Showing papers on "Escherichia coli published in 1987"
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01 Jun 1987
4,940 citations
TL;DR: To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform.
2,483 citations
TL;DR: Neither the isozyme-converting activity nor labeled Iap proteins were detected in the osmotic-shock fluid of cells carrying a multicopy iap plasmid, and the Iap protein seems to be associated with the membrane.
Abstract: The iap gene in Escherichia coli is responsible for the isozyme conversion of alkaline phosphatase. We analyzed the 1,664-nucleotide sequence of a chromosomal DNA segment that contained the iap gene and its flanking regions. The predicted iap product contained 345 amino acids with an estimated molecular weight of 37,919. The 24-amino-acid sequence at the amino terminus showed features characteristic of a signal peptide. Two proteins of different sizes were identified by the maxicell method, one corresponding to the Iap protein and the other corresponding to the processed product without the signal peptide. Neither the isozyme-converting activity nor labeled Iap proteins were detected in the osmotic-shock fluid of cells carrying a multicopy iap plasmid. The Iap protein seems to be associated with the membrane.
1,715 citations
01 Jan 1987
1,338 citations
TL;DR: The role of protein C in modulation of Escherichia coli shock in baboons is examined and the coagulopathic, hepatotoxic, and lethal responses in this latter case were prevented by infusion of exogenous protein C.
Abstract: Gram-negative septicemia elicits multiple abnormalities of the coagulation system. Although products of coagulation can lead to clot formation, thereby potentiating organ damage, recent work has shown that low concentrations of thrombin can protect animals from the shock state. Because these amounts of thrombin also lead to formation in vivo of the anticoagulant enzyme, activated protein C, we examined the role of protein C in modulation of Escherichia coli shock in baboons. First, we infused activated protein C and lethal concentrations of E. coli organisms, which prevented the coagulopathic, hepatotoxic, and lethal effects of E. coli. Second, using an antibody to protein C we blocked protein C activation in vivo to determine if this influenced the response to lethal and sublethal concentrations of E. coli organisms. Under these conditions the response to lethal concentrations of E. coli organisms was made more severe and the response to sublethal concentrations of E. coli was made lethal. The coagulopathic, hepatotoxic, and lethal responses in this latter case were prevented by infusion of exogenous protein C.
878 citations
01 Jan 1987
708 citations
TL;DR: The patch-clamp electrical recording technique is used on giant spheroplasts of Escherichia coli and pressure-activated ion channels are discovered, believing that these channels may be involved in bacterial osmoregulation and osmotaxis.
Abstract: We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherichia coli and have discovered pressure-activated ion channels. The channels have the following properties: activation by slight positive or negative pressure; voltage dependence; large conductance; selectivity for anions over cations; dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.
627 citations
TL;DR: A total of 516 Escherichia coli strains randomly isolated from coprocultures of 154 Chilean children with diarrhea and 66 controls were examined with DNA probes and tested for adherence to HEp-2 cells, and the aggregative pattern appears to signify a new, distinct class of diarrheagenic E. coli (enteroadherent-aggregative E coli).
Abstract: A total of 516 Escherichia coli strains randomly isolated from coprocultures of 154 Chilean children with diarrhea and 66 controls were examined with DNA probes and tested for adherence to HEp-2 cells. Three adherence patterns were distinguished, localized, true diffuse and “aggregative.” Enteropath
623 citations
TL;DR: The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures.
Abstract: When the growth temperature of an exponential culture of Escherichia coli is abruptly decreased from 37 to 10 degrees C, growth stops for several hours before a new rate of growth is established. During this growth lag the number of proteins synthesized is dramatically reduced, and at one point only about two dozen proteins are made; 13 of these are made at differential rates that are 3 to 300 times increased over the rates at 37 degrees C. The protein with the highest rate of synthesis during the lag is not detectably made at 37 degrees C. The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures. Images
599 citations
TL;DR: In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine, consistent with the deduced specificity of the enzyme based on the analysis of known amino- terminal sequences of intracellular proteins.
Abstract: Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).
TL;DR: It is shown that expression of F in a CD4+ cell line down-regulates the CD4 (T4) antigen and suggests that F is important in the pathophysiology of AIDS (aquired immune deficiency syndrome).
Abstract: Apart from the retroviral gag, pol and env1,2, the HIV genome contains the F (3'orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown3,4. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, auto-phosphorylation and GTP-binding activities reported for the ras gene product6,7. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4 (T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (aquired immune deficiency syndrome).
TL;DR: It is demonstrated that it is possible to change the fermentation products of an organism, such as E. coli, by the addition of genes encoding appropriate enzymes which form an alternative system for the regeneration of NAD+.
Abstract: The genes encoding essential enzymes of the fermentative pathway for ethanol production in Zymomonas mobilis, an obligately ethanologenic bacterium, were inserted into Escherichia coli under the control of a common promoter. Alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis were expressed at high levels in E. coli, resulting in increased cell growth and the production of ethanol as the principal fermentation product from glucose. These results demonstrate that it is possible to change the fermentation products of an organism, such as E. coli, by the addition of genes encoding appropriate enzymes which form an alternative system for the regeneration of NAD+.
TL;DR: A ceramide trihexoside species, consistent with a globotriosyl ceramide structure was shown to be the major verotoxin-binding glycolipid of cultured vero cells which are routinely used to measure the cytotoxicity of toxin samples.
TL;DR: Toxin lacking domain Ia is about 100-fold less toxic to mice than intact PE and should be a useful molecule for the construction of immunotoxins.
TL;DR: The nucleotide sequence of the Shiga-like toxin type II (SLT-II) structural genes cloned from bacteriophage 933W of the enterohemorrhagic Escherichia coli O157:H7 strain 933 was determined and the regulation proposed for the SLT -II operon is similar to that previously proposed for SLT-I.
Abstract: The nucleotide sequence of the Shiga-like toxin type II (SLT-II) structural genes cloned from bacteriophage 933W of the enterohemorrhagic Escherichia coli O157:H7 strain 933 was determined. This sequence was compared with the published sequence for the structural genes of the antigenically distinct Shiga-like toxin type I (SLT-I) encoded by bacteriophage 933J. The SLT-I and SLT-II structural genes shared 58% overall nucleotide and 56% amino acid sequence homologies. The A and B subunits of SLT-I and SLT-II were nearly identical in size and had similar secondary structures and hydropathy plots. The regulation proposed for the SLT-II operon is similar to that previously proposed for SLT-I.
TL;DR: K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains.
Abstract: SUMMARY: Seven phages highly active in vitro and in vivo against one or other of seven bovine enteropathogenic strains of Escherichia coli belonging to six different serotypes were isolated from sewage. Severe experimentally induced E. coli diarrhoea in calves could be cured by a single dose of 105 phage organisms. It could be prevented by doses as low as 102, by spraying the litter in the calf rooms with aqueous phage suspensions or simply by keeping the calves in uncleaned rooms previously occupied by calves whose E. coli infections had been treated with phage. Microbiological examinations of calves used in these experiments revealed that the phage organisms multiplied rapidly and profusely after gaining entry to the E. coli-infected small intestine, quickly reducing the E. coli to numbers that were virtually harmless. The only phage-resistant E. coli that emerged in the studies on calves infected with one or other of the seven E. coli strains were K~. These organisms were much less virulent than the K+ organisms from which they were derived and did not present a serious problem in calves given adequate amounts of colostrum. Infections produced by oral inoculation of a mixture of six strains of the E. coli could be controlled by administration of a pool of the six phages that were active against them but, in general, the control was less complete than that observed in the single-strain infections. K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains. Infections produced by these K+ mutants and their parents could be controlled by the use of mutant phages derived from phages that were active on their parents. During the experiments with mixed E. coli infection, an extraneous phage active against one of the six E. coli strains suddenly appeared in calves kept in the same rooms. Microbiological examinations revealed that this phage was effectively controlling the multiplication of organisms of that particular strain of E. coli in the small intestines of the calves.
TL;DR: The sequences of the arabinose-H+ and xylose- H+ membrane transport proteins of Escherichia coli are determined and are homologous with each other and with the glucose transporters of human hepatoma1 and rat brain2 cells.
Abstract: The uptake of a sugar across the boundary membrane is a primary event in the nutrition of most cells, but the hydrophobic nature of the transport proteins involved makes them difficult to characterize. Their amino-acid sequences can, however, be determined by cloning and sequencing the corresponding gene (or complementary DNA). We have determined the sequences of the arabinose-H+ and xylose-H+ membrane transport proteins of Escherichia coli. They are homologous with each other and, unexpectedly, with the glucose transporters of human hepatoma and rat brain cells. All four proteins share similarities with the E. coli citrate transporter. Comparisons of their sequences and hydropathic profiles yield insights into their structure, functionally important residues and possible evolutionary relationships. There is little apparent homology with the lactose-H+ (LacY) or melibiose-Na+ (MelB) transport proteins of E. coli.
TL;DR: A physical map of a genome is the structure of its DNA, and the resulting ordered set of fragments is a macrorestriction map that facilitates genetic and molecular studies on E. coli and serves as a model for further endeavors on larger genomes.
Abstract: A physical map of a genome is the structure of its DNA. Construction of such a map is a first step in the complete characterization of that DNA. The restriction endonuclease Not I cuts the genome of Escherichia coli K12 into 22 DNA fragments ranging from 20 kilobases (20,000 base pairs) to 1000 kilobases. These can be separated by pulsed field gel electrophoresis. The order of the fragments in the genome was determined from available E. coli genetic information and analysis of partial digest patterns. The resulting ordered set of fragments is a macrorestriction map. This map facilitates genetic and molecular studies on E. coli, and its construction serves as a model for further endeavors on larger genomes.
TL;DR: The chapter describes a general method for producing eukaryotic proteins in Escherichia coli using the fusion protein expression vector pLcII(nic – ) and subsequent purification and cleavage methods using blood coagulation factor X a .
Abstract: Publisher Summary The chapter describes a general method for producing eukaryotic proteins in Escherichia coli using the fusion protein expression vector pLcII(nic – ) and subsequent purification and cleavage methods using blood coagulation factor X a The foreign coding sequence can be joined in phase to a short coding sequence of a highly expressed E coli gene so that a hybrid protein is produced The short segment of E coli gene directs the folding of the mRNA in the vicinity of the ribosome-binding site, and thereby ensures high translational efficiency If the fusion protein, thus, produced can be cleaved specifically at the junction of the two sequences by an appropriate enzymatic or chemical method, the authentic protein will then be liberated A fusion protein expression vector pLclI(nic-) is constructed This plasmid has multiple cloning sites downstream of a short coding sequence from the λ cII gene, which is under the control of the strong leftward (PL) promoter of λ phage The following proteins have been produced in this way at the level of 5–10% of total E coli cellular protein: human β-globin and human α-globin, human myoglobin In a second stage, a sequence encoding the tetrapeptide Ile-Glu-Gly-Arg at the junction in the fusion proteins is inserted The chapter discusses cloning into the pLclI(nic – ) fusion protein expression vector, protein purification, the isolation of factor X from bovine blood, and several other related concepts The pLcII(nic – ) fusion protein expression vector is a straightforward and general method for production of eukaryotic proteins in E coli
TL;DR: Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC, and a DNA probe prepared from a 3.4-kilobase segment of the EH EC plasmid is evaluated.
Abstract: Enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), make potent cytotoxins (Verotoxins [VT] or Shiga-like toxins), and possess a plasmid (approximately 60 megadaltons) that encodes a new fimbrial antigen and promotes attachment to epithelial cells. We evaluated the use of a DNA probe, prepared from a 3.4-kilobase segment of the EHEC plasmid, to detect EHEC. The probe hybridized with 106 (99%) of 107 O157:H7 and 34 (77%) of 44 O26:H11, VT-positive strains from patients with colitis, HUS, and diarrheal disease and hybridized with 21 (81%) of 26 VT-positive E. coli of serotypes other than O157:H7 or O26:H11 from patients with hemorrhagic colitis and HUS. We examined 601 other strains, including 18 serotype O26 isolates of H types other than H11, 306 enteropathogenic E. coli, 60 enteroinvasive E. coli, 119 enterotoxigenic E. coli, and 20 isolates from the urinary tract and 77 isolates from the normal intestinal flora; only one (O127:H-) was positive (specificity, 99.8%). Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC.
TL;DR: The systems described serve as a fine illustration for the biological role of lectin‐carbohydrate interactions and will lead to a deeper understanding of the molecular basis of infectious diseases, and perhaps also to new approaches for their prevention.
TL;DR: Deletion analysis of the region upstream of the gene fusion is used to localize the promoter of the slt operon and to show that a region of DNA between the -35 and -10 boxes is necessary for iron regulation of slt expression.
Abstract: Shiga-like toxin is an iron-regulated cytotoxin quite similar to Shiga toxin from Shigella dysenteriae 1. The structural genes for Shiga-like toxin in Escherichia coli (sltA and sltB) appear to be transcribed as an operon from a promoter upstream of sltA. We used a gene fusion between the promoter and proximal portion of sltA with the gene for bacterial alkaline phosphatase to assess the regulation of toxin expression. Growth in low-iron conditions resulted in a 13- to 16-fold increase in alkaline phosphatase activity. In the presence of a null mutation in the fur locus, however, alkaline phosphatase activity was constitutively high regardless of the iron concentration. These data indicate negative regulation of the slt operon by the fur gene product. We used deletion analysis of the region upstream of the gene fusion to localize the promoter of the slt operon and to show that a region of DNA between the -35 and -10 boxes is necessary for iron regulation of slt expression. In this region, there is a 21-base-pair dyad repeat that is homologous to similar dyads in the promoter regions of three other fur-regulated genes. This region of dyad symmetry may represent an operator binding site for the Fur protein in the presence of iron.
TL;DR: The sperm whale myoglobin expressed from the synthetic gene constituted approximately 10% of the total soluble protein as holo-protein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E. coli.
Abstract: Sperm whale myoglobin was expressed in Escherichia coli from a totally synthetic gene inserted in the expression vector pUC19. The gene was constructed as 23 overlapping oligonucleotides encoding both strands of the DNA. Gene synthesis provides several advantages over traditional eukaryotic gene-cloning techniques, allowing the incorporation of an efficient ribosome binding site, appropriate initiation and termination sequences, restriction enzyme sites for convenient subcloning and future mutagenesis, and frequently used codons for highly expressed E. coli genes. The sperm whale myoglobin expressed from the synthetic gene constituted approximately 10% of the total soluble protein as holo-protein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E. coli. We credit the use of frequently used E. coli codons for the observed high-level expression. The sperm whale myoglobin produced is stable, easily purified to homogeneity, and indistinguishable from commercially available sperm whale myoglobin by optical and magnetic spectroscopic methods.
TL;DR: A vector strategy that allows transfer of plasmid DNA by conjugation from Escherichia coli to various Gram-positive bacteria in which transformation via natural competence has not been demonstrated is developed.
Abstract: We have developed a vector strategy that allows transfer of plasmid DNA by conjugation from Escherichia coli to various Gram-positive bacteria in which transformation via natural competence has not been demonstrated. The prototype vector constructed, pAT187, contains the origins of replication of pBR322 and of the broad host range streptococcal plasmid pAMβ1, a kanamycin resistance gene known to be expressed in both Gram-negative and Gram-positive bacteria, and the origin of transfer of the IncP plasmid RK2. This shuttle plasmid can be mobilised efficiently by the self-transferable IncP plasmid pRK212.1 co-resident in the E. coli donors, and was successfully transferred by filter matings at frequencies of 2 × 10−8 to 5 × 10−7 to Enterococcus faecalis, Streptococcus lactis, Streptococcus agalactiae, Bacillus thuringiensis, Listeria monocytogenes and Staphylococcus aureus.
TL;DR: The nucleotide sequence of the sltA and sltB genes that encode the Shiga-like toxin (SLT) produced by Escherichia coli phage H19B is determined, providing evidence for the hypothesis that certain prokaryotic toxins may be evolutionarily related to eukaryotic enzymes.
Abstract: We have determined the nucleotide sequence of the sltA and sltB genes that encode the Shiga-like toxin (SLT) produced by Escherichia coli phage H19B. The amino acid composition of the A and B subunits of SLT is very similar to that previously established for Shiga toxin from Shigella dysenteriae 1, and the deduced amino acid sequence of the B subunit of SLT is identical with that reported for the B subunit of Shiga toxin. The genes for the A and B subunits of SLT apparently constitute an operon, with only 12 nucleotides separating the coding regions. There is a 21-base-pair region of dyad symmetry overlapping the proposed promoter of the slt operon that may be involved in regulation of SLT production by iron. The peptide sequence of the A subunit of SLT is homologous to the A subunit of the plant toxin ricin, providing evidence for the hypothesis that certain prokaryotic toxins may be evolutionarily related to eukaryotic enzymes.
TL;DR: The DNA sequence changes in 487 spontaneous mutations in the N-terminal part of the lacI gene in mutH, mutL, and mutS strains of Escherichia coli are determined.
Abstract: We have determined the DNA sequence changes in 487 spontaneous mutations in the N-terminal part of the lacI gene in mutH, mutL, and mutS strains of Escherichia coli. These strains display elevated spontaneous mutation rates because of a deficiency in the process of postreplicative mismatch correction. As a consequence the mutational spectra reveal the nature of spontaneous DNA replication errors. The spectra consist of base substitutions (75%) and single-base deletions (25%). Among the base substitutions, transitions (both A.T----G.C and G.C----A.T) are strongly favored over transversions (96% versus 4%). Large site-to-site differences are observed among identical base substitutions, presumably reflecting the modulating effects of neighboring bases. The single-base-deletion spectrum is dominated by a large hotspot at a run of adjacent identical base pairs, implying a Streisinger-slippage mechanism. The data, when compared to a previously determined wild-type spectrum, also provide information on the specificity of the mismatch repair system.
TL;DR: The region of the fnbp‐gene encoding the fibronectin‐binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene and the resulting product in E. coli is an extracellular fusion protein consisting of two IgG‐binding domains of protein A followed by a fibronECTin‐ binding region.
Abstract: The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.
TL;DR: The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL- 2 receptor are resistant to IL-1-toxin action.
Abstract: We have genetically replaced the diphtheria toxin receptor binding domain with a synthetic gene encoding interleukin-2 (IL-2) and a translational stop signal. The diphtheria toxin-related T-cell growth factor fusion gene encodes a 70 586-d polypeptide, pro-IL-2-toxin. The mature form of IL-2-toxin has a deduced mol. wt of 68,086 and is shown to be exported to the periplasmic compartment of Escherichia coli (pABI508), and contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components. IL-2-toxin has been purified from periplasmic extracts of recombinant strains of E. coli (pABI508) by immunoaffinity chromatography using immobilized anti-IL-2. The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL-2 receptor are resistant to IL-2-toxin action.
TL;DR: The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA, consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecAprotein to these regions.