A Survey of Escherichia coli O157:H7 Virulence Factors: The First 25 Years and 13 Genomes

Holly A. Reiland, Morrine A. Omolo, Timothy J. Johnson, David J. Baumler

Department of Food Science and Nutrition University of Minnesota-Twin Cities, St. Paul and Minneapolis, USA.
Department of Veterinary and Biomedical Sciences University of Minnesota-Twin Cities, St. Paul and Minneapolis, USA.

DOI: 10.4236/aim.2014.47046                

Abstract

Escherichia coli O157:H7 is a human pathogen that was first identified from a foodborne outbreak in 1982, and in the 25 years that followed, many new strains were identified and emerged in numerous outbreaks of human disease. Extensive research has been conducted to identify virulence factor genes involved in the pathogenesis of E. coli O157:H7 and many genome sequences of E. coli O157:H7 strains have become available to the scientific community. Here, we provide a comprehensive overview of the research that has been conducted over the first 25 years to identify 394 known or putative virulence factor genes present in the genomes of E. coli O157:H7 strains. Finally, an examination of the conservation of these 394 virulence factor genes across additional genomes of E. coli O157:H7 is provided which summarizes the first 25 years and 13 genomes of this human pathogen.

Keywords

Escherichia coli, O157:H7, Enterohaemorrhagic Escherichia coli (EHEC), Virulence Factors, Genomics

1. Introduction

Escherichia coli O157:H7 is a human pathogen that was first identified from a foodborne outbreak involving ground beef in Michigan, USA in 1982. This organism sickened a number of individuals and led to more severe conditions such as Hemolytic Uremic Syndrome (HUS) and renal kidney failure. The O157:H7 serotype of E. coli was the first strain assigned to a pathotype that came to be known as Enterohaemorrhagic Escherichia coli (EHEC) due to symptoms of sickened individuals, which typically include bloody diahaerria. Since the initial identification of an O157:H7 strain in a hamburger outbreak in 1982 that sickened 47 individuals with 33 requiring hospitalization [1] , numerous strains of E. coli O157:H7 have emerged in foodborne outbreaks ranging from various meat products (although typically ground beef) to organic produce (including various leafy greens such as lettuce and spinach). In 1996, the largest outbreak occurred to date occurred in Sakai City, Japan which sickened 5,000-12,680 people, requiring 398 - 425 hospitalizations, and mainly involved school children [2] [3] [4] . The implicated food in the Sakai City outbreak was contaminated radish sprouts. Surveillance of E. coli O157:H7 has since increased dramatically and scientific efforts have intensified to understand the virulence determinants of this pathogen, and to implement control parameters in efforts to reduce the incidence of E. coli O157:H7 outbreaks. Despite these efforts, outbreaks due to EHEC still occur.

Michigan outbreak strain ATCC 43895, also called EDL933, was the first E. coli O157:H7 genome to be sequenced [5] . Shortly thereafter, the Sakai City strain was also sequenced (RIMD) [6] . Since these hallmark accomplishments, scientists have sought to identify virulence factors through genome comparisons, to understand variations that relate to the observed differences in occurrence and virulence, and to understand the overall evolution of this pathogen. With the advent of whole genome comparisons, many interesting differences have been brought to light. Recently, a large number of additional strains belonging to the O157:H7 serotype have become publicly available. New strains and their associated genomes have emerged nearly monthly from foodborne outbreaks and also from community-associated outbreaks involving locales such as swimming pools, petting zoos, and daycare centers. In this work, we have conducted a thorough examination and identified known and putative virulence factors in the published literature for E. coli O157:H7 and insight derived from orthologous genes from other pathogenic members of the Enterobacteriacaea, as this family is thought to have derived from a common ancestor. We attempt to provide a current update of virulence factors and genome variations with a goal of understanding variations that correspond to epidemiology and geographical differences.

Virulence factors can be broadly subdivided as “known” or “putative” (Figure 1). Since this human pathogen does not evoke the same disease responses in other mammalian hosts, no ideal experimental animal model exists, and therefore much of the insight is derived using mouse models, and other various assay methods. The types of virulence factors that have multiple testable methods are involved in attachment and adhesion, and also for puta-

Figure 1. Known or putative virulence factors for E.coli O157:H7 separated into categories (n = 394 genes).

tive effectors molecules via protein translocation into human tissue cell cultures. Here, we provide a summary of all recently derived insight for virulence factors genes and those genes associated with the organism’s ability to cause disease in the host. All annotations from this comprehensive literature survey are accessible in the ASAP database, and represent one of the most thorough overviews of E. coli O157:H7 from 1982 through 2007 which represents the first 25 years of research since this organism was first recognized as a human pathogen from an outbreak that occurred in 1982.

Overall this approach to identify candidate putative or known virulence factors has dramatically increased the numbers of identified genes to include 394 coding sequences, and 5 pseudogenes, representing ~7.3% (394/5401) of the complete genome of the EHEC strain (EDL933) (Supplemental data 1). The most abundant categories of these are genes involved in adhesion or effector molecules, since numerous experimental methods exist for assays using human tissue cell lines (Supplemental information 1). This is a dramatic increase from the previous review of Spears et al. [7] that provided a summary of E. coli O157:H7 virulence genes. It should be noted that some of the genes identified in this work as putative virulence factors, are involved in regulation and metabolism and have used recent experimental gene expression data from comparisons of human clinical isolates and isolates of bovine origin. It may be that some genes encode proteins that may have multiple functions, pleiotrophic effects, or increase the competitive abilities of the organism to compete with intestinal flora in the host, and are therefore included. There is a great deal of work that remains to fully understand the ability of E. coli O157:H7 to cause human disease, and this work provides new additional insight about candidate virulence genes and the variation and conservation of these 394 genes in the 13 new genomes that have been sequenced through 2007.

2. Colonization Factors

“To adhere is essential”—author unknown.

2.1. Adhesion

The adherence ability of E. coli O157:H7 is pivotal to its transmission from fresh produce items and cattle to its eventual human host. The ability to adhere to the human intestine is an early component of EHEC infection. Adhesion is one category of virulence factors that has made much experimental progress, due to the ability to conduct adherence assays that determine if a mutation strain of a certain gene affects the ability of the organism to adhere to human tissue culture cell lines such as epithelial intestinal cells. The most obvious genes involved in adherence are those that encode the machinery to produce fimbria. In addition to fimbrial genes, there are many genes that have been implicated to be involved in adherence, yet in many of these cases it is undetermined how these genes are involved in the process of adherence. These genes are included in this review since they play a role in the EHEC’s ability to adhere to cell tissue cell lines and are therefore identified as virulence factors or putative virulence factors.

There are 16 predicted gene clusters or operons in the genome of E. coli O157:H7 strain EDL933 that contain genes either known or predicted to be involved in fimbrial assembly [8] (Table 1). It is unknown whether genes in different loci can function with gene products of other clusters/operons to produce fimbria, therefore this review effort is not limited to loci that encode a complete fimbrial operon, and also includes standalone genes identified based on protein sequence similarity. Of the 16 gene adhesion clusters/operons, the work of Low et al. [8] used promoter fusion assays and demonstrated that only four fusions (loc4, loc7, loc8, and loc9) demonstrated promoter activity above the baseline level in laboratory growth conditions. Yet the other gene clusters/ operons should not be overlooked, since many of the genes contained in these loci have experimental evidence for gene mutant phenotypes demonstrating a role in adhesion. Loci that contain these genes are loc5, loc7, loc12, loc13, loc14, MAT, and ppdD (Table 1). In addition, loc7 was the only adherence gene cluster that had both promoter activity demonstrated experimentally, and was found to be up-regulated in clinical human isolates of EHEC compared to isolates of bovine origin. It is unclear what the precise role of the loc7 operon may serve for EHEC pathogenesis in humans, as this orthologous operon is conserved in many other E. coli genomes including the two non-pathogenic E. coli K-12 strains, Salmonella spp., and Shigella spp. In EHEC strains genes contained in loc7 have the most supporting experimental evidence to indicate a possible point of control to target for future prevention of the ability of O157:H7 to attach and adhere to the intestinal tract of humans. Collectively, 10 of the 16 adhesion loci have at least one line of experimental evidence to indicate that they are expressed or

Table 1. Known or putative virulence factor genes involved in attachment and adhesion of E. coli O157:H7 EHEC strains.

involved in adhesion, yet strains need to be developed with all 16 loci deleted, and then through a systematic addition of each loci individually to the mutant strain would permit the investigation of the role in adhesion of each individual fimbrial loci.

The best-characterized loci involved in adhesion are the long polar filament operons lpf1 (loc12) and lpf2 (loc13). The lpfABCC’DE operon was characterized by Torres et al. [9] where they showed that this polycistronic operon is present in O157:H7 strains as well as the ancestral O55:H7 strains. Generation of a lpfA mutant strain resulted in a decreased ability to adhere to human HeLa cells indicating that the lpf loci is involved in adherence to human tissue cells. A second lpf operon, lpf2ABCDD’, was also characterized and found that a lpfA2 mutant strain had decreased adherence to Caco-2 cells, yet no difference was observed in adherence to HeLa cells in a Fas Actin Staining (FAS) assay [10] . To further investigate the role of lpf for adherence to animals, Torres et al. [11] found that both lpfA1 and lpfA2 mutants displayed diminished ability to persist in the intestine of infected 6-week-old lambs. In the same study, they determined that lpf1 was expressed in response to temperature (37˚C), growth phase (late-logarithmic and stationary growth phase), pH of the growth medium (pH 6.5), and also osmolarity (0.2 M NaCl), whereas the lpf2 operon was expressed in conditions of late-logarithmic phase of growth and also during iron starvation [11] . Collectively it seems that the lpf fimbriae may have roles for adherence during different environmental cues, yet follow up studies should focus on the generation of anlpf1 and lpf2 double mutant strain to systematically determine the role of each in adherence and EHEC virulence.

The sfm operon (loc3), consists of 5 genes, sfmACDHF which are predicted to encode the machinery to assemble fimbria on the outer surface of the cell (Table 1). This fimbrial gene cluster is important for disease, since they are involved in the attachment of the organism to the host’s intestinal epithelial layer in the small intestine [12] [13] . In Salmonella strains, there are 13 fimbrial operons present in the genome, yet only two of these operons, fim and agf have been shown to mediate expression of fimbrial filaments on the cellular surface, which may indicate a similar role for these genes in E. coli EHEC strains.

The fim operon from Salmonella is orthologous to the sfm operon in E. coli O157:H7. Humphries et al. [12] revealed that in S. enterica serotype Typhimurum that out of the 13 predicted fimbrial operons identified in the genome, the gene product for fimA was the only one expressed and detected by Western blotting. Furthermore in animal studies with chickens, a fimD mutant of Salmonella increased its ability to enter the bloodstream (bacteraemia), yet modified the organism’s ability to cause disease in the reproductive tract, which consequently led to a decline in egg shell contamination in laying hens [13] . Combined, these studies highlight the fimBEAICDFGH operon as a target for various control parameters to control dissemination of Salmonella in poultry products. There is a possibility that similar regulation factors and cellular machinery exist in E. coli O157:H7 for fimbrial filaments.

In addition the hcpABC operon, which was recently found to encode the hemorrhagic coli pilus, generation of a hcpA mutant strain of E. coli O157:H7 led to decreased adherence to Caco-2, T84, HT-29, HeLa, Hep-2, and MBDK human cell lines compared to the parent strain in cell adherence assays and also when tested with porcine and bovine gut explants [14] . Another locus found to be involved in adhesion to human tissue cells is the operon ecpEDCBAR (E. coli common pilus). An ecpE mutant strain that represented an operon mutant for the ecp loci displayed reduced adherence to HEp-2 and HeLa cells compared to the parent strain in celladherence assays [15] . Further experimentation is required to examine which fimbrial operons are expressed in E. coli O157:H7 strains, since identification of expressed fimbrial filaments represent a target for vaccine development.

Many additional gene clusters/operons predicted to encode additional machinery for fimbrial assembly have been identified based on protein sequence similarity and include loc1 (Z0020/Z0021/Z0022/Z0023/Z0024), loc2 (yadCKLMhtrEecpDyadN), loc4 (ybgOPQD), loc5 (ycbQRTUVF), loc6 (Z1534/Z1535/Z1536/Z1537/Z1538/Z1539), loc8 (FmlABydeSRfmlD), loc9 (Z3276/yehBCDE), loc10 (Z3596/Z3597/Z3598/yfcSUV), and loc11 (yraHIJ/ Z4501/Z4502/ABH-0285237). Of these predicted fimbriae genes, a random mutagenesis library found that the ycbR mutant (loc5) lost the ability to adhere to Hep-2 cells [16] , yet all of the other lack experimental evidence to further support their role in EHEC pathogenesis. There are some genes not in clusters that are predicted to encode for fimbrial machinery based on protein sequence similarity and include Z4321, Z1536, Z5029, and Z5223, and it is unknown if these are expressed, or work in concert with other fimbrial machinery in E. coli O157:H7 strains.

Other genes and clusters not thought to encode for fimbrial machinery, but that have mutant phenotypes that displayed reduced adhesion to human tissue cell lines were fliC, iha, efa-1’, ompA, dsbA, wcaM, yeeJ, yhiF, and gadE. fliC is involved in the generation of flagella and has been found to affect the ability of the cell to adhere to HeLa tissue culture cells [9] . In addition, a fliC mutant of STEC O113:H21 strain displayed reduced virulence in a mouse model [17] . Combined these studies reveal that fliC may play a role in virulence in human infections. The gene iha (irgA homolog adhesion) was found to confer reduced adherence to HeLa cells in a mutant strain and additionally when iha was introduced into non-pathogenic E. coli, the resulting strain had the ability to adhere to kidney cells [18] . In addition the mutant strains for the genes toxB and efa-1’(efa1_1 and efa1_2 are truncated fragments of efa-1, and are collectively referred to as efa-1') displayed diminished adherence phenotypes to HeLa cells as compared to the parent strains. In O157:H7 strains, the efa gene is disrupted and is represented as two truncated fragments, yet in O157:H^- strains it encodes a large (>3000 AA protein) [19] . It is unknown if the efa gene product is involved virulence of H^- strains, yet both truncated fragments demonstrated a role in adherence and an efa_1 and efa_2 double mutant strain had even greater reduction in adherence than the single efa_1 or efa_2 mutant strains [20] . The gene ompA produces an outer membrane protein that plays a role in adherence in a HeLa cell adherence assay [21] . Finally, the gene dsbA was found to play a role in biofilm formation and virulence since a mutant strain for ompA displayed reduced adherence to PVC surfaces and HT-29 epithelial cells [22] .

The wcaM gene is part of the colonic acid operon (wca), and is involved in thermal tolerance and acid stress in E. coli O157:H7 strains [23] . In Salmonella Typhimurium strain BJ2710, a wcaM mutant generated a reduced thickness of biofilm to Hep-2 cells, indicating that the wcaM gene in E. coli O157:H7 may also play a role in adhesion to human cells. yeeJ was initially identified as a gene that produces an adhesin antigen based on sequence homology to E. coli surface adhesion antigen 43 that was found to play a role in biofilm formation [24] . Although a yeeJ mutant strain displayed no difference in biofilm formation on PVC microtiter plates or in fermenters, the predicted protein produced by yeeJ contains 13 bacterial immunoglobulin-like domains that may mediate the interaction with the host. Therefore the need to test the interaction with human tissue cells in vitro is warranted to determine the role of this gene in adhesion and possibly virulence of E. coli O157:H7 strains.

The yhiF gene encodes for a transcriptional regulator, and the gadE (yhiE) gene produces an acid-induced positive regulator. The contribution of these genes to virulence of EHEC strains may not be direct, but rather through the regulation of other virulence genes, since yhiF and gadE mutant strains displayed increased adherence to Caco-2 intestinal epithelial cells as compared to the parent strain [25] . In addition, there is a homologous gene from Clostridium difficile, toxB, which is present in the genome of E. coli O157:H7 strains that may contribute to EHEC adherence to epithelial cells. Characterization of the function of toxB demonstrated that mutant strains displayed a reduction in adherence to Caco-2 cells, thought to occur through the promotion, production, and/or secretion of type III secreted effector proteins [26] . A separate study determined that a toxB mutant formed less EspA filaments, secreted less EspD, and displayed a reduction in adhesion to HeLa cells further supporting the role of toxB in adherence [20] . Overall, the work in the area of virulence factors involved in adhesion has increased tremendously, yet still more work is required to further the understanding to the point where it is feasible to design and test control parameters to prevent adherence to human cells.

2.2. Attachment and Effacement

The difference between virulence genes classified as adherence compared to attachment and effacement, is that the latter causes a well-known characteristic of attaching and effacing (AE) lesions on the host epithelial cells. AE lesions are not found to be essential to cause bloody diarrhea and/or HUS in the humans, yet surveys of E. coli O157:H7 strains found that most strains found to cause these disease stages in humans contain intact genes encoding products conferring AE lesions. To date there have only been two virulence genes identified to cause AE lesions, one that affects humans (eae) and the other affects porcine hosts (paa). The eae gene is found in the locus of enterocyte effacement (LEE), which has been found in most E. coli O157:H7 strains and by definition in all enteropathogenic E. coli strains (EPEC). Recently the work of Deng et al. [27] , utilized a relative of E. coli, the mouse pathogen Citrobacter rodentium, to further elucidate the roles of many of the genes contained in the LEE island. In the case of eae, it was found that an eae mutant of C. rodentium was avirulent in a mouse model assay [27] . It is well established that ler is a regulator for eae expression, yet recently the work of Nadler et al. [28] determined that the transcriptional expression of eae is also affected by ydeOP and evgA.

The other gene involved in AE lesion formation is paa (porcine A/E-associated gene). In the work of Batisson et al. [29] screening of a random mutagenesis library in non-enterotoxigenic porcine E. coli O45 led to the identification of a mutant that did not induce typical lesions in a pig ileal explant model. The authors make the correlation that the presence of paa and eae sequences in porcine E. coli O45 strains is important for generation of AE lesions. The protein sequence similarity is 100% identical for E. coli (EHEC) EDL933 and Sakai strains and is found intact in seven additional genomes of E. coli O157:H7, which may suggest that this gene may play a role in AE formation in a human host.

3. Effectors

Bacterial effector proteins are part virulence mechanisms of many microbial pathogens, and in the case of E. coli O157:H7, much attention has been focused on these proteins that are injected into host cells, often via type three secretion systems (TTSS) (Figure 2). Initially the first effectors identified in E. coli O157:H7 were found in the LEE locus and much of the understanding of these virulence factor genes was elucidated using the mouse pathogen C. rodentium. With complete genomes available for E. coli O157:H7, recent attention has focused on identifying homologous new candidate effector genes based on similarity searching using known effector gene/ protein sequences. Initial research was followed up by investigation of tagged-putative effector proteins and their ability to translocate into human tissue culture cells. Here we provide an update on the genes that encode effectors and putative effectors virulence factor genes (Table 2). Although the mode of action of these effector proteins once inside of the host varies, effectors represent a large percentage of the virulence genes identified as candidates in E. coli O157:H7 due to their identification via sequence similarity and can be further examined with in vitro assays for that examine protein translocation into human tissue culture cells.

Table 2. Effectors known and predicted in E. coli O157:H7 EHEC strains.

Figure 2. Schematic of the effector entering the intestinal epithelial cell.

3.1. Locus of Enterocyte Effacement

The LEE pathogenicity island has been the subject of much recent research, and since it is found also in the mouse pathogen C. rodentium, an organism for which there is an established experimental model to study the involvement of virulence genes in a mouse host, Deng et al. [27] mutagenized all 41 coding sequences that comprise the entire LEE locus, and tested each mutant strain in mice. The results of this work are extensive and further elucidate the association of many of these genes to cause virulence in the mouse model as an effort to better understand the role of the LEE locus in human EHEC illness. Since many of the effectors in pathogenic E. coli are injected into the host via type three secretion systems, the LEE locus was the center of the initial identification of effectors, in part since there is also an extensive repertoire of genes that encode a functional TTSS adjacent to the effectors genes. There are currently six genes identified that encode effector protein in the LEE island, and in the following section an overview of the current work is provided.

3.2. Locus of Enterocyte Effacement Effectors

In the work of Crane et al. [30] an espF mutant was found to display reduced lethality to human tissue culture cells yet retained the adherence phenotype similar to the wild-type strain, and they determined that the expression of EspF in HeLa cells is toxic in a dose-dependent manner. It was also determined that EspF secretion is reliant on the TTSS machinery and that another LEE gene, cesF, had a significant effect on the translocation of EspF, as a cesF mutant had reduced amounts of EspF translocated into human tissue cells [31] . Additional LEE TTSS gene products were found to interact with EspF and CesT [32] . More supporting evidence for the role of espF in virulence came from the work of Deng et al. [27] , which found that an espF mutant in C. rodentium had attenuated virulence in a mouse host.

The tir gene encodes a translocated intimin receptor protein, which translocates into human tissue cells [33] , and also integrates into the host cell membrane and binds intimin to promote bacterial adhesion to host cells [34] . Mutant strains lacking a functional tir gene show greatly reduced adhesion to HeLa cells, do not induce the formation of actin filaments by HeLa cells [34] , and show reduced ability to invade HeLa cells [35] . In animal models, tir mutant strains are avirulent in mice thus further supporting its role in the ability to cause human illness [27] .

Map (mitochondrial associated type III secreted effector protein) is required for E. coli O157:H7 invasion of host cells. There are numerous experiments to support the role of map in virulence, since mutant strains show a reduced ability to invade HeLa cells [35] , are found to be required for filipodia formation [36] , and were found to have decreased competitive abilities in calf intestine [37] , thus illustrating that Map is important in the ability of E. coli O157:H7 to persist in bovine hosts, and likely to cause disease in human hosts.

There are numerous other effector genes that have been to play an important role in the ability to cause disease in mouse models, and these include: espH, espZ, and espG [27] [38] [39] . Additional roles of the espG gene in virulence have been demonstrated with experiments that found that the encoded protein, EspG, binds to tubulin which causes localized microtubule depolymerization resulting in actin stress fiber formation through an unknown mechanism [40] .

3.3. NonLocus of Enterocyte Effacement Effectors

Aside from the work on LEE effectors genes, there are numerous candidate virulence factor genes thought to function as effectors in host environments and many of these have been determined through assays that demonstrated effector protein translocation in human cells and include: espY4, nleB1, espW, nleG8-2, espM2, espR4, nleA, nleH1-2, espM1, nleG2-2, nleG6-1, nleG5-1, espK, espX2, espY1, nleG8-1, nleD, and nleH1-1 [33] [41] . Studies with gene mutants in mice animal models have also identified some non-LEE effectors as virulence genes such as: nleA, nleB1, nleF, nleC, and nleD [27] [42] [43] . Additional virulence genes that produce effectors were identified through a combination of research approaches that examined adherence phenotypes of mutant strains to HeLa cells, and also in the ability of mutant strains to persist in calves, and in conjuncture provide further support of these virulence genes in E. coli O157:H7 such as espR4, nleG8-1, and nleG2-3 [37] . With a lack of experimental evidence, there are a number of candidate effector virulence genes that have been identified based on sequence homology to known effector genes and these include: espX6, espX5, espX4, espL4, nleE, espR3, espL1, espR1, nleG5-2, nleG6-2, espN, espX7, nleB2-1, espY3, espY2, espX1, and nleG2-3 [33] [44] .

One of the most studied effector molecule genes is tccP which encodes for the Tir-cytoskeleton coupling protein (TccP), and this virulence gene has been shown to be present in 100% of EHEC O157:H7 strains from around the world (n = 365) based on PCR amplification, yet some variation in the size of the tccP gene was noted to range from 700 to 1150 bp [45] . TccP is also required for EHEC-induced actin polymerization, Ncklike (EPEC) activity that facilitates interactions of Tir and actin-signaling molecules, and cooperates with Tir to induce actin polymerization at the site of bacterial attachment [46] . With regards to the protein sequence of TccP, the number of proline-rich repeats in TccP directly correlates with the binding affinity to N-WASP, and the N-terminal amino acid residues 1 - 21 are required for TccP translocation into HeLa cells, while the N-terminal amino acid residues 1 - 181 are required for actin polymerization of epithelial cells and during in vitro assays [47] . tccP mutant strains were found to compete equally well with the parent strain in mixed oral infection experiments of lambs and calves, thus do not play a significant role in E. coli O157:H7’s competiveness to colonize mammalian hosts [48] . Even though the first genome sequence for E. coli O157:H7 was for strain EDL933 by Perna et al. 2001, another group resequenced the tccP gene of E. coli O157:H7 strain EDL933 and found that its sequence is identical to that of the E. coli O157:H7 Sakai strain (i.e., 1014 bp and five and a half [rather than six and a half] proline-rich repeats), thus correcting initial genome sequencing errors [49] . The two genes tccP and espJ constitute an operon, but espJ expression was not regulated by Ler [50] .

A second gene similar to tccP, called tccP2, has also received much research attention, since it was found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement a tccP mutant, and that 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated [49] . These results suggest that TccP2 is a functional equivalent to TccP and can be used by O157 and non-O157 EHEC to trigger actin polymerization via the Nck pathway [49] .

4. Secretion Systems

4.1. Type Two Secretion Systems

E. coli O157:H7 strains harbor a large plasmid termed pO157, and this plasmid contains a number of genes thought to contribute to the virulence in humans such as the hemolysin (ehxCABD), a periplasmic bifunctional catalase/peroxidase (katP), an extracellular serine protease (espP), and homologue to a toxin from Clostridium difficile thought to contribute to cell adherence to epitheial cells (toxB). The pO157 plasmid also has an operon containing 13 ORFs that comprise a type II secretion system known as the etp cluster (Table 3). The type II secretion system encoded by the etp operon has been found to contribute to the virulence of E. coli O157:H7 strains by secreting a zinc metalloprotease protein called StcE. In addition, strains cured of the pO157 plasmid had reduced secretion of EspA, EspB, and Tir, and re-introduction of mini-pO157 plasmid composed of the toxB gene and the ori regions restored production and secretion of the effectors EspA, EspB, and Tir [25] . It was also determined that a toxB mutant formed less EspA filament, secreted less EspD, displayed reduced adhesion to HeLa cells, and colonized at a similar rate to the wild type strain [20] . Collectively, it is evident that there are obvious advantages to strains of E. coli O157:H7 to maintain and express the genes on the pO157 plasmid, since every strain with a sequenced genome in this review harbors this plasmid, and has highly conserved sequence homology for all of the putative or known virulence factors genes contained within.

Table 3. Type two and three secretion system genes known and predicted in E. coli O157:H7 EHEC strains.

4.2. Type Three Secretion Systems

Type III secretion systems (TTSS) are part of the main machinery that E. coli O157:H7 cells use to adhere and permit translocation of effectors into host environments (Table 3). Many genes are involved in the assembly of the TTSS machinery and here we provide a summary of those genes and other homologous genes thought to also produce functional TTSS components. Some of these TTSS genes are found in the LEE region of the genome, but many also exist in other regions of the E. coli O157:H7 genomes. The genes eprK, eprJ, eprH, epaSR2, eprR1, epaQ, epaP, epaO, elvJ, elvI, elvC, elvA, elvE, elvG, and elvF are involved in the production of functional TTSS in E. coli O157:H7 and are believed to be involved in virulence [51] . Strains that were eprHIJK null mutants were injected intraperitoneally into 1-day-old chicks in addition to eprH null mutants, and these mutants lost the ability to adhere to HEp-2 cells in vitro supporting a role in virulence of strains of E. coli O157:H7 [51] [52] .

4.3. Locus of Enterocyte Effacement Regions

There are also numerous genes that play a role in the TTSS systems in E. coli O157:H7 strains and many of these are found in the LEE region of the genome. These genes have either been examined through experimentation or through sequence homology to be candidate virulence factors genes. Of these, the gene escC produces the type III needle complex subunit, the genes sepL, escD, escQ, encode TTSS components, and escF, escN, escV, Orf12, escJ, escU, escS, escR, escL, Orf4, escE, and Rorf1 produce TTSS factors (Table 3).

Then there are also numerous genes contained in the LEE genomic region that are predicted to be TTSS components based on sequence homology and include genes for type III secretion system components such as escI and sepD, and also the genes Orf29, Orf16, Orf15, and cesD that are predicted type III secretion system factors.

Many of these LEE region TTSS virulence genes have supporting experimental evidence to support their role in virulence. Among these the escQ product forms the ring within the basal body of the LEE-encoded type-III secretion system and EscT interacts with EscU as a structural component in the structural machinery of the LEEencoded type-III secretion system [53] . Mutant stains deficient for escQ fail to secrete type III effectors and are avirulent in mice [27] , have decreased competitive abilities in calf intestines, do not adhere to HeLa cells, and do not secrete EspD [37] .

There are also virulence genes demonstrated to be involved in virulence using assays of mutant strains that exhibited phenotypes that fail to secrete type III effectors and/or be strains that are now avirulent in mice such as escU, escT, escS, escR, escL, Orf4, escE, and eesA [27] . The latter of these, cesA, is required for proper translocation of type III proteins EspA and EspB, and mutant strains showed impaired filament formation and an inability to induce lesions on and lyse host cells in addition to failing to secrete EspA and EspB [32] . The LEEencoded chaperone CesA has also been found to interact with EspA; as it was experimentally show to co-crystallize with EspA [54] . Another gene, Rorf1, which produces a LEE-encoded type III secretion system factor interacts with EspD was shown to bind with EspD in yeast two-hybrid assays [55] . Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTSS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTSS apparatus [56] .

5. Toxins

There are two main sets of gene clusters that are found in E. coli O157:H7 genomes that encode the Shiga-Liketoxin A and B protein components and are known as stx1AB and stx2AB, and is established that there is an association of Stx toxins and disease in humans [57] . The first strains identified contained one copy of each of these toxin clusters, and were the subjects of immense experimentation. Of the two toxins, Stx2 toxin was found to be 1000 times more toxic than Stx1 based on assays in baboons [58] , and Stx2 is 1000 times more cytotoxic to human renal microvascular endothelial cells than Stx1 [59] . In addition, studies determined that the Stx1 toxin has a higher binding affinity to the human GB3 receptors, therefore competing with Stx2 toxin binding in human hosts [60] . Many new outbreak strains of E. coli O157:H7 were found to lack the stx1AB gene cluster, but instead carry two copies of stx2AB gene clusters that may differ in a few amino acids and have been classified based on classes of stx2a, stx2b, stx2c, stx2d, stx2e, and stx2f. We feel this is important information to help the scientific community better understand the severity of human EHEC disease, since in E. coli O157:H7 strains without stx1AB, the more potent Stx2 and/or Stx2c toxins will bind to host GB3 receptors and start to cause human disease, which may offer an explanation regarding the epidemiological observations that the more recent emerging strains caused a higher rate of HUS and lethality based on the total numbers of individuals sickened in the outbreaks.

6. Interaction with Host Factors

There are many ways that E. coli O157:H7 virulence genes contribute to interactions with hosts (Table 4), and here we provide a summary of the various ways that experiments have determined how these genes may play a role in human disease. The gene stcE was disrupted and this mutant strain was three-fold lower than the wild type strain in forming actin bundles on HEp-2 cells [61] . The gene espP produces a protein that contributes to the bloody diarrhea in many patients suffering from E. coli O157:H7 infections through its role as a serine protease that may degrade host protein [62] .

Table 4. Known or putative genes involved in interaction with host factors, survival though host barriers, LEE-island noneffectors, or regulators in E. coli O157:H7 EHEC strains.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Riley, L.W., Remis, R.S., Helgerson, S.D., McGee, H.B., Wells, J.G., Davis, B.R., Hebert, R.J., Olcott, E.S., Johnson, L.M., Hargrett, N.T., Blake, P.A. and Cohen, M.L. (1983) Hemorrhagic Colitis Associated with a Rare Escherichia coli Serotype. The New England Journal of Medicine, 308, 681-685. http://dx.doi.org/10.1056/NEJM198303243081203
[2]	Michino, H., Araki, K., Minami, S., Takaya, S., Sakai, N., Miyazaki, M., Ono, A. and Yanagawa, H. (1999) Massive Outbreak of Escherichia coli O157:H7 Infection in Schoolchildren in Sakai City, Japan, Associated with Consumption of White Radish Sprouts. American Journal of Epidemiology, 150, 787-796. http://dx.doi.org/10.1093/oxfordjournals.aje.a010082
[3]	Fukushima, H., Hashizume, T., Morita, Y., Tanaka, J., Azuma, K., Mizumoto, Y., Kaneno, M., Matsu-Ura, M.O., Konma, K. and Kitani, T. (1999) Clinical Experiences in Sakai City Hospital during the Massive Outbreak of Enterohemorrhagic Escherichia coli O157 Infections in Sakai City, 1996. Pediatrics International, 41, 213-217. http://dx.doi.org/10.1093/oxfordjournals.aje.a010082
[4]	Higami, S., Nishimoto, K., Kawamura, T., Tsuruhara, T., Isshiki, G. and Ookita, A. (1998) Retrospective Analysis of the Relationship between HUS Incidence and Antibiotics among Patients with Escherichia coli O157 Enterocolitis in the Sakai Outbreak. Kansenshogaku Zasshi, 72, 266-272.
[5]	Perna, N.T., Plunkett 3rd, G., Burland, V., Mau, B., Glasner, J.D., Rose, D.J., Mayhew, G.F., Evans, P.S., Gregor, J., Kirkpatrick, H.A., Posfai, G., Hackett, J., Klink, S., Boutin, A., Shao, Y., Miller, L., Grotbeck, E.J., Davis, N.W., Lim, A., Dimalanta, E.T., Potamousis, K.D., Apodaca, J., Anantharaman, T.S., Lin, J., Yen, G., Schwartz, D.C., Welch, R.A. and Blattner, F.R. (2001) Genome Sequence of Enterohaemorrhagic Escherichia coli O157:H7. Nature, 409, 529-533. http://dx.doi.org/10.1038/35054089
[6]	Hayashi, T., Makino, K., Ohnishi, M., Kurokawa, K., Ishii, K., Yokoyama, K., Han, C.G., Ohtsubo, E., Nakayama, K., Murata, T., Tanaka, M., Tobe, T., Iida, T., Takami, H., Honda, T., Sasakawa, C., Ogasawara, N., Yasunaga, T., Kuhara, S., Shiba, T., Hattori, M. and Shinagawa, H. (2001) Complete Genome Sequence of Enterohemorrhagic Escherichia coli O157:H7 and Genomic Comparison with a Laboratory Strain K-12. DNA Research, 8, 11-22. http://dx.doi.org/10.1093/dnares/8.1.11
[7]	Spears, K.J., Roe, A.J. and Gally, D.L. (2006) A Comparison of Enteropathogenic and Enterohaemorrhagic Escherichia coli Pathogenesis. FEMS Microbiology Letters, 255, 187-202. http://dx.doi.org/10.1111/j.1574-6968.2006.00119.x
[8]	Low, A.S., Holden, N., Rosser, T., Roe, A.J., Constantinidou, C., Hobman, J.L., Smith, D.G.E., Low, J.C. and Gally, D.L. (2006) Analysis of Fimbrial Gene Clusters and Their Expression in Enterohaemorrhagic Escherichia coli O157: H7. Environmental Microbiology, 8, 1033-1047. http://dx.doi.org/10.1111/j.1462-2920.2006.00995.x
[9]	Giron, J.A., Torres, A.G., Freer, E. and Kaper, J.B. (2002) The Flagella of Enteropathogenic Escherichia coli Mediate Adherence to Epithelial Cells. Molecular Microbiology, 44, 361-379. http://dx.doi.org/10.1046/j.1365-2958.2002.02899.x
[10]	Torres, A.G., Kanack, K.J., Tutt, C.B., Popov, V. and Kaper, J.B. (2004) Characterization of the Second Long Polar (LP) Fimbriae of Escherichia coli O157: H7 and Distribution of LP Fimbriae in Other Pathogenic E. coli Strains. FEMS Microbiology Letters, 238, 333-344.
[11]	Torres, A.G., Milflores-Flores, L., Garcia-Gallegos, J.G., Patel, S.D., Best, A., La Ragione, R.M., Martinez-Laguna, Y. and Woodward, M.J. (2007) Environmental Regulation and Colonization Attributes of the Long Polar Fimbriae (LPF) of Escherichia coli O157: H7. International Journal of Medical Microbiology, 297, 177-185. http://dx.doi.org/10.1016/j.ijmm.2007.01.005
[12]	Dorsey, C.W., Laarakker, M.C., Humphries, A.D., Weening, E.H. and Baumler, A.J. (2005) Salmonella enterica Serotype Typhimurium MisL Is an Intestinal Colonization Factor That Binds Fibronectin. Molecular Microbiology, 57, 196-211. http://dx.doi.org/10.1111/j.1365-2958.2005.04666.x
[13]	De Buck, J., Van Immerseel, F., Haesebrouck, F. and Ducatelle, R. (2004) Effect of Type 1 Fimbriae of Salmonella enterica Serotype Enteritidis on Bacteraemia and Reproductive Tract Infection in Laying Hens. Avian Pathology, 33, 314-320. http://dx.doi.org/10.1080/0307945042000220561
[14]	Xicohtencatl-Cortes, J., Monteiro-Neto, V., Ledesma, M.A., Jordan, D.M., Francetic, O., Kaper, J.B., Puente, J.L. and Giron, J.A. (2007) Intestinal Adherence Associated with Type IV Pili of Enterohemorrhagic Escherichia coli O157:H7. The Journal of Clinical Investigation, 117, 3519-3529.
[15]	Rendon, M.A., Saldana, Z., Erdem, A.L., Monteiro-Neto, V., Vazquez, A., Kaper, J.B., Puente, J.L. and Giron, J.A. (2007) Commensal and Pathogenic Escherichia coli Use a Common Pilus Adherence Factor for Epithelial Cell Colonization. Proceedings of the National Academy of Sciences of the United States of America, 104, 10637-10642. http://dx.doi.org/10.1073/pnas.0704104104
[16]	Gonnet, P., Rudd, K.E. and Lisacek, F. (2004) Fine-Tuning the Prediction of Sequences Cleaved by Signal Peptidase II: A Curated Set of Proven and Predicted Lipoproteins of Escherichia coli K-12. Proteomics, 4, 1597-1613. http://dx.doi.org/10.1002/pmic.200300749
[17]	Rogers, T.J., Paton, J.C., Wang, H., Talbot, U.M. and Paton, A.W. (2006) Reduced Virulence of an fliC Mutant of Shiga-Toxigenic Escherichia coli O113:H21. Infection and Immunity, 74, 1962-1966. http://dx.doi.org/10.1128/IAI.74.3.1962-1966.2006
[18]	Tarr, P.I., Bilge, S.S., Vary, J.C., Jelacic, S., Habeeb, R.L., Ward, T.R., Baylor, M.R. and Besser, T.E. (2000) Iha: A Novel Escherichia coli O157:H7 Adherence-Conferring Molecule Encoded on a Recently Acquired Chromosomal Island of Conserved Structure. Infection and Immunity, 68, 1400-1407. http://dx.doi.org/10.1128/IAI.68.3.1400-1407.2000
[19]	Janka, A., Bielaszewska, M., Dobrindt, U. and Karch, H. (2002) Identification and Distribution of the Enterohemorrhagic Escherichia coli Factor for Adherence (Efa1) Gene in Sorbitol-Fermenting Escherichia coli O157: H-. International Journal of Medical Microbiology, 292, 207-214. http://dx.doi.org/10.1078/1438-4221-00206
[20]	Stevens, M.P., Roe, A.J., Vlisidou, I., van Diemen, P.M., La Ragione, R.M., Best, A., Woodward, M.J., Gally, D.L., and Wallis, T.S. (2004) Mutation of ToxB and a Truncated Version of the Efa-1 Gene in Escherichia coli O157: H7 Influences the Expression and Secretion of Locus of Enterocyte Effacement-Encoded Proteins but Not Intestinal Colonization in Calves or Sheep. Infection and Immunity, 72, 5402-5411. http://dx.doi.org/10.1128/IAI.72.9.5402-5411.2004
[21]	Torres, A.G. and Kaper, J.B. (2003) Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells. Infection and Immunity, 71, 4985-4995. http://dx.doi.org/10.1128/IAI.71.9.4985-4995.2003
[22]	Lee, Y., Kim, Y., Yeom, S., Kim, S., Park, S., Jeon, C.O. and Park, W. (2008) The Role of Disulfide Bond Isomerase A (DsbA) of Escherichia coli O157:H7 in Biofilm Formation and Virulence. FEMS Microbiology Letters, 278, 213-222. http://dx.doi.org/10.1111/j.1574-6968.2007.00993.x
[23]	Mao, Y., Doyle, M.P. and Chen, J. (2001) Insertion Mutagenesis of Wca Reduces Acid and Heat Tolerance of Enterohemorrhagic Escherichia coli O157:H7. Journal of Bacteriology, 183, 3811-3815. http://dx.doi.org/10.1128/JB.183.12.3811-3815.2001
[24]	Roux, A., Beloin, C. and Ghigo, J.M. (2005) Combined Inactivation and Expression Strategy to Study Gene Function under Physiological Conditions: Application to Identification of New Escherichia coli Adhesins. Journal of Bacteriology, 187, 1001-1013. http://dx.doi.org/10.1128/JB.187.3.1001-1013.2005
[25]	Tatsuno, I., Nagano, K., Taquchi, K., Rong, L., Mori, H., and Sasakawa, C. (2003) Increased Adherence to Caco-2 Cells Caused by Disruption of the yhiE and yhiF Genes in Enterohemorrhagic Escherichia coli O157:H7. Infection and Immunity, 71, 2598-2606. http://dx.doi.org/10.1128/IAI.71.5.2598-2606.2003
[26]	Tatsuno, I., Horie, M., Abe, H., Miki, T., Makino, K., Shinagawa, H., Taguchi, H., Kamiya, S., Hayashi, T. and Sasakawa, C. (2001) ToxB Gene on pO157 of Enterohemorrhagic Escherichia coli O157:H7 Is Required for Full Epithelial Cell Adherence Phenotype. Infection and Immunity, 69, 6660-6669. http://dx.doi.org/10.1128/IAI.69.11.6660-6669.2001
[27]	Deng, W., Puente, J.L., Gruenheid, S., Li, Y., Vallance, B.A., Vazquez, A., Barba, J., Ibarra, A., O’Donnel, P., Metalnikov, P., Ashman, K., Lee, S., Goode, D., Pawson, T. and Finlay, B.B. (2004) Dissecting Virulence: Systematic and Functional Analyses of a Pathogenicity Island. Proceedings of the National Academy of Sciences of the United States of America, 101, 3597-3602. http://dx.doi.org/10.1073/pnas.0400326101
[28]	Nadler, C., Shifrin, Y., Nov, S., Kobi, S. and Rosenshine, I. (2006) Characterization of Enteropathogenic Escherichia coli Mutants That Fail to Disrupt Host Cell Spreading and Attachment to Substratum. Infection and Immunity, 74, 839-849. http://dx.doi.org/10.1128/IAI.74.2.839-849.2006
[29]	Batisson, I., Guimond, M.P., Girard, F., An, H., Zhu, C., Oswald, E., Fairbrother, J.M., Jacques, M. and Harel, J. (2003) Characterization of the Novel Factor Paa Involved in the Early Steps of the Adhesion Mechanism of Attaching and Effacing Escherichia coli. Infection and Immunity, 71, 4516-4525. http://dx.doi.org/10.1128/IAI.71.8.4516-4525.2003
[30]	Crane, J.K., McNamara, B.P. and Donnenberg, M.S. (2001) Role of EspF in Host Cell Death Induced by Enteropathogenic Escherichia coli. Cellular Microbiology, 3, 197-211. http://dx.doi.org/10.1046/j.1462-5822.2001.00103.x
[31]	Elliott, S.J., Sperandio, V., Giron, J.A., Shin, S., Mellies, J.L., Wainwright, L., Hutchseon, S.W., McDaniel, T.K. and Kaper, J.B. (2000) The Locus of Enterocyte Effacement (LEE)-Encoded Regulator Controls Expression of Both LEE- and Non-LEE-Encoded Virulence Factors in Enteropathogenic and Enterohemorrhagic Escherichia coli. Infection and Immunity, 68, 6115-6126. http://dx.doi.org/10.1128/IAI.68.11.6115-6126.2000
[32]	Creasey, E.A., Delahay, R.M., Daniell, S.J. and Frankel, G. (2003) Yeast Two-Hybrid System Survey of Interactions between LEE-Encoded Proteins of Enteropathogenic Escherichia coli. Microbiology, 149, 2093-2106. http://dx.doi.org/10.1099/mic.0.26355-0
[33]	Tobe, T., Beatson, S.A., Taniguchi, H., Abe, H., Bailey, C.M., Fivian, A., Younis, R., Matthews, S., Marches, O., Frankel, G., Hayashi, T. and Pallen, M.J. (2006) An Extensive Repertoire of Type III Secretion Effectors in Escherichia coli O157 and the Role of Lambdoid Phages in Their Dissemination. Proceedings of the National Academy of Sciences of the United States of America, 103, 4941-4946. http://dx.doi.org/10.1073/pnas.0604891103
[34]	DeVinney, R., Stein, M., Reinscheid, D., Abe, A., Ruschkowski, S. and Finlay, B.B. (1999) Enterohemorrhagic Escherichia coli O157:H7 Produces Tir, Which Is Translocated to the Host Cell Membrane but Is Not Tyrosine Phosphorylated. Infection and Immunity, 67, 2389-2398.
[35]	Jepson, M.A., Pellegrin, S., Peto, L., Banbury, D.N., Leard, A.D., Mellor, H. and Kenny, B. (2003) Synergistic Roles for the Map and Tir Effector Molecules in Mediating Uptake of Enteropathogenic Escherichia coli (EPEC) into Non-Phagocytic Cells. Cellular Microbiology, 5, 773-783. http://dx.doi.org/10.1046/j.1462-5822.2003.00315.x
[36]	Kenny, B., Ellis, S., Leard, A.D., Warawa, J., Mellor, H. and Jepson, M.A. (2002) Co-Ordinate Regulation of Distinct Host Cell Signalling Pathways by Multifunctional Enteropathogenic Escherichia coli Effector Molecules. Molecular Microbiology, 44, 1095-1107. http://dx.doi.org/10.1046/j.1365-2958.2002.02952.x
[37]	Dziva, F., van Diemen, P.M., Stevens, M.P., Smith, A.J. and Wallis, T.S. (2004) Identification of Escherichia coli O157:H7 Genes Influencing Colonization of the Bovine Gastrointestinal Tract Using Signature-Tagged Mutagenesis. Microbiology, 150, 3631-3645. http://dx.doi.org/10.1099/mic.0.27448-0
[38]	Kanack, K.J., Crawford, J.A., Tatsuno, I., Karmali, M.A. and Kaper, J.B. (2005) SepZ/EspZ Is Secreted and Translocated into HeLa Cells by the Enteropathogenic Escherichia coli Type III Secretion System. Infection and Immunity, 73, 4327-4337. http://dx.doi.org/10.1128/IAI.73.7.4327-4337.2005
[39]	Elliott, S.J., Krejany, E.O., Mellies, J.L., Robins-Browne, R.M., Sasakawa, C. and Kaper, J.B. (2001) EspG, a Novel Type III System-Secreted Protein from Enteropathogenic Escherichia coli with Similarities to VirA of Shigella flexneri. Infection and Immunity, 69, 4027-4033. http://dx.doi.org/10.1128/IAI.69.6.4027-4033.2001
[40]	Hardwidge, P.R., Deng, W., Vallance, B.A., Rodriguez-Escudero, I., Cid, V.J., Molina, M. and Finlay, B.B. (2005) Modulation of Host Cytoskeleton Function by the Enteropathogenic Escherichia coli and Citrobacter Rodentium Effector Protein EspG. Infection and Immunity, 73, 2586-2594. http://dx.doi.org/10.1128/IAI.73.5.2586-2594.2005
[41]	Gruenheid, S., Sekirov, I., Thomas, N.A., Deng, W., O’Donnell, P., Goode, D., Li, Y., Frey, E.A., Brown, N.F., Metalnikov, P., Pawson, T., Ashman, K. and Finlay, B.B. (2004) Identification and Characterization of NleA, a Non-LEE-Encoded Type III Translocated Virulence Factor of Enterohaemorrhagic Escherichia coli O157:H7. Molecular Microbiology, 51, 1233-1249. http://dx.doi.org/10.1046/j.1365-2958.2003.03911.x
[42]	Kelly, M., Hart, E., Mundy, R., Marchès, O., Wiles, S., Badea, L., Luck, S., Tauschek, M., Frankel, G., Robins-Browne, R.M. and Hartland, E.L. (2006) Essential Role of the Type III Secretion System Effector NleB in Colonization of Mice by Citrobacter rodentium. Infection and Immunity, 74, 2328-2337. http://dx.doi.org/10.1128/IAI.74.4.2328-2337.2006
[43]	Marches, O., Wiles, S., Dziva, F., La Ragione, R.M., Schuller, S., Best, A., Phillips, A.D., Hartland, E.L., Woodward, M.L., Stevens, M.P. and Frankel, G. (2005) Characterization of Two Non-Locus of Enterocyte Effacement-Encoded Type III-Translocated Effectors, NleC and NleD, in Attaching and Effacing Pathogens. Infection and Immunity, 73, 8411-8417. http://dx.doi.org/10.1128/IAI.73.12.8411-8417.2005
[44]	Wickham, M. E., Lupp, C., Vazquez, A., Mascarenhas, M., Coburn, B., Coombes, B.K., Karmali, M.A., Puente, J.L., Deng, W. and Finlay, B.B. (2007) Citrobacter rodentium Virulence in Mice Associates with Bacterial Load and the Type III Effector NleE. Microbes and Infection, 9, 400-407. http://dx.doi.org/10.1016/j.micinf.2006.12.016
[45]	Garmendia, J., Ren, Z., Tennant, S., Viera, M.A.M., Chong, Y., Whale, A., Azzopardi, K., Dahan, S., Sircili, M.P., Franzolin, M.R., Trabulsi, L.R., Phillips, A., Gomes, T.A.T., Xu, J., Robins-Browne, R. and Frankel, G. (2005) Distribution of tccP in Clinical Enterohemorrhagic and Enteropathogenic Escherichia coli Isolates. Journal of Clinical Microbiology, 43, 5715-5720. http://dx.doi.org/10.1128/JCM.43.11.5715-5720.2005
[46]	Garmendia, J., Phillips, A.D., Carlier, M.F., Chong, Y., Schuller, S., Marches, O., and Shaw, R.K., Knutton, S. and Frankel, G. (2004) TccP Is an Enterohaemorrhagic Escherichia coli O157:H7 Type III Effector Protein That Couples Tir to the Actin-Cytoskeleton. Cellular Microbiology, 6, 1167-1183. http://dx.doi.org/10.1111/j.1462-5822.2004.00459.x
[47]	Garmendia, J., Carlier, M.F., Egile, C., Didry, D. and Frankel, G. (2006) Characterization of TccP-Mediated N-WASP Activation during Enterohaemorrhagic Escherichia coli Infection. Cellular Microbiology, 8, 1444-1455. http://dx.doi.org/10.1111/j.1462-5822.2006.00723.x
[48]	Vlisidou, I., Dziva, F., La Ragione, R.M., Best, A., Garmendia, J., Hawes, P., Monaghan, P., Cawthraw, S.A., Frankel, G., Woodward, M.J. and Stevens, M.P. (2006) Role of Intimin-Tir Interactions and the Tir-Cytoskeleton Coupling Protein in the Colonization of Calves and Lambs by Escherichia coli O157:H7. Infection and Immunity, 74, 758-764. http://dx.doi.org/10.1128/IAI.74.1.758-764.2006
[49]	Ogura, Y., Ooka, T., Whale, A., Garmendia, J., Beutin, L., Tennant, S., Krause, G., Morabito, S., Chinen, I., Tobe, T., Abe, H., Tozzoli, R., Caprioli, A., Rivas, M., Robins-Browne, R., Hayasha, T. and Frankel, G. (2007) TccP2 of O157: H7 and Non-O157 Enterohemorrhagic Escherichia coli (EHEC): Challenging the Dogma of EHEC-Induced Actin Polymerization. Infection and Immunity, 75, 604-612. http://dx.doi.org/10.1128/IAI.01491-06
[50]	Garmendia, J. and Frankel, G. (2005) Operon Structure and Gene Expression of the EspJ-TccP Locus of Enterohaemorrhagic Escherichia coli O157:H7. FEMS Microbiology Letters, 247, 137-145. http://dx.doi.org/10.1016/j.femsle.2005.04.035
[51]	Ideses, D., Gophna, U., Paitan, Y., Chaudhuri, R.R., Pallen, M.J. and Ron, E.Z. (2005) A Degenerate Type III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis. Journal of Bacteriology, 187, 8164-8171. http://dx.doi.org/10.1128/JB.187.23.8164-8171.2005
[52]	Han, Y., Zhou Z.J. and Zhang, Y.J. (2005) Genes of E. coli O157 Associated with Adherence to Hep-2 Cells. Chinese Journal of Veterinary Science, 3, 266-269.
[53]	Pallen, M.J., Beatson, S.A. and Bailey, C.M. (2005) Bioinformatics Analysis of the Locus for Enterocyte Effacement Provides Novel Insights into Type-III Secretion. BMC Microbiology, 5, 9. http://dx.doi.org/10.1186/1471-2180-5-9
[54]	Yip, C.K., Finlay, B.B. and Strynadka, N.C. (2005) Structural Characterization of a Type III Secretion System Filament Protein in Complex with Its Chaperone. Nature Structural & Molecular Biology, 12, 75-81. http://dx.doi.org/10.1038/nsmb879
[55]	Elliott, S.J., Hutcheson, S.W., Dubois, M.S., Mellies, J.L., Wainwright, L.A., Batchelor, M., Frankel, G., Knutton, S. and Kaper, J.B. (1999) Identification of CesT, a Chaperone for the Type III Secretion of Tir in Enteropathogenic Escherichia coli. Molecular Microbiology, 33, 1176-1189. http://dx.doi.org/10.1046/j.1365-2958.1999.01559.x
[56]	Ogino, T., Ohno, R., Sekiya, K., Kuwae, A., Matsuzawa, T., Nonaka, T., Fukuda, H., Imajoh-Ohmi, S. and Abe, A. (2006) Assembly of the Type III Secretion Apparatus of Enteropathogenic Escherichia coli. Journal of Bacteriology, 188, 2801-2811. http://dx.doi.org/10.1128/JB.188.8.2801-2811.2006
[57]	Boerlin, P., McEwen, S.A., Boerlin-Petzold, F., Wilson, J.B., Johnson, R.P. and Gyles, C.L. (1999) Associations between Virulence Factors of Shiga Toxin-Producing Escherichia coli and Disease in Humans. Journal of Clinical Microbiology, 37, 497-503.
[58]	Siegler, R.L., Obrig, T.G., Pysher, T.J., Tesh, V.L., Denkers, N.D. and Taylor, F.B. (2003) Response to Shiga Toxin 1 and 2 in a Baboon Model of Hemolytic Uremic Syndrome. Pediatric Nephrology, 18, 92-96.
[59]	Louise, C.B. and Obrig, T.G. (1995) Specific Interaction of Escherichia coli O157:H7-Derived Shiga-Like Toxin II with Human Renal Endothelial Cells. The Journal of Infectious Diseases, 172, 1397-401. http://dx.doi.org/10.1093/infdis/172.5.1397
[60]	Ashkenazi, S. and Cleary, T.G. (1989) Rapid Method to Detect Shiga Toxin and Shiga-Like Toxin I Based on Binding to Globotriosyl Ceramide (Gb3), Their Natural Receptor. Journal of Clinical Microbiology, 27, 1145-1150.
[61]	Grys, T.E., Siegel, M.B., Lathem, W.W. and Welch, R.A. (2005) The StcE Protease Contributes to Intimate Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Host Cells. Infection and Immunity, 73, 1295-1303. http://dx.doi.org/10.1128/IAI.73.3.1295-1303.2005
[62]	Brunder, W., Schmidt, H. and Karch, H. (1997) EspP, a Novel Extracellular Serine Protease of Enterohaemorrhagic Escherichia coli O157:H7 Cleaves Human Coagulation Factor V. Molecular Microbiology, 24, 767-778. http://dx.doi.org/10.1128/IAI.73.3.1295-1303.2005
[63]	Wandersman, C. and Delepelaire, P. (1990) TolC, an Escherichia coli Outer Membrane Protein Required for Hemolysin Secretion. Proceedings of the National Academy of Sciences of the United States of America, 87, 4776-4780. http://dx.doi.org/10.1073/pnas.87.12.4776
[64]	Fong, K.P., Chung, W.O., Lamont, R.J. and Demuth, D.R. (2001) Intra- and Interspecies Regulation of Gene Expression by Actinobacillus actinomycetemcomitans LuxS. Infection and Immunity, 69, 7625-7634. http://dx.doi.org/10.1128/IAI.69.12.7625-7634.2001
[65]	Bauer, M.E. and Welch, R.A. (1996) Characterization of an RTX Toxin from Enterohemorrhagic Escherichia coli O157:H7. Infection and Immunity, 64, 167-175.
[66]	Dowd, S.E. and Ishizaki, H. (2006) Microarray Based Comparison of Two Escherichia coli O157:H7 Lineages. BMC Microbiology, 6, 30. http://dx.doi.org/10.1186/1471-2180-6-30
[67]	Lloyd, A.L., Rasko, D.A. and Mobley, H.L. (2007) Defining Genomic Islands and Uropathogen-Specific Genes in Uropathogenic Escherichia coli. Journal of Bacteriology, 189, 3532-3546. http://dx.doi.org/10.1128/JB.01744-06
[68]	Lopez-Solanilla, E., Garcia-Olmedo, F. and Rodriguez-Palenzuela, P. (1998) Inactivation of the SapA to SapF Locus of Erwinia Chrysanthemi Reveals Common Features in Plant and Animal Bacterial Pathogenesis. The Plant Cell, 10, 917-924.
[69]	Lopez-Solanilla, E., Llama-Palacios, A., Collmer, A., Garcia-Olmedo, F. and Rodriguez-Palenzuela, P. (2001) Relative Effects on Virulence of Mutations in the Sap, Pel, and Hrp Loci of Erwinia chrysanthemi. Molecular Plant-Microbe Interactions, 14, 386-393. http://dx.doi.org/10.1094/MPMI.2001.14.3.386
[70]	De Koning-Ward, T.F. and Robins-Browne, R.M. (1995) Contribution of Urease to Acid Tolerance in Yersinia enterocolitica. Infection and Immunity, 63, 3790-3795.
[71]	Friedrich, A.W., Lukas, R., Mellmann, A., Kock, R., Zhang, W., Mathys, W., Bielaszewska, M., Karch, H. (2006) Urease Genes in Non-O157 Shiga Toxin-Producing Escherichia coli: Mostly Silent but Valuable Markers for Pathogenicity. Clinical Microbiology and Infection, 12, 483-486. http://dx.doi.org/10.1111/j.1469-0691.2006.01379.x
[72]	Sanfrigari, F.J., Seoane, A., Rodriguez, M.C., Aquero, J. and Garcia Lobo, J.M. (2007) Characterization of the Urease operon of Brucella abortus and Assessment of Its Role in Virulence of the Bacterium. Infection and Immunity, 75, 774-780. http://dx.doi.org/10.1128/IAI.01244-06
[73]	Nakano, M., Iida, T., Ohnishi, M., Kurokawa, K., Takahashi, A., Tsukamoto, T., Yasunaga, T., Hayasha, T. and Honda, T. (2001) Association of the Urease Gene with Enterohemorrhagic Escherichia coli Strains Irrespective of Their Serogroups. Journal of Clinical Microbiology, 39, 4541-4543. http://dx.doi.org/10.1128/JCM.39.12.4541-4543.2001
[74]	Nakano, M., Iida, T. and Honda, T. (2004) Urease Activity of Enterohaemorrhagic Escherichia coli Depends on a Specific One-Base Substitution in UreD. Microbiology, 150, 3483-3489. http://dx.doi.org/10.1099/mic.0.27280-0
[75]	Fleming, A. and Young, M.Y. (1940) The Inhibitory Action of Potassium Tellurite on Coliform Bacteria. The Journal of Pathology and Bacteriology, 51, 29-35. http://dx.doi.org/10.1002/path.1700510106
[76]	Taylor, D.E., Rooker, M., Keelan, M., Ng, L.K., Martin, I., Perna, N.T., Burland, N.T. and Blattner, F.R. (2002) Genomic Variability of O Islands Encoding Tellurite Resistance in Enterohemorrhagic Escherichia coli O157:H7 Isolates. Journal of Bacteriology, 184, 4690-4698. http://dx.doi.org/10.1128/JB.184.17.4690-4698.2002
[77]	Summers, A.O. and Jacoby, G.A. (1977) Plasmid-Determined Resistance to Tellurium Compounds. Journal of Bacteriology, 129, 276-281.
[78]	Valkova, D., Valkovicova, L., Vavrova, S., Kovacova, E., Mravec, J. and Turna, J. (2007) The Contribution of Tellurite Resistance Genes to the Fitness of Escherichia coli Uropathogenic Strains. Central European Journal of Biology, 2, 182-191. http://dx.doi.org/10.2478/s11535-007-0019-9
[79]	Wilson, R.K., Shaw, R.K., Daniell, S., Knutton, S. and Frankel, G. (2001) Role of EscF, a Putative Needle Complex Protein, in the Type III Protein Translocation System of Enteropathogenic Escherichia coli. Cell Microbiology, 3, 753-762. http://dx.doi.org/10.1046/j.1462-5822.2001.00159.x
[80]	Brunder, W., Schmidt, H. and Karch, H. (1996) KatP, a Novel Catalase-Peroxidase Encoded by the Large Plasmid of Enterohaemorrhagic Escherichia coli O157:H7. Microbiology, 142, 3305-3315. http://dx.doi.org/10.1099/13500872-142-11-3305
[81]	Raetz, C.R.H. and Whitfield, C. (2002) Lipopolysaccharide Endotoxins. Annual Review of Biochemistry, 71,635-700. http://dx.doi.org/10.1146/annurev.biochem.71.110601.135414
[82]	Neves, B.C., Mundy, R., Petrovska, L., Dougan, G., Knutton, S. and Frankel, G. (2003) CesD2 of Enteropathogenic Escherichia coli Is a Second Chaperone for the Type III Secretion Translocator Protein EspD. Infection and Immunity, 71, 2130-2141. http://dx.doi.org/10.1128/IAI.71.4.2130-2141.2003
[83]	Shaw, R.K., Berger, C.N., Feys, B., Knutton, S., Pallen, M.J. and Frankel, G. (2008) Enterohemorrhagic Escherichia coli Exploits EspA Filaments for Attachment to Salad Leaves. Applied and Environmental Microbiology, 74, 2908-2914. http://dx.doi.org/10.1128/AEM.02704-07
[84]	Kresse, A.U., Beltrametti, F., Muller, A., Ebel, F. and Guzman, C.A. (2000) Characterization of SepL of Enterohemorrhagic Escherichia coli. Journal of Bacteriology, 182, 6490-6498. http://dx.doi.org/10.1128/JB.182.22.6490-6498.2000
[85]	Deng, W., Li, Y., Hardwidge, P.R., Frey, E.A., Pfuetzner, R.A., Lee, S., Gruendheid, S., Styrnakda, N.C.J., Puente, J.L. and Finlay, B.B. (2005) Regulation of Type III Secretion Hierarchy of Translocators and Effectors in Attaching and Effacing Bacterial Pathogens. Infection and Immunity, 73, 2135-2146. http://dx.doi.org/10.1128/IAI.73.4.2135-2146.2005
[86]	Jerse, A.E., Yu, J., Tall, B.D. and Kaper, J.B. (1990) A Genetic Locus of Enteropathogenic Escherichia coli Necessary for the Production of Attaching and Effacing Lesions on Tissue Culture Cells. Proceedings of the National Academy of Sciences of the United States of America, 87, 7839-7843. http://dx.doi.org/10.1073/pnas.87.20.7839
[87]	Elliott, S.J., O’Connell, C.B., Koutsouris, A., Brinkley, C., Donnenberg, M.S., Hecht, G. and Kaper, J.B. (2002) A Gene from the Locus of Enterocyte Effacement That Is Required for Enteropathogenic Escherichia coli to Increase Tight-Junction Permeability Encodes a Chaperone for EspF. Infection and Immunity, 70, 2271-2277. http://dx.doi.org/10.1128/IAI.70.5.2271-2277.2002
[88]	Gauthier, A., Puente, J.L. and Finlay, B.B. (2003) Secretin of the Enteropathogenic Escherichia coli Type III Secretion System Requires Components of the Type III Apparatus for Assembly and Localization. Infection and Immunity, 71, 3310-3319. http://dx.doi.org/10.1128/IAI.71.6.3310-3319.2003
[89]	O’Connell, C.B., Creasey, E.A., Knutton, S., Elliott, S., Crowther, L.J., Luo, W., Albert, M.J., Kaper, J.B., Frankel, G. and Donnenberg, M.S. (2004) SepL, a Protein Required for Enteropathogenic Escherichia coli Type III Translocation, Interacts with Secretion Component SepD. Molecular Microbiology, 52, 1613-1625. http://dx.doi.org/10.1111/j.1365-2958.2004.04101.x
[90]	Reading, N.C., Torres, A.G., Kendall, M.M., Hughes, D.T., Yamamoto, K. and Sperandio, V. (2007) A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin. Journal of Bacteriology, 189, 2468-2476. http://dx.doi.org/10.1128/JB.01848-06
[91]	Torres, A.G., Lopez-Sanchez, G.N., Milflores-Flores, L., Patel, S.D., Rojas-Lopez, M., de la Pena, C.F.M., Arenas-Hernandez, M.M.P. and Martinez-Laguna, Y. (2007) Ler and H-NS, Regulators Controlling Expression of the Long Polar Fimbriae of Escherichia coli O157:H7. Journal of Bacteriology, 189, 5916-5928. http://dx.doi.org/10.1128/JB.00245-07
[92]	Ogierman, M.A., Paton, A.W. and Paton, J.C. (2000) Up-Regulation of Both Intimin Andeae-Independent Adherence of Shiga Toxigenic Escherichia coli O157 by ler and Phenotypic Impact of a Naturally Occurring ler Mutation. Infection and Immunity, 68, 5344-5353. http://dx.doi.org/10.1128/IAI.68.9.5344-5353.2000
[93]	Kim, J., Nietfeldt, J. and Benson, A.K. (1999) Octamer-Based Genome Scanning Distinguishes a Unique Subpopulation of Escherichia coli O157:H7 Strains in Cattle. Proceedings of the National Academy of Sciences of the United States of America, 96, 13288-13293. http://dx.doi.org/10.1073/pnas.96.23.13288
[94]	Lee, M.S., Kaspar, C.W., Brosch, R., Shere, J. and Luchansky, J.B. (1996) Genomic Analysis Using Pulsed-Field Gel Electrophoresis of Escherichia coli O157:H7 Isolated from Dairy Calves during the United States National Dairy Heifer Evaluation Project (1991-1992). Veterinary Microbiology, 48, 223-230. http://dx.doi.org/10.1016/0378-1135(95)00135-2
[95]	Dahan, S., Wiles, S., La Ragione, R.M., Best, A., Woodward, M.J., Stevens, M.P., Shaw, R.K., Chong, Y., Knutton, S., Phillips, A. and Frankel, G. (2005) EspJ Is a Prophage-Carried Type III Effector Protein of Attaching and Effacing Pathogens That Modulates Infection Dynamics. Infection and Immunity, 73, 679-686. http://dx.doi.org/10.1128/IAI.73.2.679-686.2005
[96]	Jay, M.T., Cooley, M., Carychao, D., Wiscomb, G.W., Sweitzer, R.A., Crawford-Miksza, L., Farrar, J.A., Lau, D.K., O’Connel, J., Millington, A., Asmundson, R.V., Atwill, E.R. and Mandrell, R.E. (2007) Escherichia coli O157: H7 in Feral Swine Near Spinach Fields and Cattle, Central California Coast. Emerging Infectious Diseases, 13, 1908. http://dx.doi.org/10.3201/eid1312.070763
[97]	Baker, D.R., Moxley, R.A., Steele, M.B., Lejeune, J.T., Christopher-Hennings, J., Chen, D.G., Hardwidge, P.R. and Francis, D.H. (2007) Differences in Virulence among Escherichia coli O157:H7 Strains Isolated from Humans during Disease Outbreaks and from Healthy Cattle. Applied and Environmental Microbiology, 73, 7338-7346. http://dx.doi.org/10.1128/AEM.00755-07
[98]	Donohue-Rolfe, A., Kondova, I., Oswald, S., Hutto, D. and Tzipori, S. (2000) Escherichia coli O157:H7 Strains That Express Shiga Toxin (Stx) 2 Alone Are More Neurotropic for Gnotobiotic Piglets than Are Isotypes Producing only Stx1 or Both Stx1 and Stx2. The Journal of Infectious Diseases, 181, 1825-1829. http://dx.doi.org/10.1086/315421
[99]	Ritchie, J.M., Brady, M.J., Riley, K.N., Ho, T.D., Campellone, K.G., Herman, I.M., Donohue-Rolfe, A., Tzipori, S., Waldor, M.K. and Leong, J.M. (2008) EspFU, a Type III-Translocated Effector of Actin Assembly, Fosters Epithelial Association and Late-Stage Intestinal Colonization by E. coli O157:H7. Cell Microbiology, 10, 836-847. http://dx.doi.org/10.1111/j.1462-5822.2007.01087.x
[100]	Moreira, C.G., Carneiro, S.M., Nataro, J.P., Trabulsi, L.R. and Elias, W.P. (2003) Role of Type I Fimbriae in the Aggregative Adhesion Pattern of Enteroaggregative Escherichia coli. FEMS Microbiology Letters, 226, 79-85. http://dx.doi.org/10.1016/S0378-1097(03)00561-5
[101]	Schmidt, H., Henkel, B. and Karch, H. (1997) A Gene Cluster Closely Related to Type II Secretion Pathway Operons of Gram-Negative Bacteria Is Located on the Large Plasmid of Enterohemorrhagic Escherichia coli O157 Strains. FEMS Microbiology Letters, 148, 265-272. http://dx.doi.org/10.1111/j.1574-6968.1997.tb10299.x
[102]	Lathem, W.W., Grys, T.E., Witowski, S.E., Torres, A.G., Kaper, J.B., Tarr, P.I. and Welch, R.A. (2002) StcE, a Metalloprotease Secreted by Escherichia coli O157:H7 Specifically Cleaves C1 Esterase Inhibitor. Molecular Microbiology, 45, 277-288. http://dx.doi.org/10.1046/j.1365-2958.2002.02997.x
[103]	Ledeboer, N.A. and Jones, B.D. (2005) Exopolysaccharide Sugars Contribute to Biofilm Formation by Salmonella enterica Serovar Typhimurium on HEp-2 Cells and Chicken Intestinal Epithelium. Journal of Bacteriology, 187, 3214-3226. http://dx.doi.org/10.1128/JB.187.9.3214-3226.2005
[104]	Makino, S.I., Tobe, T., Asakura, H., Watarai, M., Ikeda, T., Takeshi, K. and Sasakawa, C. (2003) Distribution of the Secondary Type III Secretion System Locus Found in Enterohemorrhagic Escherichia coli O157:H7 Isolates among the Shiga Toxin-Producing E. coli Strains. Journal of Clinical Microbiology, 41, 2341-2347. http://dx.doi.org/10.1128/JCM.41.6.2341-2347.2003
[105]	Baumler, D.J., Banta, L.M., Hung, K.F., Schwarz, J.A., Cabot, E.C., Glasner, J.D. and Perna, N.T. (2012) Using Comparative Genomics for Inquiry-Based Learning to Dissect Virulence of Escherichia coli O157:H7. CBE-Life Sciences Education, 11, 81-93.
[106]	Jandu, N., Ho, N.K., Donato, K.A., Karmali, M.A., Mascarenhas, M., Duffy, S.P., Tailor, C. and Sherman, P.M. (2009) Enterohemorrhagic Escherichia coli O157:H7 Gene Expression Profiling in Response to Growth in the Presence of Host Epithelia. PLoS ONE, 4, e4889. http://dx.doi.org/10.1371/journal.pone.0004889
[107]	Bansal, T., Englert, D., Lee, J., Hegde, M., Wood, T.K. and Jayaraman, A. (2007) Differential Effects of Epinephrine, Norepinephrine, and Indole on Escherichia coli O157:H7 Chemotaxis, Colonization, and Gene Expression. Infection and Immunity, 75, 4597-4607. http://dx.doi.org/10.1128/IAI.00630-07
[108]	Mukherjee, A., Mammel, M.K., LeClerc, J.E. and Cebula, T.A. (2008) Altered Utilization of N-acetyl-D-Galactosamine by Escherichia coli O157:H7 from the 2006 Spinach Outbreak. Journal of Bacteriology, 190, 1710-1717. http://dx.doi.org/10.1128/JB.01737-07
[109]	Chin, N., Frey, J., Chang, C.F. and Chang, Y.F. (1996) Identification of a Locus Involved in the Utilization of Iron by Actinobacillus pleuropneumoniae. FEMS Microbiology Letters, 143, 1-6. http://dx.doi.org/10.1111/j.1574-6968.1996.tb08452.x
[110]	Baumler, D.J., Peplinski, R.G., Reed, J.L., Glasner, J.D. and Perna, N.T. (2011) The Evolution of Metabolic Networks of E. coli. BMC Systems Biology, 5, 182.
[111]	Monk, J.M., Charusanti, P., Ramy, K.A., Lerman, J.A., Premyodhin, N., Orth, J.D., Feist, A.M. and Palsson, B.O. (2013) Genome-Scale Metabolic Reconstructions of Multiple Escherichia coli Strains Highlight Strain-Specific Adaptations to Nutritional Environments. PNAS, 110, 20338-20343.