Showing papers on "Escherichia coli published in 1980"

Expression of a bacterial gene in mammalian cells

Abstract: Transfection of cultured monkey kidney cells with recombinant DNA constructed with a cloned Escherichia coli gene that codes for xanthine-guanine phosphoribosyltransferase and several different SV40 DNA-based vectors, results in the synthesis of readily measurable quantities of the bacterial enzyme. Moreover, the physiological defect in purine nucleotide synthesis characteristic of human Lesch-Nyhan cells can be overcome by the introduction of the bacterial gene into these cells.

Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli.

Abstract: Tetracycline resistance encoded by four genetically different determinants residing on plasmids in Escherichia coli was shown to be associated in each case with an energy-dependent decrease in accumulation of the antibiotic in whole cells in which resistance had been induced. The different class determinants examined were those on plasmids RP1 (class A), R222 (class B), R144 (class C), and RA1 (class D). This decrease in accumulation was attributable to an active efflux, because everted (inside-out) membrane vesicles made from tetracycline-induced E. coli cells containing any one of the four plasmids were shown to concentrate tetracycline by an active influx. This active uptake was not seen in inside-out vesicles from sensitive cells or uninduced R222-containing cells. In vesicles from induced R222-containing cells, the efflux appeared to be carrier-mediated with a Km of about 6 microM. These results demonstrate that active export of tetracycline is a common component of the mechanism for tetracycline resistance encoded by different plasmid-borne determinants in bacteria.

Cleavage of the Escherichia coli lexA protein by the recA protease.

Abstract: The recA and lexA proteins of EScherichia coli are involved in a complex regulatory circuit that allows the expression of a diverse set of functions after DNA damage or inhibition of DNA replication. Exponentially growing cells contain a low level of recA protein, and genetic evidence suggests that lexA protein is involved in its regulation, perhaps as a simple repressor. Recent models for recA derepression after DNA damage have suggested that an early event in this process is the proteolytic cleavage of lexA protein, leading to high-level expression of recA. We present several lines of evidence that the specific protease activity of the recA protein, previously described with the lambda repressor as substrate, is capable of cleaving the wild-type lexA+ protein. First, lexA protein can be cleaved in vitro under the same conditions as prevously described for lambda repressor cleavage in a reaction requring both recA protease and ATP or an analogue, adenosine 5'-[lambda-thio]-triphosphate. Second, lexA protein can be observed in vivo as a physical entity after infection with lambda lexA+ transducing phage of host strains containing ittle or no active protease, but not in strains containing high levels of active protease. Finally, infection of host cells containing active protease with a lambda lexA+ transducing phage does not lead to repression of recA, but does so in cells lacking active protease. In all of these conditions the mutant lexA3 protein is largely resistant to inactivation or cleavage; this resistance can explain the dominant phenotype of lexA3 over lexA+. We discuss models for recA derepression and re-establishment of repression which propose that modulation of the protease activity of recA protein regulates both of these transitions.

Human leukocyte interferon produced by E. coli is biologically active

Abstract: A human leukocyte interferon cDNA was enzymatically synthesized, inserted into the vector pBR322, and cloned in Escherichia coli. The DNA sequence codes for a 23-amino acid signal peptide followed by an interferon polypeptide of 165 amino acids. An expression plasmid was constructed which permits the synthesis in E. coli of 2.5 x 10(8) units of interferon per litre of culture. This LeIF protected squirrel monkeys from lethal encephalomyocarditis virus infection.

DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli.

Abstract: Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector [Casadaban, M.J. & Cohen, S.N. (1979) Proc. Natl. Acad. Sci. USA 76, 4530-4533] to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

Synthesis in E. coli of a polypeptide with human leukocyte interferon activity

Abstract: Double-stranded cDNA prepared from the 12S fraction of poly(A) RNA from interferon (IF)-producing human leukocytes was cloned in Escherichia coli using the pBR322 vector. One of the resulting clones had a 910-base pair insert which could hybridise to IF mRNA and was responsible for the production of a polypeptide with biological IF activity. Up to 10,000 units IF activity per g of cells was obtained from some clones.

The structure of one of the eight or more distinct chromosomal genes for human interferon- α

Abstract: The 12 Interferon (IFN)-related sequences detected in a human gene bank fall into not less than eight distinct classes, indicating that there are at least eight IFN-related genes. Most, if not all, of these direct the synthesis of an IFN in Escherichia coli. The sequence of one chromosomal gene and its flanking regions was identical to that deduced for the cDNA corresponding to IFN-αl mRNA. No evidence was found for the existence of an intron, in either the coding or the non-coding segments of the gene.

Genetic diversity and structure in Escherichia coli populations.

Abstract: A survey of electrophoretic variation in 20 enzymes from 109 clones of escherichia coli from natural populations yielded an estimate of mean genetic diversity approximately twice that reported in an earlier study and four to five times larger than estimates fro most eukaryotic species. Despite this extensive variability, the number of distinctive genotypes apparently is rather limited. Identical clones were obtained from unassociated hosts, and a clone that is electrophoretically indistinguishable from the laboratory strain Escherichia coli K-12 was isolated from a human infant. The results suggest that rates of genetic recombination in natural populations of Escherichia coli are low. These findings have implications for our understanding of the genetic structure of Escherichia coli populations and the factors determining the amount of neutral gene variability in this bacterial species.

Pathogenesis of escherichia coli gastroenteritis in man--another mechanism.

Abstract: ESCHERICHIA COLI infection of the gastrointestinal tract may cause diarrhea by two pathogenic mechanisms that have been well described in laboratory animals and man: direct invasion of the intestin...

Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies.

Abstract: Two mRNA species that produce biologically active interferon were isolated from human fibroblasts and studied by size fractionation and cloning in Escherichia coli plasmid pBR322. The major fibroblast interferon (Hu IFN-beta 1) is coded for by the smaller of the two mRNAs, an 11S species, 900 nucleotides long, which in cell-free systems yields a 20,000 Mr protein. The second interferon mRNA species (Hu IFN-beta 2) is 14S, about 1300 nucleotides long, and codes for another protein of 23,000-26,000 Mr. The two interferon mRNAs do not cross-hybridize. Both are induced by poly(rI.rC), but IFN-beta 2 mRNA is induced to about 10% in cells by cycloheximide treatment alone whereas under these conditions IFN-beta 1 is not induced.

Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli.

Abstract: The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method. This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols. The analogous sugars were not transported. However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell. The glpF protein allowed the rapid efflux of preequilibrated xylitol. Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol. The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.

Amino acid sequence homology between cholera toxin and Escherichia coli heat-labile toxin

Abstract: Cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT) are functionally, structurally and immunologically similar enterotoxins. Both toxins cause the elevation of cyclic AMP levels in gut epithelial cells1,2 by catalysing the NAD-dependent ADP ribosylation of membrane proteins3–6. Each toxin is composed of two dissimilar subunits7,8. The A subunit has an enzymatic activity and is the adenylate cyclase-activating component of the enterotoxin4–11. The B subunit recognizes membrane components and binds the holotoxin to the target cell juxtaposing the A subunit with its substrates9,12. Binding studies and competition experiments indicate that the membrane receptors for cholera toxin B subunit (CT-B) and LT-B are similar but not identical13 (these studies were performed before LT was purified to homogeneity). The monosialosylganglioside GMI has been shown to be the receptor for the cholera toxin14, and it probably composes part of the receptor for LT. Gyles and Barnum15 first reported that LT and cholera toxin were immunologically related, and it has subsequently been shown that they share common antigenic determinants in both A and B subunits16. The primary structure of CT-B has been determined17,18. We report here a comparison between the amino acid sequences of LT-B and CT-B. The nucleotide sequence of the LT-B cistron (eltB) was determined using a recombinant plasmid encoding LT9. Translation of this sequence revealed that LT-B and CT-B show significant amino acid sequence homology. In addition, several features of the eltB cistron were revealed by the sequence analysis.

Are growth rates of Escherichia coli in batch cultures limited by respiration

Abstract: Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h. The respiration rates of the cultures were measured continuously. With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value. The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis. The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation. This rate was closely related to the respiration rate. We suggest that aerobic growth of E. coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.

Direct transfer of cloned genes from bacteria to mammalian cells

Abstract: Induction of a virus infection by cloned simian virus 40 DNA was chosen as a test system to detect transfer of genes from bacteria to cultured mammalian cells. Escherichia coli cells containing a recombinant plasmid with three tandem inserts of simian virus 40 DNA were able to infect CV-1 monkey cells under various conditions. The gene transfer was resistant to DNase I and therefore seems not to occur via free DNA but most likely via uptake of whole bacteria, followed by release of plasmid DNA and generation of infectious circular simian virus 40 DNA in a recombination-excision process. Spontaneous transfer was found to be infrequent, 4 x 10(9) bacteria yielding one infection per 10(7) monkey cells. The frequency was greatly increased by adding bacteria as a calcium phosphate coprecipitate or by fusion of lysozyme-treated bacteria (protoplasts) with monkey cells in the presence of polyethylene glycol. With the latter technique, 10(4) protoplasts gave rise to one infection per 15 monkey cells. Experiments with other cell lines of human, monkey, and mouse origin, and also with bacteria harboring another recombinant plasmid, indicate that DNA transfer from bacteria to mammalian cells is a general phenomenon.

Sequences of the recA gene and protein

Abstract: We have determined the nucleotide sequence of the recA gene of Escherichia coli; this permits the formulation of the primary structure for the recA protein. This structure is consistent with the amino acid composition of the tryptic peptides obtained from the recA protein. The coding region of the recA gene has 1059 base pairs, which specify 352 amino acids. The recA protein has alanine and phenylalanine as its NH2- and COOH-terminal amino acids, respectively, and has the following amino acid composition: Cys3 Asp20 Asn15 Met9 Thr17 Ser20 Glu30 Gln13 Pro10 Gly35 Ala38 Val22 Ile27 Leu31 Tyr7 Phe10 His2Lys27 Trp2 Arg14. Of the three cysteine residues, only two can be alkylated under reducing and denaturing conditions. The molecular weight of the recA polypeptide is 37,842.

Influence of the Escherichia coli capsule on complement fixation and on phagocytosis and killing by human phagocytes.

Abstract: To define mechanisms by which polysaccharide capsules confer enhanced virulence on gram-negative bacteria, we examined the effect of the Escherichia coli capsule on complement fixation to the bacterial surface and on phagocytosis and killing of these bacteria by mouse macrophages and human polymorphonuclear leukocytes (PMN) and monocytes. When E. coli were attached to mouse macrophages with concanavalin A, the macrophages readily phagocytosed unencapsulated but not encapsulated bacteria even in the presence of fresh mouse serum; macrophages did not phagocytose encapsulated E. coli unless antibacterial or anti-Con A antibody was added. Similarly, when these bacteria were attached to human PMN with Con A, PMN ingested unencapsulated but not encapsulated E. coli. PMN phagocytosed and killed encapsulated serum-resistant E. coli only in the presence of both complement and antibacterial antibody; PMN phagocytosed and killed unencapsulated E. coli of the same strain in the presence of complement alone. Fluorescence microscopy showed that antibody had to be present for encapsulated but not unencapsulated E. coli to fix complement to its surface. To examine the role of the complement receptors of human PMN and monocytes in phagocytosis and killing of encapsulated E. coli, we used human and rabbit antibacterial immunoglobulin (Ig)M to fix complement to the bacteria. PMN and monocytes phagocytosed and killed encapsulated E. coli in the presence of both IgM and complement, but not in the presence of either serum opsonin alone. In the presence of antibacterial IgG, PMN and monocytes required complement to effectively phagocytose and kill the E. coli. We conclude that (a) attachment by itself results in ingestion of unencapsulated but not encapsulated E. coli; (b) under physiologic conditions, E. coli are not phagocytosed or killed the absence of antibody, the E. coli capsule blocks complement fixation to the bacterial surface probably by masking surface components, such as lipopolysaccharide, capable of activating the complement pathway; (d) the E. coli capsule imposes a requirement for specific antibacterial antibody for complement fixation; and (e) the complement receptor of human PMN and monocytes mediates phagocytoses of complement-coated encapsulated bacteria and is the primary mediator of phagocytosis and killing of these bacteria.

Nucleotide sequence of the bacterial transposon Tn1681 encoding a heat-stable (ST) toxin and its identification in enterotoxigenic Escherichia coli strains

Abstract: the Escherichia coli heat-stable toxin (ST I) is encoded within a transposon (Tn1681) flanked by inverted repeats of insertion sequence 1 (IS1) [So, M., Heffron, F. & McCarthy, B. J. (1979) Nature (London) 277, 453-456]. By subcloning restriction fragments and by insertion mutagenesis, we located precisely the gene for ST I within the transposon. We determined the complete nucleotide sequence of the central portion of Tn1681 (i.e., that part flanked by IS1) and identified the coding sequence of the toxin. From the nucleotide sequence, we deduced a probable amino acid sequence for ST I. The NH2-terminal portion of the amino acid sequence is extremely hydrophobic and bears a striking resemblance to the signal sequence of the fd phage minor coat protein. By using a subcloned restriction fragment containing the gene for ST I but no IS1 sequences, we determined (i) that the ST toxin with activity assayable in suckling mice (ST I) is genetically distinct from the St toxin assayable in ligated ileal loops (ST II) and (ii) that ST I can be responsible for diarrheal disease in different animals.

Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli

Abstract: The maltose binding protein of Escherichia coli is secreted into the external periplasmic compartment of the cell by virtue of an amino-terminal signal sequence. Using DNA sequencing, we have determined the precise nature of mutations in the signal sequence which prevent the export of the maltose binding protein, causing it to accumulate in the cytoplasm in its precursor form. In most cases, the change of a single hydrophobic or uncharged amino acid to a charged amino acid within the signal sequence is sufficient to block the secretion process.

Plasmid-mediated tissue invasiveness in Yersinia enterocolitica.

Abstract: Plasmids have an important role in the pathogenicity of certain bacterial species, and Escherichia coli provides the most complete example of the relationship involved. Enterotoxigenic strains of E. coli, in addition to producing heat-stable and/or heat-labile enterotoxins, may also produce a haemolysin and fimbriate cell surface antigens which facilitate the adherence of the bacterial cell to the mucosa of the small bowel. Numerous studies have shown that these properties are plasmid-mediated1–5 and that the plasmids act in concert to confer on the host bacterium the ability to produce enteric disease in man and in animals. Moreover, studies with invasive strains of E. coli have shown that the Col V plasmid, which codes for the synthesis of colicin V, significantly enhances the pathogenicity of its host bacterium6,7. Although the relationship between Col V plasmids and virulence is unclear, reports indicate that Col V-containing strains of E. coli are better able to survive in the alimentary tract and that colicine V itself inhibits macrophage function7,8. It is probable that bacterial virulence is a complex phenomenon involving both chromosomal and plasmid genes. We describe here a virulence plasmid which mediates tissue invasiveness in human pathogenic strains of Yersinia enterocolitica.

recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.

Abstract: The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo.

The primary structure of 16S rDNA from Zea mays chloroplast is homologous to E. coli 16S rRNA.

Abstract: The nucleotide sequence of ribosomal DNA coding for 16S rRNA from Zea mays chloroplast has been determined. A comparison with the 16S rRNA sequence from Escherichia coli reveals strong homology and thereby demonstrates the prokaryotic nature of chloroplast ribosomes from a higher plant.

Eukaryotic signal sequence transports insulin antigen in Escherichia coli.

Abstract: We made a series of plasmids with unique Pst restriction sites within or near the DNA that encodes the penicillinase signal sequence. Inserted DNA can be read in all three frames both within and immediately after the signal sequence. We cloned Pst-terminated DNA copies of the structural information for rat proinsulin and preproinsulin into these plasmids, forming a large number of hybrid penicillinase (bacterial) and insulin (eukaryotic) signal sequences. We then compared the levels of insulin antigen in the Escherichia coli periplasm with those inside the cells. We conclude that either the bacterial or the eukaryotic signal is sufficient to transport rat insulin antigen into the periplasmic space.

Comparison of Escherichia coli fimbrial antigen F7 with type 1 fimbriae.

Abstract: Two Escherichia coli O6:K2:H1 strains, C1212 and C1214, isolated from urinary tract infections, were compared for their capacity to adhere to various cells. After growth on solid medium, only C1212 bacteria agglutinate human erythrocytes and attach to urinary epithelial cells. Both of these reactions are mannose resistant. In contrast, C1214 bacteria cause a mannose-sensitive agglutination of guinea pig erythrocytes, show a mannose-sensitive attachment to buccal epithelial cells, and attach to urinary mucus. Immunoelectron microscopy revealed that C1214 bacteria possess type 1 fimbriae (mannose sensitive), which are not present in C1212 bacteria when this strain is grown on solid medium. The fimbriae of C1212 (mannose resistant) were also demonstrated by immunoelectron microscopy. We call these fimbriae demonstrated in C1212 the E. coli F7 antigen. Urinary mucus, and probably mucous material elsewhere, may function as a trap for Enterobacteriaceae with type 1 fimbriae by the specific adherence of such bacteria. We consider this a nonimmune resistance mechanism against disease caused by Enterobacteriaceae.