Reduced synthesis of β-galactosidase in Escherichia coli infected with phage ϕX174

1977 ◽  
Vol 23 (8) ◽  
pp. 1069-1077 ◽  
Author(s):  
Amit Ghosh ◽  
Ramendra K. Poddar
Keyword(s):  
Escherichia Coli ◽  
Single Stranded Dna ◽  
E Coli ◽  
Infected Cells ◽  
Specific Mrna

The synthesis of β-galactosidase (EC 3.2.1.23; β-D-galactoside galactohydrolase) in E. coli was repressed as a result of infection with single-stranded DNA phage [Formula: see text]. Evidence is presented to show that this repression was not due to the restricted entry of the inducer molecules into the infected cells but to some phage-specified product(s). It was further shown that either the infected cells synthesized a fewer number of enzyme-specific mRNA or all such molecules were translated with a reduced efficiency; the half-lives of the mRNA's remained more or less unaffected.

scholarly journals Escherichia coli Endoribonucleases Involved in Cleavage of Bacteriophage T4 mRNAs

2003 ◽  
Vol 185 (3) ◽  
pp. 983-990 ◽  
Author(s):  
Yuichi Otsuka ◽  
Hiroyuki Ueno ◽  
Tetsuro Yonesaki
Keyword(s):  
Escherichia Coli ◽  
Bacteriophage T4 ◽  
Stable Form ◽  
Rnase E ◽  
E Coli ◽  
Mutant Strains ◽  
Allele Specific ◽  
Infected Cells ◽  
Encoding Genes ◽  
Specific Mrna

ABSTRACT The dmd mutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying various soc alleles, we could detect cleavages of soc RNA in uninfected cells identical to those found in dmd mutant-infected cells. We isolated five Escherichia coli mutant strains in which the dmd mutant was able to grow. One of these strains completely suppressed the dmd mutant-specific cleavages of soc RNA. The loci of the E. coli mutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing the dmd mutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5′-truncated soc RNA, a stable form in T4-infected cells regardless of the presence of a dmd mutation, is generated by RNase E.

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis

1992 ◽  
Vol 38 (4) ◽  
pp. 350-353 ◽  
Author(s):  
A. Moreau ◽  
F. W. Paradis ◽  
R. Morosoli ◽  
F. Shareck ◽  
D. Kluepfel
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Shuttle Vector ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
E Coli ◽  
Streptomyces Spp ◽  
Ampicillin Resistance Gene

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective markerin E. coli. Vector pIAFS20 also contains the phage fl intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screeningof the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis. Key words: shuttle vector, single-stranded DNA, site-directed mutagenesis, Streptomyces spp., Escherichia coli.

scholarly journals The Colibactin Genotoxin Generates DNA Interstrand Cross-Links in Infected Cells

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Nadège Bossuet-Greif ◽  
Julien Vignard ◽  
Frédéric Taieb ◽  
Gladys Mirey ◽  
Damien Dubois ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Colon Cancer ◽  
Cross Linking ◽  
High Fat ◽  
E Coli ◽  
High Fat Diets ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Infected Cells ◽  
Fat Diets

ABSTRACTColibactins are hybrid polyketide-nonribosomal peptides produced byEscherichia coli,Klebsiella pneumoniae, and otherEnterobacteriaceaeharboring thepksgenomic island. These genotoxic metabolites are produced bypks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinalE. coli. Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in aclbPmutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observedin cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCEColorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinalEscherichia colibacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxicE. coli, we showed that colibactins directly generate DNA cross-linksin cellulo. Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role ofE. coliin colon cancer and engineering new anticancer agents.

SSB Protein—Its Interaction with DNA and Use as a Tool in Studying Genome Structure

1981 ◽  
Vol 39 ◽  
pp. 448-451
Author(s):  
Susan Chrysegelos ◽  
Kathi Dunn ◽  
Jack Griffith ◽  
Marcia Manning ◽  
Claire Moore
Keyword(s):  
Genome Structure ◽  
Duplex Dna ◽  
Single Stranded Dna ◽  
Functional Roles ◽  
E Coli ◽  
Gene 5 Protein ◽  
Nucleoprotein Complex ◽  
Infected Cells ◽  
Gene 32

A protein which binds tightly to single stranded DNA but not duplex DNA was first isolated from Escherichia coli (E. coli) by Sigal et. al and is called SSB for single stranded DNA binding protein. Together with SSB the gene 32 protein of T4 infected E. coli cells, and the gene 5 protein of phage M13 infected cells, are the best characterized members of the helix destabilizing family of proteins. They all share the properties (reviewed by Kbrnberg) of binding very tightly and cooperatively to single stranded DNA, of binding somewhat less well to single stranded RNA, and of binding poorly if at all to duplex DNA or RNA. In binding single stranded polynucleotides these proteins disrupt all secondary structure yielding a linear nucleoprotein complex. The details of binding however are very different from one protein to another and must reflect their functional roles in vivo.Physical studies of SSB have showi it to exist as a 75,000 dalton tetramer in solution which is assumed to be the active unit.

LYSOGENY IN ENTEROPATHOGENIC ESCHERICHIA COLI: III. THE EFFECTS OF TEMPERATURE AND CHLORAMPHENICOL ON THE LYSOGENY OF D-1 PHAGE-INFECTED CELLS

1961 ◽  
Vol 7 (6) ◽  
pp. 915-920 ◽  
Author(s):  
John P. Glynn ◽  
W. Robert Bailey
Keyword(s):  
Escherichia Coli ◽  
Survival Rate ◽  
Enteropathogenic Escherichia Coli ◽  
E Coli ◽  
Appropriate Treatment ◽  
Infected Cells ◽  
Effects Of Temperature

Evidence is presented to show that cells of Escherichia coli O26:B6 infected with E. coli O111 phage D-1 exhibited a greater lysogenic response if the temperature, shortly after infection, was lowered from 37 °C. Under the same conditions, lysogeny among survivors was reduced markedly at 45 °C. Cooling the system to 20 °C prior to infection increased both the survival rate and lysogenic response among phage-infected survivors. Appropriate treatment with chloramphenicol increased both survival rate and lysogenic frequency.

scholarly journals Lytic and Lysogenic Infection of DiverseEscherichia coli and Shigella Strains with a Verocytotoxigenic Bacteriophage

2001 ◽  
Vol 67 (9) ◽  
pp. 4335-4337 ◽  
Author(s):  
Chloe E. James ◽  
Karen N. Stanley ◽  
Heather E. Allison ◽  
Harry J. Flint ◽  
Colin S. Stewart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Plaque Assay ◽  
Kanamycin Resistance ◽  
Lytic Infection ◽  
Lytic Cycle ◽  
Animal Origin ◽  
Kanamycin Resistance Gene ◽  
E Coli ◽  
Infected Cells

ABSTRACT A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2 ), was used to infect Enterobacteriaceae strains. A number ofShigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

scholarly journals Intestinal Shiga Toxin-Producing Escherichia coli Bacteria Mitigate Bovine Leukemia Virus Infection in Experimentally Infected Sheep

2006 ◽  
Vol 74 (5) ◽  
pp. 2906-2916 ◽  
Author(s):  
Witold A. Ferens ◽  
Rowland Cobbold ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Antiviral Activity ◽  
Shiga Toxin ◽  
Bovine Leukemia Virus ◽  
Mononuclear Cells ◽  
Leukemia Virus ◽  
Average Weight ◽  
E Coli ◽  
Infected Cells ◽  
The Impact

ABSTRACTRuminants often carry gastrointestinal Shiga toxin (Stx)-producingEscherichia coli(STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses ofE. coliO157:H7 (an STEC) or an isogenicstxmutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates orstx-negativeE. coli. Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >104CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower (P< 0.05) and syncytia were fewer (P < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep (P< 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease.

scholarly journals A late exclusion of bacteriophage T4 can be suppressed by Escherichia coli GroEL or Rho.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 613-625
Author(s):  
C H Linder ◽  
K Carlson ◽  
F Albertioni ◽  
J Söderström ◽  
C Påhlson
Keyword(s):  
Escherichia Coli ◽  
Low Temperatures ◽  
Transcription Termination ◽  
Bacteriophage T4 ◽  
E Coli ◽  
Rho Protein ◽  
Phage Assembly ◽  
Infected Cells ◽  
In Trans ◽  
Lit Protein

Abstract A litCon mutation in Escherichia coli TU6 results in exclusion of bacteriophage T4 during the late, morphogenetic stage of its development at low temperatures. DNA was synthesized continuously in the infected cells, but less than 10% of the DNA made by 90 min after infection was packaged into DNAase-resistant particles, few viable phage were formed, and the cells lysed poorly. The exclusion could be relieved by conditions leading to elevated levels, determined immunologically, of the E. coli Rho protein (believed to be involved in regulation of T4 transcription), or chromosomally encoded E. coli GroEL (a chaperone known to be involved in phage assembly), or by supplying GroEL in trans from a plasmid. The two suppressing proteins appeared to act independently of each other. GroEL-suppression restored packaging to normal levels, perhaps by preventing GP23 from activating the host Lit protein; in addition DNA synthesis was delayed and reduced and cell lysis enhanced, demonstrating involvement of GroEL in both these processes. Rho suppression was less efficient. Since both transcription-termination-proficient and transcription-termination-deficient Rho suppressed, the results raise the possibility that Rho has a role during T4 development not directly involving transcription regulation.

scholarly journals Increased Levels of Intracellular Calcium Are Not Required for the Formation of Attaching and Effacing Lesions by Enteropathogenic and Enterohemorrhagic Escherichia coli

1998 ◽  
Vol 66 (8) ◽  
pp. 3900-3908 ◽  
Author(s):  
Christopher Bain ◽  
Rogeria Keller ◽  
Georgina K. Collington ◽  
Luiz R. Trabulsi ◽  
Stuart Knutton
Keyword(s):  
Escherichia Coli ◽  
Intracellular Calcium ◽  
Bacterial Adhesion ◽  
Enterohemorrhagic Escherichia Coli ◽  
Live Cells ◽  
Free Calcium ◽  
Lesion Formation ◽  
E Coli ◽  
Infected Cells ◽  
Temporal And Spatial

ABSTRACT Elevated concentrations of intracellular calcium ([Ca]i) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic Escherichia coli (EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]i in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]i at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic E. coli (EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]i in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]iare not required for A/E lesion formation by EPEC and EHEC.

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