. Benham, L. M. Adams, K. G. Whithear, K. A. Bettelheim, B. S. Crabb, and G. F. Browning. 2000. Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid. Infect. Immun. 68:1535-1541.[Abstract/Free Full Text]
    16. 16
  16. Hantke, K., G. Nicholson, W. Rabsch, and G. Winkelmann. 2003. Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN. Proc. Natl. Acad. Sci. USA 100:3677-3682.[Abstract/Free Full Text]
    17. 17
  17. Heidelberg, J. F., I. T. Paulsen, K. E. Nelson, E. J. Gaidos, W. C. Nelson, T. D. Read, J. A. Eisen, R. Seshadri, N. Ward, B. Methe, R. A. Clayton, T. Meyer, A. Tsapin, J. Scott, M. Beanan, L. Brinkac, S. Daugherty, R. T. DeBoy, R. J. Dodson, A. S. Durkin, D. H. Haft, J. F. Kolonay, R. Madupu, J. D. Peterson, L. A. Umayam, O. White, A. M. Wolf, J. Vamathevan, J. Weidman, M. Impraim, K. Lee, K. Berry, C. Lee, J. Mueller, H. Khouri, J. Gill, T. R. Utterback, L. A. McDonald, T. V. Feldblyum, H. O. Smith, J. C. Venter, K. H. Nealson, and C. M. Fraser. 2002. Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis. Nat. Biotechnol. 20:1118-1123.[CrossRef][Medline]
    18. 18
  18. Horne, S. M., S. J. Pfaff-McDonough, C. W. Giddings, and L. K. Nolan. 2000. Cloning and sequencing of the iss gene from a virulent avian Escherichia coli. Avian Dis. 44:179-184.[CrossRef][Medline]
    19. 19
  19. Ike, K., K. Kawahara, H. Danbara, and K. Kume. 1992. Serum resistance and aerobactin iron uptake in avian Escherichia coli mediated by conjugative 100-megadalton plasmid. J. Vet. Med. Sci. 54:1091-1098.[Medline]
    20. 20
  20. Janakiraman, A., and J. M. Slauch. 2000. The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of Salmonella typhimurium. Mol. Microbiol. 35:1146-1155.[CrossRef][Medline]
    21. 21
  21. Janben, T., C. Schwarz, P. Preikschat, M. Voss, H. C. Philipp, and L. H. Wieler. 2001. Virulence-associated genes in avian pathogenic Escherichia coli (APEC) isolated from internal organs of poultry having died from colibacillosis. Int. J. Med. Microbiol. 291:371-378.[CrossRef][Medline]
    22. 22
  22. Johnson, J. R., and J. J. Brown. 1996. A novel multiply primed polymerase chain reaction assay for identification of variant papG genes encoding the Gal(alpha 1-4)Gal-binding PapG adhesins of Escherichia coli. J. Infect. Dis. 173:920-926.[Medline]
    23. 23
  23. Johnson, T. J., C. W. Giddings, S. M. Horne, P. S. Gibbs, R. E. Wooley, J. Skyberg, P. Olah, R. Kercher, J. S. Sherwood, S. L. Foley, and L. K. Nolan. 2002. Location of increased serum survival gene and selected virulence traits on a conjugative R plasmid in an avian Escherichia coli isolate. Avian Dis. 46:342-352.[CrossRef][Medline]
    24. 24
  24. Johnson, T. J., J. Skyberg, and L. K. Nolan. 2004. Multiple antimicrobial resistance region of a putative virulence plasmid from an Escherichia coli isolate incriminated in avian colibacillosis. Avian Dis. 48:351-360.[CrossRef][Medline]
    25. 25
  25. Johnson, T. J., K. E. Siek, S. J. Johnson, and L. K. Nolan. 2005. DNA sequence and comparative genomics of pAPEC-O2-R, an avian pathogenic Escherichia coli transmissible R plasmid. Antimicrob. Agents Chemother. 49:4681-4688.[Abstract/Free Full Text]
    26. 26
  26. Kahn, P. L. Isolation of high-frequency recombining strains from Escherichia coli containing the V colicinogenic factor. J. Bacteriol. 96:205-214.
    27. 27
  27. Lawley, T. D., W. A. Klimke, M. J. Gubbins, and L. S. Frost. 2003. F factor conjugation is a true type IV secretion system. FEMS Microbiol. Lett. 224:1-15.[CrossRef][Medline]
    28. 28
  28. Li, G., C. Laturnus, C. Ewers, and L. H. Wieler. 2005. Identification of genes required for avian Escherichia coli septicemia by signature-tagged mutagenesis. Infect. Immun. 73:2818-2827.[Abstract/Free Full Text]
    29. 29
  29. Loh, S., R. Skurray, J. Celerier, M. Bagdasarian, A. Bailone, and R. Devoret. 1990. Nucleotide sequence of the psiA (plasmid SOS inhibition) gene located on the leading region of plasmids F and R6-5. Nucleic Acids Res. 18:4597.[Free Full Text]
    30. 30
  30. Moss, J. E., T. J. Cardozo, A. Zychlinsky, and E. A. Groisman. 1999. The selC-associated SHI-2 pathogenicity island of Shigella flexneri. Mol. Microbiol. 33:74-83.[CrossRef][Medline]
    31. 31
  31. Nolan, L. K., S. M. Horne, C. W. Giddings, S. L. Foley, T. J. Johnson, A. M. Lynne, and J. Skyberg. 2003. Resistance to serum complement, iss, and virulence of avian Escherichia coli. Vet. Res. Commun. 27:101-110.[Medline]
    32. 32
  32. Oelschlaeger, T. A., U. Dobrindt, and J. Hacker. 2002. Pathogenicity islands of uropathogenic E. coli and the evolution of virulence. Int. J. Antimicrob. Agents 19:517-521.[CrossRef][Medline]
    33. 33
  33. Pfaff-McDonough, S. J., S. M. Horne, C. W. Giddings, J. O. Ebert, C. Doetkott, M. H. Smith, and L. K. Nolan. 2000. Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis. Avian Dis. 44:23-33.[CrossRef][Medline]
    34. 34
  34. Provence, D. L., and R. Curtiss III. 1994. Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. Infect. Immun. 62:1369-1380.[Abstract/Free Full Text]
    35. 35
  35. Purdy, G. E., and S. M. Payne. 2001. The SHI-3 iron transport island of Shigella boydii 0-1392 carries the genes for aerobactin synthesis and transport. J. Bacteriol. 183:4176-4182.[Abstract/Free Full Text]
    36. 36
  36. Quackenbush, R. L., and S. Falkow. 1979. Relationship between colicin V activity and virulence in Escherichia coli. Infect. Immun. 24:562-564.[Abstract/Free Full Text]
    37. 37
  37. Rodriguez-Siek, K. E., C. W. Giddings, C. Doetkott, T. J. Johnson, and L. K. Nolan. 2005. Characterizing the APEC pathotype. Vet. Res. 36:241-256.[CrossRef][Medline]
    38. 38
  38. Rodriguez-Siek, K. E., C. W. Giddings, C. Doetkott, T. J. Johnson, M. K. Fakhr, and L. K. Nolan. 2005. Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis. Microbiology 151:2097-2110.[Abstract/Free Full Text]
    39. 39
  39. Russo, T. A., and J. R. Johnson. 2000. Proposal for a new inclusive designation for extraintestinal pathogenic isolates of Escherichia coli: ExPEC. J. Infect. Dis. 181:1753-1754.[CrossRef][Medline]
    40. 40
  40. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    41. 41
  41. Sanderson, K. E., and D. R. Zeigler. 1991. Storing, shipping, and maintaining records on bacterial strains. Methods Enzymol. 204:248-264.[CrossRef][Medline]
    42. 42
  42. Saul, D., A. J. Spiers, J. McAnulty, M. G. Gibbs, P. L. Bergquist, and D. F. Hill. 1989. Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids. J. Bacteriol. 171:2697-2707.[Abstract/Free Full Text]
    43. 43
  43. Schouler, C., F. Koffmann, C. Amory, S. Leroy-Setrin, and M. Moulin-Schouleur. 2004. Genomic subtraction for the identification of putative new virulence factors of an avian pathogenic Escherichia coli strain of O2 serogroup. Microbiology 150:2973-2984.[Abstract/Free Full Text]
    44. 44
  44. Skyberg, J., S. M. Horne, C. W. Giddings, R. E. Wooley, P. S. Gibbs, and L. K. Nolan. 2003. Characterizing avian Escherichia coli isolates with multiplex polymerase chain reaction. Avian Dis. 47:1441-1447.[CrossRef][Medline]
    45. 45
  45. Smith, H. W., and M. B. Huggins. 1980. The association of the O18, K1 and H7 antigens and the ColV plasmid of a strain of Escherichia coli with its virulence and immunogenicity. J. Gen. Microbiol. 121:387-400.[Abstract/Free Full Text]
    46. 46
  46. Snedecor, G. W., and W. G. Cochran. 1980. Statistical methods, 7th ed. Iowa State University Press, Ames, Iowa.
    47. 47
  47. Sorsa, L. J., S. Dufke, J. Heesemann, and S. Schubert. 2003. Characterization of an iroBCDEN gene cluster on a transmissible plasmid of uropathogenic Escherichia coli: evidence for horizontal transfer of a chromosomal virulence factor. Infect. Immun. 71:3285-3293.[Abstract/Free Full Text]
    48. 48
  48. Tettelin, H., D. Radune, S. Kasif, H. Khouri, and S. L. Salzberg. 1999. Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project. Genomics 62:500-507.[CrossRef][Medline]
    49. 49
  49. Tivendale, K. A., J. L. Allen, C. A. Ginns, B. S. Crabb, and G. F. Browning. 2004. Association of iss and iucA, but not tsh, with plasmid-mediated virulence of avian pathogenic Escherichia coli. Infect. Immun. 72:6554-6560.[Abstract/Free Full Text]
    50. 50
  50. Vandekerchove, D., F. Vandemaele, C. Adriaensen, M. Zaleska, J. P. Hernalsteens, L. D. Baets, P. Butaye, F. V. Immerseel, P. Wattiau, H. Laevens, J. Mast, B. Goddeeris, and F. Pasmans. 2005. Virulence-associated traits in avian Escherichia coli: comparison between isolates from colibacillosis-affected and clinically healthy layer flocks. Vet. Microbiol. 108:75-87.[CrossRef][Medline]
    51. 51
  51. Vidotto, M. C., J. M. Cacao, C. R. Goes, and D. S. Santos. 1991. Plasmid coding for aerobactin production and drug resistance is involved in virulence of Escherichia coli avian strains. Braz. J. Med. Biol. Res. 24:677-685.[Medline]
    52. 52
  52. Wang, Z., and T. G. Rossman. 1994. Large-scale supercoiled plasmid preparation by acidic phenol extraction. BioTechniques 16:460-463.[Medline]
    53. 53
  53. Waters, V. L., and J. H. Crosa. 1991. Colicin V virulence plasmids. Microbiol. Rev. 55:437-450.[Abstract/Free Full Text]
    54. 54
  54. Welch, R. A., V. Burland, G. Plunkett III, P. Redford, P. Roesch, D. Rasko, E. L. Buckles, S. R. Liou, A. Boutin, J. Hackett, D. Stroud, G. F. Mayhew, D. J. Rose, S. Zhou, D. C. Schwartz, N. T. Perna, H. L. Mobley, M. S. Donnenberg, and F. R. Blattner. 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 99:17020-17024.[Abstract/Free Full Text]
    55. 55
  55. Wooley, R. E., P. S. Gibbs, T. P. Brown, J. R. Glisson, W. L. Steffens, and J. J. Maurer. 1998. Colonization of the chicken trachea by an avirulent avian Escherichia coli transformed with plasmid pHK11. Avian Dis. 42:194-198.[CrossRef][Medline]
    56. 56
  56. Wooley, R. E., L. K. Nolan, J. Brown, P. S. Gibbs, C. W. Giddings, and K. S. Turner. 1993. Association of K-1 capsule, smooth lipopolysaccharides, traT gene, and colicin V production with complement resistance and virulence of avian Escherichia coli. Avian Dis. 37:1092-1096.[CrossRef][Medline]
    57. 57
  57. Zhou, D., W. D. Hardt, and J. E. Galan. 1999. Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island. Infect. Immun. 67:1974-1981.[Abstract/Free Full Text]

Journal of Bacteriology, January 2006, p. 745-758, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.745-758.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lefebvre, B., Gattuso, M., Moisan, H., Malouin, F., Diarra, M. S. (2009). Genotype comparison of sorbitol-negative Escherichia coli isolates from healthy broiler chickens from different commercial farms. Poult. Sci. 88: 1474-1484 [Abstract] [Full Text]  
  • Peigne, C., Bidet, P., Mahjoub-Messai, F., Plainvert, C., Barbe, V., Medigue, C., Frapy, E., Nassif, X., Denamur, E., Bingen, E., Bonacorsi, S. (2009). The Plasmid of Escherichia coli Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic E. coli Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model. Infect. Immun. 77: 2272-2284 [Abstract] [Full Text]  
  • Fisher, C. R., Davies, N. M. L. L., Wyckoff, E. E., Feng, Z., Oaks, E. V., Payne, S. M. (2009). Genetics and Virulence Association of the Shigella flexneri Sit Iron Transport System. Infect. Immun. 77: 1992-1999 [Abstract] [Full Text]  
  • Christenson, J. K., Gordon, D. M. (2009). Evolution of colicin BM plasmids: the loss of the colicin B activity gene. Microbiology 155: 1645-1655 [Abstract] [Full Text]  
  • Zhao, L., Gao, S., Huan, H., Xu, X., Zhu, X., Yang, W., Gao, Q., Liu, X. (2009). Comparison of virulence factors and expression of specific genes between uropathogenic Escherichia coli and avian pathogenic E. coli in a murine urinary tract infection model and a chicken challenge model. Microbiology 155: 1634-1644 [Abstract] [Full Text]  
  • Tivendale, K. A., Noormohammadi, A. H., Allen, J. L., Browning, G. F. (2009). The conserved portion of the putative virulence region contributes to virulence of avian pathogenic Escherichia coli. Microbiology 155: 450-460 [Abstract] [Full Text]  
  • Ewers, C., Antao, E.-M., Diehl, I., Philipp, H.-C., Wieler, L. H. (2009). Intestine and Environment of the Chicken as Reservoirs for Extraintestinal Pathogenic Escherichia coli Strains with Zoonotic Potential. Appl. Environ. Microbiol. 75: 184-192 [Abstract] [Full Text]  
  • Johnson, T. J., Wannemuehler, Y., Doetkott, C., Johnson, S. J., Rosenberger, S. C., Nolan, L. K. (2008). Identification of Minimal Predictors of Avian Pathogenic Escherichia coli Virulence for Use as a Rapid Diagnostic Tool. J. Clin. Microbiol. 46: 3987-3996 [Abstract] [Full Text]  
  • Johnson, T. J., Wannemuehler, Y., Johnson, S. J., Stell, A. L., Doetkott, C., Johnson, J. R., Kim, K. S., Spanjaard, L., Nolan, L. K. (2008). Comparison of Extraintestinal Pathogenic Escherichia coli Strains from Human and Avian Sources Reveals a Mixed Subset Representing Potential Zoonotic Pathogens. Appl. Environ. Microbiol. 74: 7043-7050 [Abstract] [Full Text]  
  • Fricke, W. F., Wright, M. S., Lindell, A. H., Harkins, D. M., Baker-Austin, C., Ravel, J., Stepanauskas, R. (2008). Insights into the Environmental Resistance Gene Pool from the Genome Sequence of the Multidrug-Resistant Environmental Isolate Escherichia coli SMS-3-5. J. Bacteriol. 190: 6779-6794 [Abstract] [Full Text]  
  • Caza, M., Lepine, F., Milot, S., Dozois, C. M. (2008). Specific Roles of the iroBCDEN Genes in Virulence of an Avian Pathogenic Escherichia coli O78 Strain and in Production of Salmochelins. Infect. Immun. 76: 3539-3549 [Abstract] [Full Text]  
  • Johnson, T. J., Wannemuehler, Y. M., Nolan, L. K. (2008). Evolution of the iss Gene in Escherichia coli. Appl. Environ. Microbiol. 74: 2360-2369 [Abstract] [Full Text]  
  • Li, G., Ewers, C., Laturnus, C., Diehl, I., Alt, K., Dai, J., Antao, E.-M., Schnetz, K., Wieler, L. H. (2008). Characterization of a yjjQ mutant of avian pathogenic Escherichia coli (APEC). Microbiology 154: 1082-1093 [Abstract] [Full Text]  
  • Sabri, M., Caza, M., Proulx, J., Lymberopoulos, M. H., Bree, A., Moulin-Schouleur, M., Curtiss, R. III, Dozois, C. M. (2008). Contribution of the SitABCD, MntH, and FeoB Metal Transporters to the Virulence of Avian Pathogenic Escherichia coli O78 Strain {chi}7122. Infect. Immun. 76: 601-611 [Abstract] [Full Text]  
  • Jeziorowski, A., Gordon, D. M. (2007). Evolution of Microcin V and Colicin Ia Plasmids in Escherichia coli. J. Bacteriol. 189: 7045-7052 [Abstract] [Full Text]  
  • Johnson, T. J., Kariyawasam, S., Wannemuehler, Y., Mangiamele, P., Johnson, S. J., Doetkott, C., Skyberg, J. A., Lynne, A. M., Johnson, J. R., Nolan, L. K. (2007). The Genome Sequence of Avian Pathogenic Escherichia coli Strain O1:K1:H7 Shares Strong Similarities with Human Extraintestinal Pathogenic E. coli Genomes. J. Bacteriol. 189: 3228-3236 [Abstract] [Full Text]  
  • Johnson, T. J., Wannemuehler, Y. M., Johnson, S. J., Logue, C. M., White, D. G., Doetkott, C., Nolan, L. K. (2007). Plasmid Replicon Typing of Commensal and Pathogenic Escherichia coli Isolates. Appl. Environ. Microbiol. 73: 1976-1983 [Abstract] [Full Text]  
  • Restieri, C., Garriss, G., Locas, M.-C., Dozois, C. M. (2007). Autotransporter-Encoding Sequences Are Phylogenetically Distributed among Escherichia coli Clinical Isolates and Reference Strains. Appl. Environ. Microbiol. 73: 1553-1562 [Abstract] [Full Text]  
  • Johnson, T. J., Wannemeuhler, Y. M., Scaccianoce, J. A., Johnson, S. J., Nolan, L. K. (2006). Complete DNA Sequence, Comparative Genomics, and Prevalence of an IncHI2 Plasmid Occurring among Extraintestinal Pathogenic Escherichia coli Isolates. Antimicrob. Agents Chemother. 50: 3929-3933 [Abstract] [Full Text]  
  • Skyberg, J. A., Johnson, T. J., Johnson, J. R., Clabots, C., Logue, C. M., Nolan, L. K. (2006). Acquisition of Avian Pathogenic Escherichia coli Plasmids by a Commensal E. coli Isolate Enhances Its Abilities To Kill Chicken Embryos, Grow in Human Urine, and Colonize the Murine Kidney. Infect. Immun. 74: 6287-6292 [Abstract] [Full Text]  
  • Herren, C. D., Mitra, A., Palaniyandi, S. K., Coleman, A., Elankumaran, S., Mukhopadhyay, S. (2006). The BarA-UvrY Two-Component System Regulates Virulence in Avian Pathogenic Escherichia coli O78:K80:H9.. Infect. Immun. 74: 4900-4909 [Abstract] [Full Text]  
  • Johnson, T. J., Johnson, S. J., Nolan, L. K. (2006). Complete DNA Sequence of a ColBM Plasmid from Avian Pathogenic Escherichia coli Suggests that It Evolved from Closely Related ColV Virulence Plasmids.. J. Bacteriol. 188: 5975-5983 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Johnson, T. J.
Right arrow Articles by Nolan, L. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, T. J.
Right arrow Articles by Nolan, L. K.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»