Showing papers on "Escherichia coli published in 2014"
TL;DR: The different approaches for the synthesis of recombinant proteins in E. coli are reviewed and recent progress in this ever-growing field is discussed.
Abstract: Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.
1,883 citations
TL;DR: This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.
Abstract: Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.
499 citations
TL;DR: Analyses of the genome sequences of a large number of E. coli strains and of strains from > 100 other bacterial genera indicate a value of 79-80% dDDH as the most promising threshold for delineating subspecies, which in turn suggests the presence of five subspecies withinE.
Abstract: Although Escherichia coli is the most widely studied bacterial model organism and often considered to be the model bacterium per se, its type strain was until now forgotten from microbial genomics. As a part of the G enomic E ncyclopedia of B acteria and A rchaea project, we here describe the features of E. coli DSM 30083T together with its genome sequence and annotation as well as novel aspects of its phenotype. The 5,038,133 bp containing genome sequence includes 4,762 protein-coding genes and 175 RNA genes as well as a single plasmid. Affiliation of a set of 250 genome-sequenced E. coli strains, Shigella and outgroup strains to the type strain of E. coli was investigated using digital DNA:DNA-hybridization (dDDH) similarities and differences in genomic G+C content. As in the majority of previous studies, results show Shigella spp. embedded within E. coli and in most cases forming a single subgroup of it. Phylogenomic trees also recover the proposed E. coli phylotypes as monophyla with minor exceptions and place DSM 30083T in phylotype B2 with E. coli S88 as its closest neighbor. The widely used lab strain K-12 is not only genomically but also physiologically strongly different from the type strain. The phylotypes do not express a uniform level of character divergence as measured using dDDH, however, thus an alternative arrangement is proposed and discussed in the context of bacterial subspecies. Analyses of the genome sequences of a large number of E. coli strains and of strains from > 100 other bacterial genera indicate a value of 79-80% dDDH as the most promising threshold for delineating subspecies, which in turn suggests the presence of five subspecies within E. coli.
367 citations
TL;DR: Colibactin-producing E. coli enhanced tumour growth in both xenograft and AOM/DSS models, revealing a new paradigm for carcinogenesis, in which colibactIn-induced senescence has an important role.
Abstract: Background Escherichia coli strains harbouring the pks island ( pks + E. coli ) are often seen in human colorectal tumours and have a carcinogenic effect independent of inflammation in an AOM/IL-10 −/− (azoxymethane/interleukin) mouse model. Objective To investigate the mechanism sustaining pks + E. coli -induced carcinogenesis. Method Underlying cell processes were investigated in vitro and in vivo (xenograft model) using intestinal epithelial cells infected by pks + E. coli or by an isogenic mutant defective for pks ( pks − E. coli ). The results were supported by data obtained from an AOM/DSS (azoxymethane/dextran sodium sulphate) colon cancer mouse model and from human colon cancer biopsy specimens colonised by pks + E. coli or pks− E. coli . Results Colibactin-producing E. coli enhanced tumour growth in both xenograft and AOM/DSS models. Growth was sustained by cellular senescence (a direct consequence of small ubiquitin-like modifier (SUMO)-conjugated p53 accumulation), which was accompanied by the production of hepatocyte growth factor (HGF). The underlying mechanisms involve microRNA-20a-5p, which targets SENP1, a key protein regulating p53 deSUMOylation. These results are consistent with the expression of SENP1, microRNA-20a-5p, HGF and phosphorylation of HGF receptor found in human and mouse colon cancers colonised by pks + E. coli . Conclusion These data reveal a new paradigm for carcinogenesis, in which colibactin-induced senescence has an important role.
341 citations
TL;DR: This work demonstrates that E. coli can be engineered into living diagnostics capable of nondestructively probing the mammalian gut and lays a foundation for the use of synthetic genetic circuits as monitoring systems in complex, ill-defined environments.
Abstract: The mammalian gut is a dynamic community of symbiotic microbes that interact with the host to impact health, disease, and metabolism. We constructed engineered bacteria that survive in the mammalian gut and sense, remember, and report on their experiences. Based on previous genetic memory systems, we constructed a two-part system with a “trigger element” in which the lambda Cro gene is transcribed from a tetracycline-inducible promoter, and a “memory element” derived from the cI/Cro region of phage lambda. The memory element has an extremely stable cI state and a Cro state that is stable for many cell divisions. When Escherichia coli bearing the memory system are administered to mice treated with anhydrotetracycline, the recovered bacteria all have switched to the Cro state, whereas those administered to untreated mice remain in the cI state. The trigger and memory elements were transferred from E. coli K12 to a newly isolated murine E. coli strain; the stability and switching properties of the memory element were essentially identical in vitro and during passage through mice, but the engineered murine E. coli was more stably established in the mouse gut. This work lays a foundation for the use of synthetic genetic circuits as monitoring systems in complex, ill-defined environments, and may lead to the development of living diagnostics and therapeutics.
337 citations
TL;DR: Findings support that pathogenic E. coli could be a cofactor in pathogenesis of colorectal cancer.
Abstract: Purpose: The intestinal microbiota is potentially involved in the development of colorectal carcinoma via various mechanisms. Escherichia coli are commensal bacteria of the human gut microbiota, but some pathogenic strains have acquired the ability to induce chronic inflammation and/or produce toxins, such as cyclomodulin, which could participate in the carcinogenesis process. Here, we analyzed the E. coli population associated with mucosa of patients with colon cancer in relation to clinicopathologic characteristics. We assessed carcinogenic properties of a colon cancer–associated E. coli strain in multiple intestinal neoplasia (Min) mice. Experimental design: Mucosa-associated or internalized E. coli were quantified and characterized from tumors and mucosa of patients with colon cancer and the healthy mucosa of diverticulosis controls. Min mice were inoculated with a colon cancer–associated E. coli strain (11G5). The number of colonic polyps was evaluated at 7 weeks after infection. Results: An increased level of mucosa-associated and internalized E. coli was observed in the tumors compared with normal tissue. A relationship between poor prognostic factors for colon cancer (tumor–node–metastasis stage) and colonization of mucosa by E. coli was observed. Pathogenic cyclomodulin-positive E. coli strains were more prevalent on mucosa of patients with stages III/IV than those with stage I colon cancer. Proliferative index and E. coli colonization level of the mucosa distant from the tumor significantly correlated. Min mice infected with the E. coli strain 11G5 displayed a marked increase in the number of visible colonic polyps compared with controls. Conclusion: These findings support that pathogenic E. coli could be a cofactor in pathogenesis of colorectal cancer. Clin Cancer Res; 20(4); 859–67. ©2013 AACR .
330 citations
TL;DR: The prototype toxin for each group is now designated Stx1a or Stx2a, which exhibit differences in cytotoxicity to various cell types, bind dissimilarly to receptor analogs or mimics, induce differential chemokine responses, and have several distinctive structural characteristics.
Abstract: Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dysenteriae 1 and in some serogroups of Escherichia coli (called Stx1 in E. coli). In addition to or instead of Stx1, some E. coli strains produce a second type of Stx, Stx2, that has the same mode of action as Stx/Stx1 but is antigenically distinct. Because subtypes of each toxin have been identified, the prototype toxin for each group is now designated Stx1a or Stx2a. The Stxs consist of two major subunits, an A subunit that joins noncovalently to a pentamer of five identical B subunits. The A subunit of the toxin injures the eukaryotic ribosome and halts protein synthesis in target cells. The function of the B pentamer is to bind to the cellular receptor, globotriaosylceramide, Gb3, found primarily on endothelial cells. The Stxs traffic in a retrograde manner within the cell, such that the A subunit of the toxin reaches the cytosol only after the toxin moves from the endosome to the Golgi and then to the endoplasmic reticulum. In humans infected with Stx-producing E. coli, the most serious manifestation of the disease, hemolytic-uremic syndrome, is more often associated with strains that produce Stx2a rather than Stx1a, and that relative toxicity is replicated in mice and baboons. Stx1a and Stx2a also exhibit differences in cytotoxicity to various cell types, bind dissimilarly to receptor analogs or mimics, induce differential chemokine responses, and have several distinctive structural characteristics.
322 citations
TL;DR: In this paper, the authors used 16S rRNA sequencing of luminal microbiota from ex-germ-free mice to show that inflamed mice maintain a higher abundance of Enterobacteriaceae than healthy wild-type mice.
Abstract: Enterobacteria, especially Escherichia coli, are abundant in patients with inflammatory bowel disease or colorectal cancer (CRC). However, it is unclear whether cancer is promoted by inflammation-induced expansion of E. coli and/or changes in expression of specific microbial genes. Here we use longitudinal (2, 12 and 20 weeks) 16S rRNA sequencing of luminal microbiota from ex-germ-free mice to show that inflamed Il10(-/-) mice maintain a higher abundance of Enterobacteriaceae than healthy wild-type mice. Experiments with mono-colonized Il10(-/-) mice reveal that host inflammation is necessary for E. coli cancer-promoting activity. RNA-sequence analysis indicates significant changes in E. coli gene catalogue in Il10(-/-) mice, with changes mostly driven by adaptation to the intestinal environment. Expression of specific genes present in the tumour-promoting E. coli pks island are modulated by inflammation/CRC development. Thus, progression of inflammation in Il10(-/-) mice supports Enterobacteriaceae and alters a small subset of microbial genes important for tumour development.
298 citations
TL;DR: The findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods, and suggest that cepinghalosporain resistance genes are mainly disseminated in animals and humans via distinct plasmids.
Abstract: Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.
269 citations
TL;DR: In this article, an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) was constructed and applied to the synthesis of vanillin from vanillate and succeeded in preventing formation of vanillyl alcohol.
Abstract: Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.
219 citations
TL;DR: In bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs) and provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.
TL;DR: Overall, the data reveal a new paradigm for carcinogenesis in which pks+ E. coli infection induces cellular senescence characterized by the production of growth factors that promote the proliferation of uninfected cells and, subsequently, tumor growth.
Abstract: The gut microbiota is suspected to promote colorectal cancer (CRC). Escherichia coli are more frequently found in CCR biopsies than in healthy mucosa; furthermore, the majority of mucosa-associated E. coli isolated from CCR harbors the pks genomic island (pks+ E. coli) that is responsible for the synthesis of colibactin, a genotoxic compound. We have recently reported that transient contact of a few malignant cells with colibactin-producing E. coli increases tumor growth in a xenograft mouse model. Growth is sustained by cellular senescence that is accompanied by the production of growth factors. We demonstrated that cellular senescence is a consequence of the pks+ E. coli-induced alteration of p53 SUMOylation, an essential post-translational modification in eukaryotic cells. The underlying mechanisms for this process involve the induction of miR-20a-5p expression, which targets SENP1, a key protein in the regulation of the SUMOylation process. These results are consistent with the expression of SENP1, miR-20a-5p and growth factors that are observed in a CRC mouse model and in human CCR biopsies colonized by pks+ E. coli. Overall, the data reveal a new paradigm for carcinogenesis in which pks+ E. coli infection induces cellular senescence characterized by the production of growth factors that promote the proliferation of uninfected cells and, subsequently, tumor growth.
30 Apr 2014
TL;DR: The severity of the illness it causes combined with its apparent low infectious dose qualifies E. coli O157:H7 to be among the most serious of known foodborne pathogens.
Abstract: Diarrheagenic Escherichia coli isolates are categorized into specific groups (pathotypes) based on virulence properties, mechanisms of pathogenicity, clinical syndromes, and distinct O:H serotypes. This chapter focuses on enterohemorrhagic E. coli (EHEC), which among the E. coli strains that cause foodborne illness in the United States, is the most significant group based on frequency and severity of illness. Since E. coli O157:H7 is the most common serotype of the EHEC and because more is known about this serotype than other serotypes of EHEC, the chapter focuses on E. coli O157:H7. E. coli O157:H7 strains isolated from humans, animals, and food have developed resistance to multiple antibiotics, with streptomycin-sulfisoxazoletetracycline being the most common resistance profile. Details of many reported foodborne and waterborne outbreaks of EHEC infections are provided in the chapter. The nomenclature of the Shiga toxins (Stx) family and their important characteristics are listed. Stx and Stx1 production is negatively regulated at the transcriptional level by an iron-Fur protein corepressor complex which binds at the stx1
promoter but is unaffected by temperature. The severity of the illness it causes combined with its apparent low infectious dose qualifies E. coli O157:H7 to be among the most serious of known foodborne pathogens. E. coli O157: H7 is still by far the most important serotype of Shiga toxin-producing E. coli (STEC) in North America. Isolation of non-O157:H7 STEC requires techniques not generally used in clinical laboratories; hence, these bacteria are infrequently sought or detected in routine practice.
TL;DR: The present work enables the production of small to medium volatile esters by designing several ester pathways in E. coli that generated acetate esters of ethyl, propyl, isobutyl, 2- methyl-1-butyl, 3-methyl-1,butyl and 2-phenylethyl alcohols.
Abstract: The use of CoA thioester intermediates drives formation of small- and medium-sized esters in metabolically engineered E. coli cells, including doubly branched chains generated with enzymes from amino acid degradation pathways.
TL;DR: In this article, a fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating colorectal cancer (CRC) isolate.
Abstract: Objective: Colonic mucosa-associated Escherichia coli are increased in Crohn's disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates.
Design: A fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome).
Results: 454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin.
Conclusions: IBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.
TL;DR: The results indicated that ɛ-poly-lysine has good potential to be as a natural food preservative and could damage bacterial cells through the destruction of cellular proteins.
30 Apr 2014
TL;DR: This chapter reviews the genetics and mapping of fla-che-mot genes, then the mechanisms of motility and chemotaxis, and finally, what is known about regulation of gene expression in theFla-Che-mot system.
Abstract: This chapter reviews the genetics and mapping of fla-che-mot genes, then the mechanisms of motility and chemotaxis, and finally, what is known about regulation of gene expression in the fla-che-mot system. It also concentrates on some important classes of mutations. Several mutations have been isolated on the basis of their abilities to reduce the level of autolytic enzyme expression. These strains were obtained by minimal mutagenesis and found to contain a single mutation that gave rise to a Lyt- Fla- phenotype. Chemotaxis in enteric bacteria is mediated through sets of cellular receptors that bind specific attractants. Such binding initiates a signal that is transduced through a series of protein intermediates by specific methylation-demethylation and phosphorylation-dephosphorylation. The most detailed information about the mechanisms involved in sensory transduction comes from studies of enteric bacteria, in which the majority of the genes involved in structural and regulatory roles are probably known and have been sequenced. As in Escherichia coli, the methylated chemotaxis proteins (MCPs) of Bacillus subtilis are integral membrane proteins that are methyl esterified on glutamate side chains as the result of methyl transfer from S-adenosylmethionine. Expression of the genes of the sensory pathway is regulated in both B. subtilis and enteric bacteria by the composition of the growth medium, by the stage of cell growth, and by the expression of other fla-che-mot genes needed for a functional sensory system.
TL;DR: The complete 4,631,469-bp genome sequence of this strain and the key variations from the type strain E. coli MG1655 are reported.
Abstract: Escherichia coli BW25113 is the parent strain of the Keio collection comprising nearly 4,000 single-gene deletion mutants. We report the complete 4,631,469-bp genome sequence of this strain and the key variations from the type strain E. coli MG1655.
TL;DR: This review summarizes the different biochemical strategies that are employed in the three key steps for methionine biosynthesis from homoserine and discusses the presence of one gene in a large number of species that appear to lack the gene encoding the enzyme for the preceding step in the pathway, as it is understood in E. coli.
Abstract: Methionine is essential in all organisms, as it is both a proteinogenic amino acid and a component of the cofactor, S-adenosyl methionine. The metabolic pathway for its biosynthesis has been extensively characterized in Escherichia coli; however, it is becoming apparent that most bacterial species do not use the E. coli pathway. Instead, studies on other organisms and genome sequencing data are uncovering significant diversity in the enzymes and metabolic intermediates that are used for methionine biosynthesis. This review summarizes the different biochemical strategies that are employed in the three key steps for methionine biosynthesis from homoserine (i.e. acylation, sulfurylation and methylation). A survey is presented of the presence and absence of the various biosynthetic enzymes in 1593 representative bacterial species, shedding light on the non-canonical nature of the E. coli pathway. This review also highlights ways in which knowledge of methionine biosynthesis can be utilized for biotechnological applications. Finally, gaps in the current understanding of bacterial methionine biosynthesis are noted. For example, the paper discusses the presence of one gene (metC) in a large number of species that appear to lack the gene encoding the enzyme for the preceding step in the pathway (metB), as it is understood in E. coli. Therefore, this review aims to move the focus away from E. coli, to better reflect the true diversity of bacterial pathways for methionine biosynthesis.
TL;DR: This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs.
Abstract: Streptococcus pneumoniae (S. pneumoniae) causes several serious diseases including pneumonia, septicemia and meningitis. The World Health Organization estimates that streptococcal pneumonia is the cause of approximately 1.9 million deaths of children under five years of age each year. The large number of serotypes underlying the disease spectrum, which would be reflected in the high production cost of a commercial vaccine effective to protect against all of them and the higher level of amino acid sequence conservation as compared to polysaccharide structure, has prompted us to attempt to use conserved proteins for the development of a simpler vaccine. One of the most prominent proteins is pneumolysin (Ply), present in almost all the serotypes known at the moment, which shows an effective protection against S. pneumoniae infections. We have cloned the pneumolysin gene from S. pneumoniae serotype 14 and studied the effects of eight variables related to medium composition and induction conditions on the soluble expression of rPly in Escherichia coli (E. coli) and a 28-4 factorial design was applied. Statistical analysis was carried out to compare the conditions used to evaluate the expression of soluble pneumolysin; rPly activity was evaluated by hemolytic activity assay and served as the main response to evaluate the proper protein expression and folding. The optimized conditions, validated by the use of triplicates, include growth until an absorbance of 0.8 (measured at 600 nm) with 0.1 mM IPTG during 4 h at 25°C in a 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose medium, with addition of 30 μg/mL kanamycin. This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs. It was possible to recover the protein in its active form with 75% homogeneity.
TL;DR: Assessment and characterized cell-free protein synthesis (CFPS) in crude S30 cell lysates derived from a genomically recoded Escherichia coli strain lacking RF1, and demonstrated benefits of CFPS from the RF1-deficient strains for incorporating pPaF at two- and five-sites per sfGFP protein.
Abstract: Site-specific incorporation of nonstandard amino acids (NSAAs) into proteins enables the creation of biopolymers, proteins, and enzymes with new chemical properties, new structures, and new functions. To achieve this, amber (TAG codon) suppression has been widely applied. However, the suppression efficiency is limited due to the competition with translation termination by release factor 1 (RF1), which leads to truncated products. Recently, we constructed a genomically recoded Escherichia coli strain lacking RF1 where 13 occurrences of the amber stop codon have been reassigned to the synonymous TAA codon (rEc.E13.ΔprfA). Here, we assessed and characterized cell-free protein synthesis (CFPS) in crude S30 cell lysates derived from this strain. We observed the synthesis of 190 ± 20 μg/mL of modified soluble superfolder green fluorescent protein (sfGFP) containing a single p-propargyloxy-l-phenylalanine (pPaF) or p-acetyl-l-phenylalanine. As compared to the parent rEc.E13 strain with RF1, this results in a mod...
TL;DR: It is demonstrated that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential.
Abstract: Objectives: To discern the relevance of ST648 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli as a putative new group of multiresistant and extraintestinal pathogenic strains in animals, its frequency, ESBL types, antimicrobial resistance patterns and virulence gene (VG) profiles should be determined and compared with ST131 strains from the same collection of strains. Methods: ESBL-producing E. coli isolates (n=1152), consecutively sampled from predominantly dogs, cats and horses between 2008 and 2011, were assigned to a phylogenetic group by PCR. Partial multilocus sequence typingwas performed for group D and B2 strains and strains presumed to be D-ST648 and B2-ST131 were fully typed. ESBL genes and extraintestinal pathogenic E. coli (ExPEC)-like VGs were characterized by PCR and sequence analysis and antimicrobial resistance was determined by broth dilution. Clonal analysis was done by PFGE. Results: Forty (3.5%) ESBL-producing E. coli were determined as D-ST648, whereas B2-ST131 isolates occurred less frequently (2.8%). Although the predominant ESBL type in both groups was CTX-M-15 (72.5% versus 46.9%), ST648 strains from companion animals and horses displayed a lower variety of ESBL types (CTX-M-1,-3,-14,-15 and-61 versus CTX-M-1,-2,-14,-15,-27 and-55 and SHV-12). In contrast to ST131 strains, a higher proportion of ST648 strains showed resistance to most non-b-lactam antibiotics. Overall, VGs were less abundant in ST648 strains, although some strains had VG profiles comparable to those of ST131 strains. ExPEC-associated serotype O1:H6 was predominant (46.8%) among the ST648 strains. Some PFGE clusters comprised ST648 isolates from pets, horses and wild birds and humans included from previous studies. Conclusions: Our findings demonstrate that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
TL;DR: The values of both yield and productivity of 2,3-BD obtained with the optimized biological system are the highest ever achieved with an engineered E. coli strain.
TL;DR: In vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation.
Abstract: AIM: To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients.
METHODS: Phylogroups and the presence of cyclomodulin-encoding genes of mucosa-associated E. coli from colon cancer and diverticulosis specimens were determined by PCR. Adhesion and invasion experiments were performed with I-407 intestinal epithelial cells using gentamicin protection assay. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression in T84 intestinal epithelial cells was measured by enzyme-linked immunosorbent assay and by Western Blot. Gut colonization, inflammation and pro-carcinogenic potential were assessed in a chronic infection model using CEABAC10 transgenic mice. Cell proliferation was analyzed by real-time mRNA quantification of PCNA and immunohistochemistry staining of Ki67.
RESULTS: Analysis of mucosa-associated E. coli from colon cancer and diverticulosis specimens showed that whatever the origin of the E. coli strains, 86% of cyclomodulin-positive E. coli belonged to B2 phylogroup and most harbored polyketide synthase (pks) island, which encodes colibactin, and/or cytotoxic necrotizing factor (cnf) genes. In vitro assays using I-407 intestinal epithelial cells revealed that mucosa-associated B2 E. coli strains were poorly adherent and invasive. However, mucosa-associated B2 E. coli similarly to Crohn’s disease-associated E. coli are able to induce CEACAM6 expression in T84 intestinal epithelial cells. In addition, in vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation.
CONCLUSION: In conclusion, these data bring new insights into the ability of E. coli isolated from patients with colon cancer to establish persistent colonization, exacerbate inflammation and trigger carcinogenesis.
TL;DR: This review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli.
Abstract: Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.
TL;DR: A novel biosensor for the detection of E. coli O157:H7 is described here using a film composed of ferrocene-peptide conjugates, in which the antimicrobial peptide magainin I has been incorporated as the biorecognition element.
TL;DR: This unit describes methods to produce disulfide‐bonded proteins in E. coli in either the naturally oxidizing periplasm or the cytoplasm of appropriately engineered cells, with the aim of helping the researcher optimize expression conditions for a protein of interest.
Abstract: Production of recombinant proteins at high yields in Escherichia coli requires extensive optimization of expression conditions. Production is further complicated for proteins that require specific post-translational modifications for their eventual folding. One common and particularly important post-translational modification is oxidation of the correct pair of cysteines to form a disulfide bond. This unit describes methods to produce disulfide-bonded proteins in E. coli in either the naturally oxidizing periplasm or the cytoplasm of appropriately engineered cells. The focus is on variables key to improving the oxidative folding of disulfide-bonded proteins, with the aim of helping the researcher optimize expression conditions for a protein of interest.
TL;DR: Uropathogenic E. coli have the capacity to subvert this immune response of the host by means of actively impacting on pro-inflammatory signalling pathways, or by physical masking of immunogenic structures.
Abstract: Urinary tract infections (UTIs) belong to the most common infectious diseases worldwide. The most frequently isolated pathogen from uncomplicated UTIs is Escherichia coli. To establish infection in the urinary tract, E. coli has to overcome several defence strategies of the host, including the urine flow, exfoliation of urothelial cells, endogenous antimicrobial factors and invading neutrophils. Thus, uropathogenic E. coli (UPEC) harbour a number of virulence and fitness factors enabling the bacterium to resist and overcome these different defence mechanisms. There is no particular factor which allows the identification of UPEC among the commensal faecal flora apart from the ability to enter the urinary tract and cause an infection. Many of potential virulence or fitness factors occur moreover with high redundancy. Fimbriae are inevitable for adherence to and invasion into the host cells; the type 1 pilus is an established virulence factor in UPEC and indispensable for successful infection of the urinary tract. Flagella and toxins promote bacterial dissemination, while different iron-acquisition systems allow bacterial survival in the iron-limited environment of the urinary tract. The immune response to UPEC is primarily mediated by toll-like receptors recognising lipopolysaccharide, flagella and other structures on the bacterial surface. UPEC have the capacity to subvert this immune response of the host by means of actively impacting on pro-inflammatory signalling pathways, or by physical masking of immunogenic structures. The large repertoire of bacterial virulence and fitness factors in combination with host-related differences results in a complex interaction between host and pathogen in the urinary tract.
TL;DR: The design, synthesis, and characterization of enterobactin–antibiotic conjugates, hereafter Ent-Amp/Amx, where the β-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized enterobactsin scaffold via a stable poly(ethylene glycol) linker are reported provide enhanced antibacterial activity against Escherichia coli strains under conditions of iron limitation.
Abstract: The design, synthesis, and characterization of enterobactin-antibiotic conjugates, hereafter Ent-Amp/Amx, where the β-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized enterobactin scaffold via a stable poly(ethylene glycol) linker are reported. Under conditions of iron limitation, these siderophore-modified antibiotics provide enhanced antibacterial activity against Escherichia coli strains, including uropathogenic E. coli CFT073 and UTI89, enterohemorrhagic E. coli O157:H7, and enterotoxigenic E. coli O78:H11, compared to the parent β-lactams. Studies with E. coli K-12 derivatives defective in ferric enterobactin transport reveal that the enhanced antibacterial activity observed for this strain requires the outer membrane ferric enterobactin transporter FepA. A remarkable 1000-fold decrease in minimum inhibitory concentration (MIC) value is observed for uropathogenic E. coli CFT073 relative to Amp/Amx, and time-kill kinetic studies demonstrate that Ent-Amp/Amx kill this strain more rapidly at 10-fold lower concentrations than the parent antibiotics. Moreover, Ent-Amp and Ent-Amx selectively kill E. coli CFT073 co-cultured with other bacterial species such as Staphylococcus aureus, and Ent-Amp exhibits low cytotoxicity against human T84 intestinal cells in both the apo and iron-bound forms. These studies demonstrate that the native enterobactin platform provides a means to effectively deliver antibacterial cargo across the outer membrane permeability barrier of Gram-negative pathogens utilizing enterobactin for iron acquisition.
TL;DR: The results indicate that UPEC isolate from hospital patients differ from archetypal community-acquired isolates from uncomplicated UTIs by their spectrum of virulence traits, and calls for a reevaluation of the strict pathotype classification of EAEC.
Abstract: Uropathogenic Escherichia coli (UPEC) is the most common cause of community- and hospital-acquired urinary tract infections (UTIs). Isolates from uncomplicated community-acquired UTIs express a variety of virulence traits that promote the efficient colonization of the urinary tract. In contrast, nosocomial UTIs can be caused by E. coli strains that differ in their virulence traits from the community-acquired UTI isolates. UPEC virulence markers are used to distinguish these facultative extraintestinal pathogens, which belong to the intestinal flora of many healthy individuals, from intestinal pathogenic E. coli (IPEC). IPEC is a diarrheagenic pathogen with a characteristic virulence gene set that is absent in UPEC. Here, we characterized 265 isolates from patients with UTIs during inpatient or outpatient treatment at a hospital regarding their phylogenies and IPEC or UPEC virulence traits. Interestingly, 28 of these isolates (10.6%) carried typical IPEC virulence genes that are characteristic of enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), and atypical enteropathogenic E. coli (aEPEC), although IPEC is not considered a uropathogen. Twenty-three isolates harbored the astA gene coding for the EAEC heat-stable enterotoxin 1 (EAST1), and most of them carried virulence genes that are characteristic of UPEC and/or EAEC. Our results indicate that UPEC isolates from hospital patients differ from archetypal community-acquired isolates from uncomplicated UTIs by their spectrum of virulence traits. They represent a diverse group, including EAEC, as well as other IPEC pathotypes, which in addition contain typical UPEC virulence genes. The combination of typical extraintestinal pathogenic E. coli (ExPEC) and IPEC virulence determinants in some isolates demonstrates the marked genome plasticity of E. coli and calls for a reevaluation of the strict pathotype classification of EAEC.