Showing papers on "Escherichia coli published in 1973"

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

Abstract: A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.

Iron-Binding Catechols and Virulence in Escherichia coli.

Abstract: Previous work suggested that virulent bacteria, which can grow rapidly in serum, must possess a specific mechanism for removing iron from its transferrin complex. Two strains of Escherichia coli were examined with this in mind. Strain O141, which showed inoculum-dependent growth in serum and multiplied in the mouse peritoneum, secreted iron-binding catechols into both synthetic medium and serum. One of these compounds has an association constant for iron similar to that of transferrin. Both transferrin and ethylenediamine-di-o-hydroxyphenyl acetic acid (EDDA), which have very high affinities for ferric iron, induced catechol synthesis in growing cultures of strain O111. This organism was inhibited by normal horse serum. Further work showed that traces of specific antibody inhibited catechol synthesis by O111 exposed to EDDA; therefore, the existence of this inhibitory process means that the organism can no longer obtain Fe3+, which all remains bound to transferrin in serum. In vivo, the inhibition of O111 is similar to that produced by serum in vitro. Neither phagocytosis nor killing by complement appeared to be of any significance during the first 4 h of the infections. Significantly, the purified catechol was capable of abolishing bacteriostasis in vivo. Since these results show that the production of iron-binding catechols is essential for rapid bacterial growth both in vitro and in vivo, these compounds should therefore be considered as true virulence factors. Conversely, any interference by the host with the production or activity of these compounds would constitute an important aspect of antibacterial defense. Images

Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Abstract: Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N6-methyladenine (N6-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict λ. None of the mutations had the effect of being lethal.

Escherichia coli k-12*

Abstract: ~~~~~~~c~t;;m~+C~+ NAY-+ 3 and D-1-amino-2-propanol dehydrogenase F? D 1 amino-2-propanol + NA&. Each enzyme has been obtained in purified form free of the other; the nature of the reaction catalyzed by the latter dehydrogenase alone and in a coupled system with the former enzyme has been studied. The results provide an explanation on the enzymological level for the utilization of L-threonine by cell suspensions of certain microorganisms for the biosynthesis of the D-1-amino-2-propanol moiety of Vitamin B 12'

Transport of Vitamin B12 in Escherichia coli: Common Receptor Sites for Vitamin B12 and the E Colicins on the Outer Membrane of the Cell Envelope

Abstract: The first step in the transport of cyanocobalamin (CN-B(12)) by cells of Escherichia coli was shown previously to consist of binding of the B(12) to specific receptor sites located on the outer membrane of the cell envelope. In this paper, evidence is presented that these B(12) receptor sites also function as the receptors for the E colicins, and that there is competition between B(12) and the E colicins for occupancy of these sites. The cell strains used were E. coli KBT001, a methionine/B(12) auxotroph, and B(12) transport mutants derived from strain KBT001. Colicins E1 and E3 inhibited binding of B(12) to the outer membrane B(12) receptor sites, and CN-B(12) protected cells against these colicins. Half-maximal protection was given by CN-B(12) concentrations in the range of 1 to 6 nM, depending upon the colicin concentration used. Colicin E1 competitively inhibited the binding of (57)Co-labeled CN-B(12) to isolated outer membrane particles. Functional colicin E receptor sites were found in cell envelopes from cells of only those strains that possessed intact B(12) receptors. Colicin K did not inhibit the binding of B(12) to the outer membrane receptor sites, and no evidence was found for any identity between the B(12) and colicin K receptors. However, both colicin K and colicin E1 inhibited the secondary phase of B(12) transport, which is believed to consist of the energy-coupled movement of B(12) across the inner membrane.

Treatment of E. Coli and Klebsiella Bacteremia in Agranulocytic Animals with Antiserum to a UDP-GAL Epimerase-Deficient Mutant

Abstract: In order to produce antiserum with broad protection against different Gramnegative bacteria we immunized rabbits with a UDP-galactose-deficient mutant (J5) of Escherichia coli O111 that is unencumbered by O antigen since it cannot incorporate galactose into core LPS. Without O side chains, core is accessible for producing antiserum to a wide range of bacteria with similar cores. Such antiserum has previously been shown to prevent unrelated endotoxins from producing dermal and generalized Shwartzman reactions, and death. The current study reports the results of treating bacteremia with this antiserum. Bacteremia was produced in rabbits made granulocytopenic with nitrogen mustard and fed either Klebsiella pneumoniae , E. coli O17 (multiply antibiotic-resistant), or E. coli O4. Since most rabbits are coliform-free, the organisms colonized the gut and overwhelming bacteremia occurred as granulocytes disappeared. When nonimmune serum was given intravenously (i.v.) at the onset of bacteremia with any of these three organisms, only 3.1% to 5.7% survived. In contrast, survival rates in animals given antiserum to the J5 mutant were 33.3% from E. coli O4, 40% from Klebsiella, and 69.2% from the multiply antibiotic-resistant E. coli O17 (combined p E. coli O111, (with O antigen intact) provided no significant protection. The protection against death from E. coli O17 by J5 antiserum was greater than that obtained from gentamicin (50%), the only antibiotic to which this strain was sensitive. Rabbits given J5 antiserum cleared labeled bacteria more rapidly than those given nonimmune sera. Antiserum to the J5 mutant is thought to prevent lethal bacteremia from unrelated Gram-negative bacteria by acting as both antitoxin and opsonin.

Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man.

Abstract: Enterotoxin preparations derived from Escherichia coli strain H-10407 were shown to contain vascular permeability factor (PF) activity as well as diarrheagenic activity. Intradermal injection of E. coli enterotoxin (ECT) caused localized induration and permeability of small blood vessels of the skin to intravenously administered Evans blue dye. The PF assay described here demonstrated a linear dose response and was at least as sensitive as the adult rabbit ileal loop assay for detecting ECT. E. coli PF activity was heat labile and was neutralized by homologous antiserum. PF production was enhanced by the addition of yeast extract (up to 0.6%) to a Casamino Acids-salts medium. PF activity was detectable as early as 6 h in aerated (shake) cultures in the Casamino Acids-yeast extract-salts medium, pH 8.5, maximal at 18 h and essentially unchanged at 48 h. The skin test (PF) assay for ECT has numerous advantages over current assay methods which involve gastrointestinal challenge of experimental animals.

Recircularization and Autonomous Replication of a Sheared R-Factor DNA Segment in Escherichia coli Transformants

Abstract: Controlled shearing of R-factor DNA leads to formation of fragments carrying an antibiotic resistance gene present on, but not expressed by, the intact R-factor. Transformation of CaCl2-treated E. coli by such fragments yields an autonomously replicating tetracycline-resistance plasmid (Tc6-5) that contains only a small fraction of the genome of the parent R-factor, and lacks both its fertility functions and its other drug-resistance determinants. Although the Tc6-5 plasmid is not self-transmissible, it can interact and/or recombine with conjugally-proficient plasmids that promote its transfer to other bacteria.

Cyclic adenosine 3',5'-monophosphate in Escherichia coli.

Abstract: The concentration of cyclic adenosine 3′,5′-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of β-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.

Immunochemical Similarity Between Polysaccharide Antigens of Escherichia Coli 07:K1(L):NM and Group B Neisseria Meningitidis

Abstract: Polysaccharide antigens from Escherichia coli 07:K1(L):NM and group B Neisseria meningitidis have been isolated and purified. Like the meningococcal group B polysaccharide, the sialic acid antigen from E. coli was acidic in nature, was susceptible to neuraminidase degradation and lacked O-acetyl groups. Immunologically these two polysaccharides were indistinguishable in assays of hemagglutination inhibition and they produced lines of identity by immunodiffusion. Investigation of the immunogenicity of the E. coli polysaccharide showed that it induced antibody in rabbits after multiple injections but not in human volunteers given one or two injections of 50 µg each.

Lipopolysaccharide Layer Protection of Gram-Negative Bacteria Against Inhibition by Long-Chain Fatty Acids

Abstract: Growth, amino acid transport, and oxygen consumption of Escherichia coli and Salmonella typhimurium are inhibited by short-chain (C(2)-C(6)) but not by medium or long-chain fatty acids (C(10)-C(18)) at concentrations at which these processes are completely inhibited in Bacillus subtilis. The resistance of gram-negative organisms is not correlated with their ability to metabolize fatty acids, since an E. coli mutant unable to transport oleic acid is still resistant. However, mutants of both E. coli and S. typhimurium in which the lipopolysaccharide layer does not contain the residues beyond the 2-keto-3-deoxyoctonate core are inhibited by medium (C(10)) but not by long-chain (C(18)) fatty acids. Furthermore, removal of a portion of the lipopolysaccharide layer by ethylenediaminetetraacetate treatment renders the organisms sensitive to medium and partially sensitive to long-chain fatty acids. The intact lipopolysaccharide layer of gram-negative organisms apparently screens the cells against medium and long-chain fatty acids and prevents their accumulation on the inner cell membrane (site of amino acid transport) at inhibitory concentrations. These results are relevant to the use of antimicrobial food additives, and they allow the characterization of gram-positive versus gram-negative bacteria and their lipopolysaccharide mutants.

Biochemical and genetic studies on two different types of erythromycin resistant mutants of Escherichia coli with altered ribosomal proteins.

Abstract: Ribosomes from nine E. coli mutants with high level resistance to the antibiotic erythromycin were isolated and their proteins were compared with those of the parental strains by two-dimensional polyacrylamide gel electrophoresis, by carboxymethylcellulose column chromatography and by immunological techniques. Two 50S proteins were found to be altered in the mutants: either L 4 or L 22.

Superoxide Dismutases of Escherichia coli: Intracellular Localization and Functions

Abstract: Escherichia coli B contains two superoxide dismutases which differ with respect to their localization within the cell, the nature of their prosthetic metals, their responses to changes in pO2, and their functions. One of these enzymes, which was liberated from the cells by osmotic shock and which was therefore presumed to be localized in the periplasmic space, is an iron-containing superoxide dismutase. The amount of this iron enzyme did not vary in response to changes in pO2 during growth. In contrast, the other superoxide dismutase was not solubilized by osmotic shock, was a mangano-protein, and was found in greater amounts in cells which had been grown at high pO2. E. coli, which had low levels of the iron-enzyme and high levels of the mangano-enzyme, as a consequence of growth in iron-deficient aerated medium, was killed by exposure to an exogenous flux of O2− which was generated either photochemically or enzymatically. The addition of bovine superoxide dismutase to the suspending medium protected these cells against this stress. On the other hand, E. coli, which had high levels of the iron-enzyme and low levels of the mangano-enzyme, as a consequence of growth in iron-rich anaerobic medium, was resistant to exogeneous O2−. On the basis of these and of previously reported results (4a, Yost, F. J. and I. Fridovich, J. Biol. Chem., 1973, in press), it appears that the iron superoxide dismutase, of the periplasmic space, serves as a defense against exogenous O2−, whereas the mangano-superoxide dismutase, in the matrix of these cells, serves to counter the toxicity of endogenous O2−.

Isolation of a Specialized Lambda Transducing Bacteriophage Carrying the Beta Subunit Gene for Escherichia coli Ribonucleic Acid Polymerase

Abstract: A heat-inducible lysis-defective lambda prophage has been integrated directly into the E. coli chromosome at a site (bfe) very closely linked to the ribonucleic acid polymerase mutation rif(d), a dominant rifampin resistance allele. This unusual lysogen has facilitated the isolation of specialized transducing phages conferring rifampin resistance to sensitive cells, and carrying at least the beta subunit gene of RNA polymerase in intact form.

Immunology of Escherichia coli: K Antigen and Its Relation to Urinary-Tract Infection

Abstract: The question as to whether or not the bacteria that cause urinary-tract infection (UTI) have special characteristics making them more virulent for the urinary tract has long been discussed. Thus, some are of the opinion that Escherichia coli strains of a few serotypes are especially nephropathogenic strains and more apt to cause UTI [1], while others are more sceptical of such characterization of strains [2, 3].

Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Abstract: Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.

Mutants of Escherichia coli with a defect in the degradation of nonsense fragments.

Abstract: Escherichia coli possesses a protein degradation system which can distinguish between normal and aberrant polypeptides1–8. For example, whereas the enzyme β-galactosidase, coded by the z gene of the lac operon, is itself stable, all the polypeptides produced by nonsense mutations in its structural gene are rapidly degraded in vivo1, 3. This degradation system for abnormal proteins is as sensitive to inhibition of the cell's energy metabolism by substances such as cyanide as is protein synthesis (ref. 4; and B. Shineberg, unpublished observations). Clearly, it is of considerable interest to characterize this system, which we refer to as the Deg system; the genetic locus or loci for this system is denoted by deg.

Cleavage of Adenovirus Type 2 DNA into Six Unique Fragments by Endonuclease R·RI

Abstract: The DNA of adenovirus type 2 was cleaved by restriction endonuclease R·RI into six fragments. These fragments were separated by electrophoresis on composite agarose-polyacrylamide gels. Their molecular weights ranged from 1.1 × 106 to 13.6 × 106, as measured by electron microscopy. Each fragment represented a unique segment of adenovirus type 2 DNA since: (i) the fragments were obtained in equimolar amounts; (ii) the sum of their molecular weights was equal to the molecular weight of complete adenovirus DNA; and (iii) each fragment exhibited a rate of renaturation that was inversely proportional to its size.

Genetic transformation in Escherichia coli K12.

Abstract: An auxotrophic strain of E. coli K12 treated with CaCl2 was transformed for several markers at a frequency of up to 10-6 per recipient cell by a DNA preparation isolated from a prototrophic strain. The transforming activity of the DNA preparation was eliminated by treatment with DNase, heat, or sonication, whereas RNase or Pronase treatment had little effect. Two closely linked genetic markers (leu and ara) showed a high degree of cotransformation linkage when high molecular weight DNA was used, but the linkage was almost completely eliminated when sheared, smaller molecular weight DNA was used. There is genetic evidence that the transformation is a result of the replacement of the preexisting genetic marker on the chromosome by that of the donor DNA.

Effect of cis-Platinum(II)Diamminodichloride on Wild Type and Deoxyribonucleic Acid Repair-Deficient Mutants of Escherichia coli

Abstract: The anti-tumor drug cis-platinum(II)diamminodichloride (PDD) induced extensive filamentation in wild-type Escherichia coli and in mutants lacking certain deoxyribonucleic acid (DNA) repair functions (uvrA, recB, recC, and polA); viability of repair-deficient mutants treated with PDD was significantly less than that of wild-type cells. PDD was highly toxic to lex1, lex1 uvrA6 (where its effect was cummulative), and recA13 mutants, all of which were killed without formation of filaments. (3)H-thymine incorporated into DNA of cells subsequently treated with PDD became trichloroacetic acid-soluble at rates similar to those observed after exposure to comparable doses of ultraviolet light (UV) or mitomycin C. PDD, like UV, induced extensive degradation of DNA in recA organisms. After a 30-min lag, PDD inhibited significantly the synthesis of DNA but not of ribonucleic acid or protein in E. coli. However, the relative differences between rates of DNA synthesis observed in PDD-treated and control cells decreased substantially when the duration of pulses ((3)H-thymine) was prolonged from 2 to 5 min. These observations suggest that PDD-induced damage to DNA is reversible, possibly by defined mechanisms of excision and recombination repair.

Synthesis of a large precursor to ribosomal RNA in a mutant of Escherichia coli.

Abstract: A mutant of E. coli, isolated by Kindler and Hofschneider as a strain defective in RNase III activity, forms a 30S precursor of ribosomal RNA (“30S pre-rRNA”). The half-life of the 30S pre-rRNA in growing cells at 30°, estimated by the rate of specific 3[H]uridine incorporation, is about 1 min. In rifampicin-treated cells, the RNA is metabolized to mature rRNA with a half-life of about 2 min. The 30S pre-rRNA has been highly purified. DNA-RNA hybridization tests demonstrate that it contains both 16S and 23S rRNA sequences. Also, in cultures treated with rifampicin, the cleavage products of radioactive 30S pre-rRNA include 25S and 17.5S RNA species, destined to becomes 23S and 16S rRNA. Thus, each 30S chain probably contains one 16S and one 23S RNA sequence, as well as additional sequences. Two independent techniques indicate that the additional portions account for about 27% of the total lenght: (1) By comparison to the sedimentation rate and electrophoretic mobility of marker RNAs, the 30S pre-RNA has an apparent molecular weight of 2.3 × 106 ± 5%, or 28% more than the sum of 16S and 23S rRNA; (2) 27% of the 30S pre-rRNA is not competed away from hybridization by mature 16S and 23S rRNA. Thus, bacteria appear to make a pre-rRNA similar in some respects to that observed in eukaryotes; though in normal E. coli cells, the pre-rRNA is ordinarily cleaved endonucleolytically during its formation.

Resistance to Arsenic Compounds Conferred by a Plasmid Transmissible Between Strains of Escherichia coli

Abstract: A plasmid, R773, which confers resistance to arsenic compounds, is transmissible between strains of Escherichia coli. It is a member of compatibility group F(1).