Showing papers on "Escherichia coli published in 1997"
TL;DR: The roles of the rhl and las quorum-sensing systems in virulence gene expression are characterized and RNA analysis of the wild-type strain revealed that rhlAB is organized as an operon.
Abstract: Two quorum-sensing systems (las and rhl) regulate virulence gene expression in Pseudomonas aeruginosa. The las system consists of a transcriptional activator, LasR, and LasI, which directs the synthesis of the autoinducer N-(3-oxododecanoyl) homoserine lactone (PAI-1). Induction of lasB (encoding elastase) and other virulence genes requires LasR and PAI-1. The rhl system consists of a putative transcriptional activator, RhlR, and RhlI, which directs the synthesis of N-butyryl homoserine lactone (PAI-2). Rhamnolipid production in P. aeruginosa has been reported to require both the rhl system and rhlAB (encoding a rhamnosyltransferase). Here we report the generation of a delta lasI mutant and both delta lasI delta rhlI and delta lasR rhlR::Tn501 double mutants of strain PAO1. Rhamnolipid production and elastolysis were reduced in the delta lasI single mutant and abolished in the double-mutant strains. rhlAB mRNA was not detected in these strains at mid-logarithmic phase but was abundant in the parental strain. Further RNA analysis of the wild-type strain revealed that rhlAB is organized as an operon. The rhlAB transcriptional start was mapped, and putative sigma 54 and sigma 70 promoters were identified upstream. To define components required for rhlAB expression, we developed a bioassay in Escherichia coli and demonstrated that PAI-2 and RhlR are required and sufficient for expression of rhlA. To characterize the putative interaction between PAI-2 and RhlR, we demonstrated that [3H]PAI-2 binds to E. coli cells expressing RhlR and not to those expressing LasR. Finally, the specificity of the las and rhl systems was examined in E. coli bioassays. The las system was capable of mildly activating rhlA, and similarly, the rhl system partly activated lasB. However; these effects were much less than the activation of rhlA by the rhl system and lasB by the las system. The results presented here further characterize the roles of the rhl and las quorum-sensing systems in virulence gene expression.
978 citations
TL;DR: A simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies and proper expression of the lacZ transgene in the brain has been observed.
Abstract: Escherichia coli-based artificial chromosomes have become important tools for physical mapping and sequencing in various genome projects. The lack of a general method to modify these large bacterial clones, however, has limited their utility in functional studies. We developed a simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies. The IRES-LacZ marker gene was introduced into a 131 kb BAC containing the murine zinc finger gene, RU49. No rearrangements or deletions were detected in the modified BACs. Furthermore, transgenic mice were generated by pronuclear injection of the modified BAC, and germline transmission of the intact BAC has been obtained. Proper expression of the lacZ transgene in the brain has been observed, which could not be obtained with conventional transgenic constructs.
640 citations
TL;DR: A rapid method to improve the response time and provide specific bacterial strain identification for the typing of Escherichia coli O157:H7 and other gram-negative organisms in a single day is described.
Abstract: Genomic DNA patterns generated by pulsed-field gel electrophoresis are highly specific for different strains of an organism and have significant value in epidemiologic investigations of infectious-disease outbreaks. Unfortunately, time-consuming and tedious specimen processing is an inherent problem which limits the use of this powerful technology as a real-time epidemic investigational tool. Here, I describe a rapid method to improve the response time and provide specific bacterial strain identification for the typing of Escherichia coli O157:H7 and other gram-negative organisms in a single day.
509 citations
TL;DR: It is shown that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP).
Abstract: Pathogenic Escherichia coli are responsible for a variety of diseases, including diarrhoea, haemolytic uraemic syndrome, kidney infection, septicaemia, pneumonia and meningitis. Toxins called cytotoxic necrotizing factors (CNFs) are among the virulence factors produced by uropathogenic (CNF1)1 or enteropathogenic (CNF2)2 E. coli strains that cause diseases in humans and animals, respectively. CNFs induce an increase in the content of actin stress fibres and focal contacts in cultured cells3,4. Effects of CNFs on the actin cytoskeleton correlated with a decrease in the electrophoretic mobility of the GTP-binding protein Rho4,5 and indirect evidence indicates that CNF1 might constitutively activate Rho6. Here we show that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP). Thus, this deamidation of glutamine 63 by CNF1 leads to the constitutive activation of Rho, and induces the reorganization of actin stress fibres. To our knowledge, CNF1 is the first example of a bacterial toxin acting by deamidation of a specific target protein.
493 citations
TL;DR: This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.
Abstract: Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.
471 citations
TL;DR: E. coli O157 isolates from cattle were verocytotoxigenic, eaeA gene positive, contained a 92 kb plasmid and were thus typical of strains causing infections in man and would, therefore, be unlikely to be a source of infection for man.
Abstract: Samples of rectal faeces were collected immediately after slaughter from 400 cattle each month for a 1-year period and from 1000 each of sheep, pigs and poultry over the same period. Samples were examined for Escherichia coli O157 by enrichment culture in buffered peptone water with vancomycin, cefixime and cefsulodin followed by immunomagnetic separation and culture of magnetic particles onto cefixime tellurite sorbitol MacConkey agar. E. coli O157 was isolated from 752 (15.7%) of 4800 cattle, 22 (2.2%) of 1000 sheep and from 4 (0.4%) of 1000 pigs, but not from any of 1000 chickens. Of the cattle sampled. 1840 (38.4%) were prime beef animals, 1661 (34.6%) were dairy animals being culled and the status could not be determined for the other 1299 (27%) animals. E. coli O157 was found in 246 (13.4%) of the 1840 beef cattle and 268 (16.1%) of the 1661 dairy cattle. The monthly prevalence of E. coli O157 in cattle was 4.8-36.8% and was at its highest in spring and late summer. Seventeen of the 22 isolates from sheep were also made over the summer period. All E. coli O157 isolates from sheep and 749 (99.6%) of the 752 E. coli O157 isolates from cattle were verocytotoxigenic as determined by Vero cell assay and DNA hybridization, eaeA gene positive, contained a 92 kb plasmid and were thus typical of strains causing infections in man. In contrast isolates from pigs were non-toxigenic, eaeA gene negative and did not contain a 92 kb plasmid and would, therefore, be unlikely to be a source of infection for man.
431 citations
TL;DR: In this article, the authors compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. coelicolor and S. lividans 66 using two E. coli donors: the standard, methylation proficient strain S17-1.
Abstract: Many streptomycetes, including S. coelicolor A3(2), possess a potent methyl-specific restriction system which can present an effective barrier to the introduction of heterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. coelicolor and S. lividans 66 using two E. coli donors: the standard, methylation proficient strain S17-1. and the methylation deficient donor, ET12567(pUB307). We demonstrate that the methylation deficient donor can yield > 104-fold more S. coelicolor exconjugants than the standard donor. In the case of pSET152 derivatives, which integrate into the host chromosome by site-specific recombination, up to 10% of streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is efficient enough to obtain exconjugants with ‘suicide’ delivery plasmids and therefore provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes, and possibly other bacteria.
423 citations
TL;DR: CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression, which explains previous observations on the homologous system in Erwinia carotovora.
423 citations
TL;DR: E scherichia coli in humans is a commensal inhabitant of the gastrointestinal tract as well as one of the most frequently isolated bacterial pathogens, and in studying food- borne E. coli pathogens and the ECOR collection of natural isolates, LeClerc et al. found that mutant E.coli are found.
Abstract: E scherichia coli in humans is a commensal inhabitant of the gastrointestinal tract as well as one of the most frequently isolated bacterial pathogens ([1][1]). In studying food- borne E. coli pathogens, as well as the ECOR collection of natural isolates ([2][2]), LeClerc et al . found that mutant
391 citations
TL;DR: Determination of the structure of the substrate binding domain of the Escherichia coli Hsp70 chaperone, DnaK, and the biochemical characterisation of the motif it recognizes within substrates provide insights into the principles governing H Sp70 interaction with polypeptide chains.
Abstract: Determination of the structure of the substrate binding domain of the Escherichia coli Hsp70 chaperone, DnaK, and the biochemical characterisation of the motif it recognizes within substrates provide insights into the principles governing Hsp70 interaction with polypeptide chains. DnaK recognizes extended peptide strands composed of up to five consecutive hydrophobic residues within and positively charged residues outside the substrate binding cavity.
387 citations
TL;DR: The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome, which was disrupted by insertion of a kanamycin gene through homologous recombination.
Abstract: The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA∷kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.
TL;DR: Bacterial adhesion to enterocytes, fecal colonization, or both seem to be valuable selection criteria for immunomodulation, and antiinfective mechanisms of defense can be enhanced after ingestion of specific lactic acid bacteria strains.
01 Jan 1997
TL;DR: The first Zn(II)-translocating P-type AT-Pase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome as discussed by the authors.
Abstract: The first Zn(II)-translocating P-type AT- Pase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65 Zn(II) and 109 Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA::kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.
TL;DR: The σE (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities and is further modulated by the products of the remaining genes in this operon, rseB and rseC.
Abstract: Summary The s E (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The s E reg
TL;DR: The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from the majority of EH EC strains.
Abstract: PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.
TL;DR: The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria and appear to inhibit bacterial translation at the initiation phase of protein synthesis.
Abstract: The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.
TL;DR: The results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P, which is identical to dinB, an SOS gene previously localized near the lac locus.
Abstract: dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P.
TL;DR: The results suggest that active efflux, presumably coded by a chromosomal gene(s), may play a major role in silver resistance, which is likely to be enhanced synergistically by decreases in OM permeability.
Abstract: Silver-resistant mutants were selected by stepwise exposure of silver-susceptible clinical strains of Escherichia coli, two of which did not contain any plasmids, to either silver nitrate or silver sulfadiazine. These mutants showed complete cross-resistance to both compounds. They showed low-level cross-resistance to cephalosporins and HgCl2 but not to other heavy metals. The Ag-resistant mutants had decreased outer membrane (OM) permeability to cephalosporins, and all five resistant mutants tested were deficient in major porins, either OmpF or OmpF plus OmpC. However, the well-studied OmpF- and/or OmpC-deficient mutants of laboratory strains K-12 and B/r were not resistant to either silver compound. Resistant strains accumulated up to fourfold less (110m)AgNO3 than the parental strains. The treatment of cells with carbonyl cyanide m-chlorophenylhydrazone increased Ag accumulation in Ag-susceptible and -resistant strains, suggesting that even the wild-type Ag-susceptible strains had an endogenous Ag efflux activity, which occurred at higher levels in Ag-resistant mutants. The addition of glucose as an energy source to starved cells activated the efflux of Ag. The results suggest that active efflux, presumably coded by a chromosomal gene(s), may play a major role in silver resistance, which is likely to be enhanced synergistically by decreases in OM permeability.
Journal Article•
TL;DR: New safety recommendations for destroying enterohemorrhagic E. coli (EHEC) include cooking hamburgers thoroughly, incorporating a procedure that kills EHEC in the manufacture of raw fermented sausage, such as salami, and pasteurizing or using an equivalent processing method for apple cider.
Abstract: New safety recommendations for destroying enterohemorrhagic E. coli (EHEC) include cooking hamburgers thoroughly, incorporating a procedure that kills EHEC in the manufacture of raw fermented sausage, such as salami, and pasteurizing or using an equivalent processing method for apple cider. Public health problems with EHEC are being recognized throughout the world. The need for consumer education on the safe handling of foods has never been more acute.
TL;DR: It is suggested that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.
Abstract: Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.
TL;DR: For all herds the highest prevalence occurred in the summer months, which resulted in most of the positive faecal samples being collected on a minority of sampling visits.
Abstract: Escherichia coli O157 shedding in 14 cattle herds was determined by faecal culture at intervals of approximately 1 month for up to 13 months. The overall prevalence was 1.0% (113/10832 faecal samples) and 9 of the 14 herds were detected as positive. Herds positive 2 years previously (n = 5) had a higher prevalence of positive cattle (median = 1.9%) than herds which had been negative on a previous sampling (n = 8, median = 0.2%). Weaned heifers had a higher prevalence (1.8%) than did unweaned calves (0.9%) or adults (0.4%). For all herds the highest prevalence occurred in the summer months, which resulted in most of the positive faecal samples being collected on a minority of sampling visits.
TL;DR: The results suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form, and that the genus Shigella is shown to comprise a group of closely related pathogenic E. Escherichia coli strains.
Abstract: Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.
TL;DR: Under anaerobic conditions, Escherichia coli converts pyruvate to D-lactate by means of an NADH-linked lactate dehydrogenase (LDH), which increases approximately 10-fold at low pH, suggesting a shortage of 3-carbon metabolic intermediates under low pH.
Abstract: Under anaerobic conditions, especially at low pH, Escherichia coli converts pyruvate to D-lactate by means of an NADH-linked lactate dehydrogenase (LDH). This LDH is present in substantial basal levels under all conditions but increases approximately 10-fold at low pH. The IdhA gene, encoding the fermentative lactate dehydrogenase of E. coli, was cloned using λ10E6 of the Kohara collection as the source of DNA. The IdhA gene was subcloned on a 2.8 kb MIuI-MIuI fragment into a multicopy vector and the region encompassing the gene was sequenced. The IdhA gene of E. coli was highly homologous to genes for other D-lactate-specific dehydrogenases but unrelated to those for the L-lactate-specific enzymes. We constructed a disrupted derivative of the IdhA gene by inserting a kanamycin resistance cassette into the unique KpnI site within the coding region. When transferred to the chromosome, the IdhA::Kan construct abolished the synthesis of the D-LDH completely. When present in high copy number, the IdhA gene was greatly overexpressed, suggesting escape from negative regulation. Cells expressing high levels of the D-LDH grew very poorly, especially in minimal medium. This poor growth was largely counteracted by supplementation with high alanine or pyruvate concentrations, suggesting that excess LDH converts the pyruvate pool to lactate, thus creating a shortage of 3-carbon metabolic intermediates. Using an IdhA-cat gene fusion construct we isolated mutants which no longer showed pH-dependent regulation of the IdhA gene. Some of these appeared to be in the pta gene, which encodes phosphotransacetylase, suggesting the possible involvement of acetyl phosphate in IdhA regulation.
TL;DR: These findings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies.
Abstract: Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection Here we show that, in contrast to E coli subjected to opsonic interactions stimulated by the host's immune response, E coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidification during FimH-mediated internalization, both of which are triggered by antibody-mediated internalization This different processing of non-opsonized bacteria is supported by morphological evidence of tight-fitting phagosomes compared with looser, antibody-mediated phagosomes We propose that non-opsonized FimH-expressing E coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents filipin, nystatin and methyl beta-cyclodextrin specifically block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage membranes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization Our findings bring the traditionally extracellular E coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies
TL;DR: Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system, and compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.
Abstract: The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.
TL;DR: The results presented illustrate the remarkable capacity of E. coli to adapt to environmental challenges, and have significant implications for the survival of spoilage and pathogenic bacteria, and hence for food safety.
TL;DR: Heterologous expression of epsD in Escherichia coli showed that its gene product is a glucosyltransferase linking the first sugar of the repeating unit to the lipid carrier.
Abstract: Lactococcus lactis strain NIZO B40 produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose. A 40 kb plasmid encoding exopolysaccharide production was isolated through conjugal transfer of total plasmid DNA from strain NIZO B40 to the plasmid-free L. lactis model strain MG1614 and subsequent plasmid curing. A 12 kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified downstream of an iso-IS982 element. The predicted gene products of epsABCDEFGHIJK show sequence homologies with gene products involved in exopolysaccharide, capsular polysaccharide, lipopolysaccharide, or teichoic acid biosynthesis of other bacteria. Transcriptional analysis of the eps gene cluster revealed that the gene cluster is transcribed as a single 12 kb mRNA. The transcription start site of the promoter was mapped upstream of the first gene epsR. The involvement of epsD in exopolysaccharide (EPS) biosynthesis was demonstrated through a single gene disruption rendering an exopolysaccharide-deficient phenotype. Heterologous expression of epsD in Escherichia coli showed that its gene product is a glucosyltransferase linking the first sugar of the repeating unit to the lipid carrier.
TL;DR: A purified thermal hysteresis protein, with up to 100 times the specific activity of fish antifreeze proteins, from the common yellow mealworm beetle, Tenebrio molitor, is purified.
Abstract: We have purified a thermal hysteresis (antifreeze) protein, with up to 100 times the specific activity of fish antifreeze proteins, from the common yellow mealworm beetle, Tenebrio molitor. It is a threonine- and cysteine-rich protein, of relative molecular mass 8,400, composed largely of 12-amino-acid repeats. We estimate that a concentration of roughly 1 mg ml−1 of this protein can account for the 5.5 °C of thermal hysteresis found in Tenebrio larvae (Fig. 1).
TL;DR: The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks and was analyzed kinetically in the forward and reverse directions.
Abstract: NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks.
TL;DR: A dairy herd associated with Escherichia coli O157 infection in humans was studied for the 15 months following the outbreak to examine seasonal, age and management factors affecting faecal excretion of the organism and to determine the mode and frequency of milk contamination with the organism.
Abstract: A dairy herd associated with Escherichia coli O157 infection in humans was studied for the 15 months following the outbreak to examine seasonal, age and management factors affecting faecal excretion of the organism and to determine the mode and frequency of milk contamination with the organism. Between May 1993 and July 1994, 28 visits were made to the farm to collect a total of 3593 rectal swabs from cows, heifers and calves and 329 milk samples. E. coli O157:H7 was isolated from 153 (4.3%) of 3593 bovine rectal swabs. The maximum prevalence at any one visit was 14% in lactating cows, 40% in non-lactating cows, 56% in calves and 68% in heifers. The prevalence in lactating cows, which was significantly lower than in the other groups, peaked during May-July 1993 and again briefly after the cattle were housed during November 1993 and then again during May 1994. Excretion rates of E. coli O157:H7 in lactating cows were highest during the first month after calving, falling during lactation and rising to another peak at 7 months postpartum. Between November 1993 and May 1994 there was no evidence of excretion in any group. Eighty-seven (74%) of the animals which excreted E. coli O157:H7 did so on only one occasion but 23 (32%) of 73 cows and heifers and 7 (16%) of 44 calves which excreted the organism did so on more than one occasion. E. coli O157:H7 was not isolated from milk taken from the bulk tank but it was isolated from individual milk samples (one milk jar and one fore-milk) from two animals previously shown to be faecal excretors of the organism. All isolates of E. coli O157:H7 obtained were of the same phage type, toxin genotype and plasmid profile.