scholarly journals A MUTANT OF E. COLI THAT RESTRICTS GROWTH OF BACTERIOPHAGE T4 AT ELEVATED TEMPERATURES

Genetics ◽  
1980 ◽  
Vol 94 (2) ◽  
pp. 301-325
Author(s):  
Jean L Jensen ◽  
Millard Susman
Keyword(s):  
Elevated Temperatures ◽  
Bacteriophage T4 ◽  
Temperature Shift ◽  
Wild Type ◽  
E Coli ◽  
Early Protein ◽  
Phage Dna ◽  
Phage Growth ◽  
Spontaneous Mutants ◽  
Phage Proteins

ABSTRACT After nitrosoguanidine mutagenesis, a Phage Host Defective (phd) mutant of E. coli HfrH was isolated that supported the growth of T4D wild-type bacteriophage at 30",but not at 40"or higher. Eleven independent spontaneous mutants of T4 (go mutants) were isolated that overcame the growth restriction at high temperature. All of these mutants were located within three percent recombination of a gene 39 amber mutation in the clockwise direction on the standard map. In mixed infections, the representative go mutant chosen for further study seems to be recessive to its wild-type allele. Temperature-shift experiments suggested that the mutated host function involved in phage growth is a "late" function, beginning in mid-eclipse.—Electrophoresis of phage proteins labelled early and late in infection showed that under restrictive conditions early protein synthesis was normal, but that certain late proteins were absent. However, measurements ofDNA synthesis showed that under restrictive conditions the amount of phage DNA synthesized, and especially the amount of DNA sedimenting as high molecular weight replicative intermediate, was reduced. Pulse-chase experiments showed that the phage DNA made under restrictive conditions was not rapidly degraded.

scholarly journals BACTERIOPHAGE T4 ENDONUCLEASES II AND IV, OPPOSITELY AFFECTED BY dCMP HYDROXYMETHYLASE ACTIVITY, HAVE DIFFERENT ROLES IN THE DEGRADATION AND IN THE RNA POLYMERASE-DEPENDENT REPLICATION OF T4 CYTOSINE-CONTAINING DNA

Genetics ◽  
1986 ◽  
Vol 114 (3) ◽  
pp. 669-685
Author(s):  
Karin Carlson ◽  
Aud Ȗvervatin
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Bacteriophage T4 ◽  
The Other ◽  
Wild Type ◽  
E Coli ◽  
Phage Dna ◽  
Dna Template ◽  
Do So

ABSTRACT Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.—Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.—The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33  -  55  -) RNA polymerase was less sensitive to endonuclease IV action.

scholarly journals Model for Bacteriophage T4 Development inEscherichia coli

1999 ◽  
Vol 181 (5) ◽  
pp. 1677-1683 ◽  
Author(s):  
Avinoam Rabinovitch ◽  
Hilla Hadas ◽  
Monica Einav ◽  
Zeev Melamed ◽  
Arieh Zaritsky
Keyword(s):  
Standard Deviation ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
Growth Conditions ◽  
Wild Type ◽  
E Coli ◽  
Phage Development ◽  
Dependent Parameter ◽  
Mathematical Relations ◽  
Infected Bacterium

ABSTRACT Mathematical relations for the number of mature T4 bacteriophages, both inside and after lysis of an Escherichia coli cell, as a function of time after infection by a single phage were obtained, with the following five parameters: delay time until the first T4 is completed inside the bacterium (eclipse period, ν) and its standard deviation (ς), the rate at which the number of ripe T4 increases inside the bacterium during the rise period (α), and the time when the bacterium bursts (μ) and its standard deviation (β). Burst size [B = α(μ − ν)], the number of phages released from an infected bacterium, is thus a dependent parameter. A least-squares program was used to derive the values of the parameters for a variety of experimental results obtained with wild-type T4 inE. coli B/r under different growth conditions and manipulations (H. Hadas, M. Einav, I. Fishov, and A. Zaritsky, Microbiology 143:179–185, 1997). A “destruction parameter” (ζ) was added to take care of the adverse effect of chloroform on phage survival. The overall agreement between the model and the experiment is quite good. The dependence of the derived parameters on growth conditions can be used to predict phage development under other experimental manipulations.

scholarly journals A Mutation within the β Subunit of Escherichia coli RNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010 ◽  
Vol 192 (21) ◽  
pp. 5580-5587 ◽  
Author(s):  
Tamara D. James ◽  
Michael Cashel ◽  
Deborah M. Hinton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Β Subunit ◽  
Subunit Gene ◽  
Wild Type ◽  
Promoter Activation ◽  
E Coli ◽  
Growth Phenotype

ABSTRACT During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with σ70, the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its β-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the β subunit may influence interactions among MotA, AsiA, and RNA polymerase.

scholarly journals Borrelia burgdorferi bba74 Is Expressed Exclusively during Tick Feeding and Is Regulated by Both Arthropod- and Mammalian Host-Specific Signals

2009 ◽  
Vol 191 (8) ◽  
pp. 2783-2794 ◽  
Author(s):  
Vishwaroop B. Mulay ◽  
Melissa J. Caimano ◽  
Radha Iyer ◽  
Star Dunham-Ems ◽  
Dionysios Liveris ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Elevated Temperatures ◽  
Temperature Shift ◽  
Mammalian Host ◽  
Wild Type ◽  
Tick Feeding ◽  
Reciprocal Regulation ◽  
Heterogeneous Expression ◽  
Membrane Spanning

ABSTRACT Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in the Borrelia burgdorferi tick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA, ospC, and ospE) and their proteins. In vitro, transcripts for bba74 were expressed at 23°C and further enhanced by a temperature shift (37°C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-type B. burgdorferi grown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type and rpoS mutant spirochetes, revealed that, like ospA, bba74 is transcribed by σ70 and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type and rpoS mutant spirochetes was conducted to determine the transcriptional and translational profiles of bba74 during the tick-mouse cycle. Results from these studies revealed (i) that bba74 is transcribed by σ70 exclusively during the larval and nymphal blood meals and (ii) that transcription of bba74 is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of B. burgdorferi to complete its infectious life cycle, the temporal compartmentalization of this gene's transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation of ospA and ospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue as B. burgdorferi transitions between its arthropod vector and mammalian host.

scholarly journals THERMAL RESCUE OF UV-IRRADIATED BACTERIOPHAGE T4 AND BIPHASIC MODE OF ACTION OF THE WXY SYSTEM

Genetics ◽  
1984 ◽  
Vol 107 (4) ◽  
pp. 525-536 ◽  
Author(s):  
Mark A Conkling ◽  
John W Drake
Keyword(s):  
Temperature Sensitivity ◽  
Late Stage ◽  
Bacteriophage T4 ◽  
Temperature Shift ◽  
Pyrimidine Dimer ◽  
Mutation Rates ◽  
Cold Sensitivity ◽  
Wild Type ◽  
In The Wild ◽  
Increasing Temperature

ABSTRACT When ultraviolet-irradiated bacteriophage T4 is assayed at plating temperatures ranging from 20° to 40°, its survival increases at the higher temperatures. This "thermal rescue" requires an intact WXY system but not the denV pyrimidine dimer excision system. Mutation rates decrease with increasing temperature, indicating that some lesions processed in a mutagenic manner at lower temperatures are accurately repaired or circumvented at high temperatures. When both the cold sensitivity of UV survival in the wild type and the temperature sensitivity of newly isolated ts mutants of uvsX and uvsY were used, expression of the WXY system was monitored in temperature shift UV survival experiments and was found to be biphasic: the uvsX and uvsY functions increase UV survival in two increments, one at an early and another at a late stage of infection. The uvsW function, however, increases UV survival only early in infection.

scholarly journals Two Arginine Repressors Regulate Arginine Biosynthesis in Lactobacillus plantarum

2004 ◽  
Vol 186 (18) ◽  
pp. 6059-6069 ◽  
Author(s):  
Hervé Nicoloff ◽  
Florence Arsène-Ploetze ◽  
Cédric Malandain ◽  
Michiel Kleerebezem ◽  
Françoise Bringel
Keyword(s):  
Lactobacillus Plantarum ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
Carbamoyl Phosphate ◽  
Gram Positive ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Arginine Repressor ◽  
In The Wild ◽  
Spontaneous Mutants

ABSTRACT The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine. This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway. Fourteen mutants were genetically characterized for constitutive arginine production. Mutations were located either in one of the arginine repressor genes (argR1 or argR2) present in L. plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involved in arginine biosynthesis. Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis. In L. plantarum, arginine repression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1. A123, equivalent to the conserved residue A124 in E. coli ArgR involved in arginine binding, was different in the wild-type ArgR2. Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which have only 35% identical residues. Other mutants harbored wild-type argR genes, and 20 mutants have lost their ability to grow in normal air without carbon dioxide enrichment; this revealed a link between arginine biosynthesis and a still-unknown CO2-dependent metabolic pathway. In many gram-positive bacteria, the expression and interaction of different ArgR-like proteins may imply a complex regulatory network in response to environmental stimuli.

scholarly journals UvsW Protein Regulates Bacteriophage T4 Origin-Dependent Replication by Unwinding R-Loops

2001 ◽  
Vol 21 (8) ◽  
pp. 2706-2715 ◽  
Author(s):  
Kathleen C. Dudas ◽  
Kenneth N. Kreuzer
Keyword(s):  
Bacteriophage T4 ◽  
Dna Helicase ◽  
Wild Type ◽  
Recombination Proteins ◽  
Phage Dna ◽  
Early Expression ◽  
Replicative Intermediates ◽  
Type Infection

ABSTRACT The UvsW protein of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including repair, recombination, and recombination-dependent replication. UvsW has also been implicated in the repression of origin-dependent replication at late times of infection, when UvsW is normally synthesized. Two well-characterized T4 origins, ori(uvsY) andori(34), are believed to initiate replication through an R-loop mechanism. Here we provide both in vivo and in vitro evidence that UvsW is an RNA-DNA helicase that catalyzes the dissociation of RNA from origin R-loops. Two-dimensional gel analyses show that the replicative intermediates formed atori(uvsY) persist longer in a uvsWmutant infection than in a wild-type infection. In addition, the inappropriate early expression of UvsW protein results in the loss of these replicative intermediates. Using a synthetic origin R-loop, we also demonstrate that purified UvsW functions as a helicase that efficiently dissociates RNA from R-loops. These and previous results from a number of studies provide strong evidence that UvsW is a molecular switch that allows T4 replication to progress from a mode that initiates from R-loops at origins to a mode that initiates from D-loops formed by recombination proteins.

scholarly journals FUNCTIONAL INTERACTIONS BETWEEN THE DNA LIGASE OF ESCHERICHIA COLI AND COMPONENTS OF THE DNA METABOLIC APPARATUS OF T4 BACTERIOPHAGE

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 177-189
Author(s):  
J D Karam ◽  
M Leach ◽  
L J Heere
Keyword(s):  
Dna Ligase ◽  
Wild Type ◽  
Wild Type Level ◽  
E Coli ◽  
Functional Interactions ◽  
Mutant Strains ◽  
T4 Bacteriophage ◽  
T4 Phage ◽  
Gene 32 ◽  
Phage Growth

ABSTRACT T4 phage completely defective in both gene 30 (DNA ligase) and the rll gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rll (T4 30- rll- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rll- phage on ligase-defective hosts. One class mapped in T4 gene su30 (KRYLO1V9 72) and improved T4 30- rll- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (heliz-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the rde of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.

scholarly journals Shigella flexneri DegP Facilitates IcsA Surface Expression and Is Required for Efficient Intercellular Spread

2002 ◽  
Vol 70 (11) ◽  
pp. 6355-6364 ◽  
Author(s):  
Georgiana E. Purdy ◽  
Mei Hong ◽  
Shelley M. Payne
Keyword(s):  
Actin Polymerization ◽  
Elevated Temperatures ◽  
Cultured Cells ◽  
Shigella Flexneri ◽  
Surface Expression ◽  
Wild Type ◽  
Cell Monolayers ◽  
E Coli ◽  
Induced Apoptosis ◽  
Intercellular Spread

ABSTRACT A degP mutant of Shigella flexneri was identified in a screen for insertion mutants that invaded cultured cells but did not form wild-type plaques in monolayers. The degP mutant SM1100 invaded Henle cells at wild-type levels and induced apoptosis in macrophages but formed smaller plaques than those formed by wild-type S. flexneri in confluent monolayers of Henle and Caco-2 cells. The proportion of SM1100 bacteria with IcsA localized to the bacterial pole, a process required for actin polymerization into actin “tails,” was reduced compared to results with wild-type bacteria. The reduction in proper IcsA localization may account for the reduced plaque size of the degP mutant. Although DegP is a protease, the protease activity of S. flexneri DegP was not required for IcsA localization or the formation of plaques in Henle cell monolayers. DegP was also required for efficient polar IcsA localization in E. coli expressing icsA. In addition, the growth or survival of SM1100 was compromised compared to that of the wild type at elevated temperatures and in acidic conditions.

Replication of Bacteriophage 186 DNA

1972 ◽  
Vol 30 ◽  
pp. 194-195
Author(s):  
Dhruba K. Chattoraj ◽  
Ross B. Inman
Keyword(s):  
Electron Microscopy ◽  
Dna Replication ◽  
Frame Of Reference ◽  
Dna Molecules ◽  
E Coli ◽  
Phage Dna ◽  
Partial Denaturation

Electron microscopy of replicating intermediates has been quite useful in understanding the mechanism of DNA replication in DNA molecules of bacteriophage, mitochondria and plasmids. The use of partial denaturation mapping has made the tool more powerful by providing a frame of reference by which the position of the replicating forks in bacteriophage DNA can be determined on the circular replicating molecules. This provided an easy means to find the origin and direction of replication in λ and P2 phage DNA molecules. DNA of temperate E. coli phage 186 was found to have an unique denaturation map and encouraged us to look into its mode of replication.

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