Showing papers on "Escherichia coli published in 1974"

R factor transfer in Rhizobium leguminosarum.

Abstract: SUMMARY: R factors of the compatibility class P were transferred between strains of Escherichia coli k12 and Rhizobium leguminosarum. These R factors were stable in R. leguminosarum and conferred similar levels of antibiotic resistance to those in the corresponding R+ E. coli k12 hosts, with the exception of carbenicillin resistance which was greatly reduced. Transfer between R. leguminosarum strains was by conjugation and was stimulated by conditions favouring spheroplast formation. R factor mediated recombination could not be demonstrated.

Transformation of Salmonella typhimurium by Plasmid Deoxyribonucleic Acid

Abstract: A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described. Transformed bacteria acquire a circular deoxyribonucleic acid species having the genetic and molecular characteristics of the transforming plasmid.

Cyclic Adenosine Monophosphate and Alteration of Chinese Hamster Ovary Cell Morphology: a Rapid, Sensitive In Vitro Assay for the Enterotoxins of Vibrio cholerae and Escherichia coli

Abstract: The major limitation to our understanding of the clinical importance of enterotoxigenic Escherichia coli in diarrheal illness has been the lack of a simple rapid assay for the enterotoxin produced by certain E. coli. On the basis of the activation of adenylate cyclase by heat-labile enterotoxin of E. coli (LT) and by cholera toxin (CT) in intestinal and other tissues, cultured Chinese hamster ovary (CHO) cells with known morphological responses to dibutyryl cyclic adenosine 5'-monophosphate (AMP) were exposed to these enterotoxins. Crude culture filtrates of LT-producing E. coli and CT stimulated cyclic AMP accumulation and cell elongation in CHO cells. The similarity of time course, concentration dependence, and potentiation by phosphodiesterase inhibitors suggested cyclic AMP mediation of the morphological change. Heat inactivated CT and LT in this system. Choleragenoid inhibited CT; antiserum against CT inhibited both enterotoxin effects. In contrast to culture filtrates of 16 strains of E. coli known to produce LT, culture filtrates from 13 E. coli that do not produce LT did not alter CHO cell morphology. The morphological change is a simple, specific assay for these enterotoxins and detect 3 x 10(-17) mol of CT or a 1:250 dilution of crude culture filtrate of LT-producing E. coli 334.

The effect of silver ions on the respiratory chain of Escherichia coli.

Abstract: Silver ions inhibited the oxidation of glucose, glycerol, fumarate, succinate, D- and L-lactate, and endogenous substrates by intact cell suspensions of Escherichia coli. Silver ions reacted with t...

Escherichia coli K1 capsular polysaccharide associated with neonatal meningitis.

Abstract: Examination of 77 strains of Escherichia coli from the cerebrospinal fluid of neonates with meningitis revealed 65 (84 per cent) with the capsular (K1) polysaccharide The Esch coli K1 ca

Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture.

Abstract: Cell-free culture filtrates of heat-labile enterotoxin-producing strains of Escherichia coli are capable of inducing morphological changes and steroidogenesis in monolayer cultures of adrenal cells. These tissue culture changes are simiar to those induced by cholera enterotoxin and cannot be effected by culture filtrates of other enterotoxigenic or enteropathogenic types of bacteria. The results of the tissue culture studies correlated well with those done in the standard intestinal-loop systems and suggest that this tissue culture system could be used to significantly aid epidemiological and molecular studies with heat-labile Escherichia coli enterotoxin.

Replication and transcription of eukaryotic DNA in Escherichia coli.

Abstract: Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA.

Characteristics of PRD1, a Plasmid-Dependent Broad Host Range DNA Bacteriophage

Abstract: Several distinctive properties of PRD1, an icosahedral plasmid-dependent phage, are described. The drug-resistance plasmid-dependent host range of PRD1 extends beyond the P incompatibility group and includes gram-negative bacteria containing plasmids of incompatibility groups N and W. PRD1 phage will infect pseudomonads and Enterobacteriaceae containing either a P or W incompatibility group plasmid. PRD1 adsorbs to the cell wall of R(+) bacteria and thus its infectivity indicates cell wall alterations by these drug-resistance plasmid groups. PRD1 nucleic acid is duplex DNA with an estimated molecular weight of 24 x 10(6). The appearance of PRD1 in electron micrographs is suggestive of lipid content in addition to its buoyant density of 1.348 in CsCl and its sensitivity to chloroform. The latent period of PRD1 varies with the R(+) host bacterial strain used for growth of the phage.

Total Reconstitution of Functionally Active 50S Ribosomal Subunits from Escherichia coli

Abstract: Total reconstitution of 50S subunits from E. coli was achieved by a two-step incubation procedure. In the first step, 23S RNA, 5S RNA, and the total proteins from 50S subunits were incubated for 20 min at 40° in the presence of 4 mM Mg++ and 400 mM NH4Cl. In the second step, the Mg++ concentration was raised to 20 mM and the incubation was performed for 90 min at 50°. No requirement for 30S subunits or other components (e.g., polyamine) was found. The reconstituted particle has the same sedimentation coefficient as the native 50S subunit and is highly active in protein synthesis with natural (R17 RNA) and artificial [poly(U)] messengers as well as in tests for peptidyltransferase (fragment assay) and for binding of antibiotics (chloramphenicol).

The Enterotoxin Plasmids of Escherichia Coli

Abstract: The genetic and molecular nature of the Ent plasmids that determine enterotoxin biosynthesis in strains of Escherichia coli isolated from man and domestic animals has been investigated. At least one transmissible plasmid was present in 90% of 96 toxigenic strains compared with 36% of 204 nontoxigenic strains. Ent plasmids were demonstrated in 17% of the toxigenic strains and often appeared to be selftransmissible. A single Ent plasmid was isolated from 15 enterotoxigenic strains by conjugation experiments. The class of Ent plasmids that determines the production of both heat-stable and heat-labile enterotoxins was homogeneous and consisted of a single DNA species with an approximate molecular weight of 6.0 x 107 daltons. The class of Ent plasmids that codes for heat-stable enterotoxins only was heterogeneous and consisted of a single species of DNA that ranged in size from 2.1 to 8.0 x 107 daltons.

Thermal enhancement of ultraviolet mutability in a tif-1 uvrA derivative of Escherichia coli B-r: evidence that ultraviolet mutagenesis depends upon an inducible function.

Abstract: Inhibition of DNA replication in E. coli results in coordinate expression of a cluster of diverse functions, including prophage induction and filamentous growth, many of which are thermally inducible, without inhibition of DNA synthesis, in strains carrying a mutation in the “tif” locus. It has been proposed that one of the inducible functions in this cluster is responsible for the ultraviolet mutability of E. coli. As a test of this hypothesis, the effect of postirradiation temperature elevation on ultraviolet mutability was compared in strains differing by a tif mutation. In a tif+ strain, raising the temperature to 42° after irradiation did not change the frequency of induced mutations to tryptophan-independence. Similar treatment of a tif-1 strain resulted in as much as a 10-fold increase in the induced mutation yield. Thermal enhancement of ultraviolet mutability in the tif-1 strain was prevented or promoted by agents known to exert parallel effects on thermal induction of lambda prophage in tif lysogens. The results support the hypothesis that ultraviolet mutability in E. coli depends upon an inducible function that is normally expressed only when DNA replication is inhibited.

Comparison of the Deoxyribonucleic Acid Molecular Weights and Homologies of Plasmids Conferring Linked Resistance to Streptomycin and Sulfonamides

Abstract: Bacterial strains showing linked resistance to streptomycin (Sm) and sulfonamides (Su) were chosen representing a wide taxonomic and geographical range. Their SmSu resistances were transferred to Escherichia coli K-12 and then plasmid deoxyribonucleic acid (DNA) was isolated by ethidium bromide CsCl centrifugation. The plasmid DNA was examined by electron microscopy and analyzed by sedimentation through 5 to 20% neutral sucrose gradients. Plasmid DNA from strains having transmissible SmSu resistance consisted of two or three molecular species, one of which had a molecular mass of about 5.7 Mdal (106 daltons), the others varying between 20 to 60 Mdal. By using transformation or F′ mobilization, we isolated the SmSu-resistance determinant from any fellow resident plasmids in each strain and again isolated the plasmid DNA. Cosedimentation of each of these with a differently labeled reference plasmid DNA (R300B) showed 9 out of 12 of the plasmids to have a molecular mass not significantly different from the reference (5.7 Mdal); two others were 6.3 and 9.2 Mdal, but PB165 consisted of three plasmids of 7.4, 14.7, and 21.4 Mdal. Three separate isolations of the SmSu determinant from PB165 gave the same three plasmids, which we conclude may be monomer, dimer, and trimer, respectively. DNA-DNA hybridizations at 75 C demonstrated 80 to 93% homology between reference R300B DNA and each isolated SmSu plasmid DNA, except for the 9.2-Mdal plasmid which had 45% homology and PB165 which had 35%. All the SmSu plasmids were present as multiple copies (about 10) per chromosome. The conjugative plasmid of R300 (present as 1.3 copies per chromosome) has been shown to have negligible effect on the number of copies of its accompanying SmSu plasmid R300B. We conclude that the SmSu plasmids are closely related and probably have a common evolutionary origin.

Insect Immunity I. Characteristics of an Inducible Cell-Free Antibacterial Reaction in Hemolymph of Samia cynthia Pupae

Abstract: Pupae of the silk moth, Samia cynthia, were found to contain an inducible antibacterial activity in their hemolymph. This immunity response was provoked by primary infections with either Escherichia coli K-12 or Enterobacter cloacae. In both cases the antibacterial activity was directed chiefly towards E. coli. During standard conditions, 1% of hemolymph could kill 103 to 104 viable E. coli, strain D31, within 5 min. A lower level of antibacterial activity was induced by injections of a sterile salt solution. The killing of strain D31 followed single-hit kinetics, and increasing rate constants were obtained for increasing amounts of hemolymph. The reaction was sensitive to pretreatment with trypsin and it was protected by reducing agents. The activity was inhibited by microgram quantities of lipopolysaccharide (LPS) prepared from certain LPS mutants of E. coli K-12. A comparison of the susceptibility showed that “heptose-less” LPS mutants were more sensitive to killing than other strains. During standard conditions hemolymph will lyse both E. coli and Micrococcus lysodeikticus. Lysis of E. coli followed a multi-hit kinetics and it was inhibited by LPS, whereas lysis of M. lysodeikticus was unaffected by LPS. Hemolymph was fractionated on Sephadex G-200, and the lytic activities were recovered in partly overlapping peaks. Reconstitution with pooled fractions gave synergistic effects with the killing assay.

Purification and properties of reconstitutively active and inactive adenosinetriphosphatase from Escherichia coli.

Abstract: The Mg2+- and Ca2+-stimulated ATPase (EC 3.6.1.3; ATP phosphohydrolase) (bacterial coupling factor) was purified from two strains of E. coli by two different procedures: (a) method of Nelson, Kanner, and Gutnick [Proc. Nat. Acad. Sci. USA (1974) 71, 2720-2724] and (b) a modified procedure described in this paper. The ATPase purified from E. coli K12 (λ) by the first procedure had 4 subunits (α, β, γ, and e). It did not bind to a deficient membrane, nor did it reconstitute ATP-driven transhydrogenase activity. Our modified procedure (b) yielded 5 subunits (α, β, γ, δ, and e). This ATPase could bind to a deficient membrane and reconstitute ATP-driven transhydrogenase. This finding suggests that the δ subunit is required for the reaction with the membrane. The molecular weight of the 4-subunit ATPase was significantly lower than that of the 5-subunit ATPase, as judged by equilibrium centrifugation. The specific ATPase activities of both preparations were about the same. These two procedures were also applied to E. coli ML308-225.

Outer Membrane Proteins of Escherichia coli III. Evidence that the Major Protein of Escherichia coli O111 Outer Membrane Consists of Four Distinct Polypeptide Species

Abstract: Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.

Con--mutants: class of mutants in Escherichia coli K-12 lacking a major cell wall protein and defective in conjugation and adsorption of a bacteriophage.

Abstract: We describe a new class of mutants of Escherichia coli K-12 defective in conjugation (Con− mutants). They lack a major protein of the outer membrane and are defective as recipients with Hfr and F′ donors and as recipients for several F-like R factors and Col factors. In the case of crosses with an Hfr donor, we have shown that the Con− recipient is defective in pair formation. The mutants are resistant to certain phages due to loss of receptor activity.

Macromolecular Composition During Steady-State Growth of Escherichia coli B/r

Abstract: By using Escherichia coli B/r, the cellular amounts of ribonucleic acid (RNA) and protein were determined as a function of the steady-state growth rate (0.67 to 2.40 doublings per h) by a method which combines measurements of the RNA to deoxyribonucleic acid (DNA) ratio and the differential rate of ribosomal protein synthesis with the Cooper and Helmstetter theory of DNA replication. The results indicate that the ratios RNA/DNA, RNA/protein, and protein/DNA give linear relationships with the growth rate (above 1.2 doublings per h), whereas RNA/cell and protein/cell show a more complex growth rate dependency. The significance of these relationships is discussed. Finally, a detailed description of the growth parameters and composition of E. coli B/r is presented.

Conditional Mutator Gene in Escherichia coli: Isolation, Mapping, and Effector Studies

Abstract: A mutator gene, mutD5, whose phenotype is conditional, has been identified in Escherichia coli By P1 transduction it has been shown to lie at about 57 min on the chromosome, being co-transduced with proA and argF In rich medium, streptomycin- and nalidixic acid-resistant mutation frequencies are 50 to 100 times higher than those in minimal medium In minimal medium, the mutD5-induced mutation frequencies are still 50 to 100 times above co-isogenic wild-type (mut+) levels Similar results were obtained with all markers tested Mutant frequencies can be raised by thymidine in the medium at concentrations as low as 004 μM, or by the endogenous generation of thymidine from thymine plus a deoxyribosyl donor Deoxyadenosine, various ribonucleosides, thymine, and 2-deoxyribose do not stimulate mutation None of these effects are related to growth rate, since growth rate and mutation rate can be decoupled completely

Volatilisation of mercury and organomercurials determined by inducible R-factor systems in enteric bacteria

Abstract: WE have screened a large number of R plasmid-bearing Escherichia coli and obtained a few that confer resistance to the organomercurials phenylmercuric acetate (PMA) and methylmercuric chloride (MMA). Resistance to cationic Hg(II) in E. coli, Staphylococcus aureus and Pseudomonas aeruginosa has invariably been associated with plasmids that also mediate resistance to various antibiotics (R plasmids)1–6. The mechanism of mercury resistance, is the enzymatic reduction of Hg(II) to Hg(0), which is volatile2,7,8. Previously, no organomercurial-reducing E. coli strains have been reported. The only organomercurial-reducing strain studied in detail is a PMA-resistant soil pseu-domonad7–9. As organomercurials including PMA are a human health problem10, it will be of interest to see if the mercury(ial) resistance of the bacteria in our alimentary canal influences the fate of ingested mercury(ials).

Genome Construction Between Bacterial Species In Vitro: Replication and Expression of Staphylococcus Plasmid Genes in Escherichia coli

Abstract: Genes carried by EcoRI endonuclease-generated fragments of Staphylococcus plasmid DNA have been covalently joined to the E. coli antibiotic-resistance plasmid pSC101, and the resulting hybrid molecules have been introduced into E. coli by transformation. The newly constructed plasmids replicate as biologically functional units in E. coli, and express genetic information carried by both of the parent DNA molecules. In addition, electron microscope heteroduplex analysis of the recombinant plasmids indicate that they contain DNA sequences derived from E. coli and Staphylococcus aureus. Recombinant molecules can transform other E. coli cells for penicillin-resistance markers originally carried by the staphylococcal plasmid, and can be transferred among E. coli strains by conjugally proficient transfer plasmids.

A search for transmissible pathogenic characters in invasive strains of Escherichia coli: the discovery of a plasmid-controlled toxin and a plasmid-controlled lethal character closely associated, or identical, with colicine V.

Abstract: SUMMARY: Most invasive strains of Escherichia coli from man and domestic animals were lethal for chickens and mice. The lethal characteristic was not, in general, transferred when invasive strains were grown in mixed culture with non-pathogenic strains of E. coli, although two transmissible plasmids coding for pathogenic properties were discovered. One plasmid, designated Vir, was found in an E. coli strain causing bacteraemia in a lamb. It transferred at high rate to several strains of E. coli, including a rec A-KI2 strain, to Salmonella typhi, Salm. typhimurium and Shigella sonnei. Culture filtrates and, especially, bacterial ultrasonicates of Vir+ strains were toxic for chickens, mice and rabbits. The toxin was heat-sensitive, acid-sensitive and non-diffusible. Organisms producing it were agglutinated by specific Vir+ antisera; their toxic activity was not neutralized. The transfer factor of the Vir plasmid was fi+ and could transfer antibiotic-resistance determinants in addition to the Vir determinant. The other plasmid was first discovered in an E. coli strain F120, isolated from an outbreak of bacteraemia in chickens. Organisms of E. coli K12 and of other E. coli strains acquiring this plasmid during mixed culture with FI20 were increased in lethality for chickens and mice; this was associated not with toxic activity but with greater ability to survive in blood and peritoneal fluids. Strain FI20 possessed transmissible ColV and Collb plasmids; increased lethality was closely associated with the ColV plasmid. When the ColV plasmids of another six wild strains of E. coli of varied origin were transferred to organisms of E. coli KI2, the lethality increase was similar to that for ColV transfer from FI20. No lethality change accompanied transfer of other Col plasmids. It was concluded that colicine V itself might be responsible for the increased lethality. Strains of E. coli associated with bacteraemia in man and animals commonly produced colicine V.

24. The Glutamine Synthetase of Escherichia coli: Structure and Control

Abstract: Publisher Summary This chapter discusses the structure the glutamine synthetase of Escherichia coli. The glutamine synthetase from E. coli and that from other gram-negative microorganisms tested exist in two forms: an unmodified form and a form possessing covalently bound adenylyl groups (10-Ig). Under appropriate conditions, both forms of the enzyme are catalytically active; however, they differ significantly with respect to catalytic potential, pH optimum, divalent cation specificity, and susceptibility to feedback inhibition. Covalent modification of the E. coli enzyme is the basis of an elaborate mechanism for the fine control of glutamine synthetase activity in this organism. E. coli glutamine synthetase has a molecular weight of 600,000 and is composed of 12 apparently identical subunits. Electron microscopic examination of preparations negatively stained with sodium silicotungstate disclosed that the subunits are arranged in two hexagonal rings that lie one on top of the other in a face-to-face fashion. In hydrodynamic studies, the enzyme in solution behaves as a compact, essentially spherical particle. It was calculated from these data that the β parameter for describing the shapes of kinetic units in solution is 2.1 X 106 or about the same value as that for a spherical particle. The chapter also discusses the amino acid composition of E. coli glutamine synthetase. The composition is expressed in terms of the number of amino acid residues per 50,000 g since it is believed that all 12 subunits of the enzyme are identical. It is especially noteworthy that each subunit contains 5 cysteinyl, 15 tryosyl, 3-tryptophanyl, 15-16 methionyl, 26 lysyl, and 25 arginyl residues.

Characterization of Escherichia coli Flagellar Mutants That Are Insensitive to Catabolite Repression

Abstract: In Escherichia coli, the synthesis of the flagellar organelle is sensitive to catabolite repression. Synthesis requires the presence of the cyclic adenosine monophosphate receptor protein (Crp) and 3′,5′-cyclic adenosine monophosphate (cAMP); i.e., mutants that lack Crp or adenylcyclase (Cya) synthesize no flagella. We isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli. The mutations in these strains were transferred onto episomes and they were then introduced into a variety of other strains. The presence of the mutation resulted in flagella synthesis in Cya and Crp strains as well as in the wild type grown under conditions of catabolite repression. Deletion analysis and other genetic studies indicated that: (i) the cfs mutations had a dominant effect when they were in the transconfiguration in merodiploids: (ii) they occurred in or very close to the flaI gene: and (iii) their expression required the presence of an intact flaI gene adjacent to the cfs mutation. Biochemical studies showed that the synthesis of at least two flagellar polypeptides, the hook subunit and an amber fragment of flagellin, were absent in strains that carried a cya mutation. Their synthesis was depressed in strains grown under conditions of catabolite repression. The presence of the cfs mutation restored the specific synthesis of these two polypeptides. We suggest that the formation of the flaI gene product is the step in flagellar synthesis that is catabolite sensitive and requires cAMP. We propose a regulatory function for the product of the flaI gene.

The association of K88 antigen with haemagglutinating activity in porcine strains of Escherichia coli.

Abstract: SUMMARY: All of 108 strains of Escherichia coli that synthesized K88 antigen caused mannose-resistant and eluting (m.r.e.) haemagglutination of guinea-pig erythrocytes in a microhaemagglutination test; none of 23 representative strains did so in a tile haemagglutination test which requires firmer binding. It was concluded that the K88 antigen was the m.r.e. haemagglutinin since (i) only K88-positive strains caused m.r.e. haemagglutination (ii) K88-positive strains grown at 18°C failed to produce both haemagglutinin and K88 antigen (iii) haemagglutinating activity was not detected in K88-negative mutants of a K88-positive enteropathogenic strain, and (iv) extracts of K88 antigen possessed haemagglutinating activity which could not be separated from the K88 antigen by the fractionation and serological procedures examined. Haemagglutination appears to resemble the attachment of K88-positive bacteria to the gut wall in enteric disease and the haemagglutination test may assist in characterizing this mechanism.