Escherichia coli, one of the most abundant bacterial species in the human gut microbiota, has developed a mutualistic relationship with its host, regulating immunological responses. In contrast, enterotoxigenic E. coli (ETEC), one of the main etiologic agents of diarrheal morbidity and mortality in children under the age of five in developing countries, has developed mechanisms to reduce the immune-activator effect to carry out a successful infection. Following infection, the host cell initiates the shutting-off of protein synthesis and stress granule (SG) assembly. This is mostly mediated by the phosphorylation of translation initiator factor 2α (eIF2α). We therefore evaluated the ability of a non-pathogenic E. coli strain (E. coli HS) and an ETEC strain (ETEC 1766a) to induce stress granule assembly, even in response to exogenous stresses. In this work, we found that infection with E. coli HS or ETEC 1766a prevents SG assembly in Caco-2 cells treated with sodium arsenite (Ars) after infection. We also show that this effect occurs through an eIF2α phosphorylation (eIF2α-P)-dependent mechanism. Understanding how bacteria counters host stress responses will lay the groundwork for new therapeutic strategies to bolster host cell immune defenses against these pathogens.

The gaseous headspace above naïve Escherichia Coli (E. coli) cultures and whole human blood inoculated with E. coli were collected and analyzed for the presence of trace gases that may have the potential to be used as novel, non-invasive markers of infectious disease.

Escherichia coli (E. coli) is the most common cause of urinary tract infection (UTI) and typically treated with antibiotics. Unrestricted use of antibiotics may lead to the emergence of antibiotic-resistant bacteria. The present study aimed to isolate and characterize phages against E. coli from infected urine samples and to determine the lytic activity of phages against E. coli in vitro.

Classification of pathogenic Escherichia coli (E. coli) has traditionally relied on detecting specific virulence associated genes (VAGs) or combinations thereof. For E. coli isolated from faecal samples, the presence of specific genes associated with different intestinal pathogenic pathovars will determine their classification and further course of action. However, the E. coli genome is not a static entity, and hybrid strains are emerging that cross the pathovar definitions. Hybrid strains may show gene contents previously associated with several distinct pathovars making the correct diagnostic classification difficult. We extended the analysis of routinely submitted faecal isolates to include known virulence associated genes that are usually not examined in faecal isolates to detect the frequency of possible hybrid strains.

We hereby present the first descriptions of human-invasive infections caused by Escherichia marmotae, a recently described species that encompasses the former "Escherichia cryptic clade V." We describe four cases, one acute sepsis of unknown origin, one postoperative sepsis after cholecystectomy, one spondylodiscitis, and one upper urinary tract infection. Cases were identified through unsystematic queries in a single clinical lab over 6 months. Through genome sequencing of the causative strains combined with available genomes from elsewhere, we demonstrate Es. marmotae to be a likely ubiquitous species containing genotypic virulence traits associated with Escherichia pathogenicity. The invasive isolates were scattered among isolates from a range of nonhuman sources in the phylogenetic analyses, thus indicating inherent virulence in multiple lineages. Pan genome analyses indicate that Es. marmotae has a large accessory genome and is likely to obtain ecologically advantageous traits, such as genes encoding antimicrobial resistance. Reliable identification might be possible by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), but relevant spectra are missing in commercial databases. It can be identified through 16S rRNA gene sequencing. Escherichia marmotae could represent a relatively common human pathogen, and improved diagnostics will provide a better understanding of its clinical importance. IMPORTANCE Escherichia coli is the most common pathogen found in blood cultures and urine and among the most important pathogenic species in the realm of human health. The notion that some of these isolates are not Es. coli but rather another species within the same genus may have implications for what Es. coli constitutes. We only recently have obtained methods to separate the two species, which means that possible differences in important clinical aspects, such as antimicrobial resistance rates, virulence, and phylogenetic structure, may exist. We believe that Es. marmotae as a common pathogen is new merely because we have not looked or bothered to distinguish between the thousands of invasive Escherichia passing through microbiological laboratories each day.

Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications.

Escherichia coli (E. coli) bacteria can damage DNA of the gut lining cells and may encourage the development of colon cancer according to recent reports. Genetic switches are specific sequence motifs and many of them are drug targets. It is interesting to know motifs and their location in sequences. At the present study, Gibbs sampler algorithm was used in order to predict and find functional motifs in E. coli NC101 contig 1. The whole genomic sequence of Escherichia coli NC101 contig 1 were retrieved from http://www.ncbi.nlm.nih.gov (NCBI Reference sequence: NZ_AEFA01000001.1) in order to be analyzed with DAMBE software and BLAST. The results showed that the 6-mer motif is CUGGAA in most sequences (genes1-3, 8, 9, 12, 14-18, 20-23, 25, 27, 29, 31-34), CUUGUA for gene 4 , CUGUAA for gene 5, CUGAUG for gene 6, CUGAUA for gene7, CUGAAA for genes 10, 11, 13, 26, 28, and CUGGAG for gene 19, and CUGGUA for gene30 in E. coli NC101 contig 1. It is concluded that the 6-mer motif is CUGGAA in most sequences in E. coli NC101 contig1. The present study may help experimental studies on elucidating the pharmacological and phylogenic functions of the motifs in E. coli.

No abstract available

Methanol represents an attractive substrate for biotechnological applications. Utilization of reduced one-carbon compounds for growth is currently limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. Here we apply an in silico-guided multiple knockout approach to engineer a methanol-essential Escherichia coli strain, which contains the ribulose monophosphate cycle for methanol assimilation. Methanol conversion to biomass was stoichiometrically coupled to the metabolization of gluconate and the designed strain was subjected to laboratory evolution experiments. Evolved strains incorporate up to 24% methanol into core metabolites under a co-consumption regime and utilize methanol at rates comparable to natural methylotrophs. Genome sequencing reveals mutations in genes coding for glutathione-dependent formaldehyde oxidation (frmA), NAD(H) homeostasis/biosynthesis (nadR), phosphopentomutase (deoB), and gluconate metabolism (gntR). This study demonstrates a successful metabolic re-routing linked to a heterologous pathway to achieve methanol-dependent growth and represents a crucial step in generating a fully synthetic methylotrophic organism.

Shiga toxin-producing Escherichia coli (STEC) are important enteric pathogens worldwide, causing diarrhea with or without blood visibly present and hemolytic uremic syndrome. STEC are unique among diarrheogenic E coli in producing Shiga toxin type 1 and type 2, the virulence factors responsible for bloody diarrhea and hemolytic uremic syndrome. Cattle and other ruminants are the natural reservoir of STEC as their normal intestinal flora. Humans become infected by consumption of foods contaminated with cattle feces. Early diagnosis of STEC infection is important because of the contraindication for treating STEC using antimicrobial agents, and the intense supportive care needed if renal failure occurs.

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped β-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events.

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.

To analyze the immunochemical structure of Escherichia coli ribosomal protein S13 and its organization in situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13 N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S131-22; three monoclonal antibodies bound to S131-40; the binding sites of three other antibodies have been located in S1323-80, with epitopes possibly associated with residues 40-80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whose in situ accessibility may reveal the S13 organization on the ribosome.

Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E. coli O104:H4 in Europe in 2011. We assessed the opportunities for E. coli carrying the aggR and stx genes to emerge in 'backyard' farms in south-east Asia.

In bacterial genomes, the compactly encoded genes and operons are well organized, with genes in the same biological pathway or operons in the same regulon close to each other on the genome sequence. In addition, the linearly close genes have a higher probability of co-expression and their protein products tend to form protein-protein interactions. However, the organization features of bacterial genomes in a three-dimensional space remain elusive. The DNA interaction data of Escherichia coli, measured by the genome conformation capture (GCC) technique, have recently become available, which allowed us to investigate the spatial features of bacterial genome organization.

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mβ1, mβ1'β2, mβ1-β5, mβ1-β6 and mβ4-β5) by transplanting β1, β1'β2, β1-β5, β1-β6 and β4-β5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mβ1'β2(ESWR) SSB was generated by mutating the MtuSSB specific 'PRIY' sequence in the β2 strand of mβ1'β2 SSB to EcoSSB specific 'ESWR' sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mβ1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mβ1-β6, MtuSSB, mβ1'β2 and mβ1-β5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1'β2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

IPEC-J2, a promising in vitro model system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenic Escherichia coli (ETEC), nonpathogenic E. coli, and E. coli endotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured with E. coli strains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction.

To develop an attenuated vaccine candidate against K88ac enterotoxigenic Escherichia coli (ETEC), a novel Escherichia coli (E. coli) K88ac LT(S63K)ΔSTb with LT(S63K) mutation and ST1 deletion was generated using site mutagenesis and λ-Red homologous recombination based on wild paternal ETEC strain C83902. E. coli K88ac LT(S63K)ΔSTb showed very similar fimbriae expression and growth kinetics to the wild strain C83902, but it was significantly attenuated according to the results of a rabbit ligated ileal loop assay and mouse infection study. Oral inoculation with E. coli K88ac LT(S63K)ΔSTb stimulated the mucosa immune response and induced the secretion of IgA to K88ac in the intestines in mice. A challenge experiment revealed that the attenuated strain provided efficient protection against C83902 in the following 7 days and at the 24th day post-inoculation, suggesting that the attenuated isolate could act as an ecological protectant and vaccine in preventing K88ac ETEC.

Recent developments in high-throughput reverse genetics1,2 have revolutionized our ability to map gene function and interactions3-6. The power of these approaches depends on their ability to identify functionally associated genes, which elicit similar phenotypic changes across several perturbations (chemical, environmental or genetic) when knocked out7-9. However, owing to the large number of perturbations, these approaches have been limited to growth or morphological readouts10. Here we use a high-content biochemical readout, thermal proteome profiling11, to measure the proteome-wide protein abundance and thermal stability in response to 121 genetic perturbations in Escherichia coli. We show that thermal stability, and therefore the state and interactions of essential proteins, is commonly modulated, raising the possibility of studying a protein group that is particularly inaccessible to genetics. We find that functionally associated proteins have coordinated changes in abundance and thermal stability across perturbations, owing to their co-regulation and physical interactions (with proteins, metabolites or cofactors). Finally, we provide mechanistic insights into previously determined growth phenotypes12 that go beyond the deleted gene. These data represent a rich resource for inferring protein functions and interactions.

Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency kcat/Km, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration.