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1

Systematic mutation of bacteriophage T4 lysozyme  

Microsoft Academic Search

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber

Dale Rennell; Suzanne E. Bouvier; Larry W. Hardy; Anthony R. Poteete

1991-01-01

2

Thermodynamic and Structural Compensation in “Size-switch” Core Repacking Variants of Bacteriophage T4 Lysozyme  

Microsoft Academic Search

Previous analysis of randomly generated multiple mutations within the core of bacteriophage T4 lysozyme suggested that the “large-to-small” substitution Leu121 to Ala (L121A) and the spatially adjacent “small-to-large” substitution Ala129 to Met (A129M) might be mutually compensating. To test this hypothesis, the individual variants L121A and A129M were generated, as well as the double “size-switch” mutant L121A\\/A129M. To make the

Enoch Baldwin; Jian Xu; Omid Hajiseyedjavadi; Walter A. Baase; Brian W. Matthews

1996-01-01

3

Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage.  

PubMed

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E. coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E. coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour. PMID:15464594

Tanji, Yasunori; Furukawa, Chiaki; Na, Suk-Hyun; Hijikata, Tomonori; Miyanaga, Kazuhiko; Unno, Hajime

2004-10-19

4

Control of bacteriophage T4 tail lysozyme activity during the infection process.  

PubMed

Bacteriophage T4 has an efficient mechanism for injecting the host Escherichiacoli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E.coli, two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 degrees C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E.coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane. PMID:15701513

Kanamaru, Shuji; Ishiwata, Yasutaka; Suzuki, Toshiharu; Rossmann, Michael G; Arisaka, Fumio

2005-01-25

5

Control of Bacteriophage T4 Tail Lysozyme Activity During the Infection Process  

SciTech Connect

Bacteriophage T4 has an efficient mechanism for injecting the host Escherichia coli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E. coli, two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E. coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane.

Kanamaru, Shuji; Ishiwata, Yasutaka; Suzuki, Toshiharu; Rossmann, Michael G.; Arisaka, Fumio (Tokyo I); (Purdue)

2010-07-19

6

Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage  

Microsoft Academic Search

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid.

Yasunori Tanji; Chiaki Furukawa; Suk-Hyun Na; Tomonori Hijikata; Kazuhiko Miyanaga; Hajime Unno

2004-01-01

7

Escherichia coli Mutants Permissive for T4 Bacteriophage with Deletion in e Gene (Phage Lysozyme)  

PubMed Central

Escherichia coli mutants have been isolated that are permissive for the infection by T4 phage with deletion in the cistron for the phage lysozyme, the e gene. Some, but not all, of these mutants are simultaneously permissive for the infection by T4 phage defective in the t gene, the product of which has also been implicated in the release of progeny phages. Most of these mutants shared the following properties: temperature sensitivity in growth and cell division, increased sensitivity towards a number of unrelated antibiotics and colicins, and increased sensitivity towards anionic detergents (sodium dodecyl sulfate and sodium deoxycholate). The possible biochemical basis for these phenotypes is discussed.

Raj, C. V. Sundar; Wu, H. C.

1973-01-01

8

SRLS analysis of 15N relaxation from bacteriophage T4 lysozyme: a tensorial perspective that features domain motion.  

PubMed

Bacteriophage T4L lysozyme (T4L) comprises two domains connected by a helical linker. Several methods detected ns domain motion associated with the binding of the peptidoglycan substrate. An ESR study of nitroxide-labeled T4L, based on the slowly relaxing local structure (SRLS) approach, detected ns local motion involving the nitroxide and the helix housing it. (15)N?H spin relaxation data from T4L acquired at magnetic fields of 11.7 and 18.8 T, and 298 K, were analyzed previously with the model-free (MF) method. The results did not detect domain motion. SRLS is the generalization of MF. Here, we apply it to the same data analyzed previously with MF. The restricted local N?H motion is described in terms of tilted axial local ordering (S) and local diffusion (D(2)) tensors; dynamical coupling to the global tumbling is accounted for. We find that D(2,?) is 1.62 × 10(7) (1.56 × 10(7)) s(?1) for the N-terminal (C-terminal) domain. This dynamic mode represents domain motion. For the linker D(2,?) is the same as the rate of global tumbling, given by (1.46 ± 0.04) × 10(7) s(?1). D(2,?) is 1.3 × 10(9), 1.8 × 10(9) and 5.3 × 10(9) s(?1) for the N-terminal domain, the C-terminal domain, and the linker, respectively. This dynamic mode represents N?H bond vector fluctuations. The principal axis of D(2) is virtually parallel to the N?H bond. The order parameter, S(0)(2), is 0.910 ± 0.046 for most N?H bonds. The principal axis of S is tilted from the C(i?1)(?) ?C(i)(?) axis by ?2° to 6° for the N-, and C-terminal domains, and by 2.5° for the linker. The tensorial-perspective-based and mode-coupling-based SRLS picture provides new insights into the structural dynamics of bacteriophage T4 lysozyme. PMID:22568692

Meirovitch, Eva

2012-05-21

9

In vitro Synthesis of Bacteriophage Lysozyme  

Microsoft Academic Search

Active lysozyme of bacteriophage T4 has been synthesized in a cell free system programmed by RNA from cells infected with the phage. The appearance of this lysozyme activity has the characteristics of de novo synthesis.

W. Salser; R. F. Gesteland; A. Bolle

1967-01-01

10

Structure of the bacteriophage T4 baseplate  

Microsoft Academic Search

My Ph.D. research involved studying the process of infection of Escherichia coli bacteria by bacteriophage T4. Bacteriophage T4 is a large, double-stranded DNA, tailed virus belonging to the Myoviridae subfamily. The bacteriophage T4 tail is composed of two concentric cylinders: the inner rigid tail tube and the outer contractile tail sheath. One end of the tail is associated with the

Petr Gennadievich Leiman

2003-01-01

11

T4 LYSOZYME AND ATTACIN GENES ENHANCE RESISTANCE OF TRANSGENIC 'GALAXY' APPLE AGAINST ERWINIA AMYLOVORA (BURR.)  

Technology Transfer Automated Retrieval System (TEKTRAN)

Genes encoding T4 lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of 'Galaxy' apple, enha...

12

Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins.  

PubMed Central

A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on 15N (or 13C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-15N (or 13C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from 15N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-15N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl[15N]alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

McIntosh, L P; Griffey, R H; Muchmore, D C; Nielson, C P; Redfield, A G; Dahlquist, F W

1987-01-01

13

Identification of T4 gene 25 product, a component of the tail baseplate, as a 15K lysozyme  

Microsoft Academic Search

The proteins synthesized in Escherichia coli B cells after infection with various T4 bacteriophage tail baseplate mutants were analysed by the immunoblotting method for the presence of the 15 Kilodalton lysozyme found in phage T4 particles. Using three different antisera: anti-phage, anti-baseplate and anti-15K lysozyme, it has been found that the 15K lysozyme is not present in lysates of bacteria

Boguslaw Szewczyk; Krystyna Bienkowska-Szewczyk; Lloyd M. Kozloff

1986-01-01

14

Recombination in Bacteriophage T4: A Mechanism  

Microsoft Academic Search

Genetic recombination between rII mutants of T4 bacteriophage grown in Escherichia coli can occur under conditions where DNA synthesis is strongly inhibited by 5-fluorodeoxyuridine. The small amount of DNA synthesized under these conditions cannot account for the observed frequency of recombinants. The major mechanism of recombination in this system is a process of breakage and rejoining.

E. Simon

1965-01-01

15

Influence of transgenic T4-lysozyme-producing potato plants on potentially beneficial plant-associated bacteria  

Microsoft Academic Search

A field release of genetically engineered potato plants that produce bacteriophage T4-lysozyme for enhanced bacterial resistance was monitored for changes in plant-associated bacterial populations, in the functions of potentially beneficial bacteria and in the diversity of antagonistic bacterial species. These parameters have been analyzed for two T4-lysozyme-expressing lines, a transgenic control and a non-transgenic line, over a period of 2

Jana Lottmann; Holger Heuer; Kornelia Smalla; Gabriele Berg

1999-01-01

16

Model for Bacteriophage T4 Development in Escherichia coli  

Microsoft Academic Search

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, n) and its standard deviation (s), the rate at which

AVINOAM RABINOVITCH; HILLA HADAS; MONICA EINAV; ZEEV MELAMED

1999-01-01

17

Multicomponent anthrax toxin display and delivery using bacteriophage T4  

Microsoft Academic Search

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to

Sathish B. Shivachandra; Qin Li; Kristina K. Peachman; Gary R. Matyas; Stephen H. Leppla; Carl R. Alving; Mangala Rao; Venigalla B. Rao

2007-01-01

18

Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme  

PubMed Central

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Yang, Guoliang; Cecconi, Ciro; Baase, Walter A.; Vetter, Ingrid R.; Breyer, Wendy A.; Haack, Julie A.; Matthews, Brian W.; Dahlquist, Frederick W.; Bustamante, Carlos

2000-01-01

19

High-frequency generalised transduction by bacteriophage T4  

Microsoft Academic Search

GENERALISED transduction-the transfer of bacterial genes from one cell to another via bacteriophages-is one of a small number of processes by which bacteria can acquire exogenous genetic information. Transduction was first demonstrated in Salmonella typhimurium with the phage P22 by Zinder and Lederberg1,2 and subsequently in Escherichia coli with the phages P13 and T14. Bacteriophage T4 is the largest, and

Geoffrey G. Wilson; Karen K. Y. Young; Gordon J. Edlin

1979-01-01

20

Defining a Bacteriophage T4 Late Promoter: Bacteriophage T4 Gene 55 Protein Suffices for Directing Late Promoter Recognition  

Microsoft Academic Search

The RNA polymerase from bacteriophage T4-infected Escherichia coli, which specifically initiates transcription at phage T4 late promoters, is extensively modified by ADP-ribosylation of core subunits and by binding several virus-encoded subunits. We show here that one of these subunits, the phage T4 gene 55 protein, designated gp55, alone endows unmodified RNA polymerase core enzyme from uninfected E. coli with the

George A. Kassavetis; E. Peter Geiduschek

1984-01-01

21

Structure of the bacteriophage T4 DNA adenine methyltransferase  

Microsoft Academic Search

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains

Zhe Yang; John R Horton; Lan Zhou; Xu Jia Zhang; Aiping Dong; Xing Zhang; Samuel L Schlagman; Valeri Kossykh; Xiaodong Cheng

2003-01-01

22

Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy  

PubMed Central

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ?15 ?s relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.

Yirdaw, Robel B.; Mchaourab, Hassane S.

2012-01-01

23

Direct observation of T4 lysozyme hinge-bending motion by fluorescence correlation spectroscopy.  

PubMed

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ? 15 ?s relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants. PMID:23062345

Yirdaw, Robel B; McHaourab, Hassane S

2012-10-02

24

A restriction map of the bacteriophage T4 genome  

Microsoft Academic Search

We report a detailed restriction map of the bacteriophage T4 genome and the alignment of this map with the genetic map. The sites cut by the enzymes BglII, XhoI, KpnI, SalI, PstI, EcoRI and HindIII have been localized. Several novel approaches including two-dimensional (double restriction) electrophoretic separations were used.

Patrick H. O'Farrell; Elizabeth Kutter; Mikiye Nakanishi

1980-01-01

25

A Restriction Map of the Bacteriophage T4 Genome  

PubMed Central

Summary We report a detailed restriction map of the bacteriophage T4 genome and the alignment of this map with the genetic map. The sites cut by the enzymes BglII, XhoI, KpnI, SalI, PstI, EcoRI and HindIII have been localized. Several novel approaches including two-dimensional (double restriction) electrophoretic separations were used.

O'Farrell, Patrick H.; Kutter, Elizabeth; Nakanishi, Mikiye

2010-01-01

26

DNA polymerase of the T4-related bacteriophages  

Microsoft Academic Search

The DNA polymerase of bacteriophage T4, product of phage gene 43 (gp43), has served as a model replicative DNA polymerase in nucleic acids research for nearly 40 years. The base-selection (polymerase, or Pol) and editing (3?-exonuclease, or Exo) functions of this multifunctional protein, which have counterparts in the replicatioe polymerases of other organisms, are primary determinants of the high fidelity

Jim D. Karam; William H. Konigsberg

2000-01-01

27

The molecular architecture of the bacteriophage T4 neck.  

PubMed

A hexamer of the bacteriophage T4 tail terminator protein, gp15, attaches to the top of the phage tail stabilizing the contractile sheath and forming the interface for binding of the independently assembled head. Here we report the crystal structure of the gp15 hexamer, describe its interactions in T4 virions that have either an extended tail or a contracted tail, and discuss its structural relationship to other phage proteins. The neck of T4 virions is decorated by the "collar" and "whiskers", made of fibritin molecules. Fibritin acts as a chaperone helping to attach the long tail fibers to the virus during the assembly process. The collar and whiskers are environment-sensing devices, regulating the retraction of the long tail fibers under unfavorable conditions, thus preventing infection. Cryo-electron microscopy analysis suggests that twelve fibritin molecules attach to the phage neck with six molecules forming the collar and six molecules forming the whiskers. PMID:23434847

Fokine, Andrei; Zhang, Zhihong; Kanamaru, Shuji; Bowman, Valorie D; Aksyuk, Anastasia A; Arisaka, Fumio; Rao, Venigalla B; Rossmann, Michael G

2013-02-19

28

Multicomponent anthrax toxin display and delivery using bacteriophage T4.  

PubMed

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to Hoc can be displayed on the same capsid and such particles can elicit broad immunological responses. Anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their functional domains, were fused to Hoc with an N-terminal hexa-histidine tag and the recombinant proteins were over-expressed in E. coli and purified. Using a defined in vitro assembly system, the anthrax-Hoc fusion proteins were efficiently displayed on T4 capsid, either individually or in combinations. All of the 155 Hoc binding sites can be occupied by one antigen, or they can be split among two or more antigens by varying their molar ratio in the binding reaction. Immunization of mice with T4 phage carrying PA, LF, and EF elicited strong antigen-specific antibodies against all antigens as well as lethal toxin neutralization titers. The triple antigen T4 phage elicited stronger PA-specific immune responses than the phage displaying PA alone. These features offer novel avenues to develop customized multicomponent vaccines against anthrax and other pathogenic diseases. PMID:17069938

Shivachandra, Sathish B; Li, Qin; Peachman, Kristina K; Matyas, Gary R; Leppla, Stephen H; Alving, Carl R; Rao, Mangala; Rao, Venigalla B

2006-10-17

29

Effect of alpha particles on bacteriophage T4Br(+)  

SciTech Connect

The effects of heavy particle radiation, which is believed to be responsible for the high relative biological effectiveness (RBE) of space hadrons, on bacteriophages are investigated. Dry film cultures of bacteriophage T4 were irradiated with 5.3 MeV Po-210 alpha particles to doses from 5 to 60 Gray, and compared with cultures irradiated by Co-60 gamma radiation. Examination of the exponential dose-response curves for bacteriophage survival indicates an RBE of 4.68 for the alpha particles. The r-mutation frequency per 10,000 surviving phages is found to peak at 7.1 at doses between 65 and 85 Gray for gamma radiation, however it declines steadily from a level of 10.2 per 10,000 survivors with increasing dose of alpha radiation. Comparison of the mutation frequencies at the same levels of lethality and the spectra of mutations produced by the two types of radiation indicates alpha and gamma radiation to differ as well in the mechanisms of mutation production. It is concluded that the observed high RBE of space hadrons cannot be explained by the presence of high-energy particles in the secondary radiation. 13 references.

Leonteva, G.A.; Akoev, I.G.; Grigorev, A.E.

1983-01-01

30

Structure and assembly of bacteriophage T4 head  

PubMed Central

The bacteriophage T4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. The past twenty years of research has greatly elevated the understanding of phage T4 head assembly and DNA packaging. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as that found in phage HK97 and several other icosahedral bacteriophages. Folding of gp23 requires the assistance of two chaperones, the E. coli chaperone GroEL and the phage coded gp23-specific chaperone, gp31. The capsid also contains two non-essential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. The structure of Soc shows two capsid binding sites which, through binding to adjacent gp23 subunits, reinforce the capsid structure. Hoc and Soc have been extensively used in bipartite peptide display libraries and to display pathogen antigens including those from HIV, Neisseria meningitides, Bacillus anthracis, and FMDV. The structure of Ip1*, one of the components of the core, has been determined, which provided insights on how IPs protect T4 genome against the E. coli nucleases that degrade hydroxymethylated and glycosylated T4 DNA. Extensive mutagenesis combined with the atomic structures of the DNA packaging/terminase proteins gp16 and gp17 elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. Cryo-EM structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at a rate of up to 2000 bp/sec, the fastest reported to date of any packaging motor. FRET-FCS studies indicate that the DNA gets compressed during the translocation process. The current evidence suggests a mechanism in which electrostatic forces generated by ATP hydrolysis drive the DNA translocation by alternating the motor between tensed and relaxed states.

2010-01-01

31

Halogenated Benzenes Bound within a Non-polar Cavity in T4 Lysozyme Provide Examples of I?S and I?Se Halogen-bonding  

Microsoft Academic Search

We showed earlier that the mutation of Leu99 to alanine in bacteriophage T4 lysozyme creates an internal cavity of volume ?150 Å3 that binds benzene and a variety of other ligands. As such, this cavity provides an excellent target to study protein-ligand interaction. Here, we use low-temperature crystallography and related techniques to analyze the binding of halogen-incorporated benzenes typified by C6F5X,

Lijun Liu; Walter A. Baase; Brian W. Matthews

2009-01-01

32

Latent injury in frozen-thawed bacteriophage T4Bo.  

PubMed

Two interesting new phenomena have been observed in suspensions of T4Bo bacteriophage which were frozen to temperatures below the eutectic temperature of the salt (sodium chloride) in the suspending medium. Approximately 10% of the phage appeared to survive such a phase change as determined by plaque titre. However, exposure of these survivors to ultrasonic vibration or repeated freezing showed them to be hypersensitive and thus latently injured. The hypersensitivity was lost on incubating the phage at 37 degrees C. for 3 hr. Furthermore, following a eutectic phase change, the surviving phage could be inactivated by rapid cooling to -90 degrees C. followed by slow rewarming. Such inactivation cannot be accounted for by accepted theories of freezing injury. PMID:1068188

Steele, P R

1976-08-01

33

T4 DNA topoisomerase: a new ATP-dependent enzyme essential for initiation of T4 bacteriophage DNA replication  

NASA Astrophysics Data System (ADS)

A novel ATP-dependent DNA topoisomerase which makes reversible double-strand breaks in the DNA double helix has been purified to near homogeneity from T4 bacteriophage-infected Escherichia coli cells. Genetic data suggest that this activity is essential for initiating T4 DNA replication forks in vivo.

Liu, Leroy F.; Liu, Chung-Cheng; Alberts, Bruce M.

1979-10-01

34

T4 DNA topoisomerase: a new ATP-dependent enzyme essential for initiation of T4 bacteriophage DNA replication  

Microsoft Academic Search

A novel ATP-dependent DNA topoisomerase which makes reversible double-strand breaks in the DNA double helix has been purified to near homogeneity from T4 bacteriophage-infected Escherichia coli cells. Genetic data suggest that this activity is essential for initiating T4 DNA replication forks in vivo.

Leroy F. Liu; Chung-Cheng Liu; Bruce M. Alberts

1979-01-01

35

Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs.  

PubMed

The detergents used to solubilize GPCRs can make crystal growth the rate-limiting step in determining their structure. The Kobilka laboratory showed that insertion of T4-lysozyme (T4L) in the 3rd intracellular loop is a promising strategy towards increasing the solvent-exposed receptor area, and hence the number of possible lattice-forming contacts. The potential to use T4L with the olfactory-related receptors hOR17-4 and hVN1R1 was thus tested. The structure and function of native and T4L-variants were compared. Both receptors localized to the cell membrane, and could initiate ligand-activated signaling. Purified receptors not only had the predicted alpha-helical structures, but also bound their ligands canthoxal (M(W) = 178.23) and myrtenal (M(W) = 150.22). Interestingly, the T4L variants had higher percentages of soluble monomers compared to protein aggregates, effectively increasing the protein yield that could be used for structural and function studies. They also bound their ligands for longer times, suggesting higher receptor stability. Our results indicate that a T4L insertion may be a general method for obtaining GPCRs suitable for structural studies. PMID:22491779

Corin, Karolina; Pick, Horst; Baaske, Philipp; Cook, Brian L; Duhr, Stefan; Wienken, Christoph J; Braun, Dieter; Vogel, Horst; Zhang, Shuguang

2012-04-11

36

Model for DNA packaging into bacteriophage T4 heads.  

PubMed Central

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule. Images

Black, L W; Silverman, D J

1978-01-01

37

Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes  

Microsoft Academic Search

Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56,denA anddenB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA withEcoRI have been cloned using the vector plasmid pCR1.

Tom Mattson; Griet Van Houwe; Antoinette Bolle; Gerald Selzer; Richard Epstein

1977-01-01

38

Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins  

Microsoft Academic Search

The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a

Andrei Fokine; Valorie D. Bowman; Anthony J. Battisti; Qin Li; Paul R. Chipman; Venigalla B. Rao; Michael G. Rossmann

2007-01-01

39

Two-component nature of bacteriophage T4 receptor activity in Escherichia coli K-12.  

PubMed Central

Escherichia coli bacteriophage T4 uses the lipopolysaccharide of the outer cell envelope membrane as a receptor. Lipopolysaccharide from E. coli K-12 required a major outer membrane protein, polypeptide Ib, for phage inactivation.

Henning, U; Jann, K

1979-01-01

40

Excision repair and patch size in UV-irradiated bacteriophage T4  

SciTech Connect

We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control, experiments with the denV/sub 1/ excision-deificient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

Yarosh, D.B. (National Cancer Institute, Bethesda, MD); Rosenstein, B.S.; Setlow, R.B.

1981-11-01

41

Excision repair and patch size in UV-irradiated bacteriophage T4  

SciTech Connect

We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.

1981-11-01

42

Protein Flexibility and Adaptability Seen in 25 Crystal Forms of T4 Lysozyme  

Microsoft Academic Search

The structures of various mutants of T4 lysozyme have been determined in 25 non-isomorphous crystal forms. This provides an unusually diverse data base to compare the structures and dynamics of a closely related set of proteins in different crystal packing environments.In general, the more tightly packed crystals diffract better than those that are highly hydrated although the wild-type crystal form

Xue-jun Zhang; Joan A. Wozniak; Brian W. Matthews

1995-01-01

43

A Multifrequency Electron Spin Resonance Study of T4 Lysozyme Dynamics  

Microsoft Academic Search

Electron spin resonance (ESR) spectroscopy at 250GHz and 9GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10°C to 35°C. Two separate derivatives, labeled at sites 44 and 69, were analyzed. The 250-GHz ESR spectra are well described by a microscopic ordering with macroscopic disordering (MOMD)

Jeff P. Barnes; Zhichun Liang; Hassane S. Mchaourab; Jack H. Freed; Wayne L. Hubbell

1999-01-01

44

The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration  

PubMed Central

Background The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes. Results In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed. Conclusion We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.

2009-01-01

45

Stabilization of Proteins by a Bacteriophage T4 Gene Cloned in Escherichia coli  

Microsoft Academic Search

The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the lambda phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4

Lee D. Simon; Barbara Randolph; Nina Irwin; Gloria Binkowski

1983-01-01

46

Genetic diversity among five T4-like bacteriophages  

Microsoft Academic Search

BACKGROUND: Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene

James M Nolan; Vasiliy Petrov; Claire Bertrand; Henry M Krisch; Jim D Karam

2006-01-01

47

Effects of T4 Lysozyme Release from Transgenic Potato Roots on Bacterial Rhizosphere Communities Are Negligible Relative to Natural Factors  

Microsoft Academic Search

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach.

Holger Heuer; Reiner M. Kroppenstedt; Jana Lottmann; Gabriele Berg; Kornelia Smalla

2002-01-01

48

Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: a generic method to study bacteriophage/host interactions  

PubMed Central

Bacteriophages are bacterial viruses that infect bacterial cells and they have developed ingenious mechanisms to modify the bacterial RNA polymerase. Using a rapid, specific, single-step immunoisolation procedure to purify Escherichia coli RNA polymerase from bacteriophage T4 infected cells; we have identified bacteriophage T4-dependent modifications of the host RNA polymerase. We suggest that this methodology is applicable for the identification of bacteriophage-dependent alterations of the host synthesis machinery.

Westblade, Lars F.; Minakhin, Leonid; Kuznedelov, Konstantin; Tackett, Alan J.; Chang, Emmanuel J.; Mooney, Rachel A.; Vvedenskaya, Irina; Wang, Qingjun; Fenyo, David; Rout, Michael P.; Landick, Robert; Chait, Brian T.; Severinov, Konstantin; Darst, Seth A.

2008-01-01

49

Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme.  

PubMed Central

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.

Lipscomb, L. A.; Gassner, N. C.; Snow, S. D.; Eldridge, A. M.; Baase, W. A.; Drew, D. L.; Matthews, B. W.

1998-01-01

50

The Folding Pathway of T4 Lysozyme: 1. An On-Pathway Hidden Folding Intermediate  

PubMed Central

T4 Lysozyme has two easily distinguishable but energetically coupled domains: the N- and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable sub-millisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N- and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we reexamined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate of the wild type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.

Kato, Hidenori; Vu, Ngoc_Diep; Feng, Hanqiao; Zhou, Zheng; Bai, Yawen

2008-01-01

51

2Aminopurine induced mutations in T4 bacteriophage  

Microsoft Academic Search

Treatment of E. coli B bacteria by the base analogue 2-aminopurine, before infection with T4 phages, induces mutations of the transition type into the virus. Treatment of the phage-bacterium complex, only during the first four minutes of the latent period i.e. at a moment where no phage DNA is synthetized, is also mutagenic. The kinetics of acquisition and loss by

P. R. R. Vigier; H. Marcovich

1973-01-01

52

Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase  

Microsoft Academic Search

T4 phage polynucleotide kinase (PNK) displays 5¢- hydroxyl kinase, 3¢-phosphatase and 2¢,3¢-cyclic phosphodiesterase activities. The enzyme phos- phorylates the 5¢ hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonu- cleotide substrates of various lengths and sequences. In these structures, the 5¢ ribose

Jennifer H. Eastberg; John Pelletier; Barry L. Stoddard

2004-01-01

53

Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives.  

PubMed

The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies. PMID:21129204

Mueser, Timothy C; Hinerman, Jennifer M; Devos, Juliette M; Boyer, Ryan A; Williams, Kandace J

2010-12-03

54

Bacteriophage T4 Capsid: A Unique Platform for Efficient Surface Assembly of Macromolecular Complexes  

Microsoft Academic Search

We report the first description of a macromolecular complex display system using bacteriophage T4. Decorated with two dispensable outer capsid proteins, Hoc (155 copies) and Soc (810 copies), the 120 nm×86 nm T4 capsid particle offers a unique binding site-rich platform for surface assembly of hetero-oligomeric complexes. To display the 710 kDa anthrax toxin complex, two bipartite functional fusion proteins, LF-Hoc and LFn-Soc,

Qin Li; Sathish B. Shivachandra; Stephen H. Leppla; Venigalla B. Rao

2006-01-01

55

Expression of the denV Gene of Bacteriophage T4 Cloned in Escherichia coli  

Microsoft Academic Search

The denV gene of bacteriophage T4 has been cloned into Escherichia coli K-12 by inserting appropriate fragments of cytosine-containing T4 DNA into the Sal I site of the plasmid pBR322. The denV gene codes for an enzyme that initiates the excision repair of pyrimidine dimers produced in DNA by UV. In uvrA recA mutants, deficient in an early step in

R. Stephen Lloyd; Philip C. Hanawalt

1981-01-01

56

RNA Ligase Reaction Products in Plasmolyzed Escherichia coli Cells Infected by T4 Bacteriophage  

Microsoft Academic Search

Searching for a physiological role of T4 RNA ligase [polyribonucleotide synthetase (ATP); poly(ribonucleotide):poly(ribonucleotide) ligase (AMP-forming), EC 6.5.1.3] activity, we developed an acellular system of plasmolyzed Escherichia coli cells infected by T4 bacteriophage. Upon incubation of this system with [gamma -32P]ATP, 32P was transferred into a large number of polyribonucleotides, mostly up to 300-400 residues long. The bulk of 32P in

Michel David; Raya Vekstein; Gabriel Kaufmann

1979-01-01

57

The sequences and activities of RegB endoribonucleases of T4-related bacteriophages  

Microsoft Academic Search

The RegB endoribonuclease encoded by bacterio- phage T4 is a unique sequence-specific nuclease that cleaves in the middle of GGAG or, in a few cases, GGAU tetranucleotides, preferentially those found in the Shine-Dalgarno regions of early phage mRNAs. In this study, we examined the primary structures and functional properties of RegB ribonucleases encoded by T4-related bacteriophages. We show that all

Lina Piesiniene; Lidija Truncaite; Aurelija Zajanckauskaite; Rimas Nivinskas

2004-01-01

58

A promiscuous antitoxin of bacteriophage T4 ensures successful viral replication.  

PubMed

Bacteria are constantly threatened by predation from bacteriophage parasites and, in response, have evolved an array of resistance mechanisms. These resistance mechanisms then place greater selection pressure on the infecting bacteriophages, which develop counter-strategies in a perpetual 'arms race' between virus and host. Toxin-antitoxin (TA) loci are widespread in bacteria and can confer multiple benefits, including resistance to bacteriophages. The study by Otsuka and Yonesaki, published in this issue of Molecular Microbiology, describes a new plasmid-encoded TA system, lsoAB, which confers resistance to a dmd(-) mutant of bacteriophage T4 through the activity of the LsoA toxin. Infections with wild-type T4, however, are unaffected as the Dmd protein acts as an alternative antitoxin to LsoA, thus preventing its anti-bacteriophage activity. Dmd has also been shown to negate the activity of a related toxin, RnlA. This is a striking result indicating that Dmd can act as a promiscuous antitoxin, binding and inhibiting multiple toxin partners, when antitoxin activity is generally considered to be limited to a single cognate toxin. This study is an exciting addition to both the bacteriophage resistance and TA fields, and suggests a greater role for TA system-based resistance and counter-resistance in the world's oldest predator-prey relationship. PMID:22283468

Short, Francesca L; Blower, Tim R; Salmond, George P C

2012-01-29

59

Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: Evidence for a kinetic partitioning mechanism  

PubMed Central

Kinetic partitioning is predicted to be a general mechanism for proteins to fold into their well defined native three-dimensional structure from unfolded states following multiple folding pathways. However, experimental evidence supporting this mechanism is still limited. By using single-molecule atomic force microscopy, here we report experimental evidence supporting the kinetic partitioning mechanism for mechanical unfolding of T4 lysozyme, a small protein composed of two subdomains. We observed that on stretching from its N and C termini, T4 lysozyme unfolds by multiple distinct unfolding pathways: the majority of T4 lysozymes unfold in an all-or-none fashion by overcoming a dominant unfolding kinetic barrier; and a small fraction of T4 lysozymes unfold in three-state fashion involving unfolding intermediate states. The three-state unfolding pathways do not follow well defined routes, instead they display variability and diversity in individual unfolding pathways. The unfolding intermediate states are local energy minima along the mechanical unfolding pathways and are likely to result from the residual structures present in the two subdomains after crossing the main unfolding barrier. These results provide direct evidence for the kinetic partitioning of the mechanical unfolding pathways of T4 lysozyme, and the complex unfolding behaviors reflect the stochastic nature of kinetic barrier rupture in mechanical unfolding processes. Our results demonstrate that single-molecule atomic force microscopy is an ideal tool to investigate the folding/unfolding dynamics of complex multimodule proteins that are otherwise difficult to study using traditional methods.

Peng, Qing; Li, Hongbin

2008-01-01

60

Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4  

Microsoft Academic Search

Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.

U. K. Laemmli

1970-01-01

61

Carbon loss during irradiation of T4 bacteriophages and E. coli bacteria in electron microscopes  

Microsoft Academic Search

The loss of ¹⁴C due to electron irradiation has been measured on labeled T4 bacteriophages and E. coli bacteria under conditions relevant for practical electron microscopy for fixed and scanning beam exposure. During irradiation, the remaining material became less and less sensitive to further carbon loss. Surface migration of molecular fragments and adsorbed molecules is involved in the process of

J DUBOCHET

1975-01-01

62

The structure of bacteriophage T4 gene product 9: the trigger for tail contraction  

Microsoft Academic Search

Background: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the Escherichia coli host. The tail has a baseplate to whïch are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment

Victor A Kostyuchenko; Grigorii A Navruzbekov; Lidia P Kurochkina; Sergei V Strelkov; Vadim V Mesyanzhinov; Michael G Rossmann

1999-01-01

63

Inactivation of Escherichia coli and bacteriophage T4 by high levels of dissolved CO2.  

PubMed

Little information is available regarding the effectiveness of water disinfection by CO(2) at low pressure. The aim of this study was to evaluate the use of high levels of dissolved CO(2) at 0.3-0.6 MPa for the inactivation of microorganisms. Bacteriophage T4 was chosen as the model virus and Escherichia coli was selected as the representative bacterium. The results of the study showed a highly effective log inactivation of E. coli and bacteriophage T4 at low and medium initial concentrations by high levels of dissolved CO(2) at 0.3 MPa with a treatment time of 20 min. When the pressure was increased to 0.6 MPa, inactivation of both microorganisms at high initial concentrations was improved to different extents. Neither pressurized air nor O(2) effectively inactivated both E. coli and bacteriophage T4. The pH was not a key factor affecting the inactivation process by this method. The results of scanning electron microscopy of E. coli and transmission electron microscopy of bacteriophage T4 suggested that "CO(2) uptake at high pressure and bursting of cells by depressurization" were the main reasons for lethal effect on microorganisms. This technology has potential for application in the disinfection of water, wastewater, and liquid food in the future. PMID:21336924

Cheng, Xuehang; Imai, Tsuyoshi; Teeka, Jantima; Yamaguchi, Junki; Hirose, Mami; Higuchi, Takaya; Sekine, Masahiko

2011-02-20

64

Transient Activation of RNA Polymerase in Escherichia coli B after Infection with Bacteriophage T4  

Microsoft Academic Search

Skold and Buchanan1 have reported that there is a rapid loss of RNA polymerase activity in Escherichia coli B after infection with T4 bacteriophage. More recent studies on the mechanism of this inactivation have been made in this2 and other laboratories.3,\\\\ 4 In this communication, we report the observation of a transient stimulation of RNA polymerase activity when measurement is

Arland E. Oleson; Jaakko P. Pispa; John M. Buchanan

1969-01-01

65

Rapid Isolation and Identification of Bacteriophage T4Encoded Modifications of Escherichia coli RNA Polymerase: A Generic Method to Study Bacteriophage\\/Host Interactions  

Microsoft Academic Search

Bacteriophages are bacterial viruses that infect bacterial cells, and they have developed ingenious mechanisms to modify the bacterial RNA polymerase. Using a rapid, specific, single-step affinity isolation procedure to purify Escherichia coli RNA polymerase from bacteriophage T4-infected cells, we have identified bacteriophage T4-dependent modifications of the host RNA polymerase. We suggest that this methodology is broadly applicable for the identification

Lars F. Westblade; Leonid Minakhin; Konstantin Kuznedelov; Alan J. Tackett; Emmanuel J. Chang; Rachel A. Mooney; Irina Vvedenskaya; Qing Jun Wang; David Fenyö; Michael P. Rout; Robert Landick; Brian T. Chait; Konstantin Severinov; Seth A. Darst

2008-01-01

66

EPR Relaxation-Enhancement-Based Distance Measurements on Orthogonally Spin-Labeled T4-Lysozyme.  

PubMed

Lanthanide-induced enhancement of the longitudinal relaxation of nitroxide radicals in combination with orthogonal site-directed spin labeling is presented as a systematic distance measurement method intended for studies of bio-macromolecules and bio-macromolecular complexes. The approach is tested on a water-soluble protein (T4-lysozyme) for two different commercially available lanthanide labels, and complemented by previously reported data on a membrane-inserted polypeptide. Single temperature measurements are shown to be sufficient for reliable distance determination, with an upper measurable distance limit of about 5-6 nm. The extracted averaged distances represent the closest approach in Ln(III) -nitroxide distance distributions. Studies of conformational changes and of bio-macromolecule association-dissociation are proposed as possible application area of the relaxation-enhancement-based distance measurements. PMID:23775845

Razzaghi, Sahand; Brooks, Evan K; Bordignon, Enrica; Hubbell, Wayne L; Yulikov, Maxim; Jeschke, Gunnar

2013-06-14

67

Infection of Escherichia coli by T2 and T4 Bacteriophages as Seen in the Electron Microscope: T4 Head Morphogenesis  

Microsoft Academic Search

Bacteriophage T4 capsids seem to be assembled on E. coli protoplasmic membranes. This process seems to involve ``lumps'' of head protein, which convert to tau particles, which in turn give rise to empty heads. The empty heads leave the bacterial membrane and are then filled with DNA in the central region of the cell. T4 gene 16 and 17 products

Lee D. Simon

1972-01-01

68

Complete genome sequence of T4-Like Escherichia coli bacteriophage HX01.  

PubMed

Phage T4 is among the best-characterized biological systems (S. Kanamaru and F. Arisaka, Seikagaku 74:131-135, 2002; E. S. Miller et al., Microbiol. Mol. Biol. Rev. 67:86-156, 2003; W. B. Wood and H. R. Revel, Bacteriol. Rev. 40:847-868, 1976). To date, several genomes of T4-like bacteriophages are available in public databases but without any APEC bacteriophages (H. Jiang et al., Arch. Virol. 156:1489-1492, 2011; L. Kaliniene, V. Klausa, A. Zajanckauskaite, R. Nivinskas, and L. Truncaite, Arch. Virol. 156:1913-1916, 2011; J. H. Kim et al., Vet. Microbiol. 157:164-171, 2012; W. C. Liao et al., J. Virol. 85:6567-6578, 2011). We isolated a bacteriophage from a duck factory, named HX01, that infects avian pathogenic Escherichia coli (APEC). Sequence and morphological analyses revealed that phage HX01 is a T4-like bacteriophage and belongs to the family Myoviridae. Here, we announce the complete genome sequence of phage HX01 and report the results of our analysis. PMID:23166268

Tang, Fang; Li, Yanzhe; Zhang, Wei; Lu, Chengping

2012-12-01

69

Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4  

PubMed Central

The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single-strand break was determined. Between 4 and 37 °C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50–1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3?-hydroxy group and DNA at the 5?-phosphate. Since this RNA–DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 °C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of ‘DNA repair’.

Bullard, Desmond R.; Bowater, Richard P.

2006-01-01

70

Amber mutants of bacteriophage T4D: their isolation and genetic characterization.  

PubMed

We have isolated a large number of mutants of bacteriophage T4D that are unable to form plaques on strain B of Escherichia coli, but are able to grow (nearly) normally on some other strains of E. coli, in particular strain CR63. These mutants, designated amber (am), have been characterized by complementation tests, by genetic crosses, and by their response to chemical mutagens. It is concluded that a particular subclass of base substitution mutations may give rise to amber mutants and that such mutants occur in many genes, which are widely distributed over the T4 genome. PMID:22419076

Epstein, Richard H; Bolle, Antoinette; Steinberg, Charles M

2012-03-01

71

Expression of the bacteriophage T4 denV structural gene in Escherichia coli  

SciTech Connect

The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream of the bacteriophage lambda hybrid promoter-operator oLpR, was analyzed under a variety of growth parameters. Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli (uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation.

Recinos, A. III; Augustine, M.L.; Higgins, K.M.; Lloyd, R.S.

1986-11-01

72

Inactivation of bacteriophage T4 by organic and inorganic tin compounds  

Microsoft Academic Search

Summary Butyltins and inorganic tins inactivated bacteriophage T4. The effect was on the phage and not on its host,Escherichia coli. The order of effectiveness was SnCl4=monobutyltin>dibutyltin>tributyltin=SnCl2. For the butyltins and SnCl4 this was the reverse of the order of effectiveness usually observed for plants, animals, and microorganisms. This pattern suggests that degradation of tributyltin does not always detoxify it. Monobutyltin

Mark M. Doolittle; Joseph J. Cooney

1992-01-01

73

Inactivation of Escherichia coli and bacteriophage T4 by high levels of dissolved CO 2  

Microsoft Academic Search

Little information is available regarding the effectiveness of water disinfection by CO2 at low pressure. The aim of this study was to evaluate the use of high levels of dissolved CO2 at 0.3–0.6 MPa for the inactivation of microorganisms. Bacteriophage T4 was chosen as the model virus and Escherichia coli was selected as the representative bacterium. The results of the study

Xuehang Cheng; Tsuyoshi Imai; Jantima Teeka; Junki Yamaguchi; Mami Hirose; Takaya Higuchi; Masahiko Sekine

2011-01-01

74

On the receptor for bacteriophage T4 in Escherichia coli K12  

Microsoft Academic Search

Purified lipopolysaccharide (LPS) from a mutant strain ofEscherichia coli K12 altered in its LPS has been shown to serve as a receptor for bacteriophage T4, which contrasts with LPS from a wild-type strain. Studies of extragenic suppression of a mutation in the gene specifying protein 1b revealed that the galactose residue in the LPS normally masks the LPS receptor and

Ifor R. Beachamt; Roger N. Picken

1981-01-01

75

Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA  

Microsoft Academic Search

A series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli has been carried out. The results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication,

L. L. Restifo; H. H. Vogelbacker; T. Madara; S. K. Ling; A. W. Kozinski

1983-01-01

76

Interaction of bacteriophage T4 AsiA protein with Escherichia coli ? 70 and its variant  

Microsoft Academic Search

Bacteriophage T4 produces a small protein AsiA, which inhibits transcription from ?70-dependent promoters in E. coli by tightly binding to ?70 and is therefore termed as anti-sigma factor. We observed that there was no inhibition of single round transcription at lac UV5 promoter when AsiA was added to preformed open complex between RNA polymerase and template DNA. However, transcription was

Susanta Pahari; Dipankar Chatterji

1997-01-01

77

Effects of virus and host genes on recombination among ultraviolet-irradiated bacteriophage T4  

Microsoft Academic Search

The influence of the polA, uvrA, and recA genes of Escherichia coli on recombination among ultraviolet-irradiated T4 bacteriophages was determined with respect to recombination between rII markers and phage yield. The polA and uvrA gene products have no effect on these two aspects of phage DNA metabolism. A recA mutation does not significantly alter rII recombination frequencies in irradiated phage

M. M. Priemer; V. L. Chan

1978-01-01

78

Bacteriophage T4 Development in Escherichia coli is Growth Rate Dependent  

Microsoft Academic Search

Three independent parameters (eclipse and latent periods, and rate of ripening during the rise period) are essential and sufficient to describe bacteriophage development in its bacterial host. A general model to describe the classical “one-step growth” experiment [Rabinovitch et al. (1999a) J. Bacteriol.181, 1687–1683] allowed their calculations from experimental results obtained with T4 in Escherichia coli B\\/r under different growth

AVINOAM RABINOVITCH; Itzhak Fishovw; HILLA HADAS; MONICA EINAV; ARIEH ZARITSKY

2002-01-01

79

Study of the transfer RNAs coded by T2, T4, and T6 bacteriophages  

Microsoft Academic Search

T2,T4, and T6 bacteriophage tRNAs coding for arginine, leucine, proline, isoleucine, and glycine were isolated under conditions of short term and long term infection of Escherchia coli B cells. The corresponding phage tRNA species were examined for sequence homology by RNA.DNA hybridization analysis and by their relative behavior on reversed phase chromatography. The results indicate that all three T-even phages

S. M. Desai; S. B. Weiss

1977-01-01

80

ESCHERICHIA COLI RHO FACTOR IS INVOLVED IN LYSIS OF BACTERIOPHAGE T4INFECTED CELLS  

Microsoft Academic Search

A Rid (_Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho+ hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-sup- pressing rho or a multicopy plasmid expressing the rho+ allele. This suggests that the restriction may be due

CLAES H. LINDER

81

The sequences and activities of RegB endoribonucleases of T4-related bacteriophages.  

PubMed

The RegB endoribonuclease encoded by bacteriophage T4 is a unique sequence-specific nuclease that cleaves in the middle of GGAG or, in a few cases, GGAU tetranucleotides, preferentially those found in the Shine-Dalgarno regions of early phage mRNAs. In this study, we examined the primary structures and functional properties of RegB ribonucleases encoded by T4-related bacteriophages. We show that all but one of 36 phages tested harbor the regB gene homologues and the similar signals for transcriptional and post-transcriptional autogenous regulation of regB expression. Phage RB49 in addition to gpRegB utilizes Escherichia coli endoribonuclease E for the degradation of its transcripts for gene regB. The deduced primary structure of RegB proteins of 32 phages studied is almost identical to that of T4, while the sequences of RegB encoded by phages RB69, TuIa and RB49 show substantial divergence from their T4 counterpart. Functional studies using plasmid-phage systems indicate that RegB nucleases of phages T4, RB69, TuIa and RB49 exhibit different activity towards GGAG and GGAU motifs in the specific locations. We expect that the availability of the different phylogenetic variants of RegB may help to localize the amino acid determinants that contribute to the specificity and cleavage efficiency of this processing enzyme. PMID:15486207

Piesiniene, Lina; Truncaite, Lidija; Zajanckauskaite, Aurelija; Nivinskas, Rimas

2004-10-14

82

The sequences and activities of RegB endoribonucleases of T4-related bacteriophages  

PubMed Central

The RegB endoribonuclease encoded by bacteriophage T4 is a unique sequence-specific nuclease that cleaves in the middle of GGAG or, in a few cases, GGAU tetranucleotides, preferentially those found in the Shine–Dalgarno regions of early phage mRNAs. In this study, we examined the primary structures and functional properties of RegB ribonucleases encoded by T4-related bacteriophages. We show that all but one of 36 phages tested harbor the regB gene homologues and the similar signals for transcriptional and post-transcriptional autogenous regulation of regB expression. Phage RB49 in addition to gpRegB utilizes Escherichia coli endoribonuclease E for the degradation of its transcripts for gene regB. The deduced primary structure of RegB proteins of 32 phages studied is almost identical to that of T4, while the sequences of RegB encoded by phages RB69, TuIa and RB49 show substantial divergence from their T4 counterpart. Functional studies using plasmid–phage systems indicate that RegB nucleases of phages T4, RB69, TuIa and RB49 exhibit different activity towards GGAG and GGAU motifs in the specific locations. We expect that the availability of the different phylogenetic variants of RegB may help to localize the amino acid determinants that contribute to the specificity and cleavage efficiency of this processing enzyme.

Piesiniene, Lina; Truncaite, Lidija; Zajanckauskaite, Aurelija; Nivinskas, Rimas

2004-01-01

83

Effect of Myxin on Deoxyribonucleic Acid Synthesis in Escherichia coli Infected with T4 Bacteriophage 1  

PubMed Central

Exposure of Escherichia coli cells to myxin results in the almost complete inhibition of new deoxyribonucleic acid (DNA) synthesis, extensive degradation of pre-existing intracellular DNA, and a rapid loss of viability in these cells (9). After exposure to myxin for 30 min (<1% survivors and >25% degradation of DNA), infection of these cells by T4 bacteriophage results in the renewal of DNA synthesis at a rate essentially equal to that found in T4-infected cells in the absence of myxin. This DNA was characterized as T4 DNA by hybridization and by hydroxyapatite chromatography. These results suggest that the primary site of action of myxin does not involve the biochemical pathways involved in either the energy metabolism or the biosynthesis of DNA precursors in the uninfected host cell. The yield of infectious T4 particles was reduced when myxin was present during multiplication. This effect may be partly accounted for by the finding that a significant fraction of the T4 DNA synthesized in the presence of myxin is apparently not properly enclosed by the bacteriophage protein coat since it is shown to be degraded by exogenous nuclease.

Behki, R. M.; Lesley, S. M.

1971-01-01

84

Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins.  

PubMed Central

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein. Images

Benson, K H; Kreuzer, K N

1992-01-01

85

Accessibility and Dynamics of Nitroxide Side Chains in T4 Lysozyme Measured by Saturation Recovery EPR  

PubMed Central

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O2 or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128–135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.

Pyka, Janusz; Ilnicki, Jan; Altenbach, Christian; Hubbell, Wayne L.; Froncisz, Wojciech

2005-01-01

86

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant  

PubMed Central

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers)1–3. However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules4 and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta7 model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

Bouvignies, Guillaume; Vallurupalli, Pramodh; Hansen, D. Flemming; Correia, Bruno E.; Lange, Oliver; Bah, Alaji; Vernon, Robert M.; Dahlquist, Frederick W.; Baker, David; Kay, Lewis E.

2013-01-01

87

Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4  

SciTech Connect

The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single zinc-finger sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. The authors have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. The results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression.

Gauss, P.; Krassa, K.B.; McPheeters, D.S.; Nelson, M.A.; Gold, L.

1987-12-01

88

Mass distribution of a probable tail-length-determining protein in bacteriophage T4  

SciTech Connect

Analysis of dark-field scanning transmission electron micrographs of unstained freeze-dried specimens established that the interior of the intact bacteriophage T4 tail tube contains extra density that is missing in tubes artificially emptied by treatment with 3 M guanidine hydrochloride. The mass of the tail tube is 3.1 x 10W daltons, and the central channel is 3.2 nm in diameter. Quantitative analysis of the density data is consistent with the presence of up to six strands of a protein molecule in the central channel that could serve as the template or ruler structure that determines the length of the bacteriophage tail and that could be injected into the cell with the phage DNA.

Duda, R.L.; Wall, J.S.; Hainfeld, J.F.; Sweet, R.M.; Eiserling, F.A.

1985-08-01

89

Mass distribution of a probable tail-length-determining protein in bacteriophage T4.  

PubMed Central

Analysis of dark-field scanning transmission electron micrographs of unstained freeze-dried specimens established that the interior of the intact bacteriophage T4 tail tube contains extra density that is missing in tubes artificially emptied by treatment with 3 M guanidine hydrochloride. The mass of the tail tube is 3.1 X 10(6) daltons, and the central channel is 3.2 nm in diameter. Quantitative analysis of the density data is consistent with the presence of up to six strands of a protein molecule in the central channel that could serve as the template or ruler structure that determines the length of the bacteriophage tail and that could be injected into the cell with the phage DNA. Images

Duda, R L; Wall, J S; Hainfeld, J F; Sweet, R M; Eiserling, F A

1985-01-01

90

Effect of Osmotic Shock and Low Salt Concentration on Survival and Density of Bacteriophages T4B and T4Bo1  

PubMed Central

Measurements of survival and buoyant densities of bacteriophages T4B, T4Bo1, and T4D have demonstrated the following: (a) After suspension in a concentrated salt solution, T4B and T4D are sensitive both to osmotic shock and to subsequent exposure to low monovalent salt concentrations. (b) Sensitivity of T4B to dilution from a concentrated salt solution is dependent on dilution rate, that of T4D is less dependent, and that of T4Bo1 is independent. (c) Sensitivity of all three phages to low salt concentrations depends on initial salt concentrations to a variable extent. (d) Density gradient profiles indicate that nearly half of osmotically shocked T4B retain their DNA. Similar analysis demonstrates that few, if any, T4Bo1 lose DNA when subjected to a treatment causing 90% loss of infectivity. (e) The effective buoyant densities of T4B and T4Bo1 depend significantly on the dilution treatments to which the phages are subjected prior to centrifugation in CsCl gradients. These data are explicable in terms of the different relative permeabilities of the phages to water and solutes, and of alterations in the counterion distribution surrounding the DNA within the phage heads.

Leibo, Stanley P.; Mazur, Peter

1966-01-01

91

Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions.  

PubMed

Several variants of T4 lysozyme have been identified that sequester small organic ligands in cavities or clefts. To evaluate potential binding sites for non-polar molecules, we screened a number of hydrophobic large-to-small mutants for stabilization in the presence of benzene. In addition to Leu99-->Ala, binding was indicated for at least five other mutants. Variants Met102-->Ala and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further characterized using X-ray crystallography and thermal denaturation. As predicted from the shape of the cavity in the benzene complex, mutant Leu133-->Gly also bound p-xylene. We attempted to enlarge the cavity of the Met102-->Ala mutant into a deep crevice through an additional substitution, but the double mutant failed to bind ligands because an adjacent helix rearranged into a non-helical structure, apparently due to the loss of packing interactions. In general, the protein structure contracted slightly to reduce the volume of the void created by truncating substitutions and expanded upon binding the non-polar ligand, with shifts similar to those resulting from the mutations.A polar molecule binding site was also created by truncating Arg95 to alanine. This creates a highly complementary buried polar environment that can be utilized as a specific "receptor" for a guanidinium ion. Our results suggest that creating a deficiency through truncating mutations of buried residues generates "binding potential" for ligands with characteristics similar to the deleted side-chain. Analysis of complex and apo crystal structures of binding and non-binding mutants suggests that ligand size and shape as well as protein flexibility and complementarity are all determinants of binding. Binding at non-polar sites is governed by hydrophobicity and steric interactions and is relatively permissive. Binding at a polar site is more restrictive and requires extensive complementarity between the ligand and the site. PMID:9514755

Baldwin, E; Baase, W A; Zhang, X j; Feher, V; Matthews, B W

1998-03-27

92

Purification of phage display-modified bacteriophage T4 by affinity chromatography  

PubMed Central

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.

2011-01-01

93

Electrostatic Contributions to T4 Lysozyme Stability: Solvent-Exposed Charges versus Semi-Buried Salt Bridges  

Microsoft Academic Search

We carried our Poisson-Boltzmann (PB) calculations for the effects of charge reversal at five exposed sites (K16E, R119E, K135E, K147E, and R154E) and charge neutralization and proton titration of the H31-D70 semi-buried salt bridge on the stability of T4 lysozyme. Instead of the widely used solvent-exclusion (SE) surface, we used the van der Waals (vdW) surface as the boundary between

Feng Dong; Huan-Xiang Zhou

2002-01-01

94

Structural and Thermodynamic Characterization of T4 Lysozyme Mutants and the Contribution of Internal Cavities to Pressure Denaturation  

PubMed Central

Using small angle X-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured states of a series of T4 lysozyme mutants that are stabilized by high pressures. Recent studies imply that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer of hydrophobic residues into water. To investigate water penetration and the volume change associated with pressure denaturation, we studied the solution behavior of four T4 lysozyme mutants having different cavity volumes at low and neutral pH up to a pressure of 400 MPa (0.1 MPa = 0.9869 atm). At low pH, L99A T4 lysozyme expanded from a compact folded state to a partially unfolded state with a corresponding change in radius of gyration from 17 to 32 Å. The volume change upon denaturation correlated well with the total cavity volume, indicating that all of the molecule's major cavities are hydrated with pressure. As a direct comparison to high-pressure crystal structures of L99A T4 lysozyme solved at neutral pH [Collins, M. D., Hummer, G., Quillin, M. L., Matthews, B. W., and Gruner, S. M. (2005), PNAS 102, 16668-16671], pressure denaturation of L99A and the structurally similar L99G/E108V mutant was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities is responsible for the compactness of the pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation.

Ando, Nozomi; Barstow, Buz; Baase, Walter A.; Fields, Andrew; Matthews, Brian W.

2009-01-01

95

Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins.  

PubMed

The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a fusion protein of the N-terminal domain of the anthrax lethal factor (LFn) with Soc. The LFn-Soc fusion protein was complexed in vitro with Hoc(-)Soc(-)T4 phage. Subsequently, cleaved anthrax protective antigen heptamers (PA63)(7) were attached to the exposed LFn domains. A cryo-electron microscopy study of the decorated T4 particles shows the complex of PA63 heptamers with LFn-Soc on the phage surface. Although the cryo-electron microscopy reconstruction is unable to differentiate on its own between different proposed models of the anthrax toxin, the density is consistent with a model that had predicted the orientation and position of three LFn molecules bound to one PA63 heptamer. PMID:17624389

Fokine, Andrei; Bowman, Valorie D; Battisti, Anthony J; Li, Qin; Chipman, Paul R; Rao, Venigalla B; Rossmann, Michael G

2007-07-10

96

Bacteriophage Tail Components IV. Pteroyl Polyglutamate Synthesis in T4D-Infected Escherichia coli B  

PubMed Central

The nature of pteroyl polyglutamates in uninfected and T4D bacteriophage-infected Escherichia coli B has been examined. 3H-p-aminobenzoic acid has been used to label the folate compounds and gel permeation chromatography on glass beads to separate the folate compound by molecular size. It has been found that, although the major folate compound in uninfected bacteria is pteroyl triglutamate, E. coli B cells also contain folate compounds having as many as six glutamate residues. Infection with T4D stimulated the addition of glutamate residues to the lower-molecular-weight host pteroyl compounds, resulting in the conversion of the host compounds into the hexaglutamate form. This viral-induced conversion is chloramphenicol sensitive and appears to be due to a late phage gene product. The phage gene responsible for this conversion has not been identified. In cells infected with a T4D mutant defective in gene 28, there was an apparent production of the large pteroyl polyglutamates equivalent in size to pte(glu)9-12. These high-molecular-weight forms were converted into pte(glu)6 by incubation with bacterial extracts made after infection with T4D 28+. Apparently, the product of T4D gene 28+ is capable of specifically cleaving the high-molecular-weight polyglutamates to the form necessary for phage tail assembly.

Kozloff, Lloyd M.; Lute, Murl

1973-01-01

97

New epistasis group for the repair of DNA damage in bacteriophage T4: replication repair  

SciTech Connect

The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encode the major T4 ssDNA-binding protein (the scaffolding of the DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. The authors conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before of after DNA replication.

Wachsman, J.T.; Drake, J.W.

1987-03-01

98

Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins  

SciTech Connect

The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a fusion protein of the N-terminal domain of the anthrax lethal factor (LFn) with Soc. The LFn-Soc fusion protein was complexed in vitro with Hoc{sup -}Soc{sup -}T4 phage. Subsequently, cleaved anthrax protective antigen heptamers (PA63){sub 7} were attached to the exposed LFn domains. A cryo-electron microscopy study of the decorated T4 particles shows the complex of PA63 heptamers with LFn-Soc on the phage surface. Although the cryo-electron microscopy reconstruction is unable to differentiate on its own between different proposed models of the anthrax toxin, the density is consistent with a model that had predicted the orientation and position of three LFn molecules bound to one PA63 heptamer.

Fokine, Andrei; Bowman, Valorie D.; Battisti, Anthony J. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States); Li Qin [Department of Biology, Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064 (United States); Chipman, Paul R. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States); Rao, Venigalla B. [Department of Biology, Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064 (United States); Rossmann, Michael G. [Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 (United States)], E-mail: mr@purdue.edu

2007-10-25

99

Expression of a Cloned denV Gene of Bacteriophage T4 in Escherichia coli  

Microsoft Academic Search

A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC

Kristoffer Valerie; Earl E. Henderson; Jon K. de Riel

1985-01-01

100

Genome of low-temperature T4-related bacteriophage vB_EcoM-VR7  

Microsoft Academic Search

The complete genome sequence of the T4-related low-temperature Escherichia coli bacteriophage vB_EcoM-VR7 was determined. The genome sequence is 169,285 bp long, with a G+C content of 40.3%. Overall, 95%\\u000a of the phage genome is coding. It encodes 293 putative protein-encoding open reading frames (ORFs) and tRNAMet. More than half (59%) of the genomic DNA lacks significant homology with the DNA of

Laura KalinieneVytautas Klausa; Vytautas Klausa; Aurelija Zajan?kauskaite; Rimas Nivinskas; Lidija Truncaite

101

Phylogeny of the Major Head and Tail Genes of the Wide-Ranging T4Type Bacteriophages  

Microsoft Academic Search

We examined a number of bacteriophages with T4-type morphology that propagate in different genera of enterobacteria, Aeromonas, Burkholderia, and Vibrio. Most of these phages had a prolate icosahedral head, a contractile tail, and a genome size that was similar to that of T4. A few of them had more elongated heads and larger genomes. All these phages are phylogenetically related,

FRANCOISE TETART; CARINE DESPLATS; MZIA KUTATELADZE; CAROLINE MONOD; HANS-WOLFGANG ACKERMANN; H. M. Krisch

2001-01-01

102

EXPRESSION OF A DNA REPLICATION GENE CLUSTER IN BACTERIOPHAGE T4: GENETIC LINKAGE AND THE CONTROL OF GENE PRODUCT INTERACTIONS  

Microsoft Academic Search

The results of this study bear on the relationship between genetic linkage and control of interactions between the protein products of different cistrons. In T4 bacteriophage, genes 45 and 44 encode essential components of the phage DNA replication multiprotein complex. T4 gene 45 maps directly up stream of gene 44 relative to the overall direction of reading of this region

W. L. GERALD; J. D. KARAM

103

Capsid Size and Deoxyribonucleic Acid Length: the Petite Variant of Bacteriophage T4  

PubMed Central

A mutant which produces a small-headed (“petite”) variant of bacteriophage T4 is described. The mutation (E920g) maps in a new gene (66) between genes 23 and 24. Petite phage particles composed up to 70% of the phage yield. The petite phage was nonviable upon single infection but produced progeny when two or more infected a cell. Its genome was shortened by a random deletion of about 30%, and deoxyribonucleic acid (DNA) extracted from the particles was 0.68 the length of normal T4 DNA. The reduction in DNA length was accompanied by a proportional reduction in head volume. Double mutants between E920g and head-defective mutants in gene 21 produced unusually high frequencies of spherical capsidlike structures (?-particles). Images

Eiserling, Frederick A.; Geiduschek, E. Peter; Epstein, Richard H.; Metter, E. Jeffrey

1970-01-01

104

Transcription and RNA processing during expression of genes preceding DNA ligase gene 30 in T4-related bacteriophages  

Microsoft Academic Search

Early gene expression in bacteriophage T4 is controlled primarily by the unique early promoters, while T4-encoded RegB endoribonuclease promotes degradation of many early messages contributing to the rapid shift of gene expression from the early to middle stages. The regulatory region for the genes clustered upstream of DNA ligase gene 30 of T4 was known to carry two strong early

Lidija Truncaite; Aurelija Zajan?kauskaite; Aivaras Arlauskas; Rimas Nivinskas

2006-01-01

105

Effects of Bacteriophage T4Induced Modification of Escherichia coli RNA Polymerase on Gene Expression in vitro  

Microsoft Academic Search

After T4 bacteriophage infection of E. coli a complex series of events take place in the bacterium, including gross inhibition of host transcription and discrete changes in the classes of the genes of T4 that are transcribed. Accompanying these changes in the pattern of transcription one finds T4-induced changes in the RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase). The effects of

Reinhard Mailhammer; Huey-Lang Yang; Gary Reiness; Geoffrey Zubay

1975-01-01

106

The Relation between Breakdown of Superinfecting Virus Deoxyribonucleic Acid and Temporal Exclusion Induced by T4 and T5 Bacteriophages  

Microsoft Academic Search

SUMMARY Temporal exclusion and breakdown of superinfecting virus deoxyribonucleic acid were measured after infection with T 4 and T 5 bacteriophages of normal strains of Escherichia coli and strains deficient in endonuclease-I. Bacteria deficient in endonuclease-I when infected with T4 phage excluded superinfecting T4 with little solubilization of the secondary DNA. With wild-type bacteria exclusion was accompanied by extensive superinfection

PHOEBE E. FIELDING; MARY R. LUNT

1970-01-01

107

Structure, assembly, and DNA packaging of the bacteriophage T4 head.  

PubMed

The bacteriophage T4 head is an elongated icosahedron packed with 172 kb of linear double-stranded DNA and numerous proteins. The capsid is built from three essential proteins: gp23*, which forms the hexagonal capsid lattice; gp24*, which forms pentamers at 11 of the 12 vertices; and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. Intensive work over more than half a century has led to a deep understanding of the phage T4 head. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as numerous other icosahedral bacteriophages. However, phage T4 displays an unusual membrane and portal initiated assembly of a shape determining self-sufficient scaffolding core. Folding of gp23 requires the assistance of two chaperones, the Escherichia coli chaperone GroEL acting with the phage-coded gp23-specific cochaperone, gp31. The capsid also contains two nonessential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. Through binding to adjacent gp23 subunits, Soc reinforces the capsid structure. Hoc and Soc have been used extensively in bipartite peptide display libraries and to display pathogen antigens, including those from human immunodeficiency virus (HIV), Neisseria meningitides, Bacillus anthracis, and foot and mouth disease virus. The structure of Ip1*, one of a number of multiple (>100) copy proteins packed and injected with DNA from the full head, shows it to be an inhibitor of one specific restriction endonuclease specifically targeting glycosylated hydroxymethyl cytosine DNA. Extensive mutagenesis, combined with atomic structures of the DNA packaging/terminase proteins gp16 and gp17, elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. The cryoelectron microscopy structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at the highest rate known and can package multiple segments. Förster resonance energy transfer-fluorescence correlation spectroscopy studies indicate that DNA gets compressed in the stalled motor and that the terminase-to-portal distance changes during translocation. Current evidence suggests a linear two-component (large terminase plus portal) translocation motor in which electrostatic forces generated by ATP hydrolysis drive DNA translocation by alternating the motor between tensed and relaxed states. PMID:22420853

Black, Lindsay W; Rao, Venigalla B

2012-01-01

108

Genomes of the T4-related bacteriophages as windows on microbial genome evolution  

PubMed Central

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.

2010-01-01

109

Structure and location of gene product 8 in the bacteriophage T4 baseplate.  

PubMed

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure. PMID:12729757

Leiman, Petr G; Shneider, Mikhail M; Kostyuchenko, Victor A; Chipman, Paul R; Mesyanzhinov, Vadim V; Rossmann, Michael G

2003-05-01

110

Late effect of bacteriophage T4D on the permeability barrier of Escherichia coli.  

PubMed Central

Cold centrifugation of lysis-inhibited Escherichia coli B infected with wild-type T4D results in extensive lysis beginning around 20 min after infection at 37 degrees C. Infection with an e mutant, which fails to make lysozyme, prevents lysis, but does not prevent a marked loss of K+ and Mg3+. The t gene product, thought to disrupt the cytoplasmic membrane in natural lysis, is not required for this handling-induced cation loss or lysis. Three lines of evidence argue that late protein synthesis is required to develop this potential for cation loss; the potential does not develop in infections by: (i) mutants defective in DNA synthesis, (ii) mutants defective in gene 55, and (iii) wild-type T4 when chloramphenicol is added at 6 min after infection. All late mutants examined, which are blocked in the major pathways of morphogenesis, do not prevent development of the potential. The evidence argues for a new, late effect of T4 infection on the cytoplasmic membrane.

Thompson, S; Wiberg, J S

1978-01-01

111

Bacteriophage T4Induced Anticodon-Loop Nuclease Detected in a Host Strain Restrictive to RNA Ligase Mutants  

Microsoft Academic Search

The fate of host tRNAs during T4 bacteriophage infection was investigated with Escherichia coli CTr5x, the only known host strain that is restrictive to RNA ligase and polynucleotide kinase mutants. Three CTr5x tRNA species were cleaved during infection. One was leucine tRNA1, which was cleaved in the extra arm, as reported elsewhere for E. coli B infected with bacteriophage T2

Michael David; Gian Domenico Borasio; Gabriel Kaufmann

1982-01-01

112

Expression of a cloned denV gene of bacteriophage T4 in Escherichia coli  

SciTech Connect

A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC strains to be rescued by the denV gene. A uvrD (DNA helicase II) strain was also complemented, but to a lesser extent. A wild-type strain did not seem to be affected at the UV doses tested. Surprisingly, all recA, recB, and recC strains tested also showed an increased UV resistance, perhaps by reinforcement of the intact uvr system in these strains. Complementation of denV- T4 strains and host-cell reactivation of lambda phage was also observed in denV+ E. coli strains. Equilibrium sedimentation showed that DNA repair synthesis occurred in a UV-irradiated uvrA E. coli strain carrying the cloned denV gene. Southern blotting confirmed earlier results that the denV gene is located at 64 kilobases on the T4 map. Phage T2 (denV-) did not hybridize to a denV-specific probe.

Valerie, K.; Henderson, E.E.; de Riel, J.K.

1985-07-01

113

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs.  

PubMed

The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development. PMID:22499790

Durand, Sylvain; Richard, Graziella; Bontems, François; Uzan, Marc

2012-04-12

114

Complementation of Bacteriophage Induction and Recombination Defects in Escherichia coli RecA ? Mutants by Expression of the Cloned T4 Bacteriophage uvsX Gene  

Microsoft Academic Search

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction\\u000a of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these\\u000a prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency\\u000a of spontaneous induction and mitomycin C-stimulated bacteriophage

Steven A. Kuhl; Jay A. Zimmer; Pratik Rohatgi

2003-01-01

115

The dynamics of lysozyme from bacteriophage lambda in solution probed by NMR and MD simulations.  

PubMed

(15) N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H(N) -H(?) coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (? lysozyme) in solution. The lower and upper lip regions in ? lysozyme (residues 51-60 and 128-141, respectively) show reduced (1) H-(15) N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T2 indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for ? lysozyme indicate that two fluctuating ?-strands (?3 and ?4) are populated in the lower lip region while the N terminus of helix ?6 (residues 136-139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the ?-sheet and helical secondary structure in the lip regions, with populations of 40-60?%. Thus in solution ? lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein. PMID:23801644

Smith, Lorna J; Bowen, Alice M; Di Paolo, Alexandre; Matagne, André; Redfield, Christina

2013-06-25

116

Cloning of the bacteriophage T4 uvsX gene and purification and characterization of the T4 uvsX recombination protein.  

PubMed

The bacteriophage T4 uvsX gene is a nonessential gene required for normal levels of DNA repair, recombination, and replication. We demonstrate that plasmids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologically active uvsX protein. This uvsX protein imparts increased survival to UV-irradiated T4 uvsX- phage and decreases the T4 uvsX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recombination nuclease. The uvsX protein purified from cells with a uvsX+ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix-destablizing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA. These results agree with the recent characterization of uvsX protein from T4-infected cells by Yonesaki et al. (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148, 127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B.M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 363-370). In addition, we find that under some reaction conditions strand exchange is catalyzed by uvsX protein in the absence of 32 protein. The level of the uvsX protein expressed by the uvsX+ plasmids is high and independent of the orientation of the T4 DNA within the vector. This suggests that transcription promoter(s) lie upstream of the uvsX gene on the cloned T4 DNA. In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the uvsX gene and extend through the gene. PMID:3007520

Hinton, D M; Nossal, N G

1986-04-25

117

A Gene of Bacteriophage T4 whose Product Prevents True Late Transcription on Cytosine-Containing T4 DNA  

Microsoft Academic Search

T-even coliphages have 5-hydroxymethylcytosine in their DNA instead of cytosine. In some T4 mutants, the replicated DNA contains cytosine, but then no late gene products are made. We show that the inability to make late gene products with cytosine-containing T4 DNA is due to a T4 gene product. This gene product, while probably nonessential under normal conditions, interacts with an

Larry Snyder; Larry Gold; Elizabeth Kutter

1976-01-01

118

Evidence that bacteriophage T4 ephl is a missense hoc mutation  

SciTech Connect

An electrophoretic mutation of bacteriophage T4, ephl, appears to code for a missense hoc (highly antigenic outer capsid) protein. This is based on the observation that particles lacking (hoc protein hoc/sup -/ particles), after incubation in a crude extract of Escherichia coli infected with phage carrying the ephl mutation acquired the electrophoretic mobility of the ephl strain (the electrophoretic mobility of the ephl strain itself is slower than that of hoc/sup -/ particles). Thus, it is likely that during infection of E. coli with the ephl strain, a hoc protein is made that has a lower negative charge than normal hoc protein but can nevertheless bind to particles lacking hoc protein. These results confirm that ephl is a hoc mutation.

Childs, J.D.; Pilon, R.

1983-05-01

119

Atomic force microscopy images of T4 bacteriophages on silicon substrates  

SciTech Connect

A new atomic force microscope incorporating microfabricated cantilevers and employing laser beam deflection for force detection has been constructed and is being applied to studied of biological material. In this study, T4 bacteriophage virus particles were deposited from solution onto electronic grade flat silicon wafers and imaged in air with the microscope. Microliter droplets of the solution were deposited and either allowed to dry or removed with blotting paper. The images show both isolated viruses and aggregates of various sizes. The external structure as well as strands believed to be DNA streaming out of the virus could be observed. The construction of the microscope and its performance are also described. 19 refs., 4 figs.

Kolbe, W.F.; Ogletree, D.F.; Salmeron, M.B.

1991-08-01

120

Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase.  

PubMed Central

The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of DNA replication. Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32). The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex. We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex. The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32. A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction. These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase. A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6

Reddy, M K; Weitzel, S E; von Hippel, P H

1993-01-01

121

The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration  

Microsoft Academic Search

BACKGROUND: The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present

Krystyna D?browska; Grzegorz Skaradzi?ski; Paulina Jo?czyk; Aneta Kurz?pa; Joanna Wietrzyk; Barbara Owczarek; Maciej ?aczek; Kinga ?wita?a-Jele?; Janusz Boraty?ski; Gryzelda Po?niak; Magdalena Maciejewska; Andrzej Górski

2009-01-01

122

Vaccination against Very Virulent Infectious Bursal Disease Virus Using Recombinant T4 Bacteriophage Displaying Viral Protein VP2  

Microsoft Academic Search

In order to develop a desirable inexpensive, effective and safe vaccine against the very virulent infectious bursal disease virus (vvIBDV), we tried to take advantage of the emerging T4 bacteriophage surface protein display system. The major immunogen protein VP2 from the vvIBDV strain HK46 was fused to the nonessential T4 phage surface capsid protein, a small outer capsid (SOC) protein,

Yong-Chang CAO; Quan-Cheng SHI; Jing-Yun MA; Qing-Mei XIE; Ying-Zuo BI

2005-01-01

123

Membrane interaction of the portal protein gp20 of bacteriophage T4.  

PubMed

Assembly of the bacteriophage T4 head structure occurs at the cytoplasmic face of the inner membrane of Escherichia coli with the formation of proheads. The proheads contain an internal scaffolding core that determines the size and the structure of the capsid. In a mutant where the major shell protein gp23 was compromised, core structures without a shell had been detected. Such core structures were also found in the mutant T4am20am23. Since the mutation in gene 20 is at the N terminus of gp20, it was assumed that these core structures assemble in the absence of gp20. However, sequencing showed that the mutation introduces a new ribosome binding site that leads to a restart at codon 15. Although the mutant protein gp20s lacks the very N-terminal sequence, we found that it still binds to the membrane of the host cell and can initiate prohead assembly. This explains its activity to allow the assembly of core structures and proheads at the membrane surface. With a cross-linking approach, we show here that gp20 and gp20s are escorted by the chaperones DnaK, trigger factor, and GroEL and dock on the membrane at the membrane protein YidC. PMID:22855489

Quinten, Tobias A; Kuhn, Andreas

2012-08-01

124

Escherichia coli Endoribonucleases Involved in Cleavage of Bacteriophage T4 mRNAs  

PubMed Central

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.

Otsuka, Yuichi; Ueno, Hiroyuki; Yonesaki, Tetsuro

2003-01-01

125

A Mutant of E. COLI That Restricts Growth of Bacteriophage T4 at Elevated Temperatures  

PubMed Central

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 of DNA 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.

Jensen, Jean L.; Susman, Millard

1980-01-01

126

Endonuclease V from bacteriophage T4 interacts with its substrate in the minor groove.  

PubMed

The binding of bacteriophage T4 endonuclease V to its substrate has been studied using synthetic oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. Substrate analogues containing a methylphosphonate linkage with a defined configuration at the thymine dimer site were prepared, and the binding of the enzyme to each diastereomer was analyzed by the filter-binding method. The duplex containing a methylphosphonate with the SP configuration formed a complex with the enzyme, although the dissociation constant for this substrate analogue was about 8 times larger than that for the 12-mer substrate containing a phosphodiester linkage at this site. In contrast, no binding was observed when a duplex containing the RP-methylphosphonate linkage was used. The glycosyl bond of the thymine dimer in the SP isomer was cleaved by the enzyme, while no incision was detected in the case of the RP isomer, even after alkali treatment. Another substrate analogue containing a sulfur atom in place of the 3'-oxygen of the 5'-component at the thymine dimer site showed a reduced affinity for the enzyme. These results suggest that T4 endonuclease V interacts with its substrate in the minor groove. This mode of binding was confirmed by methylation protection experiments. PMID:8180181

Iwai, S; Maeda, M; Shimada, Y; Hori, N; Murata, T; Morioka, H; Ohtsuka, E

1994-05-10

127

Effects of temperature on the fitness cost of resistance to bacteriophage T4 in Escherichia coli.  

PubMed

Resistance to predation, herbivory, or disease often comes at a cost such that resistant genotypes are competitively inferior to their sensitive counterparts in the absence of predators, herbivores, or pathogens. The effects of this trade-off on natural populations depend on its sensitivity to environmental changes. We used Escherichia coli and bacteriophage T4 as a model predator/prey system to study the effects of temperature on the cost of resistance. An array of independent T4-resistant mutants, derived from a single ancestral strain of E. coli B, had a mean reduction in competitive fitness that depended strongly on environmental temperature; the cost of resistance generally increased with temperature. Genetic variance for fitness among phage-resistant mutants also depended on temperature; however, genetic variance increased at high and low thermal extremes. These results suggest that temperature is likely to be an important determinant of the consequences of predation in natural communities. We also discuss the underlying mechanistic basis for the cost of resistance in this system and its interaction with temperature. PMID:19222568

Quance, Michael A; Travisano, Michael

2009-02-13

128

The Folding Pathway of T4 Lysozyme: 2. The High-Resolution Structure and Folding of a Hidden Intermediate  

PubMed Central

Folding intermediates have been detected and characterized for many proteins. However, their structures at atomic resolution have only been determined for two small single domain proteins: Rd-apocytochrome b562 and engrailed homeo domain. T4 lysozyme has two easily distinguishable but energetically coupled domains: the N- and C-terminal domains. An early native-state hydrogen exchange experiment identified an intermediate with the C-terminal domain folded and the N-terminal domain unfolded. We have used a native-state hydrogen exchange-directed protein engineering approach to populate this intermediate and demonstrated that it is on the folding pathway and exists after the rate-limiting step. Here, we determined its high-resolution structure and the backbone dynamics by multi-dimensional NMR methods. We also characterized the folding behavior of the intermediate using stopped-flow fluorescence, protein engineering, and native-state hydrogen exchange. Unlike the folding intermediates of the two single-domain proteins, which have many non-native side chain interactions, the structure of the hidden folding intermediate of T4 lysozyme is largely native-like. It folds like many small single domain proteins. These results have implications for understanding the folding mechanism and evolution of multi-domain proteins.

Kato, Hidenori; Feng, Hanqiao; Bai, Yawen

2008-01-01

129

Bacteriophage T4 in vitro system to clone long DNA molecules. Final report, June 1, 1990--January 31, 1996.  

National Technical Information Service (NTIS)

A summary is presented of the following objectives: development of a bacteriophage T4 in vitro system, and techniques to clone long segments of foreign DNA; development of a giant prohead DNA packaging system that could potentially be used to clone even a...

V. B. Rao

1997-01-01

130

A bacteriophage T4 in vitro system to clone long DNA molecules. Final report, June 1, 1990--January 31, 1996  

SciTech Connect

A summary is presented of the following objectives: development of a bacteriophage T4 in vitro system, and techniques to clone long segments of foreign DNA; development of a giant prohead DNA packaging system that could potentially be used to clone even a megabase size DNA; and development of techniques to rapidly map the cloned DNA inserts.

Rao, V.B.

1997-09-01

131

Bacteriophage T4 capsid: a unique platform for efficient surface assembly of macromolecular complexes.  

PubMed

We report the first description of a macromolecular complex display system using bacteriophage T4. Decorated with two dispensable outer capsid proteins, Hoc (155 copies) and Soc (810 copies), the 120 nm x 86 nm T4 capsid particle offers a unique binding site-rich platform for surface assembly of hetero-oligomeric complexes. To display the 710 kDa anthrax toxin complex, two bipartite functional fusion proteins, LF-Hoc and LFn-Soc, were constructed. Using a defined in vitro binding system, sequential assembly was performed by first attaching LF-Hoc and/or LFn-Soc to hoc-soc- phage, saturating the Hoc and Soc binding sites. Trypsin-nicked PA63 was then assembled into heptamers through specific interaction with the capsid-exposed LFn domain. EF was then attached to the unoccupied sites of PA63 heptamers, completing the assembly of the tripartite anthrax toxin. Negative electron microscopy showed decoration of each capsid with a layer of heptameric PA63 rings. Up to 229 anthrax toxin complexes, equivalent to a total of 2400 protein molecules and a mass of about 133 MDa (2.7 times the mass of capsid shell), were anchored on a single particle, making it the highest density display reported on any virus. The phage T4 capsid lattice provides a stable biological platform allowing maximum display of large hetero-oligomeric complexes in vitro and offers insights for developing novel vaccines, analysis of protein-protein interactions, and structure determination of complexes. PMID:16982068

Li, Qin; Shivachandra, Sathish B; Leppla, Stephen H; Rao, Venigalla B

2006-08-23

132

Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages*  

PubMed Central

Tailed bacteriophages use powerful molecular motors to package the viral genome into a preformed capsid. Packaging at a rate of up to ?2000 bp/s and generating a power density twice that of an automobile engine, the phage T4 motor is the fastest and most powerful reported to date. Central to DNA packaging are dynamic interactions among the packaging components, capsid (gp23), portal (gp20), motor (gp17, large “terminase”), and regulator (gp16, small terminase), leading to precise orchestration of the packaging process, but the mechanisms are poorly understood. Here we analyzed the interactions between small and large terminases of T4-related phages. Our results show that the gp17 packaging ATPase is maximally stimulated by homologous, but not heterologous, gp16. Multiple interaction sites are identified in both gp16 and gp17. The specificity determinants in gp16 are clustered in the diverged N- and C-terminal domains (regions I–III). Swapping of diverged region(s), such as replacing C-terminal RB49 region III with that of T4, switched ATPase stimulation specificity. Two specificity regions, amino acids 37–52 and 290–315, are identified in or near the gp17-ATPase “transmission” subdomain II. gp16 binding at these sites might cause a conformational change positioning the ATPase-coupling residues into the catalytic pocket, triggering ATP hydrolysis. These results lead to a model in which multiple weak interactions between motor and regulator allow dynamic assembly and disassembly of various packaging complexes, depending on the functional state of the packaging machine. This might be a general mechanism for regulation of the phage packaging machine and other complex molecular machines.

Gao, Song; Rao, Venigalla B.

2011-01-01

133

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation.  

PubMed

Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called ? appropriation, the T4 co-activator AsiA structurally remodels the ?(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of ?(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the ?-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses. PMID:23902794

Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie; Waddell, M Brett; White, Stephen; Hinton, Deborah M

2013-07-31

134

Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4.  

PubMed

Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTP?S analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 61. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well. PMID:22869700

Jose, Davis; Weitzel, Steven E; Jing, Debra; von Hippel, Peter H

2012-08-06

135

Parent-to-Progeny Transfer and Recombination of T4rII Bacteriophage 1  

PubMed Central

Transfer of parental, light (not substituted with 5-bromodeoxyuridine) 32P-deoxyribonucleic acid (DNA) from rII? mutants of T4 bacteriophage to heavy (5-bromodeoxyuridine-substituted) progeny in Escherichia coli B was less homogeneous than in wild phages. The net transfer was 5 to 20% of the value for wild T4 phage, and the parental contribution per progeny DNA molecule amounted to 7 to 100% of the genome. Three classes could be distinguished, based on the density distribution of parental label in CsCl analysis of the progeny phages. “Far recombined” phages contain parental material only in semiconservatively replicated subunits covalently attached to progeny DNA, amounting to 5 to 10% parental contribution per genome. “Intermediate recombinants” contain, aside from conventional recombinant DNA, parental DNA banding at the original, light density. This DNA may be unattached to heavy progeny DNA or attached by weak bonds which are very sensitive to shearing during the extraction procedure. The parental contribution is 10 to 50% per progeny DNA molecule in this class. “Conservative” phages band close to the parental, light density in CsCl; their DNA is purely light. When the parental phage is labeled with both 3H-leucine (capsid) and 32P (DNA), the specific activity of 3H/32P in the “conservative progeny” is 10 to 40% of that in the parental, showing that at least some of the 32P in this area belongs to phages with parental DNA as the sole DNA component inside an unlabeled capsid, i.e., parental DNA which has been injected into the host and matured in a new capsid without replication or recombination. This phenomenon occurs to about the same extent in both single and multiple infection.

Carlson, Karin; Kozinski, Andrzej W.

1970-01-01

136

A systematic method for analysing the protein hydration structure of T4 lysozyme.  

PubMed

A systematic method for the analysis of the hydration structure of proteins is demonstrated on the case study of lysozyme. The method utilises multiple structural data of the same protein deposited in the protein data bank. Clusters of high water occupancy are localised and characterised in terms of their interaction with protein. It is shown that they constitute a network of interconnected hydrogen bonds anchored to the protein molecule. The high occupancy of the clusters does not directly correlate with water-protein interaction energy as was originally hypothesised. The highly occupied clusters rather correspond to the nodes of the hydration network that have the maximum number of hydrogen bonds including both the protein atoms and the surrounding water clusters. PMID:23996490

Kysilka, J; Vondrášek, J

2013-10-01

137

Recombination hotspots in bacteriophage T4 are dependent on replication origins.  

PubMed Central

Bacteriophage T4 recombination "hotspots" were first detected by the rescue of genetic markers from UV-irradiated phage particles. These hotspots have since been detected following treatments that yield other forms of DNA damage, and at least one is active in the absence of damage. The previous mapping of phage replication origins near the peaks of two recombination hotspots suggested that the origins cause the localized enhancement of recombination. Here we show that deletion of one origin eliminates the corresponding recombination hotspot, as judged by rescue of markers from UV-irradiated phage. Furthermore, insertion of either origin into a recombination "coldspot" enhances rescue of nearby markers. We conclude that these origins are necessary, and very likely sufficient, for the generation of recombination hotspots. We also show that the hotspots are active in the absence of both phage-encoded UvsY and host-encoded RecA proteins, suggesting that some of the stimulated recombination occurs by a synaptase-independent mechanism.

Yap, W Y; Kreuzer, K N

1991-01-01

138

Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA  

SciTech Connect

A series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli has been carried out. The results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication, strand displacement, and a considerable amount of DNA replication. UV irradiation of 5-bromodeoxyuridine-labeled phage results in single-stranded breaks in a linear, dose-dependent manner. This damage does not interfere with injection of the phage genome, but some of the UV-irradiated heavy phage DNA undergoes additional intracellular breakdown (also dose dependent). However, a minority (25%) of the injected parental DNA is protected, maintaining its preinjection size. Furthermore, the extent of the repair replication is greater at higher doses of UV irradiation applied to the heavy phage. This abundant synthesis results ultimately in dispersion of the parental sequences as short stretches in the midst of long segments of newly synthesized progeny DNA. Together, the extensive replication and the resulting distribution pattern of parental sequences, without significant solubilization of parental label, are most consistent with a model of repair synthesis in which the leading strand displaces, rather than ligates to, the encountered 5' end.

Restifo, L.L.; Vogelbacker, H.H.; Madara, T.; Ling, S.K.; Kozinski, A.W.

1983-07-01

139

Terminal Cross-Linking of DNA Strands by an Enzyme System from Escherichia coli Infected with Bacteriophage T4*  

PubMed Central

An enzyme system, purified 560-fold from Escherichia coli infected with bacteriophage T4, catalyzes the formation of a phosphodiester bond between the original 5?-phosphoryl end-group of a DNA strand and a 3?-hydroxyl group of the complementary strand. The product, a terminally cross-linked, spontaneously renaturable DNA duplex, has been characterized by chromatographic analysis, by sedimentation analysis, and by enzymatic digestion. Essential components of the enzyme system, which requires both ATP and Mg++, include the T4-induced DNA ligase and a component found in extracts of uninfected E. coli, which is probably an exonuclease. Images

Weiss, Bernard

1970-01-01

140

Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates  

Microsoft Academic Search

The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme\\/DNA ratios all mutants except one bound to DNA only as tetramers to two DNA substrates. The putatively

Pernilla Lagerback; Evalena Andersson; Christer Malmberg; Karin Carlson

2009-01-01

141

Stoichiometry and Domainal Organization of the Long Tail-fiber of Bacteriophage T4: A Hinged Viral Adhesin  

Microsoft Academic Search

The long-tail fibers (LTFs) form part of bacteriophage T4's apparatus for host cell recognition and infection, being responsible for its initial attachment to susceptible bacteria. The LTF has two parts, each ?70 to 75 nm long; gp34 (140 kDa) forms the proximal half-fiber, while the distal half-fiber is composed of gp37 (109 kDa), gp36 (23 kDa) and gp35 (30 kDa).

Mario E. Cerritelli; Joseph S. Wall; Martha N. Simon; James F. Conway; Alasdair C. Steven

1996-01-01

142

T4-type bacteriophage communities estimated from the major capsid genes (g23) in manganese nodules in Japanese paddy fields  

Microsoft Academic Search

The present study compared the capsid gene (g23) of T4-type bacteriophages (phages) in Mn nodules with those in the plow layer soil and subsoils of two Japanese paddy fields by applying the primers MZIA1bis and MZIA6 to DNA extracts from the nodules and soils. The deduced amino acid sequences of the g23 genes in the Mn nodules were similar to

Vita Ratri Cahyani; Jun Murase; Eiji Ishibashi; Susumu Asakawa; Makoto Kimura

2009-01-01

143

[Recombination in amber mutants of bacteriophage T4B. III. Influence of X-mutation on intragenic recombination in T4 phages].  

PubMed

Study was made of the influence of X-mutation on interallelic and intergenic recombination at the region of genes 34-38 of phage T4B. In the case of intergenic crosses in all studied recombinations of genes mutation X caused a 2-fold decrease in recombination frequency. X-mutation showed the same effect on integenic recombination in genes 36 and 38. In the case of crossing flank markers of gene 35 the frequency of recombination decreased 3.5-fold, that of gene 34--6 fold. Crosses of double am-X mutants mapped at different sites of gene 34 allowed to detect the gradient of the effect X-mutation on recombination in gene 34 in the direction from gene 33 to gene 35. The data obtained indicate a local anomaly of DNA structure of the region of genes 34 and 35 and allowed to draw a conclusion that the degree of suppressing recombination process under the influence of X-mutation depends nor on the original recombination frequency between markers to be crossed, that is on the internal structure of the given region of the genome of bacteriophage T4. Possible reasons for specific effect of X-mutations on the recombination in genes 34 and 35 of phage T4B are discussed. PMID:892435

Piruzian, E S; Zlotina, S N

1977-01-01

144

Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes  

Microsoft Academic Search

Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant.

Frank Rasche; Ester Marco-Noales; Henk Velvis; Leo S. van Overbeek; María M. López; Jan D. van Elsas; Angela Sessitsch

2006-01-01

145

High-molecular-weight DNA and the sedimentation coefficient: a new perspective based on DNA from T7 bacteriophage and two novel forms of T4 bacteriophage  

SciTech Connect

The DNA molecules from T7 bacteriophage and a recently obtained mutant form of T4D were studied. The DNA of this T4 mutant contains cytosine in place of all of the glucosylated hydroxymethylcytosines normally present in T4. Molecular weights were measured with an electron microscope technique, and sedimentation coefficients were determined in isokinetic sucrose gradients. T7 DNA was found to have an M/sub r/ of 26.5 x 10/sup 6/. The T4 mutant, which we have termed T4c, produces two distinct phage head and DNA size classes. DNA from the standard heads (T4c DNA) has an M/sub r/ of 114.9 x 10/sup 6/, and DNA from the petite heads (T4cp DNA) has an M/sub r/ of 82.9 x 10/sup 6/. This enabled the derivation of an equation of sedimentation coefficient at zero concentration corrected to water at 20/sup 0/C versus M/sub r/ for the molecular weight range of 25 x 10/sup 6/ to 115 x 10/sup 6/ that is based solely on cytosine-containing DNA standards, thereby avoiding possible anomalies introduced by the glucosylation and hydroxymethylation of cytosine. The theory of Gray et al. provided the best description of the sedimentation coefficient versus M/sub r/ relationship, based on the sedimentation coefficients and the molecular weights of the three DNA standards and other evidence.

Clark, R.W.; Wever, G.H.; Wiberg, J.S.

1980-01-01

146

Transcription and RNA processing during expression of genes preceding DNA ligase gene 30 in T4-related bacteriophages.  

PubMed

Early gene expression in bacteriophage T4 is controlled primarily by the unique early promoters, while T4-encoded RegB endoribonuclease promotes degradation of many early messages contributing to the rapid shift of gene expression from the early to middle stages. The regulatory region for the genes clustered upstream of DNA ligase gene 30 of T4 was known to carry two strong early promoters and two putative RegB sites. Here, we present the comparative analysis of the regulatory events in this region of 16 T4-type bacteriophages. The regulatory elements for control of this gene cluster, such as rho-independent terminator, at least one early promoter, the sequence for stem-loop structure, and the RegB cleavage sites have been found to be conserved in the phages studied. Also, we present experimental evidence that the initial cleavage by RegB of phages TuIa and RB69 enables degradation of early phage mRNAs by the major Escherichia coli endoribonuclease, RNase E. PMID:16225899

Truncaite, Lidija; Zajanckauskaite, Aurelija; Arlauskas, Aivaras; Nivinskas, Rimas

2005-10-12

147

Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.  

PubMed

The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast ?-pheromone receptor, a GPCR encoded by the yeast STE2 gene. Insertions were accompanied by varying extents of deletion or duplication of the loop. A set of phenotypic screens allow detection of potentially rare variant receptors that are expressed, bind to agonist and are capable of signal transduction via activation of the cognate G protein. A large fraction of screened full-length receptor variants containing at least partial duplications of the loop on either side of the inserted T4 lysozyme retain the ability to activate the downstream signaling pathway in response to binding of ligand. However, we were unable to identify any receptors with truncated C-termini that retain significant signaling function in the presence of inserted T4 lysozyme. Our results establish the feasibility of creating functional receptors containing insertions of T4 lysozyme in their third intracellular loops. PMID:23077276

Mathew, Elizabeth; Ding, Fa-Xiang; Naider, Fred; Dumont, Mark E

2012-10-16

148

Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein.  

PubMed

The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein. PMID:2995394

Hinton, D M; Silver, L L; Nossal, N G

1985-10-15

149

Radiation-Induced Damage in T4 Bacteriophage: The Effect of Superoxid Radicals and Molecular Oxygen. Progress Report, December 1, 1977--November 30, 1978.  

National Technical Information Service (NTIS)

The sensitivity of T4 bacteriophage towards gamma irradiation has been studied in phosphate buffer suspensions. The spectrum of the water radicals was controlled by a careful choice of the appropriate saturating gas and the addition of radical scavengers....

A. Samuni M. Chevion Y. S. Halpern Y. A. Ilan G. Czapski

1978-01-01

150

Studies on a Mutant Regulatory Protein Synthesized by Gene 45 of Bacteriophage T4D: Differential Functional Stabilization and Suppression of Temperature-Sensitive Characteristics.  

National Technical Information Service (NTIS)

Bacteriophage T4 diverts host cell metabolism from bacterial to viral functions after infection. The pattern is one of total, rapid, and irreversible host genome inactivation and degradation, and involves time-ordered sequences of gene expression generate...

B. R. Baser

1978-01-01

151

Bacteriophage T4 RNA ligase is gene 63 product, the protein that promotes tail fiber attachment to the baseplate.  

PubMed Central

RNA ligase and tail fiber attachment activities, normally induced following bacteriophage T4 infection of Escherichia coli, are not induced when gene 63 amber mutants of T4 infect nonpermissive host cells. Both activities are induced when these mutants infect permissive hosts, or when revertants of these mutants infect nonpermissive hosts. When one of these mutants infects a host that carries supF, both activities are more than normally heat labile. RNA ligase, purified to homogeneity, promotes the tail fiber attachment reaction in vitro with a specific activity similar to that of the most highly purified preparations of gene 63 product isolated on the basis of tail fiber attachment activity. We conclude that T4 RNA ligase is gene 63 product. The RNA ligase and tail fiber attachment reactions differ in requirements and in response to some inhibitors, suggesting that the two activities of the gene 63 product may be mechanistically unrelated.

Snopek, T J; Wood, W B; Conley, M P; Chen, P; Cozzarelli, N R

1977-01-01

152

Bacteriophage T4 DNA replication protein 61. Cloning of the gene and purification of the expressed protein.  

PubMed

In vitro, a bacteriophage T4 primase composed of T4 61 and 41 proteins, catalyzes the formation of pentaribonucleotides used to initiate DNA synthesis on single-stranded DNA. We have determined that cells containing a plasmid with the T4 DNA from 18.68 to 15.05 map units express an activity that substitutes for authentic 61 protein in vitro in catalyzing primer-dependent DNA synthesis with six other T4 DNA replication proteins. This result establishes that this region, genetically assigned to gene 61, is the structural gene for the priming protein. Cells containing a plasmid with gene 61 downstream of the strong phage lambda promoter PL and the antitermination site nutL produce 100-fold more 61 protein than T4-infected cells. We have developed an improved purification procedure which yields 100 mg of homogeneous, active protein from 178 g of these cells. In the plasmid, the T4 DNA downstream of gene 61 expresses a protein of 30,000 daltons. This protein may be the T4 DNA adenine methylase (dam) gene product, since Schlagman and Hattman (Schlagman, S. L. and Hattman, S. (1983) Gene 22, 139-156) have shown that this activity is expressed by plasmids containing T4 DNA from this region. In the PL, nutL vector, the expression of both the 30,000-dalton and 61 proteins is enhanced up to 20-fold by the presence of the phage lambda N protein, a transcription antitermination protein, suggesting that expression of the T4 DNA in the plasmid may be regulated transcriptionally. In addition, in both N+ and N- cells, the level of 61 protein, whose gene is proximal to PL on the plasmid, is lower than that of the product of the promoter distal 30,000-dalton protein gene. This result suggests that, at least in the plasmid construction, the expression of 61 protein may also be regulated after transcription. PMID:2995395

Hinton, D M; Nossal, N G

1985-10-15

153

Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.  

PubMed

The antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (Enterococcus faecalis, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Micrococcus lysodeikticus) and five gram-negative bacteria (Yersinia enterocolitica, Shigella flexneri, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella typhimurium). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). T4L, LaL and GEWL were highly pure as evaluated by silver staining of SDS-PAGE gels and zymogram analysis while CFL was only partially pure. At ambient pressure each gram-positive test organism displayed a specific pattern of sensitivity to the six lysozymes, but none of the gram-negative bacteria was sensitive to any of the lysozymes. High pressure treatment (130-300 MPa, 25 degrees C, 15 min) sensitised several gram-positive and gram-negative bacteria for one or more lysozymes. M. lysodeikticus and P. aeruginosa became sensitive to all lysozymes under high pressure, S. typhimurium remained completely insensitive to all lysozymes, and the other bacteria showed sensitisation to some of the lysozymes. The possible applications of the different lysozymes as biopreservatives, and the possible reasons for the observed differences in bactericidal specificity are discussed. PMID:16487612

Nakimbugwe, Dorothy; Masschalck, Barbara; Atanassova, Miroslava; Zewdie-Bosüner, Abebetch; Michiels, Chris W

2006-02-17

154

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts.  

PubMed

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria. PMID:16684100

Nakimbugwe, Dorothy; Masschalck, Barbara; Deckers, Daphne; Callewaert, Lien; Aertsen, Abram; Michiels, Chris W

2006-06-01

155

Reconstruction of Bacteriophage T4 DNA Replication Apparatus from Purified Components: Rolling Circle Replication Following de novo Chain Initiation on a Single-Stranded Circular DNA Template  

Microsoft Academic Search

The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells infected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside

Charles F. Morris; Navin K. Sinha; Bruce M. Alberts

1975-01-01

156

[The cotransduction of pET system plasmids by mutants of T4 and RB43 bacteriophages].  

PubMed

The study of the cotransduction of the plasmid pairs pET-3a-pLysE and pET-3a-pLysS by the mutant phage T4alc7 showed that the antibiotic resistance markers of the plasmids were cotransduced with a high frequency. The analysis of the plasmid DNA of cotransductants and cotransformants showed that the mutant phage T4alc7 can be used for obtaining the monomeric and oligomeric forms of plasmids and for the cotransduction of two-plasmid overproduction systems into E. coli strains. The plaque mutants RB43-03 and RB43-13 derived from bacteriophage RB43 were found to be able to cotransduce the antibiotic resistance markers of pET-3a and pLysE plasmids. PMID:14768546

Tianiashin, V I; Zimin, V I; Boronin, A M

157

The mono-ADP-ribosyltransferases Alt and ModB of bacteriophage T4: target proteins identified.  

PubMed

Infection of Escherichia coli by bacteriophage T4 leads to the expression of three phage mono-ADP-ribosyltransferases (namely, Alt, ModA, and ModB), each of which modifies a distinct group of host proteins. To improve understanding of these interactions and their consequences for the T4 replication cycle, we used high-resolution two-dimensional gel electrophoresis and mass-spectrometry to identify some of the putative target proteins ADP-ribosylated in vitro by Alt (total approximately 27) and ModB (total approximately 8). E. coli trigger factor and the elongation factor EF-Tu were 2 targets of ModB action, and these proteins were among the 10 identified as targets of Alt, hinting that these factors are involved in phage replication. PMID:16112649

Depping, Reinhard; Lohaus, Christiane; Meyer, Helmut E; Rüger, Wolfgang

2005-10-01

158

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation  

PubMed Central

Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context.

2010-01-01

159

Electrostatic contributions to T4 lysozyme stability: solvent-exposed charges versus semi-buried salt bridges.  

PubMed Central

We carried our Poisson-Boltzmann (PB) calculations for the effects of charge reversal at five exposed sites (K16E, R119E, K135E, K147E, and R154E) and charge neutralization and proton titration of the H31-D70 semi-buried salt bridge on the stability of T4 lysozyme. Instead of the widely used solvent-exclusion (SE) surface, we used the van der Waals (vdW) surface as the boundary between the protein and solvent dielectrics (a protocol established in our earlier study on charge mutations in barnase). By including residual charge-charge interactions in the unfolded state, the five charge reversal mutations were found to have DeltaDeltaG(unfold) from -1.6 to 1.3 kcal/mol. This indicates that the variable effects of charge reversal observed by Matthews and co-workers are not unexpected. The H31N, D70N, and H31N/D70N mutations were found to destabilize the protein by 2.9, 1.3, and 1.6 kcal/mol, and the pK(a) values of H31 and D70 were shifted to 9.4 and 0.6, respectively. These results are in good accord with experimental data of Dahlquist and co-workers. In contrast, if the SE surface were used, the H31N/D70N mutant would be more stable than the wild-type protein by 1.3 kcal/mol. From these and additional results for 27 charge mutations on five other proteins, we conclude that 1) the popular view that electrostatic interactions are generally destabilizing may have been based on overestimated desolvation cost as a result of using the SE surface as the dielectric boundary; and 2) while solvent-exposed charges may not reliably contribute to protein stability, semi-buried salt bridges can provide significant stabilization.

Dong, Feng; Zhou, Huan-Xiang

2002-01-01

160

Some properties of HU are modified after the infection of Escherichia coli by bacteriophage T4.  

PubMed Central

Escherichia coli HU, an abundant, nucleoid-associated, DNA-binding protein, plays a role in several biological processes including DNA replication. Many other bacteria have well-conserved HU homologs, and there are several more-distantly related members of the family, including TF1, encoded by Bacillus subtilis phage SPO1. We have asked whether coliphage T4, like SPO1, encodes an HU homolog or whether it alters the properties of host HU. We have been unable to detect a T4-specified HU homolog, but we have shown that E. coli HU extracted from phage-infected cells differs in some properties from that extracted from uninfected cells. First, HU from uninfected cells inhibits a reconstituted T4 DNA replication system, whereas HU from infected cells does not. Second, HU from infected cells appears to bind a T4-encoded polypeptide, as shown by coimmunoprecipitation. We propose that such binding alters HU function in T4-infected cells. Images

Bensaid, A; Uzan, M; Jacq, A; Hibner, U; Brody, E; Rouviere-Yaniv, J

1994-01-01

161

Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions 1 1 Edited by P. E. Wright  

Microsoft Academic Search

Several variants of T4 lysozyme have been identified that sequester small organic ligands in cavities or clefts. To evaluate potential binding sites for non-polar molecules, we screened a number of hydrophobic large-to-small mutants for stabilization in the presence of benzene. In addition to Leu99 ? Ala, binding was indicated for at least five other mutants. Variants Met102 ? Ala and

Enoch Baldwin; Walter A Baase; Xue-jun Zhang; Victoria Feher; Brian W Matthews

1998-01-01

162

pH-Induced denaturation of proteins: A single salt bridge contributes 3-5 kcal\\/mol to the free energy of folding of T4 lysozyme  

Microsoft Academic Search

The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pK{sub a} values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pK{sub a} value of

D. Eric Anderson; Wayne J. Becktel; Frederick W. Dahlquist

1990-01-01

163

Induction of lethality and DNA breakage by the decay of iodine-125 in bacteriophage T4  

Microsoft Academic Search

Iodine-125 was incorporated into the DNA of coliphage T4 in the form of ; 5-iododeoxyuridine (IUdR), an analogue of thymidine. ¹²⁵I decays by ; electron capture with consequent vacancy cascades and multiple ionization (Auger ; effect). This decay mode is known to cause extensive fragmentation of labeled ; small molecules. ¹²⁵Ilabeled T4 phage were stored in a glycerol-casamine ; acid

Robert E. Krisch; Ronald D. Ley

1974-01-01

164

Intervening Sequence in the Thymidylate Synthase Gene of Bacteriophage T4  

Microsoft Academic Search

The continuous sequence of 2.3 kilobases in a 3-kilobase DNA fragment encoding the structural gene for coliphage T4 thymidylate synthase 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) was determined by using the M13 dideoxy chain-termination method. From the coding information within this gene and that provided by sequence analysis of selected CNBr peptides from the protein product, the primary structure of T4 thymidylate

Frederick K. Chu; Gladys F. Maley; Frank Maley; Marlene Belfort

1984-01-01

165

Some properties of HU are modified after the infection of Escherichia coli by bacteriophage T4.  

PubMed

Escherichia coli HU, an abundant, nucleoid-associated, DNA-binding protein, plays a role in several biological processes including DNA replication. Many other bacteria have well-conserved HU homologs, and there are several more-distantly related members of the family, including TF1, encoded by Bacillus subtilis phage SPO1. We have asked whether coliphage T4, like SPO1, encodes an HU homolog or whether it alters the properties of host HU. We have been unable to detect a T4-specified HU homolog, but we have shown that E. coli HU extracted from phage-infected cells differs in some properties from that extracted from uninfected cells. First, HU from uninfected cells inhibits a reconstituted T4 DNA replication system, whereas HU from infected cells does not. Second, HU from infected cells appears to bind a T4-encoded polypeptide, as shown by coimmunoprecipitation. We propose that such binding alters HU function in T4-infected cells. PMID:8132451

Bensaid, A; Uzan, M; Jacq, A; Hibner, U; Brody, E; Rouvière-Yaniv, J

1994-03-01

166

An ORFan no more: the bacteriophage T4 39.2 gene product, NwgI, modulates GroEL chaperone function.  

PubMed

Bacteriophages are the most abundant biological entities in our biosphere, characterized by their hyperplasticity, mosaic composition, and the many unknown functions (ORFans) encoded by their immense genetic repertoire. These genes are potentially maintained by the bacteriophage to allow efficient propagation on hosts encountered in nature. To test this hypothesis, we devised a selection to identify bacteriophage-encoded gene(s) that modulate the host Escherichia coli GroEL/GroES chaperone machine, which is essential for the folding of certain host and bacteriophage proteins. As a result, we identified the bacteriophage RB69 gene 39.2, of previously unknown function and showed that homologs of 39.2 in bacteriophages T4, RB43, and RB49 similarly modulate GroEL/GroES. Production of wild-type bacteriophage T4 Gp39.2, a 58-amino-acid protein, (a) enables diverse bacteriophages to plaque on the otherwise nonpermissive groES or groEL mutant hosts in an allele-specific manner, (b) suppresses the temperature-sensitive phenotype of both groES and groEL mutants, (c) suppresses the defective UV-induced PolV function (UmuCD) of the groEL44 mutant, and (d) is lethal to the host when overproduced. Finally, as proof of principle that Gp39.2 is essential for bacteriophage growth on certain bacterial hosts, we constructed a T4 39.2 deletion strain and showed that, unlike the isogenic wild-type parent, it is incapable of propagating on certain groEL mutant hosts. We propose a model of how Gp39.2 modulates GroES/GroEL function. PMID:22234860

Ang, Debbie; Georgopoulos, Costa

2012-01-10

167

Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4  

SciTech Connect

The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. The authors examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D{sup +} or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D{sup +} or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D{sup +} strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with ({gamma}-{sup 32}P)ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly.

Drivdahl, R.H.; Kutter, E.M. (Evergreen State College, Olympia, WA (USA))

1990-05-01

168

Characterization of the major capsid genes (g23) of T4-type bacteriophages in the wetlands of northeast China.  

PubMed

To obtain genetic information and to evaluate the composition of T4-type bacteriophage (phage) communities in wetlands, environmental soil and water DNAs were obtained from two natural wetlands dominated by Carex lasiocarpa and Deyeuxia angustifolia plant species, and a neighboring paddy field in Sanjiang plain of northeast China. The biomarker gene of g23, which encodes the major capsid protein of T4-type phages, was amplified with primers MZIA1bis and MZIA6, and the PCR products were cloned and sequenced. In total, 96 and 50 different g23 clones were obtained from natural wetlands and a paddy field, respectively. A larger number of clones with low levels of identity to known sequences were found in water than in soil both in the natural wetlands and the paddy field, suggesting that many of T4-type phages in wetland water and paddy floodwater in Sanjiang plain are uncharacterized. Phylogenetic analyses showed that the g23 clones in natural wetlands, irrespective of water and soil, were distinctly different from those in marine waters, lake waters, and upland black soils, but were similar to those in paddy fields. The UniFrac analysis of g23 assemblages indicated that T4-type phage community compositions were different between soils and waters, and also were different between the natural wetlands and the paddy field. In general, the global analysis of g23 clone assemblages demonstrated that T4-type phage community compositions were different among natural wetlands, marines, lakes, paddy fields, and upland black soils. PMID:23306393

Zheng, Chunyu; Wang, Guanghua; Liu, Junjie; Song, Changchun; Gao, Hongxing; Liu, Xiaobing

2013-01-11

169

A mutation within the ? subunit of Escherichia coli RNA polymerase impairs transcription from bacteriophage T4 middle promoters.  

PubMed

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 ?(70), 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 ?-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 ? subunit may influence interactions among MotA, AsiA, and RNA polymerase. PMID:20729353

James, Tamara D; Cashel, Michael; Hinton, Deborah M

2010-08-20

170

Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage  

SciTech Connect

The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G. (CUA); (Purdue)

2011-09-16

171

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex.  

PubMed Central

Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built.

Song, H K; Sohn, S H; Suh, S W

1999-01-01

172

Bacteriophage T4 Self-Assembly: Localization of gp3 and Its Role in Determining Tail Length  

PubMed Central

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

Vianelli, A.; Wang, G. R.; Gingery, M.; Duda, R. L.; Eiserling, F. A.; Goldberg, E. B.

2000-01-01

173

In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine  

PubMed Central

The bacteriophage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56-µm-long, 170-kb viral genome into 120 nm × 86 nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, highly antigenic outer capsid protein (Hoc) and small outer capsid protein (Soc). T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein in the T4 head elicited robust antibody and cellular immune responses. This “progene delivery” approach might lead to new types of vaccines and genetic therapies.

Tao, Pan; Mahalingam, Marthandan; Marasa, Bernard S.; Zhang, Zhihong; Chopra, Ashok K.; Rao, Venigalla B.

2013-01-01

174

In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine.  

PubMed

The bacteriophage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56-µm-long, 170-kb viral genome into 120 nm × 86 nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, highly antigenic outer capsid protein (Hoc) and small outer capsid protein (Soc). T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein in the T4 head elicited robust antibody and cellular immune responses. This "progene delivery" approach might lead to new types of vaccines and genetic therapies. PMID:23530211

Tao, Pan; Mahalingam, Marthandan; Marasa, Bernard S; Zhang, Zhihong; Chopra, Ashok K; Rao, Venigalla B

2013-03-25

175

Bacteriophage T4D Gene 42 Mutants Exhibit a Defective Genetic Exclusion Phenotype.  

National Technical Information Service (NTIS)

Genetic exclusion in phage T4 is the prime responsibility of the imm and sp genes. The map region containing imm does not allow sufficient coding space to encode for proteins the size reported for the imm gp. After assaying 30 mutants of the genes adjacen...

J. W. Obringer

1991-01-01

176

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages  

PubMed Central

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a “small terminase” and a “large terminase” component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

Sun, Siyang; Gao, Song; Kondabagil, Kiran; Xiang, Ye; Rossmann, Michael G.; Rao, Venigalla B.

2012-01-01

177

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages  

SciTech Connect

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a 'small terminase' and a 'large terminase' component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

Sun, Siyang; Gao, Song; Kondabagil, Kiran; Xiang, Ye; Rossmann, Michael G.; Rao, Venigalla B. (CUA); (Purdue)

2012-04-04

178

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages.  

PubMed

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a "small terminase" and a "large terminase" component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation. PMID:22207623

Sun, Siyang; Gao, Song; Kondabagil, Kiran; Xiang, Ye; Rossmann, Michael G; Rao, Venigalla B

2011-12-29

179

Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages ?29, ?, and T4  

NASA Astrophysics Data System (ADS)

A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage ?29, we have recently extended these methods to study the E. coli bacteriophages ? and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

Smith, Douglas E.; Fuller, Derek N.; Raymer, Dorian M.; Rickgauer, Peter; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Catalano, Carlos E.; Kottadiel, Vishal; Rao, Venigalla B.

2007-09-01

180

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions  

Microsoft Academic Search

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a

C. A. Phillips; J. Gordon; E. K. Spicer

1996-01-01

181

Experimental electromagnetic effects on the model organism Escherichia coli and the bacteriophage T4  

NASA Astrophysics Data System (ADS)

This experimentally-based work was designed to answer the research question as to whether the phenomenon of nuclear magnetic resonance (NMR) can produce observable effects upon the bacterial virus activity of T4, with such activity demonstrated through the infection of its host bacterium Escherichia coli. The biological samples were placed for three hours within a coil antenna assembly propagating oscillating fields of radio frequency electromagnetic energy generated at the frequency of 5.6 MHz, and set at right angles within a magnetic field of 1450 gauss (recognizing such conditions are not set for the maximum effective resonance for hydrogen nuclei). The laboratory technique of plaque formation was the basis upon which the statistically tested data were compiled. Exposure of the bacterium alone exhibited an increase in viral activity over the control group (40--68% higher numbers of plaque formation), while exposure of T4 alone saw a decrease (approximately 23%) in infection rates. Depending on the protocol, placement of both T4 and E. coli into the coil assembly saw a decrease of either approximately 50% or 42% in infection rates. Future research must address identification of the effects being observed.

Lisiewski, Darlene Mildred

182

Molecular evolution of bacteriophages: Discrete patterns of codon usage in T4 genes are related to the time of gene expression  

Microsoft Academic Search

Summary Patterns of codon usage in certain coliphages are adapted to expression inEscherichia coli. Bacteriophage T4 may be an exception to test the rule, as it produces eight tRNAs with specificities that are otherwise rare inE. coli. A database of all known T4 DNA sequences has been compiled, comprising 174 genes and a total of 115 kb (approximately 70% of

Elizabeth Cowe; Paul M. Sharp

1991-01-01

183

Change in T4-type bacteriophage communities during the composting process of rice straw: Estimation from the major capsid gene (g23) sequences  

Microsoft Academic Search

The present study examined T4-type phage communities in rice straw (RS) under the composting process by analyzing the composition of the major capsid gene (g23) of T4-type bacteriophages. The g23 clones were obtained from RS throughout the composting process from RS materials to composting RS in the curing stage (for 124?days). Most of the g23 clones were phylogenetically closely related

Vita Ratri Cahyani; Jun Murase; Susumu Asakawa; Makoto Kimura

2009-01-01

184

Functional Analysis of the Highly Antigenic Outer Capsid Protein, Hoc, a Virus Decoration Protein from T4-like Bacteriophages  

PubMed Central

Summary Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the center of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modeling studies show that Hoc consists of a string of four domains, three immunoglobulin (Ig)-like and one non-Ig domain at the C-terminus. Biochemical data suggest that the Hoc protein has two functional modules, a capsid binding module containing domains 1 and 4 and a solvent-exposed module containing domains 2 and 3. This model is consistent with the dumbbell-shaped cryo-EM density of Hoc observed in the reconstruction of the T4 capsid. Mutagenesis localized the capsid binding site to the C-terminal 25 amino acids, which are predicted to form two ?-strands flanking a capsid binding loop. Mutations in the loop residues, ESRNG, abolished capsid binding, suggesting that these residues might interact with the major capsid protein, gp23*. With the conserved capsid binding module forming a foothold on the virus and the solvent-exposed module able to adapt to bind to a variety of surfaces, Hoc probably provides survival advantages to the phage, such as increasing the virus concentration near the host, efficient dispersion of the virus, and exposing the tail for more efficient contact with the host cell surface prior to infection.

Sathaliyawala, Taheri; Islam, Mohammad Z.; Li, Qin; Fokine, Andrei; Rossmann, Michael G.; Rao, Venigalla B.

2010-01-01

185

Using the rate of respiration to monitor events in the infection of Escherichia coli cultures by bacteriophage T4.  

PubMed

The growing interest in applications of bacteriophages creates a need for improvements in the production processes. Continuous monitoring of the phage production is an essential aspect of any control strategy and, at present, there is no completely satisfactory option. The approach presented here uses IR-spectrometry to continuously measure the rate of respiration (CO(2) released) of Escherichia coli infected by phage T4 at various multiplicities of infection (MOI). Within the trends in these data, or in other aspects of the rate of respiration, it was possible to reliably and reproducibly identify five features that reflected specific events in the infection process. These included two events in the host cell apparent growth rate and events in the magnitude of the host cell density, in the measurement of OD(600) or in the specific rate of respiration. All of these correlations were within 95% confidence showing that they are suitable for the monitoring and control of E. coli populations infected by phage T4. This method is reliable, cheap, and can be operated in-line and in real time. PMID:20039436

Sauvageau, Dominic; Allain, Béatrice; Cooper, David G

186

Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D  

PubMed Central

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.

Shah, D. B.

1976-01-01

187

Crystal structure of a heat and protease-stable part of the bacteriophage T4 short tail fibre 1 1 Edited by D. Rees  

Microsoft Academic Search

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipopolysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube.

Mark J van Raaij; Guy Schoehn; Martin R Burda; Stefan Miller

2001-01-01

188

Phylogenetic diversity and assemblage of major capsid genes (g23) of T4-type bacteriophages in paddy field soils during rice growth season in Northeast China  

Microsoft Academic Search

Although bacteriophages (phages) are ubiquitous and the most abundant biological entities on Earth, the genetic information on their diversity and community composition in natural environments, particularly in soils, is limited. This study elucidated the diversity and composition of T4-type phages by analyzing partial major capsid gene (g23) sequences in DNA extracts from five paddy field soils in Northeast China during

Junjie Liu; Guanghua Wang; Qiang Wang; Judong Liu; Jian Jin; Xiaobing Liu

2012-01-01

189

A Selective Barrier to Horizontal Gene Transfer in the T4Type Bacteriophages That Has Preserved a Core Genome with the Viral Replication and Structural Genes  

Microsoft Academic Search

Genomic analysis of bacteriophages frequently reveals a mosaic structure made up from modules that come from disparate sources. This fact has led to the general acceptance of the notion that rampant and promiscuous lateral gene transfer (LGT) plays a critical role in phage evolution. However, recent sequencing of a series of the T4-type phages has revealed that these large and

Jonathan Filee; Eric Bapteste; Edward Susko; H. M. Krisch

2006-01-01

190

The Structure of the Receptor-binding Domain of the Bacteriophage T4 Short Tail Fibre Reveals a Knitted Trimeric Metal-binding Fold  

Microsoft Academic Search

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipo-polysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube.

Ellen Thomassen; Gerrit Gielen; Michael Schütz; Guy Schoehn; Jan Pieter Abrahams; Stefan Miller; Mark J. van Raaij

2003-01-01

191

Morphological manifestations of freezing and thawing injury in bacteriophage T4Bo.  

PubMed

Electron microscopic observation of negatively stained preparations of frozen and thawed suspensions of T4Bo phage clearly separated the morphological changes produced produced by low-temperature salt denaturation from those produced by eutectic phase changes. Salt denaturation caused contraction of tail sheaths. Eutectic phase changes appeared to cause two separate lesions. Firstly the tail sheath was disjointed 18-22 nm. below the collar and the tail core was disjointed at 40-60 nm. below the collar, giving rise to separated heads with a small tail remnant, and separated tails in which the sheath remarkably remained in its extended form. Secondly, tears were seen in the head membranes of particles with collapsed empty heads. In all the experiments the percentage of normal phage particles counted electron-microscopically was close to the percentage of viable phage as determined by plaque assay. PMID:1068189

Steele, P R

1976-08-01

192

Intervening sequence in the thymidylate synthase gene of bacteriophage T4.  

PubMed Central

The continuous sequence of 2.3 kilobases in a 3-kilobase DNA fragment encoding the structural gene for coliphage T4 thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) was determined by using the M13 dideoxy chain-termination method. From the coding information within this gene and that provided by sequence analysis of selected CNBr peptides from the protein product, the primary structure of T4 thymidylate synthase was determined. The most significant finding of these studies is the presence of a 1017-base-pair interruption two-thirds of the way through the nucleotide sequence of the structural gene. The 5'- and 3'-terminal ends of this intron are demarcated by an apparent stop and start codon, respectively. The corresponding methionine preceding the second coding region of the synthase is not incorporated into the final protein product. Structural evidence confirming the presence of the intervening sequence in the phage genome was obtained by restriction and hybridization analysis. Support for the presence of the intron was also obtained at the functional level by enzyme expression studies using selected td gene fragments. This work also confirms the findings of Purohit and Mathews [ Purohit , S. & Mathews , C. K. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 1759], which reveal that the termination codon for the dihydrofolate reductase gene and the triplet initiating thymidylate synthase overlap by a four-base stretch, A-T-G-A. The implications of this unusual gene arrangement are discussed. Images

Chu, F K; Maley, G F; Maley, F; Belfort, M

1984-01-01

193

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.  

PubMed Central

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.

Zhang, X. J.; Baase, W. A.; Matthews, B. W.

1992-01-01

194

Replication of T4rII Bacteriophage in Escherichia coli K-12 (?) 1  

PubMed Central

The defect of T4rII replication in Escherichia coli K-12 (?) can be phenotypically reversed by various supplements to the growth medium. Arginine, lysine, spermidine, and a number of diamines allowed varying levels of rII replication. The best reversion was obtained with 0.4 m sucrose in 0.002 to 0.005 m Ca++. Monovalent cations severely inhibited reversion. A cell surface site of polyamine action is consistent with the fact that spermidine inhibits phage ghost-induced cell lysis and with the finding that sufficient polyamine is available within the cells to allow normal patterns of neutralization of phage deoxyribonucleic acid, as detected by the polyamine content of progeny phage. In the absence of effective supplements, rII-infected cells swelled and lost refractility. The data indicate that a leaky cell envelop is involved. No difference in mucopeptides of uninfected K-12 (?) and K-12 was detected and, because the mucopeptide in r+ infected cells was found to be at least partially hydrolyzed midway through the lytic cycle, it did not appear that the rII defect concerned mucopeptide synthesis. The pattern of cell phospholipid synthesis changes after phage infection, but no difference was detected between r+ and rII with regard to biosynthesis of phosphatidylethanolamine and phosphatidylglycerol.

Buller, Clarence S.; Astrachan, L.

1968-01-01

195

Genetic Definition of Two Functional Elements in a Bacteriophage T4 Host-Range ``cassette''  

PubMed Central

Gene 37 of T4 encodes the major subunit of the distal half of the tail fiber. The distal tip of the fiber, comprised of the carboxy-terminal ends of two molecules of gene 37 product (gp37), carries the principal determinant of the phage host range. The gp37 carboxyl termini recognize the bacterial surface during infection, and, in addition, include a site required for interaction with the product of gp38 during distal half-fiber assembly. In the absence of interaction with gp38, gp37 polypeptides do not dimerize. Eleven temperature-sensitive mutants with defects located near the promoter-distal end of gene 37 were tested at nonpermissive temperatures for production of an antigen that is diagnostic of distal half-fiber assembly. Six of the mutations prevent distal half-fiber assembly. The other five allow assembly of distal half fibers, which combine with proximal half fibers and attach to phage particles, but the resulting phage do not adsorb to bacteria. These two classes of mutations define two adjacent but separate genetic regions, corresponding to two different functional domains in gp37. These two regions and the neighboring gene 38 comprise a functional unit that can be considered as a host-range ``cassette,'' with features that are strikingly similar to corresponding functional units in other unrelated as well as related phages.

Snyder, M.; Wood, W. B.

1989-01-01

196

Determination of the Orientation of T4 Lysozyme Vectorially Bound to a Planar-Supported Lipid Bilayer Using Site-Directed Spin Labeling  

PubMed Central

Site-directed spin labeling is used to investigate the structure of adsorbed T4 lysozyme (T4L). A monolayer of T4L is prepared by tethering the protein selectively via a His-tag to the chelating headgroups (NTA Ni) of a planar quartz-supported lipid bilayer. This results in a vectorially oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance spectra. Similar measurements of spin-labeled lipid bilayers were used to characterize the structure and dynamics of the supports. Electron paramagnetic resonance line shape was analyzed using the stochastic Liouville equation approach developed by Freed and co-workers. The simulations reveal a conservation of the secondary and tertiary structure of T4L upon adsorption although slight conformational changes in the presence of the surface can be detected by probing tertiary contact sites. The orientation of the entire protein was deduced on the basis of an anisotropic motional model for the spin-labeled side chain. In addition, a polar order but azimuthal disorder of the molecules was assumed to fit the data. These results demonstrate the utility of site-directed spin labeling in combination with spectral simulation to study not only the secondary and tertiary structure of adsorbed proteins in monolayer coverage but also their orientation with respect to the surface.

Jacobsen, Kerstin; Oga, Shirley; Hubbell, Wayne L.; Risse, Thomas

2005-01-01

197

Restriction endonuclease inhibitor IPI* of bacteriophage T4: a novel structure for a dedicated target.  

PubMed

Phage T4 protects its DNA from the two-gene-encoded gmrS/gmrD (glucose-modified hydroxymethylcytosine restriction endonuclease) CT of pathogenic Escherichia coli, CT596, by injecting several hundred copies of the 76-amino-acid-residue nuclease inhibitor, IPI*, into the infected host. Here, the three-dimensional solution structure of mature IPI* is reported as determined by nuclear magnetic resonance techniques using 1290 experimental nuclear Overhauser effect and dipolar coupling constraints ( approximately 17 constraints per residue). Close examination of this oblate-shaped protein structure reveals a novel fold consisting of two small beta-sheets (beta1: B1 and B2; beta2: B3-B5) flanked at the N- and C-termini by alpha-helices (H1 and H2). Such a fold is very compact in shape and allows ejection of IPI* through the narrow 30-A portal and tail tube apertures of the virion without unfolding. Structural and dynamic measurements identify an exposed hydrophobic knob that is a putative gmrS/gmrD-binding site. A single gene from the uropathogenic E. coli UT189, which codes for a gmrS/gmrD-like UT fusion enzyme (with approximately 90% identity to the heterodimeric CT enzyme), has evolved IPI* inhibitor immunity. Analysis of the gmrS/gmrD restriction endonuclease enzyme family and its IPI* family phage antagonists reveals an evolutionary pathway that has elaborated a surprisingly diverse and specifically fitted set of coevolving attack and defense structures. PMID:18037438

Rifat, Dalin; Wright, Nathan T; Varney, Kristen M; Weber, David J; Black, Lindsay W

2007-11-01

198

The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase  

SciTech Connect

The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-{angstrom} resolution. The protein folds into an {alpha}/{beta}-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.

Cheng, X.; Pflugrath, J.W. [W.M. Keck Structural Biology Lab., Cold Spring Harbor, NY (United States); Zhang, X.; Studier F.W. [Brookhaven National Lab., Upton, NY (United States)

1994-04-26

199

Analyzing indirect secondary electron contrast of unstained bacteriophage T4 based on SEM images and Monte Carlo simulations  

SciTech Connect

The indirect secondary electron contrast (ISEC) condition of the scanning electron microscopy (SEM) produces high contrast detection with minimal damage of unstained biological samples mounted under a thin carbon film. The high contrast image is created by a secondary electron signal produced under the carbon film by a low acceleration voltage. Here, we show that ISEC condition is clearly able to detect unstained bacteriophage T4 under a thin carbon film (10-15 nm) by using high-resolution field emission (FE) SEM. The results show that FE-SEM provides higher resolution than thermionic emission SEM. Furthermore, we investigated the scattered electron area within the carbon film under ISEC conditions using Monte Carlo simulation. The simulations indicated that the image resolution difference is related to the scattering width in the carbon film and the electron beam spot size. Using ISEC conditions on unstained virus samples would produce low electronic damage, because the electron beam does not directly irradiate the sample. In addition to the routine analysis, this method can be utilized for structural analysis of various biological samples like viruses, bacteria, and protein complexes.

Ogura, Toshihiko, E-mail: t-ogura@aist.go.jp

2009-03-06

200

Crystallographic and NMR analyses of UvsW and UvsW.1 from bacteriophage T4.  

PubMed

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3'-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7A and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases. PMID:17878153

Kerr, Iain D; Sivakolundu, Sivashankar; Li, Zhenmei; Buchsbaum, Jeffrey C; Knox, Luke A; Kriwacki, Richard; White, Stephen W

2007-09-17

201

Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates.  

PubMed

The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme/DNA ratios all mutants except one bound to DNA only as tetramers to two DNA substrates. The putatively catalytic E118 residue actually interfered with DNA binding (possibly due to steric hindrance or repulsion between the glutamate side chain and DNA), as shown by the ability of E118A to bind stably also as monomer or dimer to a single substrate. The tetrameric structure of EndoII in the DNA-protein complex is surprising considering the asymmetry of the recognized sequence and the predominantly single-stranded nicking. Combining the results obtained here with those from our previous in vivo studies and the recently obtained crystal structure of EndoII E118A, we suggest a model where EndoII translocates DNA between two adjacent binding sites and either nicks one strand of one or both substrates bound by the tetramer, or nicks both strands of one substrate. Thus, only one or two of the four active sites in the tetramer is catalytically active at any time. PMID:19666720

Lagerbäck, Pernilla; Andersson, Evalena; Malmberg, Christer; Carlson, Karin

2009-08-07

202

Proteomic profiles and kinetics of development of bacteriophage T4 and its rI and rIII mutants in slowly growing Escherichia coli.  

PubMed

Bacteriophage T4 survival in its natural environment requires adjustment of phage development to the slow bacterial growth rate or the initiation of mechanisms of pseudolysogeny or lysis inhibition (LIN). While phage-encoded RI and probably RIII proteins seem to be crucial players in pseudolysogeny and LIN phenomena, the identity of proteins involved in the regulation of T4 development in slowly growing bacteria has remained unknown. In this work, using a chemostat system, we studied the development of wild-type T4 (T4wt) and its rI (T4rI) and rIII (T4rIII) mutants in slowly growing bacteria, where T4 did not initiate LIN or pseudolysogeny. We determined eclipse periods, phage propagation times, latent periods and burst sizes of T4wt, T4rI and T4rIII. We also compared intracellular proteomes of slowly growing Escherichia coli infected with either T4wt or the mutants. Using two-dimensional PAGE analyses we found 18 differentially expressed proteins from lysates of infected cells. Proteins whose amounts were different in cells harbouring T4wt and the mutants are involved in processes of replication, phage-host interactions or they constitute virion components. Our data indicate that functional RI and RIII proteins - apart from their already known roles in LIN and pseudolysogeny - are also necessary for the regulation of phage T4 development in slowly growing bacteria. This regulation may be more complicated than previously anticipated, with many factors influencing T4 development in its natural habitat. PMID:23239571

Golec, Piotr; Karczewska-Golec, Joanna; Voigt, Birgit; Albrecht, Dirk; Schweder, Thomas; Hecker, Michael; Wegrzyn, Grzegorz; ?os, Marcin

2012-12-12

203

Role of primary photoacceptors in low-power laser effects: action of He-Ne laser radiation on bacteriophage T4Escherichia coli interaction  

Microsoft Academic Search

The effect of He-Ne laser radiation (lambda = 632.8 nm) on bacteriophage T4-Escherichia coli WP2 interactions was studied. Irradiation of bacteria having respiratory chain components as primary photoacceptors accelerated their division in a dose-dependent manner, but irradiation had no effect on the properties of the phage (measured as its ability to infect host cells). At the same time, exposure of

Olga Tiphlova; Tiina Karu

1989-01-01

204

Molecular analysis of the major capsid genes ( g23 ) of T4-type bacteriophages in an upland black soil in Northeast China  

Microsoft Academic Search

Bacteriophages (phages) are the most abundant biological entities on the planet and are important as the greatest genomic\\u000a reservoirs in both marine and terrestrial environments. In this study, we analysed T4-type phage communities in an upland\\u000a black soil by monitoring g23 clones in DNA extracted from seasonal soil samples with no fertilizer, chemical fertilizers, chemical fertilizers plus manure,\\u000a and natural

Guanghua Wang; Zhenhua Yu; Junjie Liu; Jian Jin; Xiaobing Liu; Makoto Kimura

2011-01-01

205

The ?-helical domain of bacteriophage T4 controls the folding of the fragment of long tail fibers in a chimeric protein  

Microsoft Academic Search

The key stage of the infection of the Escherichia coli cell with bacteriophage T4, the binding to the surface of the host cell, is determined by the specificity of the long tail\\u000a fiber proteins of the phage, in particular, gp37. The assembly and oligomerization of this protein under natural conditions\\u000a requires the participation of at least two additional protein factors,

R. N. Chuprov-Netochin; N. M. Faizullina; N. N. Sykilinda; M. N. Simakova; V. V. Mesyanzhinov; K. A. Miroshnikov

2010-01-01

206

Functions of replication factor C and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4  

SciTech Connect

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase {delta} on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.

Tsurimoto, Toshiki; Stillman, B. (Cold Spring Harbor Laboratory, NY (USA))

1990-02-01

207

Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.  

PubMed Central

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4. Images

Tsurimoto, T; Stillman, B

1990-01-01

208

Cpl-7, a Lysozyme Encoded by a Pneumococcal Bacteriophage with a Novel Cell Wall-binding Motif*  

PubMed Central

Bacteriophage endolysins include a group of new antibacterials reluctant to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases and a C-terminal region encompassing three identical repeats of 42 amino acids (CW_7 repeats). These repeats are unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, and are responsible for the protein attachment to the cell wall. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle x-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an ?115-? long molecule with two well differentiated regions, corresponding to the CM and the cell wall binding region (CWBR), arranged in a lateral disposition. The CM displays the (??)5?3 barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly assembled three-helical bundles whose dispositions remind us of a super-helical structure. Its approximate dimensions are 60 × 20 × 20 ? and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database have been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin.

Bustamante, Noemi; Campillo, Nuria E.; Garcia, Ernesto; Gallego, Cristina; Pera, Benet; Diakun, Gregory P.; Saiz, Jose Luis; Garcia, Pedro; Diaz, J. Fernando; Menendez, Margarita

2010-01-01

209

Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis.  

PubMed

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5' phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5'-dTdCdA-3' but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5'-dTdCdA-3', which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA. PMID:16971463

Hirano, Nobutaka; Ohshima, Hiroyuki; Takahashi, Hideo

2006-09-13

210

Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 DAS mutation  

SciTech Connect

Lethal, amber mutations in T4 genes 46 and 47 cause incomplete degradation of host DNA, premature arrest of phage DNA synthesis, accumulation of abnormal DNA replication intermediates, and defective recombination. These phenotypes can be explained by the hypothesis that genes 46 and 47 control a DNA exonuclease, but in vitro demonstration of such a nuclease has not yet been reported. Membrane and supernatant fractions from 46/sup -/ and 47/sup -/ mutant-infected and 46/sup +/ 47/sup +/ control-infected cells were assayed for the presence of the protein products of these genes (i.e., gp46 and gp47) and for the ability to degrade various DNA substrates to acid-soluble products in vitro. The two proteins were found only on membranes. The membrane fraction from 46/sup -/ 47/sup -/ mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower than the membrane fraction from control-infected cells. No such effect was found in the cytoplasmic fractions. The effect on nuclease activity in membranes was the same whether 47/sup -/ and 47/sup -/ mutants were present singly or together. NaClO/sub 4/, a chaotropic agent, released both gp46 and gp47 from 46/sup +/ 47/sup +/ membranes, as well as the DNase activity controlled by genes 46 and 47. DNA cellulose chromatography of proteins released from membranes by NaClO/sub 4/ showed that gp46 and gp47 bound to the native DNAs of both E. coli and T4. Thus, the overall enrichment of gp46 and gp47 relative to total T4 protein was 600-fold (10-fold in membranes, 2-fold more upon release from membranes by NaClO/sub 4/, and 30-fold more upon elution from DNA cellulose). T4 das mutations, which partially suppress the defective phenotype of 46/sup -/ and 47/sup -/ mutants, caused a considerable increase in in vitro DNase activity in both membrane and cytoplasmic fractions.

Mickelson, C.; Wiberg, J.S.

1981-10-01

211

Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase  

SciTech Connect

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.

Saporito, S.M.; Gedenk, M.; Cunningham, R.P.

1989-05-01

212

Involvement of the Escherichia coli endoribonucleases G and E in the secondary processing of RegB-cleaved transcripts of bacteriophage T4.  

PubMed

Sequence-specific endoribonuclease RegB of bacteriophage T4 cleaves early phage mRNAs and facilitates the transition between early and subsequent phases of T4 gene expression. The great majority of RegB targets have been identified in the intergenic regions of T4 transcripts, frequently in the Shine-Dalgarno sequences. Here we show that localization of RegB targets is not restricted to intergenic regions of mRNA. We detected 30 intragenic RegB sites in T4 transcripts that are differently susceptible to cleavage. Four RegB-processed mRNAs were previously shown to undergo further processing at so-called "secondary sites". We have found three additional transcripts carrying clear targets for both RegB and another endoribonuclease. We show that secondary cuts within RegB-processed T4 mRNAs are generated mainly by Escherichia coli RNase G, but that in some cases RNase E can recognize the same targets. Using plasmid-phage systems we demonstrate that T4 infection favours cleavage by the host endoribonucleases at these sites. PMID:18395239

Zajanckauskaite, Aurelija; Truncaite, Lidija; Strazdaite-Zieliene, Zivile; Nivinskas, Rimas

2008-04-18

213

The dynamic pause-unpackaging state, an off-translocation recovery state of a DNA packaging motor from bacteriophage T4  

PubMed Central

Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ?170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor–DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the “pause-unpackaging” state is an off-translocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome.

Kottadiel, Vishal I.; Rao, Venigalla B.; Chemla, Yann R.

2012-01-01

214

A family of anti-sigma70 proteins in T4-type phages and bacteria that are similar to AsiA, a Transcription inhibitor and co-activator of bacteriophage T4.  

PubMed

Anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic RNA polymerase. The bacteriophage T4 anti-sigma70 protein, AsiA, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of Escherichia coli RNA polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 DNA sequence. In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical sigma70 -10 consensus sequence but have a MotA box sequence centered at -30 rather than the sigma70 -35 sequence. The E.coli anti-sigma70 protein Rsd also interacts with region 4.2 of sigma70 and inhibits transcription from sigma70 promoters. Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to sigma70 region 4. We show that, like T4 AsiA, Rsd binds to sigma70 in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex. Previous work has assigned sigma70 amino acid F563, within region 4.1, as a critical determinant for AsiA binding. This residue is also involved in the binding of sigma70 to the beta-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between sigma70 region 4.1 and the beta-flap. We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with sigma70 region 4 is diminished by the substitution F563Y. We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from sigma70-dependent promoters. We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary sigma factors. In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA. We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA. PMID:15561138

Pineda, Melissa; Gregory, Brian D; Szczypinski, Bridget; Baxter, Kimberly R; Hochschild, Ann; Miller, Eric S; Hinton, Deborah M

2004-12-10

215

Structural Determinants of Nitroxide Motion in Spin-Labeled Proteins: Solvent-Exposed Sites in Helix B of T4 Lysozyme  

SciTech Connect

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.

Guo,Z.; Cascio, D.; Hideg, K.; Hubbell, W.

2008-01-01

216

Structural determinants of nitroxide motion in spin-labeled proteins: Solvent-exposed sites in helix B of T4 lysozyme  

PubMed Central

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.

Guo, Zhefeng; Cascio, Duilio; Hideg, Kalman; Hubbell, Wayne L.

2008-01-01

217

Structural Determinants of Nitroxide Motion in Spin-labeled Proteins: Tertiary Contact and Solvent-inaccessible Sties in Helix G of T4 Lysozyme  

SciTech Connect

A nitroxide side chain (R1) has been substituted at single sites along a helix-turn-helix motif in T4 lysozyme (residues 114-135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two-component EPR spectra. At 115, R1 is shown to occupy two rotamers in the room temperature crystal structure, one of which has not been previously reported. The two components in the EPR spectrum apparently arise from differential interactions of the two rotamers with the surrounding structure, the most important of which is a hydrophobic interaction of the nitroxide ring. Interestingly, the crystal structure at 100 K reveals a single rotamer, emphasizing the possibility of rotamer selection in low-temperature crystal structures. Residue 118 is at a solvent-inaccessible site in the protein core, and the structure of 118R1, the first reported for the R1 side chain at a buried site, reveals how the side chain is accommodated in an overpacked core.

Guo,Z.; Cascio, D.; Hideg, K.; Kalai, T.; Hubbell, W.

2007-01-01

218

Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22  

SciTech Connect

Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H/sub 2/O/sub 2/ or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

Hartman, P.E.; Hartman, Z.; Citardi, M.J.

1988-05-01

219

Frameshift and double-amber mutations in the bacteriophage T4 uvsX gene: Analysis of mutant UvsX proteins from infected cells  

Microsoft Academic Search

The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25–32 kDa. Wild-type UvsX protein was also detected in

Myra O. Rosario; John W. Drake

1990-01-01

220

Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex.  

PubMed

More than 50 copies of a phi X174 DNA template can be made in 60 min in an in vitro DNA replication system consisting of seven purfied replication proteins isolated from T4 bacteriophage-infected cells. By transfecting with the DNA products and assaying for the reversion of specific amber mutants, the high degree of base-pairing fidelity in this system is revealed; the in vitro system is also shown to respond to the mutagenic effect of Mn2+ and to display strong base-pair context effects on fidelity, as expected from in vivo studies. PMID:6246450

Hibner, U; Alberts, B M

1980-05-29

221

[Recombination of amber mutants of bacteriophage T4B. II. Localization of amber mutants on maps of genes 30, 34, 35, 36 and 38].  

PubMed

Localization of 275 amber mutants of five genes of phage T4B (30, 34, 35, 36 and 38) on genetic maps allowed us to determine the recombination length of these genes. Gene 34 substantially differs from the rest studied genes by numbers of amber mutants isolated in each gene and by recombination frequency. In particular, according to the results of crossing the flank markers, the recombination length of gene 34 is 10 times greater than in gene 38; using the summary value of recombination frequencies between elementary intervals a 20-fold excess of the length of gene 34 compared with the length of gene 38 was receieved. Molecular weight of the product of gene 34 is only 6 times as great as in gene 38. An elevated recombination frequency was also detected in gene 35. The data obtained indicate a local recombination anomaly at the region of genes 34--35 of bacteriophage T4 genome. PMID:330318

Piruzian, E S; Zlotina, S N

1977-01-01

222

ISOLATION AND CHARACTERIZATION OF T4 BACTERIOPHAGE GP17 TERMINASE: A LARGE SUBUNIT MULTIMER WITH ENHANCED ATPASE ACTIVITY  

Technology Transfer Automated Retrieval System (TEKTRAN)

Phage T4 terminase is a two-subunit enzyme that binds to prohead portal protein and cuts and packages concatemeric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from ch...

223

NMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains.  

PubMed

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L. PMID:12654266

Liepinsh, Edvards; Généreux, Catherine; Dehareng, Dominique; Joris, Bernard; Otting, Gottfried

2003-04-01

224

Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.  

PubMed

All genetic markers from phage T2 are partially excluded from the progeny of mixed infections with the related phage T4 (general, or phage exclusion). Several loci, including gene 56 of T2, are more dramatically excluded, being present in only approximately 1% of the progeny. This phenomenon is referred to as localized marker exclusion. Gene 69 is adjacent to gene 56 of T4 but is absent in T2, being replaced by completely nonhomologous DNA. We describe SegF, a novel site-specific DNA endonuclease encoded by gene 69, which is similar to GIY-YIG homing endonucleases of group I introns. Interestingly, SegF preferentially cleaves gene 56 of T2, both in vitro and in vivo, compared with that of phage T4. Repair of the double-strand break (DSB) results in the predominance of T4 genes 56 and segF in the progeny, with exclusion of the corresponding T2 sequences. Localized exclusion of T2 gene 56 is dependent on full-length SegF and is likely analogous to group I intron homing, in which repair of a DSB results in coconversion of markers in the flanking DNA. Phage T4 has many optional homing endonuclease genes similar to segF, whereas similar endonuclease genes are relatively rare in other members of the T-even family of bacteriophages. We propose that the general advantage enjoyed by T4 phage, over almost all of its relatives, is a cumulative effect of many of these localized events. PMID:11825876

Belle, Archana; Landthaler, Markus; Shub, David A

2002-02-01

225

The initial adsorption of T4 bacteriophages to Escherichia coli cells at equivalent concentrations: Experiments and mathematical modeling  

Microsoft Academic Search

The emergence of antibiotic-resistant mutants among pathogenic bacteria has re-focused interest in alternative antibacterial treatments such as “phage therapy”, where viruses are harnessed to infect and destroy bacteria included in their host range. The first stage in bacteriophage multiplication, its adsorption to the bacterial cell surface, has not been accurately resolved before. Previous studies focused on very low phage-to-bacteria concentration

Yuval Zonenstein; Arieh Zaritsky; José Merchuk; Monica Einav; Giora Enden

2010-01-01

226

pH-Induced denaturation of proteins: A single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme  

SciTech Connect

The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pK{sub a} values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pK{sub a} value of 9.1 in the native state and 6.8 in the unfolded state at 10{degree}C in moderate salt. Similarly, the aspartate pK{sub a} is shifted to a value of about 0.5 in the native state from its value of 3.5-4.0 in the unfolded state. These shifts in pK{sub a} show that the salt bridge is stabilized 3-5 kcal/mol. These observations and consideration of the thermodynamic coupling of protonation state to folding of proteins suggest a mechanism of acid denaturation in which the unfolded state is progressively stabilized by protonation of its acid residues as pH is lowered below pH 4. The unfolded state is stabilized only if acidic groups in the folded state have lower pK{sub a} values than in the unfolded state. When the pH is sufficiently low, the acid groups of both the native and unfolded states are fully protonated, and the apparent unfolding equilibrium constant becomes pH independent. Similar arguments apply to base-induced unfolding. These observations suggest that the electrostatic contribution of each ionizable group to the stability of the folded state can be directly assessed by simply measuring its apparent pK{sub a} by NMR or other methods.

Anderson, D.E.; Becktel, W.J.; Dahlquist, F.W. (Univ. of Oregon, Eugene (USA))

1990-03-06

227

Effect of Freezing Conditions on Distances and Their Distributions Derived from Double Electron Electron Resonance (DEER): A Study of Doubly-Spin-Labeled T4 Lysozyme  

PubMed Central

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (?100 ?s) samples to those from commonly shock-frozen (slow freeze, 1s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

Georgieva, Elka R.; Roy, Aritro S.; Grigoryants, Vladimir M.; Borbat, Petr P.; Earle, Keith A.; Scholes, Charles P.; Freed, Jack H.

2012-01-01

228

Changes in major capsid genes ( g23 ) of T4-type bacteriophages with soil depth in two Japanese rice fields  

Microsoft Academic Search

Although microbial communities in soil are well known to change with soil depth, the changes in viral communities with soil\\u000a depth have not been documented. This study examined the soil depth profiles of T4-type phage communities in two Japanese rice\\u000a fields from g23 clones in soil DNA extracts to a depth of 1 m. T4-type phage communities changed with soil depth,

Guanghua Wang; Jun Murase; Katsutoshi Taki; Yoshinori Ohashi; Nanako Yoshikawa; Susumu Asakawa; Makoto Kimura

2009-01-01

229

The Cotransduction of pET System Plasmids by Mutants of T4 and RB43 Bacteriophages  

Microsoft Academic Search

The study of the cotransduction of the plasmid pairs pET-3a–pLysE and pET-3a–pLysS by the mutant phage T4alc7 showed that the antibiotic resistance markers of the plasmids were cotransduced with a high frequency. The analysis of the plasmid DNA of cotransductants and cotransformants showed that the mutant phage T4alc7 can be used for obtaining the monomeric and oligomeric forms of plasmids

V. I. Tanyashin; A. A. Zimin; A. M. Boronin

2003-01-01

230

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system  

Microsoft Academic Search

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA,

E. H. Radany; E. C. Friedberg

1982-01-01

231

In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli  

Microsoft Academic Search

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces.

Marietta Weiss; Emmanuel Denou; Anne Bruttin; Ruth Serra-Moreno; Marie-Lise Dillmann; Harald Brüssow

2009-01-01

232

FUNCTIONAL INTERACTIONS BETWEEN THE DNA LIGASE OF ESCHERZCHZA COLI AND COMPONENTS OF THE DNA METABOLIC APPARATUS OF T4 BACTERIOPHAGE  

Microsoft Academic Search

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 Zig 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

J. D. KARAM; M. LEACH; ND L. J. HEERE

233

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: Bacteriophage T4-infected Escherichia coli as a model system  

Microsoft Academic Search

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV\\/sup +\\/ or den V⁻ (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated

E. H. Radany; E. C. Friedberg

1982-01-01

234

The dynamic pause-unpackaging state, an off-translocation recovery state of a DNA packaging motor from bacteriophage T4.  

PubMed

Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ~170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor-DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the "pause-unpackaging" state is an off-translocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome. PMID:23169641

Kottadiel, Vishal I; Rao, Venigalla B; Chemla, Yann R

2012-11-19

235

A species barrier between bacteriophages T2 and T4: exclusion, join-copy and join-cut-copy recombination and mutagenesis in the dCTPase genes.  

PubMed Central

Bacteriophage T2 alleles are excluded in crosses between T2 and T4 because of genetic isolation between these two virus species. The severity of exclusion varies in different genes, with gene 56, encoding an essential dCT(D)Pase/dUT(D)Pase of these phages, being most strongly affected. To investigate reasons for such strong exclusion, we have (1) sequenced the T2 gene 56 and an adjacent region, (2) compared the sequence with the corresponding T4 DNA, (3) constructed chimeric phages in which T2 and T4 sequences of this region are recombined, and (4) tested complementation, recombination, and exclusion with gene 56 cloned in a plasmid and in the chimeric phages in Escherichia coli CR63, in which growth of wild-type T2 is not restricted by T4. Our results argue against a role of the dCTPase protein in this exclusion and implicate instead DNA sequence differences as major contributors to the apparent species barrier. This sequence divergence exhibits a remarkable pattern: a major heterologous sequence counter-clockwise from gene 56 (and downstream of the gene 56 transcripts) replaces in T2 DNA the T4 gene 69. Gene 56 base sequences bordering this substituted region are significantly different, whereas sequences of the dam genes, adjacent in the clockwise direction, are similar in T2 and in T4. The gene 56 sequence differences can best be explained by multiple compensating frameshifts and base substitutions, which result in T2 and T4 dCTPases whose amino acid sequences and functions remain similar. Based on these findings we propose a model for the evolution of multiple sequence differences concomitant with the substitution of an adjacent gene by foreign DNA: invasion by the single-stranded segments of foreign DNA, nucleated from a short DNA sequence that was complementary by chance, has triggered recombination-dependent replication by "join-copy" and "join-cut-copy" pathways that are known to operate in the T-even phages and are implicated in other organisms as well. This invasion, accompanied by heteroduplex formation between partially similar sequences, and perhaps subsequent partial heteroduplex repair, simultaneously substituted T4 gene 69 for foreign sequences and scrambled the sequence of the dCTPase gene 56. We suggest that similar mechanisms can mobilize DNA segments for horizontal transfer without necessarily requiring transposase or site-specific recombination functions.

Gary, T P; Colowick, N E; Mosig, G

1998-01-01

236

Interaction of Escherichia coli B and B/4 and Bacteriophage T4D with Berea Sandstone Rock in Relation to Enhanced Oil Recovery.  

PubMed

Much research and development is needed to recover oil reserves presently unattainable, and microbially enhanced oil recovery is a technology that may be used for this purpose. To address the problem of bacterial contamination in an oil field injection well region, we connected each end of a Teflon-sleeved Berea sandstone rock to a flask containing nutrient medium. By inoculating one flask with Escherichia coli B, we could observe bacterial growth in the uninoculated flask resulting from the transport and establishment of cells across the rock. Differences in bacterial populations occurred depending on whether bacteriophage T4D was first adsorbed to the rock. The results of these experiments indicate that the inhibition of bacterial establishment within a rock matrix is possible via lytic interaction. Some nonlytic effects are also implied by experiments with B/4 cells, which are T4D-resistant mutants of E. coli B. A 10 to 40% retention of T4 by the rock occurred when it was loaded with 10 to 10 PFU. We also describe a lysogenic system for possible use in microbially enhanced oil recovery techniques. PMID:16346492

Chang, P L; Yen, T F

1984-03-01

237

Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the ?-subunit flap domain of Escherichia coli RNA polymerase.  

PubMed

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNA-mounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 ?-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the ?-flap domain. Like domain 4 of the promoter specificity ? factor (?(4)), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the ?-flap domain. Nevertheless, gp33 and ?(4) are not structurally related. The gp33/?-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex. PMID:22135460

Twist, Kelly-Anne F; Campbell, Elizabeth A; Deighan, Padraig; Nechaev, Sergei; Jain, Vikas; Geiduschek, E Peter; Hochschild, Ann; Darst, Seth A

2011-12-01

238

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas  

Microsoft Academic Search

Upon infection of Escherichia coli with bormodeoxyuridine-labeled T4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by

S. Ling; H. H. Vogelbacker; L. L. Restifo; T. Mattson; A. W. Kozinski

1981-01-01

239

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas  

Microsoft Academic Search

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by

S. K. Ling; H. H. Vogelbacker; L. L. Restifo; T. Mattson; A. W. Kozinski

1981-01-01

240

Bacteriophage T4 mutants which propagate on E. coli K12 but not on E. coli B  

Microsoft Academic Search

Summary We have isolated and characterized 2 mutants of coliphage T4 which are able to propagate on E. coli K12 but not on E. coli B. We have assigned the mutations to genes 8 and 53, both structural genes. The products of genes 8 and 53 are found in the baseplate.

C. Georgopoulos; M. Georgiou; G. Selzer; H. Eisen

1977-01-01

241

In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli.  

PubMed

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces. Compared to germ-free control animals, oral T4 phage led to a 300-fold higher fecal phage titer in mice mono-colonized with E. coli strain WG-5. The in vivo T4 phage replication was transient and reached peak fecal titers about 8 h after oral phage application followed by a rapid titer decrease over two days. Similar data were obtained in mice colonized with E. coli strain Nissle. In contrast, orally applied T7 phage experienced a massive and sustained in vivo replication in mice mono-colonized with E. coli strain WG-5 irrespective whether phage or E. coli host was applied first. T7 phage replication occurred mainly in the large intestine. High titers of T7 phage and high E. coli cell counts coexisted in the feces. The observation of only 20% T7 phage-resistant fecal E. coli colonies suggests a refuge model where phage-sensitive E. coli cells are physically or physiologically protected from phage infection in the gut. The difference between T7 and T4 with respect to gut replication might partly reflect their distinct in vitro capacity to replicate on slowly growing cells. PMID:19699505

Weiss, Marietta; Denou, Emmanuel; Bruttin, Anne; Serra-Moreno, Ruth; Dillmann, Marie-Lise; Brüssow, Harald

2009-08-21

242

Role of primary photoacceptors in low-power laser effects: action of He-Ne laser radiation on bacteriophage T4-Escherichia coli interaction  

SciTech Connect

The effect of He-Ne laser radiation (lambda = 632.8 nm) on bacteriophage T4-Escherichia coli WP2 interactions was studied. Irradiation of bacteria having respiratory chain components as primary photoacceptors accelerated their division in a dose-dependent manner, but irradiation had no effect on the properties of the phage (measured as its ability to infect host cells). At the same time, exposure of bacteria to stimulating doses of He-Ne laser radiation (from 10(3) to 6 x 10(4) J/m2) increased their ability to promote the growth of unexposed phages. These results clearly indicate that low-power laser effects require primary photoacceptors (phage contains no chromophores for red light).

Tiphlova, O.; Karu, T.

1989-01-01

243

Localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate with colloidal gold: F(ab)2 and undecagold: Fab' conjugates.  

PubMed

We report the localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate. Proceeding on the assumption that these proteins occupy discrete locations, we have decorated baseplates and tails with immunological probes. Using 5 nm diameter colloidal gold: F(ab')2 conjugates, we show that proteins gp7 and gp10 are located directly at the vertex, with gp10 positioned in the pin directly below gp7. gp8 is located beside gp7 towards the centre of the baseplate. Using a novel undecagold: Fab' conjugate we have also determined the radial positions of gp7 and gp8 in baseplates that have transformed to stars. A mechanism for the nature of the hexagon-to-star transformation is proposed. PMID:2254933

Watts, N R; Hainfeld, J; Coombs, D H

1990-11-20

244

Interaction of Escherichia coli B and B/4 and bacteriophage T4D with Berea sandstone rock in relation to enhanced oil recovery  

SciTech Connect

Much research and development is needed to recover oil reserves presently unattainable, and microbially enhanced oil recovery is a technology that may be used for this purpose. To address the problem of bacterial contamination in an oil field injection well region, the authors connected each end of a Teflon-sleeved Berea sandstone rock to a flask containing nutrient medium. By inoculating one flask with Escherichia coli B, they could observe bacterial growth in the uninoculated flask resulting from the transport and establishment of cells across the rock. Differences in bacterial populations occurred depending on whether bacteriophage T4D was first adsorbed to the rock. Also described is a lysogenic system for possible use in microbially enhanced oil recovery techniques. 23 references.

Chang, P.L.; Yen, T.F.

1984-03-01

245

gpwac of the T4-Type Bacteriophages: Structure, Function, and Evolution of a Segmented Coiled-Coil Protein That Controls Viral Infectivity  

PubMed Central

The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the ?50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.

Letarov, A.; Manival, X.; Desplats, C.; Krisch, H. M.

2005-01-01

246

Effect of bacterial host repair systems on the viability of hydroxylamine and methyl methanesulfonate treated T4 and ? bacteriophages  

Microsoft Academic Search

Survival of HA or MMS-treated T4 and lambda phages was estimated in bacterial cells differing in their ability to repair DNA. It has been found that the mismatch repair system of the bacterial host, which involvesmutSmutRmutLuvrE anddam loci, does not excise, or does so to only a limited extent, the nonpaired bases from DNA of HA or MMS-treated phages. Mutation

Celina Janion

1982-01-01

247

Structure of Bacteriophage T4 Endonuclease II Mutant E118A, a Tetrameric GIY-YIG Enzyme  

Microsoft Academic Search

Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides

C. Evalena Andersson; Pernilla Lagerbäck; Karin Carlson

2010-01-01

248

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas  

SciTech Connect

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.

Ling, S.K.; Vogelbacker, H.H.; Restifo, L.L.; Mattson, T.; Kozinski, A.W.

1981-11-01

249

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas  

SciTech Connect

Upon infection of Escherichia coli with bormodeoxyuridine-labeled T4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, non-bromo-deoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.

Ling, S. (Univ. of Pennsylvania School of Medicine, Philadelphia); Vogelbacker, H.H.; Restifo, L.L.; Mattson, T.; Kozinski, A.W.

1981-11-01

250

Bacteriophage T4-encoded Stp can be replaced as activator of anticodon nuclease by a normal host cell metabolite.  

PubMed

The bacterial tRNALys-specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection. PMID:14507369

Amitsur, Michal; Benjamin, Sima; Rosner, Rachel; Chapman-Shimshoni, Daphne; Meidler, Roberto; Blanga, Shani; Kaufmann, Gabriel

2003-10-01

251

Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase.  

PubMed Central

The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.

Belanger, K G; Mirzayan, C; Kreuzer, H E; Alberts, B M; Kreuzer, K N

1996-01-01

252

The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain : the 'double wing' motif.  

SciTech Connect

MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters. We have determined the crystal structure of the C-terminal DNA-binding domain of MotA (MotCF) to 1.6 A resolution using multiwavelength, anomalous diffraction methods. The structure reveals a novel DNA-binding alpha/beta motif that contains an exposed beta-sheet surface that mediates interactions with the DNA. Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves. We present a model of the interaction in which beta-ribbons at opposite corners of the six-stranded beta-sheet penetrate the DNA grooves, and call the motif a 'double wing' to emphasize similarities to the 'winged-helix' motif. The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non-specific multimers on DNA.

Li, N.; Sickmier, E. A.; Zhang, R.; Joachimiak, A.; White, S. W.; Biosciences Division; St. Jude Children's Research Hospital; Univ. of Tennessee Health Science Center; Corixa Inc.

2002-01-01

253

The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging.  

PubMed

Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal-terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279-316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase-portal orientation fits better to a proposed "DNA crunching" compression packaging motor and to portal determined DNA headful cutting. PMID:24074593

Dixit, Aparna Banerjee; Ray, Krishanu; Thomas, Julie A; Black, Lindsay W

2013-09-07

254

Expression of cloned bacteriophage T4 uvsW and uvsY genes in rec+ and rec- Escherichia coli  

SciTech Connect

Chimeric plasmids containing the uvsY uvsW region of the T4 genome were examined for the expression of these genes. Certain of these plasmids were shown to express the uvsY or the uvsW gene products by their ability to complement the UV sensitivity of infecting uvsW or uvsY mutant phage. Also, a chimeric plasmid containing both the uvsW and uvsY genes increases the survival of UV-irradiated, methyl methane sulfonate- or ethyl methane sulfonate-treated recA hosts.

DeVries, J.K.; Wallace, S.S.

1983-09-01

255

Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines  

PubMed Central

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal ?-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the ?-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.

Tao, Pan; Mahalingam, Marthandan; Kirtley, Michelle L.; van Lier, Christina J.; Sha, Jian; Yeager, Linsey A.; Chopra, Ashok K.; Rao, Venigalla B.

2013-01-01

256

Optical tweezers studies of viral DNA packaging: Motor function and DNA confinement in Bacteriophages phi29, lambda, and T4  

NASA Astrophysics Data System (ADS)

In the assembly of many viruses a powerful molecular motor translocates the genome into a pre-assembled capsid. We use optical tweezers to directly measure translocation of a single DNA molecule into the viral capsid. Improved techniques allow us to measure initiation and early stages of packaging. With phi29 the DNA terminal protein was found to cause large variations in the starting point of packaging. Removal of this protein results in terminal initiation, permitting more accurate assessment of motor function and DNA confinement forces. We investigated the role of electrostatic repulsion by varying ionic screening of the DNA. The observed trends are in accord with those theoretically expected considering counter-ion competition; however the forces are larger than expected in comparison with recent theories and DNA ejection measurements. We have recently succeeded in extending our methods to study two other phages: lambda and T4. These systems have unique structural and functional features, presenting an opportunity for comparative studies in this family of molecular motors. Initial measurements show that lambda and T4 translocate DNA several times faster than the phi29 motor, but are more sensitive to applied load.

Smith, Douglas

2007-03-01

257

An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.  

PubMed Central

Numerous reports describe the phage T4 enzymes thymidylate synthase and dihydrofolate reductase as structural components of the baseplate. However, Y. Wang and C. K. Mathews (J. Virol. 63:4736-4743, 1989) reported that antisera against the respective recombinant enzymes failed to neutralize phage infectivity, in contrast to previous results. Moreover, a deletion mutant lacking the genes for these two enzymes adsorbed normally to host cells. Since these findings tended to undermine the idea of the two enzymes as structural proteins, we developed a quantitative immunoblot assay to resolve the issue directly. Our results show that both enzymes are present only as minor contaminants (< 0.05 copy per phage) and as such cannot be bona fide structural proteins.

Chen, X; Mathews, C K; Wheeler, L J; Maley, G; Maley, F; Coombs, D H

1995-01-01

258

Dmd of bacteriophage T4 functions as an antitoxin against Escherichia coli LsoA and RnlA toxins.  

PubMed

Enterohaemorrhagic Escherichia coli O157:H7 harbours a cryptic plasmid, pOSAK1, that carries only three ORFs: mobA (involved in plasmid mobilization), ORF1 and ORF2. Predicted proteins encoded by these two ORFs were found to share a weak homology with RnlA and RnlB, respectively, a toxin–antitoxin system encoded on the E. coli K-12 chromosome. Here, we report that lsoA (ORF1) encodes a toxin and lsoB (ORF2) an antitoxin. In spite of the homologies, RnlB and LsoB functioned as antitoxins against only their cognate toxins and not interchangeably with each other. Interestingly, T4 phage Dmd suppressed the toxicities of both RnlA and LsoA by direct interaction, the first example of a phage with an antitoxin against multiple toxins. PMID:22403819

Otsuka, Yuichi; Yonesaki, Tetsuro

2012-02-01

259

A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex.  

PubMed

Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks "internally" labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome-DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)(6) complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA-protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies. PMID:23578280

Lee, Wonbae; Jose, Davis; Phelps, Carey; Marcus, Andrew H; von Hippel, Peter H

2013-04-24

260

Structure of bacteriophage T4 endonuclease II mutant E118A, a tetrameric GIY-YIG enzyme.  

PubMed

Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P2(1) with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context. PMID:20156453

Andersson, C Evalena; Lagerbäck, Pernilla; Carlson, Karin

2010-02-13

261

Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences  

Microsoft Academic Search

physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent

Umender K. Sharma; Dipankar Chatterji

2008-01-01

262

Portal-large terminase interactions of the bacteriophage T4 DNA packaging machine implicate a molecular lever mechanism for coupling ATPase to DNA translocation.  

PubMed

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation. PMID:22345478

Hegde, Shylaja; Padilla-Sanchez, Victor; Draper, Bonnie; Rao, Venigalla B

2012-02-15

263

Regulation of translation of the head protein of T4 bacteriophage by specific binding of EF-Tu to a leader sequence.  

PubMed

Recent evidence indicates that translation elongation factor Tu (EF-Tu) has a role in the cell in addition to its well established role in translation. The translation factor binds to a specific region called the Gol region close to the N terminus of the T4 bacteriophage major head protein as the head protein emerges from the ribosome. This binding was discovered because EF-Tu bound to Gol peptide is the specific substrate of the Lit protease that cleaves the EF-Tu between amino acid residues Gly59 and lle60, blocking phage development. These experiments raised the question of why the Gol region of the incipient head protein binds to EF-Tu, as binding to incipient proteins is not expected from the canonical role of EF-Tu. Here, we use gol-lacZ translational fusions to show that cleavage of EF-Tu in the complex with Gol peptide can block translation of a lacZ reporter gene fused translationally downstream of the Gol peptide that activated the cleavage. We propose a model to explain how binding of EF-Tu to the emerging Gol peptide could cause translation to pause temporarily and allow time for the leader polypeptide to bind to the GroEL chaperonin before translation continues, allowing cotranslation of the head protein with its insertion into the GroEL chaperonin chamber, and preventing premature synthesis and precipitation of the head protein. Cleavage of EF-Tu in the complex would block translation of the head protein and therefore development of the infecting phage. Experiments are presented that confirm two predictions of this model. Considering the evolutionary conservation of the components of this system, this novel regulatory mechanism could be used in other situations, both in bacteria and eukaryotes, where proteins are cotranslated with their insertion into cellular structures. PMID:14623179

Snyder, Larry; Blight, Sherry; Auchtung, Jennifer

2003-11-28

264

Double-strand break repair and recombination-dependent replication of DNA in bacteriophage T4 in the absence of UvsX recombinase: replicative resolution pathway.  

PubMed

The effects of mutations in bacteriophage T4 genes uvsX and 49 on the double-strand break (DSB)-promoted recombination were studied in crosses, in which DSBs were induced site-specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i×ets1 and in three-factor crosses of the type i×ets1 a6, where ets1 is an insertion in the rIIB gene carrying the cleavage site for SegC; i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site, and a6 is rIIA point mutation located at 2040 bp from ets1. The frequency/distance relationships were obtained in crosses of the wild-type phage and of the amber mutant S17 (gene uvsX) and the double mutant S17 E727 (genes uvsX and 49). These data provide information about the frequency and distance distribution of the single-exchange (splices) and double-exchange (patches) events. The extended variant of the splice/patch coupling (SPC) model of recombination, which includes transition to the replication resolution (RR) alternative is substantiated and used for interpretation of the frequency/distance relationships. We conclude that the uvsX mutant executes recombination-dependent replication but does it by a qualitatively different way. In the absence of UvsX function, the DSB repair runs largely through the RR subpathway because of inability of the mutant to form a Holliday junction. In the two-factor crosses, the double uvsX 49- is recombinationally more proficient than the single uvsX mutant (partial suppression of the uvsX deficiency), while the patch-related double exchanges are virtually eliminated in this background. PMID:22365497

Shcherbakov, Victor P; Plugina, Lidia; Shcherbakova, Tamara; Kudryashova, Elena; Sizova, Svetlana

2012-02-23

265

Photocatalytic antimicrobial activity of thin surface films of TiO 2 , CuO and TiO 2 \\/CuO dual layers on Escherichia coli and bacteriophage T4  

Microsoft Academic Search

TiO2-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with\\u000a thin films of TiO2, CuO and hybrid CuO\\/TiO2 prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO2 prepared by a sol–gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of\\u000a viruses. The chemical oxidising activity was also

Iram B. Ditta; Alex Steele; Christopher Liptrot; Julie Tobin; Helen Tyler; Heather M. Yates; David W. Sheel; Howard A. Foster

2008-01-01

266

Inhibition of gamma-Ray-induced Degradation of E. coli Bs1 DNA by Infection with T1, T2 and T4 Bacteriophage  

Microsoft Academic Search

THE breakdown of DNA in bacteria in response to ionizing radiation has received much attention1-7. This breakdown is mediated by an enzyme or system of enzymes which has not yet been fully characterized. We have investigated the effect of DNA degradation on parental DNA of T1 bacteriophage within the infected cell in radiation conditions which usually break down host DNA.

J. D. Chapman; J. Swez; E. C. Pollard

1968-01-01

267

Bacteriophage T4 nanoparticle capsid surface SOC and HOC bipartite display with enhanced classical swine fever virus immunogenicity: a powerful immunological approach.  

PubMed

The phage T4 HOC, SOC bipartite display system is attractive for the expression of cDNA and display of peptides or proteins at high copy numbers on the phage capsid surface. Until recently, using T4 phage vector to display large foreign molecular immunogens resulted only from either an SOC or HOC single site. In this report, the main advantages of the phage T4 system over other display technologies are substantiated by using the phage T4 SOC, HOC dual site display vector T4-Zh(-) to express: (1) on the SOC site, the classical swine fever virus (CSFV) major antigenic determinant cluster mE2 (123 amino acid, aa) through gene fusion to the SOC gene C-terminus of T4 genome, and (2) on the HOC site, full-length CSFV primary antigen E2 (371 aa) through a co-transformed plasmid, hence leading to a simultaneous display of both proteins on the T4 capsid surface. The immunogenicities of these constructs were measured by ID-ELISA, dot-ELISA, Western blotting, and immunogenic response in mice including humoral and cellular immunity tests. The immunological efficiencies both in vitro and in mice of phage T4 with both single site and dual site displays, as well as conventional Escherichia coli plasmid expression, were evaluated. The animal immune response data showed that the antibody titers elicited by the T4 phage-CSFV recombinants were significantly higher than those obtained by E. coli plasmid expression, and the unpurified double site display T4 phage particles were around two times higher than either single site display or plasmid expression while being at lower phage concentrations than the single site phages. The immunogens were effective in the absence of eukaryotic protein modifications. Therefore, the phage T4 dual site display emerges as a powerful method with an enhanced immune response in animals for research and development of immunological products. PMID:17081627

Wu, Jianmin; Tu, Changchun; Yu, Xinlong; Zhang, Maolin; Zhang, Nianzu; Zhao, Minyi; Nie, Wenxian; Ren, Zhaojun

2006-11-01

268

The non-enzymatic microbicidal activity of lysozymes  

Microsoft Academic Search

T4 lysozyme was thought to destroy bacteria by its muramidase activity. However, we demonstrate here that amphipathic helix stretches in the C-terminus of T4 lysozyme mediate its bactericidal and fungistatic activities. In heat-denatured T4 lysozyme, the enzymatic activity is completely abolished but unexpectedly, the antimicrobial functions remain preserved. Small synthetic peptides corresponding to amphipathic C-terminal domains of T4 lysozyme show

Petra Porsch; Andreas Mahn; Olaf Brinkmann; Werner Gieffers

1999-01-01

269

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins  

SciTech Connect

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

Shivachandra, Sathish B. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Rao, Mangala [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Janosi, Laszlo [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Sathaliyawala, Taheri [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States); Matyas, Gary R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Alving, Carl R. [Division of Retrovirology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 (United States); Leppla, Stephen H. [Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, NIH, 30 Convent Dr., Bethesda, MD 20892 (United States); Rao, Venigalla B. [Department of Biology, 103 McCort Ward Hall, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064 (United States)]. E-mail: rao@cua.edu

2006-02-05

270

Restoration of Ultraviolet-Induced Unscheduled DNA Synthesis of Xeroderma Pigmentosum Cells by the Concomitant Treatment with Bacteriophage T4 Endonuclease V and HVJ (Sendai Virus)  

Microsoft Academic Search

Ultraviolet (UV)-induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and UV-inactivated HVJ (Sendai virus). The present results suggest that (1) T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ

Kiyoji Tanaka; Mutsuo Sekiguchi; Yoshio Okada

1975-01-01

271

A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA(N6-adenine)-methyltransferase  

Microsoft Academic Search

The fluorescence of 2-aminopurine (2A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6- adenine)-methyltransferase. With an unmethylated target (2A\\/A duplex) or its methylated derivative (2A\\/mA duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with 2A being flipped out of the DNA helix. Though neither S-adenosyl-L-homo- cysteine nor sinefungin had

Ernst G. Malygin; Alexey A. Evdokimov; Victor V. Zinoviev; Lidiya G. Ovechkina; William M. Lindstrom; Norbert O. Reich; L. Schlagman

272

Differential mechanisms of binding of anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA to E. coli RNA polymerase lead to diverse physiological consequences.  

PubMed

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to sigma(70) with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70). PMID:18359804

Sharma, Umender K; Chatterji, Dipankar

2008-03-21

273

Isolation and Characterization of Two Basic Internal Proteins from the T-Even Bacteriophages 1  

PubMed Central

Two species of basic internal proteins were found in osmotic shock supernatant solutions of bacteriophages T4B, T4D, T2H, T2L, and T6. The major species of protein isolated had a molecular weight of approximately 21,000 daltons, whereas the minor protein molecular weight was near 9,500 daltons. The two protein species exhibited unique isoelectric points and amino acid compositions. The 21,000-dalton protein of T2L showed major electrophoretic and compositional differences from the other 21,000-dalton proteins isolated. Similarities between the 21,000-dalton proteins and phage lysozyme are discussed. Images

Stone, Kenneth R.; Cummings, Donald J.

1970-01-01

274

Genetic studies on capsid-length determination in bacteriophage T4. I. Isolation and partial characterization of second-site revertants of a gene 23 mutation affecting capsid length.  

PubMed Central

The T4 mutation ptg19-80 affects the mechanism of capsid-length determination. It is located in gene 23, which encodes the major structural protein of the capsid. The mutation results in the production of abnormal-length capsids in high frequencies. This paper describes the isolation and partial characterization of second-site revertants of ptg19-80. In the course of their analysis, it was discovered that ptg19-80 is itself a double mutation consisting of a gene 23 mutation (ptg19-80c), which causes the morphogenetic defect, and a suppressor mutation located near the lysozyme gene. Phenotypic characterization of nine pseudo-wild-type revertants of this double-mutation revealed that these revertants all produced lower frequencies of abnormal capsids than did ptg19-80. Seven of these revertants were shown to contain two suppressor mutations, one mapping in or near gene 22 and done mapping in or near gene 24. Both mutations were required for suppression. These suppressors displayed no discernible phenotype in the absence of ptg19-80c. Images

Doherty, D H

1982-01-01

275

Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157:H7 levels in sheep guts  

PubMed Central

In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genome?120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5. Unlike T5, CEV2 infects O157:H7 in vitro, both aerobically and anaerobically, rapidly adsorbing and killing, but resistant mutants regrew within 24 h. When used together with T4-like CEV1 (MOI ?2 per phage), bacterial killing was longer lasting. CEV2 did not reproduce when co-infecting the same cell as CEV1, presumably succumbing to CEV1's ability to shut off transcription of cytosine-containing DNA. In vivo sheep trials to remove resident O157:H7 showed that a cocktail of CEV2 and CEV1 (?1011 total PFU) applied once orally was more effective (>99.9% reduction) than CEV1 alone (?99%) compared to the untreated phage-free control. Those sheep naturally carrying CEV2, receiving no additional phage treatment, had the lowest O157:H7 levels (?99.99% reduction). These data suggest that phage cocktails are more effective than individual phage in removing O157:H7 that have taken residence if the phage work in concert with one another and that naturally resident O157:H7-infecting phages may prevent O157:H7 gut colonization and be one explanation for the transient O157:H7 colonization in ruminants.

Raya, Raul R; Oot, Rebecca A; Moore-Maley, Ben; Wieland, Serena; Callaway, Todd R; Kutter, Elizabeth M

2011-01-01

276

Mibt and Bacteriophage Multiplication.  

National Technical Information Service (NTIS)

N-methyl isatin-beta-thiosemicarbazone, a known inhibitor of pox-virus in vitro and in vivo, is without effect upon the virulent bacteriophage T4, T4B, T4Bo1, or the temperate phage lambda grown in E. coli. At concentrations which show striking anti-viral...

D. Norman M. De La Cruz

1968-01-01

277

Assembly and dynamics of Gp59-Gp32-single-stranded DNA (ssDNA), a DNA helicase loading complex required for recombination-dependent replication in bacteriophage T4.  

PubMed

The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks. PMID:22500043

Branagan, Amy M; Maher, Robyn L; Morrical, Scott W

2012-04-12

278

Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: identification of phenylalanine-183 as the site of crosslinking  

SciTech Connect

Using ultraviolet light, both the 33,000-dalton single-stranded DNA-binding protein from T4 bacteriophage (gp32) as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:p(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.

Shamoo, Y.; Williams, K.R.; Konigsberg, W.H.

1988-01-01

279

Cloning and expression of T4 DNA polymerase.  

PubMed Central

The structural gene coding for bacteriophage T4 DNA polymerase (gene 43) has been cloned into inducible plasmid vectors, which provide a source for obtaining large amounts of this enzyme after induction. The T4 DNA polymerase produced in this fashion was purified by an innovative three-step procedure and was fully active. Images

Lin, T C; Rush, J; Spicer, E K; Konigsberg, W H

1987-01-01

280

Isolation of Lactococcus lactis Mutants Simultaneously Resistant to the Cell Wall-Active Bacteriocin Lcn972, Lysozyme, Nisin, and Bacteriophage c2  

PubMed Central

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits cell wall biosynthesis by binding to lipid II. In this work, two mutants resistant to Lcn972, Lactococcus lactis D1 and D1-20, with high (>320 arbitrary units [AU]/ml) and low (80 AU/ml) susceptibilities, respectively, have been isolated. Resistance to Lcn972 did not impose a burden to growth under laboratory conditions, nor did it substantially alter the physicochemical properties of the cell surface. However, the peptidoglycan of the mutants featured a higher content of muropeptides with tripeptide side chains than the wild-type strain, linking for the first time peptidoglycan remodelling to bacteriocin resistance. Moreover, L. lactis lacking a functional d,d-carboxypeptidase DacA (i.e., with a high content of pentapeptide side chain muropeptides) was shown to be more susceptible to Lcn972. Cross-resistance to lysozyme and nisin and enhanced susceptibility to penicillin G and bacitracin was also observed. Intriguingly, the Lcn972-resistant mutants were not infected by the lytic phage c2 and less efficiently infected by phage sk1. Lack of c2 infectivity was linked to a 22.6-kbp chromosomal deletion encompassing the phage receptor protein gene pip. The deletion also included maltose metabolic genes and the two-component system (TCS) F. However, a clear correlation between these genes and resistance to Lcn972 could not be clearly established, pointing to the presence of as-yet-unidentified mutations that account for Lcn972 resistance.

Roces, Clara; Courtin, Pascal; Kulakauskas, Saulius; Rodriguez, Ana; Chapot-Chartier, Marie-Pierre

2012-01-01

281

Receptor-recognizing proteins of T-even type bacteriophages. The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb.  

PubMed

Escherichia coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers. Virions of the T2 family use protein 38 (which is attached to the free end of protein 37) for this purpose. The corresponding areas of genes 37 belonging to TuIa and TuIb were cloned and sequenced. Comparison of the deduced protein primary structures, including those of T4 and lambda Stf (Stf most likely representing a subunit of the side tail fibers of phage lambda) showed that an area of 70 to 100 residues is characterized by very variable sequences, while the sequences of the adjacent 43 to 44 C-terminal residues as well as those upstream from the variable region are highly homologous. The variable regions are flanked and interrupted seven or eight times by the motif His-x-His-y, with x and y most often being Ser or Thr; furthermore, the locations of these repeated tetrapeptides are conserved. Using hybrid phages obtained by recombination of one phage with cloned fragments of gene 37 of another, it could be shown that the area of this gene encoding receptor specificity includes the variable area. The situation is analogous to the known receptor-recognizing region of proteins 38 belonging to the T2-type family, except that the repeating sequence is of a different nature. In T4, receptor specificity is coded for by 382 base-pairs of the 3'-end of the gene, starting exactly at the variable area. It was found that T4 can use the outer membrane protein OmpC or lipopolysaccharide as receptors with the same efficiency, and it is proposed that the 70 residues of the variable part of the protein serve to bind to both ligands. PMID:2147721

Montag, D; Hashemolhosseini, S; Henning, U

1990-11-20

282

YfiD of Escherichia coli and Y06I of Bacteriophage T4 as Autonomous Glycyl Radical Cofactors Reconstituting the Catalytic Center of Oxygen-Fragmented Pyruvate Formate-Lyase  

Microsoft Academic Search

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N–C? of Gly734. A recombinant protein comprising the core of PFL (Ser1–Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase

A. F. Volker Wagner; Sabine Schultz; Jörg Bomke; Thomas Pils; Wolf D. Lehmann; Joachim Knappe

2001-01-01

283

Endonucleolytic Cleavage of Uv-Irradiated DNA Controlled by the V(+) Gene in Phage T4.  

National Technical Information Service (NTIS)

Extracts of E. coli infected with bacteriophage T4 contain a phage-induced UV-specific endonucleolytic activity. This activity is absent from extracts of cells infected with the UV-sensitive mutant T4V1, but is present in another UV-sensitive mutant T4X. ...

E. C. Friedberg J. J. King

1969-01-01

284

Synchronized disruption of Escherichia coli cells by T4 phage infection  

Microsoft Academic Search

For development of an autolytic Escherichia coli protein expression system, T4 bacteriophage (T4)-mediated E. coli disruption was investigated. At least two types of E. coli cell lysis, “lysis from without” (LO) and “lysis from within” (LI), are known to be induced by T4. The efficiency of cell disruption was monitored by the release of ?-galactosidase from the cells. In the

Kazuhiro Asami; Xin-Hui Xing; Yasunori Tanji; Hajime Unno

1997-01-01

285

Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.  

PubMed Central

Model-free approaches (random mutagenesis, DNA shuffling) in combination with more "rational," three-dimensional information-guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage lambda, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque-forming ability. Compensating mutations in W138P T4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid lambda phage with sufficient lysozyme activity to produce normal-size plaques, even at elevated temperature (42 degrees C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target-randomized variants.

Jucovic, M.; Poteete, A. R.

1998-01-01

286

Molecular Structure of Lysozyme  

NSDL National Science Digital Library

Alexander Fleming discovered the antibiotic activity of lysozyme in 1922 when he dropped mucus into a culture of bacteria and noticed that the bacteria were killed. In humans, lysozyme is in the blood, mucus, tears and saliva. The mechanism by which lysozyme kills bacteria is by hydrolyzing the glycosidic bond of the bacterial cell wall, the protective outer coating of the organism. This causes cell lysis, hence the name lysozyme (zyme is for enzyme).

2003-01-23

287

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein.  

PubMed

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ?Ea1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ?Ea104 and ?Ea116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ?Ea1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ?Ea1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme. PMID:22456518

Müller, Ina; Gernold, Marina; Schneider, Bernd; Geider, Klaus

2012-03-27

288

Silicates as Nonspecific Adsorbents of Bacteriophage: a Model for Purification of Water from Viruses  

PubMed Central

Amorphous silicates having small particles with a large surface area were found to have high nonspecific adsorption capacity. Investigations with Escherichia coli T4 bacteriophage have shown adsorption capacity of up to 99.9% on different tested silicates. Optimal conditions for the adsorption of T4 bacteriophage on various commercially available silicates were determined. A continuous-flow adsorption of T4 bacteriophage on silicates appears to be a model system for the removal of viruses from potable water and sewage. Images

Fass, Refael; Straussman, Yochevet; Shahar, Abraham; Mizrahi, Avshalom

1980-01-01

289

Cleavage of T4Induced Proteins During Phage Morphogenesis: Characterization of Peptides  

Microsoft Academic Search

SUMMARY Polypeptides of low tool. wt. have been extracted from T4 coliphages and from Escherichia coli B cells infected with a wild type and various amber mutants of bacteriophage T4. Six peptides were fractionated by chromatography on phospho- cellulose: three of them were cleaved from proteins synthesized late in infection and related to phage head. The remaining three peptides have

L. Benchetrit; U. Bachrach

1976-01-01

290

Antimutator Mutants in Bacteriophage T4 and Escherichia coli  

Microsoft Academic Search

Antimutators are mutant strains that have reduced mutation rates compared to the corresponding wild- type strain. Their existence, along with mutator mutants that have higher mutation rates compared to the wild-type strain, are powerful evidence that mutation rates are genetically controlled. Compared to mutator mutants, antimutators have a very distinguishing property. Because they prevent normally oc- curring mutations, they, uniquely,

Roel M. Schaaper

291

Transcriptional control in the prereplicative phase of T4 development.  

PubMed

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two ? subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of ???, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of ???, which then allows the T4 activator MotA to also interact with ???. In addition, AsiA restructuring of ??? prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity. PMID:21029433

Hinton, Deborah M

2010-10-28

292

Transcriptional control in the prereplicative phase of T4 development  

PubMed Central

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two ? subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of ?70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of ?70, which then allows the T4 activator MotA to also interact with ?70. In addition, AsiA restructuring of ?70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity.

2010-01-01

293

Nanoscale bacteriophage biosensors beyond phage display  

PubMed Central

Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

2013-01-01

294

Plasticity of the Gene Functions for DNA Replication in the T4-like Phages  

Microsoft Academic Search

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter

Vasiliy M. Petrov; James M. Nolan; Claire Bertrand; Dawn Levy; Carine Desplats; H. M. Krisch; Jim D. Karam

2006-01-01

295

The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain  

PubMed Central

Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections.

Rodriguez-Rubio, Lorena; Gutierrez, Dolores; Martinez, Beatriz; Rodriguez, Ana; Gotz, Friedrich

2012-01-01

296

Bacteriophage Assembly  

PubMed Central

Bacteriophages have been a model system to study assembly processes for over half a century. Formation of infectious phage particles involves specific protein-protein and protein-nucleic acid interactions, as well as large conformational changes of assembly precursors. The sequence and molecular mechanisms of phage assembly have been elucidated by a variety of methods. Differences and similarities of assembly processes in several different groups of bacteriophages are discussed in this review. The general principles of phage assembly are applicable to many macromolecular complexes.

Aksyuk, Anastasia A.; Rossmann, Michael G.

2011-01-01

297

Bacterial genetic factors controlling the suppression of T4 phage Amber mutants  

Microsoft Academic Search

To examine the genetic factors of E. coli which determine suppression of the amber (am) mutant phenotype of T4 bacteriophage, a survey was made in which seventythree bacterial strains of diverse genetic backgrounds were tested for their abilities to support the growth of an ensemble of twenty-nine am mutants. The results of this survey were that on the basis of

Rebecca Hill Krieg; Gunther S. Stent

1968-01-01

298

Naturally occurring bacteriophages lyse a large proportion of canine and feline uropathogenic Escherichia coli isolates in vitro.  

PubMed

We investigated the feasibility of bacteriophage therapy to combat canine and feline Escherichia coli urinary tract infections (UTIs) by testing the in vitro lytic ability of 40 naturally occurring bacteriophages on 53 uropathogenic E. coli (UPEC). The mean number of UPEC strains lysed by an individual bacteriophage was 21/53 (40%, range 17-72%). In total, 50/53 (94%) of the UPEC strains were killed by one or more of the bacteriophages. Ten bacteriophages lysed 51% of UPEC strains individually and 92% of UPEC strains as a group. Electron microscopy and DNA sequencing of 5 'promising' bacteriophages revealed that 4 bacteriophages belonged to the lytic T4-like genus, while one displayed morphologic similarity to temperate P2-like bacteriophages. Overall, these results indicate that the majority of UPEC are susceptible to lysis by naturally occurring bacteriophages. Thus, bacteriophages show promise as therapeutic agents for treatment of canine and feline E. coli UTIs. PMID:17959211

Freitag, T; Squires, R A; Schmid, J

2007-10-23

299

Crystallization of Hevamine, an Enzyme with Lysozyme\\/Chitinase Activity from Hevea brasiliensis Latex  

Microsoft Academic Search

Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investigate the atomic details of the substrate specificity and the cause for hevamine’s low pH optimum (pH 4.0),

Bauke W. Dijkstra; Jaap J. Beintema; Asmini Budiani; Henriëtte J. Rozeboom

1990-01-01

300

Lysozymes in the animal kingdom  

Microsoft Academic Search

Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the ?-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme\\u000a types have been identified — the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic

Lien Callewaert; Chris W. Michiels

2010-01-01

301

Bacteriophage–bacteriophage interactions in the evolution of pathogenic bacteria  

Microsoft Academic Search

Many bacteriophages carry virulence genes encoding proteins that play a major role in bacterial pathogenesis. Recently, investigators have identified bacteriophage–bacteriophage interactions in the bacterial host cell that also contribute significantly to the virulence of bacterial pathogens. The relationships between the bacteriophages pertain to one bacteriophage providing a helper function for another, unrelated bacteriophage in the host cell. Accordingly, these interactions

E. Fidelma Boyd; Brigid M. Davis; Bianca Hochhut

2001-01-01

302

Chlamydia bacteriophages.  

PubMed

Phages are called "good viruses" due to their ability to infect and kill pathogenic bacteria. Chlamydia are small, Gram-negative (G-) microbes that can be dangerous to human and animals. In humans, these bacteria are etiological agents of diseases such as psittacosis or respiratory tract diseases, while in animals, the infection may result in enteritis in cattle and chronic bowel diseases, as well as miscarriages in sheep. The first-known representative of chlamydiaphages was Chp1. It was discovered in Chlamydia psittaci isolates. Since then, four more species of chlamydiaphages have been identified [Chp2, Chp3, ?CPG1 ?CPAR39 (?Cpn1) and Chp4]. All of them were shown to infect Chlamydia species. This paper described all known chlamydiaphages. They were characterised in terms of origin, host range, and their molecular structure. The review concerns the characterisation of bacteriophages that infects pathogenic and dangerous bacteria with unusual, intracellular life cycles that are pathogenic. In the era of antibiotic resistance, it is difficult to cure chlamydophilosis. Those bacteriophages can be an alternative to antibiotics, but before this happens, we need to get to know chlamydiaphages better. PMID:23903989

Sliwa-Dominiak, Joanna; Suszy?ska, Ewa; Pawlikowska, Ma?gorzata; Deptu?a, Wies?aw

2013-08-01

303

What's new in lysozyme research?  

Microsoft Academic Search

Summary The present review is focused on the main achievements realized in the lysozyme research field since the meeting held in 1972 to commemorate the fiftieth anniversary of the discovery of this enzyme. Despite of extensive structural, physico-chemical, crystallographic, genetic, immunological and evolutionary studies devoted to lysozymes, their biological role is still not exactly known.

Pierre Jollès; Jacqueline Jollès

1984-01-01

304

Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages  

SciTech Connect

Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a 'glue' between neighboring hexameric capsomers, forming a 'cage' that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 {angstrom} resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.

Qin, Li; Fokine, Andrei; O'Donnell, Erin; Rao, Venigalla B.; Rossmann, Michael G. (CUA); (Purdue)

2010-07-22

305

Structure of the Small Outer Capsid Protein, Soc: a Clamp for Stabilizing Capsids of T4-like Phages  

PubMed Central

Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9 kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as “glue” between neighboring hexameric capsomers, forming a “cage” that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 Å resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryo-electron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.

Qin, Li; Fokine, Andrei; O'Donnell, Erin; Rao, Venigalla B.; Rossmann, Michael G.

2009-01-01

306

Structural studies on metal-containing enzymes. T4 endonuclease VII and D. gigas formate dehydrogenase  

Microsoft Academic Search

Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease VII (EndoVII) and D. gigas fonnate dehydrogenase, which are studied by X-ray crystallography. The structure of EndoVII reveals how a

H. C. A. Raaijmakers

2001-01-01

307

DNA-Directed Synthesis in vitro of T4 Phage-Specific Enzymes  

Microsoft Academic Search

The synthesis of deoxynucleotide kinase (EC 2.7.4.2) in vitro by a preparation consisting of T4 bacteriophage DNA and a cell-free extract of Escherichia coli has been reported. A study of the role of monovalent cations in the synthesis of this enzyme as well as alpha -glucosyl transferase (EC 2.4.1.2) shows that potassium ions are required for maximal enzyme production. Examination

Peter J. Natale; John M. Buchanan

1972-01-01

308

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production  

PubMed Central

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1?, IL-1?, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-?, TNF-?, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.

Miernikiewicz, Paulina; Dabrowska, Krystyna; Piotrowicz, Agnieszka; Owczarek, Barbara; Wojas-Turek, Justyna; Kicielinska, Jagoda; Rossowska, Joanna; Pajtasz-Piasecka, Elzbieta; Hodyra, Katarzyna; Macegoniuk, Katarzyna; Rzewucka, Kamila; Kopciuch, Agnieszka; Majka, Tomasz; Letarov, Andrey; Kulikov, Eugene; Maciejewski, Henryk; Gorski, Andrzej

2013-01-01

309

Site-directed mutagenesis of the T4 endonuclease V gene: The role of arginine-3 in the target search  

Microsoft Academic Search

Endonuclease V, a pyrimidine dimer specific endonuclease in T4 bacteriophage, is able to scan DNA, recognize pyrimidine dimer photoproducts produced by exposure to ultraviolet light, and effectively incise DNA through a two-step mechanism at the damaged bases. The interaction of endonuclease V with nontarget DNA is thought to occur via electrostatic interactions between basic amino acids and the acidic phosphate

Diane R. Dowd; R. Stephen Lloyd

1989-01-01

310

COG3926 and COG5526: A tale of two new lysozyme-like protein families  

PubMed Central

We have identified two new lysozyme-like protein families by using a combination of sequence similarity searches, domain architecture analysis, and structural predictions. First, the P5 protein from bacteriophage ?8, which belongs to COG3926 and Pfam family DUF847, is predicted to have a new lysozyme-like domain. This assignment is consistent with the lytic function of P5 proteins observed in several related double-stranded RNA bacteriophages. Domain architecture analysis reveals two lysozyme-associated transmembrane modules (LATM1 and LATM2) in a few COG3926/DUF847 members. LATM2 is also present in two proteins containing a peptidoglycan binding domain (PGB) and an N-terminal region that corresponds to COG5526 with uncharacterized function. Second, structure prediction and sequence analysis suggest that COG5526 represents another new lysozyme-like family. Our analysis offers fold and active-site assignments for COG3926/DUF847 and COG5526. The predicted enzymatic activity is consistent with an experimental study on the zliS gene product from Zymomonas mobilis, suggesting that bacterial COG3926/DUF847 members might be activators of macromolecular secretion.

Pei, Jimin; Grishin, Nick V.

2005-01-01

311

Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples.  

PubMed Central

In this paper it is shown that conformation and packing of double-stranded DNA within the head of bacteriophages lambda and T4 can be assessed by cryo-electron microscopy of vitrified specimens. Electron diffraction patterns show that DNA within vitrified bacteriophages has a B conformation. Electron micrographs of vitrified bacteriophages show domains within the head formed by a approximately 2.5-nm striation and arising from the DNA packing. The number of differently oriented domains seen within a vitrified bacteriophage depends upon the geometry of the DNA container: the bacteriophage capsid. The packing of DNA within bacteriophages seems then to be governed by at least two phenomena. The first is the tendency of DNA to form local alignments (nematic liquid crystals). The second is the orientation of these liquid crystals by the bacteriophage capsid. From these observations we propose a possible packaging mechanism: constrained nematic crystallization. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5.

Lepault, J; Dubochet, J; Baschong, W; Kellenberger, E

1987-01-01

312

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli  

Microsoft Academic Search

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay.

O. Minikh; M. Tolba; L. Y. Brovko; M. W. Griffiths

2010-01-01

313

Protein determinants of phage T4 lysis inhibition.  

PubMed

Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ~20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ~80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition. PMID:22389108

Moussa, Samir H; Kuznetsov, Vladimir; Tran, Tram Anh T; Sacchettini, James C; Young, Ry

2012-03-02

314

THE CELL LYSIS ACTIVITY OF THE STREPTOCOCCUS AGALACTIAE BACTERIOPHAGE B30 ENDOLYSIN RELIES ON THE CHAP ENDOPEPTIDASE DOMAIN.  

Technology Transfer Automated Retrieval System (TEKTRAN)

The 443 amino acid Streptococcus agalactiae bacteriophage B30 endolysin gene contains a CHAP endopeptidase domain, an Acm lysozyme-like glycosidase, and a C-terminal SH3b cell wall binding domain. Although both hydrolase domains are enzymatically functional, it is unknown the degree to which each c...

315

Microneedle-mediated transdermal bacteriophage delivery.  

PubMed

Interest in bacteriophages as therapeutic agents has recently been reawakened. Parenteral delivery is the most routinely-employed method of administration. However, injection of phages has numerous disadvantages, such as the requirement of a health professional for administration and the possibility of cross-contamination. Transdermal delivery offers one potential means of overcoming many of these problems. The present study utilized a novel poly (carbonate) (PC) hollow microneedle (MN) device for the transdermal delivery of Escherichia coli-specific T4 bacteriophages both in vitro and in vivo. MN successfully achieved bacteriophage delivery in vitro across dermatomed and full thickness skin. A concentration of 2.67 × 10(6)PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 × 10(3)PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 × 10(3)PFU/ml being detected in blood 30 min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses. PMID:22750416

Ryan, Elizabeth; Garland, Martin J; Singh, Thakur Raghu Raj; Bambury, Eoin; O'Dea, John; Migalska, Katarzyna; Gorman, Sean P; McCarthy, Helen O; Gilmore, Brendan F; Donnelly, Ryan F

2012-06-30

316

Microneedle-mediated transdermal bacteriophage delivery  

PubMed Central

Interest in bacteriophages as therapeutic agents has recently been reawakened. Parenteral delivery is the most routinely-employed method of administration. However, injection of phages has numerous disadvantages, such as the requirement of a health professional for administration and the possibility of cross-contamination. Transdermal delivery offers one potential means of overcoming many of these problems. The present study utilized a novel poly (carbonate) (PC) hollow microneedle (MN) device for the transdermal delivery of Escherichia coli-specific T4 bacteriophages both in vitro and in vivo. MN successfully achieved bacteriophage delivery in vitro across dermatomed and full thickness skin. A concentration of 2.67 × 106 PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 × 103 PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 × 103 PFU/ml being detected in blood 30 min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24 h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses.

Ryan, Elizabeth; Garland, Martin J.; Singh, Thakur Raghu Raj; Bambury, Eoin; O'Dea, John; Migalska, Katarzyna; Gorman, Sean P.; McCarthy, Helen O.; Gilmore, Brendan F.; Donnelly, Ryan F.

2012-01-01

317

The tape measure protein of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has an active muramidase domain.  

PubMed

Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections. PMID:22729533

Rodríguez-Rubio, Lorena; Gutiérrez, Dolores; Martínez, Beatriz; Rodríguez, Ana; Götz, Friedrich; García, Pilar

2012-06-22

318

Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria  

PubMed Central

Bacteriophages offer interesting alternatives to antibodies for the specific capture and detection of pathogenic bacteria onto biosensing surfaces. Procedures for the optimal chemical immobilization of lytic bacteriophages onto surfaces are presented. More specifically, the removal of lysate contaminants from bacteriophage suspensions by size exclusion chromatography significantly increases the resultant planar surface density of immobilized bacteriophages. E. coli T4 and Salmonella enterica serovar Typhimurium P22 phage systems seem to undergo highly heterogeneous adsorption to the surface, possibly explaining the observed phage clustering at higher surface densities. The T4 phage and its E. coli host were initially employed as a model system where we discovered an optimal planar surface density of phages for best bacterial capture: 18.9 ± 0.8 phages/?m2 capturing 18.0 ± 0.3 bacteria/100 ?m2. Phage surface clustering ultimately limits the T4 phage-immobilized surface’s ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli reported using intact T4 bacteriophages. Two additional purified bacteriophage systems (P22 and Campylobacter jejuni phage NCTC 12673) were then similarly studied for their ability to capture their corresponding host bacteria (Salmonella enterica serovar Typhimurium and Campylobacter jejuni respectively) on a surface.

Naidoo, Ravendra; Singh, Amit; Arya, Sunil K.; Beadle, Bernadette; Glass, Nick; Tanha, Jamshid; Szymanski, Christine M.; Evoy, Stephane

2012-01-01

319

Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria.  

PubMed

Bacteriophages offer interesting alternatives to antibodies for the specific capture and detection of pathogenic bacteria onto biosensing surfaces. Procedures for the optimal chemical immobilization of lytic bacteriophages onto surfaces are presented. More specifically, the removal of lysate contaminants from bacteriophage suspensions by size exclusion chromatography significantly increases the resultant planar surface density of immobilized bacteriophages. E. coli T4 and Salmonella enterica serovar Typhimurium P22 phage systems seem to undergo highly heterogeneous adsorption to the surface, possibly explaining the observed phage clustering at higher surface densities. The T4 phage and its E. coli host were initially employed as a model system where we discovered an optimal planar surface density of phages for best bacterial capture: 18.9 ± 0.8 phages/?m(2) capturing 18.0 ± 0.3 bacteria/100 ?m(2). Phage surface clustering ultimately limits the T4 phage-immobilized surface's ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli reported using intact T4 bacteriophages. Two additional purified bacteriophage systems (P22 and Campylobacter jejuni phage NCTC 12673) were then similarly studied for their ability to capture their corresponding host bacteria (Salmonella enterica serovar Typhimurium and Campylobacter jejuni respectively) on a surface. PMID:22666653

Naidoo, Ravendra; Singh, Amit; Arya, Sunil K; Beadle, Bernadette; Glass, Nick; Tanha, Jamshid; Szymanski, Christine M; Evoy, Stephane

2012-01-01

320

Enzymatic properties of rhea lysozyme.  

PubMed

Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5-6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using beta-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53-65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B. PMID:15665474

Pooart, Jureerut; Torikata, Takao; Araki, Tomohiro

2005-01-01

321

In Vivo Inactivation of Lysozyme by Ozone  

PubMed Central

Bronchial mucus contains large quantities of the antibacterial enzyme lysozyme. Acutely exposing mice or rabbits to ozone reduced the amount of active lysozyme obtainable by bronchopulmonary lavage. The effect was proportional to ozone concentration, as well as to duration of exposure. Enzyme activity returned to normal levels during the 12 hr following exposure. Ozone (5 ?liters/liter; 5 parts per million) for 3 hr reduced lysozyme levels approximately 30%. Studies of the release of lysozyme by alveolar cells support the theory that loss of activity is due to in vivo oxidation of lysozyme.

Holzman, R. S.; Gardner, D. E.; Coffin, D. L.

1968-01-01

322

PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX  

SciTech Connect

The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. (1) Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32*I protein for this synthesis. (2) Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. (3) Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. (4) The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3{prime}-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.

Burke, R.L.; Alberts, B.M.; Hosoda, J.

1980-07-01

323

Kinetic Traps in Lysozyme Folding  

Microsoft Academic Search

Folding of lysozyme from hen egg white was investigated by using interrupted refolding experiments. This method makes use of a high energy barrier between the native state and transient folding intermediates, and, in contrast to conventional optical techniques, it enables one to specifically monitor the amount of native molecules during protein folding. The results show that under strongly native conditions

Thomas Kiefhaber

1995-01-01

324

Sweetness and enzymatic activity of lysozyme.  

PubMed

Hen egg lysozyme elicits a sweet taste sensation for human beings. Effects of reduction of disulfide bonds, heat treatment, and chemical modification of hen egg lysozyme on both sweetness and hydrolytic activity were investigated. Both the sweetness and enzymatic activities were lost when the intradisulfide linkage in a lysozyme molecule was reduced and S-3-(trimethylated amino) propylated. The sweetness and enzymatic activity of lysozyme were lost on heating at 95 degrees C for 18 h. These facts suggest that tertiary structures of lysozyme are indispensable for eliciting a sweet taste as well as enzymatic activity. Although the modification of carboxyl residues in a lysozyme by glycine methylester or aminomethansulfonic acid resulted in the loss of enzymatic activity by blocking the catalytic residues, the sweetness was fully retained. These results indicate that the sweetness of lysozyme was independent of its enzymatic activity. The lysozyme purified from goose egg white similarly elicited a sweet taste, although goose (g-type) lysozyme is quite different from hen egg lysozyme (c-type) on the basis of structural, immunological, and enzymatic properties. These findings indicate that a specific protein property of lysozyme is required for sweetness elicitation and that the enzymatic activity and carbohydrates produced by enzymatic reaction are not related to the sweet taste. PMID:11600047

Masuda, T; Ueno, Y; Kitabatake, N

2001-10-01

325

Bacteriophages infecting Propionibacterium acnes.  

PubMed

Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed. PMID:23691509

Brüggemann, Holger; Lood, Rolf

2013-04-11

326

Bacteriophages Infecting Propionibacterium acnes  

PubMed Central

Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed.

2013-01-01

327

A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals.  

PubMed Central

We describe a conserved family of bacterial gene products that includes the VirB1 virulence factor encoded by tumor-inducing plasmids of Agrobacterium spp., proteins involved in conjugative DNA transfer of broad-host-range bacterial plasmids, and gene products that may be involved in invasion by Shigella spp. and Salmonella enterica. Sequence analysis and structural modeling show that the proteins in this group are related to chicken egg white lysozyme and are likely to adopt a lysozyme-like structural fold. Based on their similarity to lysozyme, we predict that these proteins have glycosidase activity. Iterative data base searches with three conserved sequence motifs from this protein family detect a more distant relationship to bacterial and bacteriophage lytic transglycosylases, and goose egg white lysozyme. Two acidic residues in the VirB1 protein of Agrobacterium tumefaciens form a putative catalytic dyad, Each of these residues was changed into the corresponding amide by site-directed mutagenesis. Strains of A. tumefaciens that express mutated VirB1 proteins have a significantly reduced virulence. We hypothesize that many bacterial proteins involved in export of macromolecules belong to a widespread class of hydrolases and cleave beta-1,4-glycosidic bonds as part of their function. Images Fig. 2 Fig. 4

Mushegian, A R; Fullner, K J; Koonin, E V; Nester, E W

1996-01-01

328

Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination  

Microsoft Academic Search

Bacteriophage T4 homologous recombination events are promoted by presynaptic filaments of UvsX recombinase bound to single-stranded DNA (ssDNA). UvsY, the phage recombination medi- ator protein, promotes filament assembly in a concentration-dependent manner, stimulating UvsX at stoichiometric concentrations but inhibiting at higher concentrations. Recent work demon- strated that UvsX-H195Q\\/A mutants exhibit decreased ssDNA-binding affinity and altered enzy- matic properties. Here, we

Joshua N. Farb; Scott W. Morrical

2009-01-01

329

Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae  

Microsoft Academic Search

A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as

K. Valerie; G. Fronko; E. E. Henderson; J. K. de Riel

1986-01-01

330

Function of cloned T4 recombination genes, uvsX and uvsY , in cells of Escherichia coli  

Microsoft Academic Search

Genes uvsX and uvsY of bacteriophage T4 both control genetic recombination and repair of damaged DNA, and their mutant phenotypes bear a striking resemblance to each other. It has been shown recently that the uvsX gene product is analogous to the recA gene product of Escherichia coli (Yonesaki et al. 1985; Yonesaki and Minagawa 1985; Formosa and Alberts 1986), but

Teiichi Minagawa; Hisao Fujisawa; Tetsuro Yonesaki; Yeiko Ryo

1988-01-01

331

Sequence boundary T4, South China Sea  

SciTech Connect

Marginal basins of the western South China Sea were formed by rifting in the Eocene and filled during the Early and Middle Tertiary. Seismic profiling, drilling, and mapping for over a decade has revealed numerous structural and stratigraphic sequences, many bearing gas or oil. Chen et al. (1993) published a sequence stratigraphy for the Qingdongnan Basin, proposing correlations to the global onlap cycles of Haq et al. (1987). Although more than 30 wells have been completed, chronostratigraphy has been largely proprietary. Sequence boundary T4 is a regionally extensive seismic reflector. Major regressive sedimentary facies, shelf margin incision, and basin fan formation indicate that it is a type I sequence boundary. Study of calcareous nannofossils and planktonic foraminifera in wells penetrating T4 away from clastic deposits date the event as early Late Miocene (N14/NN8). In most sections, however, the magnitude of the hiatus varies considerably: erosion below the unconformity may expose units as old as the Late Oligocene (P21/NP24) and non-deposition following the unconformity may continue up to the Early Pliocene (N18/NN12). Microfossil paleobathymetry indicates that basinal sedimentary depositi on following T4 occurred at bathyal or deeper water depths. Because the T4 sequence boundary and subsequent deep-water sediments can be consistently recognized throughout the South China Sea-Phillippines-Indonesia region, T4 can be regarded as a major hydrocarbon seal.

Thompson, P.R.; Abbott, W.H. [ARCO Exploration and Production Technology Co., Plano, TX (United States)

1995-04-01

332

Direct observation of stalled fork restart via fork regression in the T4 replication system.  

PubMed

The restart of a stalled replication fork is a major challenge for DNA replication. Depending on the nature of the damage, different repair processes might be triggered; one is template switching, which is a bypass of a leading-strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching. We show that the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading-strand lesion damage by a pseudostochastic process, periodically forming and migrating a four-way Holliday junction. The initiation of the repair process requires partial replisome disassembly via the departure of the replicative helicase. The results support the role of fork regression pathways in DNA repair. PMID:23197534

Manosas, Maria; Perumal, Senthil K; Croquette, Vincent; Benkovic, Stephen J

2012-11-30

333

Evolution of T4-related phages  

Microsoft Academic Search

Much progress has been made in understanding T-even phage biology in the last 50 years. We now know the entire sequence of T4, encoding nearly 300 genes, only 69 of which have been shown to be essential under standard laboratory conditions; no specific function is yet known for about 140 of them. The origin of most phage genes is unclear,

Elizabeth Kutter; Ketevan Gachechiladze; Alexandr Poglazov; Elena Marusich; Mikhail Shneider; Pia Aronsson; Alberto Napuli; Darlene Porter; Vadim Mesyanzhinov

1995-01-01

334

Blocking the T4 lysis inhibition phenotype.  

PubMed

Nonlysogenic Escherichia coli K cells exhibit a delay in lysis when infected by T4rII phage termed lysis inhibition (LIN). E. coli K cells expressing lambda rexB from either a prophage defective for rexA, or a multicopy plasmid supported T4rII infection, but prevented the establishment of LIN. In addition, E. coli null mutations in either the periplasmic "tail-specific protease" tsp, or the 10Sa RNA ssrA, completely blocked the establishment of LIN following T4 infections. The expression of rexB in the absence of rexA resulted in several cellular phenotypes, including aberrant cell surface morphology, the partial to near complete suppression of mutations of lambda S and T4t holin genes, and lysis by cells aging on plates or growing with high rexB expression at elevated temperatures. These activities of RexB were impeded in the presence of RexA. PMID:14637004

Slavcev, Roderick A; Hayes, Sidney

2003-12-01

335

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 2013-04-01 false Lysozyme (muramidase) test system. 862...Chemistry Test Systems § 862.1490 Lysozyme (muramidase) test system. (a) Identification. A lysozyme (muramidase) test system is a...

2013-04-01

336

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 2010-04-01 false Lysozyme (muramidase) test system. 862...Chemistry Test Systems § 862.1490 Lysozyme (muramidase) test system. (a) Identification. A lysozyme (muramidase) test system is a...

2010-04-01

337

21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2010 CFR

...2009-04-01 2009-04-01 false Lysozyme (muramidase) test system. 862...Chemistry Test Systems § 862.1490 Lysozyme (muramidase) test system. (a) Identification. A lysozyme (muramidase) test system is a...

2009-04-01

338

Evolution of the mammalian lysozyme gene family  

PubMed Central

Background Lysozyme c (chicken-type lysozyme) has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme c has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known. Results Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution. Conclusions The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme c and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.

2011-01-01

339

Renal Handling of Endogenous Lysozyme in the Rat  

Microsoft Academic Search

Quantitative studies of endogenous lysozyme (low molecular weight protein) were performed in rats. Urine and plasma concentrations of lysozyme and inulin were measured spectrophotometrically. An improved lysozyme assay (standard curve established by using egg white-lysozyme) enabled us to determine the mean plasma concentration of endogenous lysozyme (4.4 ?g· ml-1) and the urinary concentrations of endogenous lysozyme (between 0.1 and 3.8

Constantin Cojocel; Karl Baumann

1983-01-01

340

A bioluminescence-based assay for enumeration of lytic bacteriophage.  

PubMed

A bioluminescence-based assay for enumeration of lytic bacteriophage was developed. The assay consists of a bioluminescent Escherichia coli as the host bacterium, the lytic bacteriophage T4 and an automated luminometer measuring luminescence over time. The assay is based on the decrease in luminescence as the bioluminescent host cells are lysed by T4. The T4 concentration, bioluminescent E. coli concentration, phage suspension medium, and temperature (25 degrees C and 37 degrees C) were varied. There was a strong negative correlation between bioluminescence intensities and T4 phage concentrations at both room temperature (R(2)=0.993) and 37 degrees C (R(2)=0.970). Phage was detected more rapidly at 37 degrees C than at 25 degrees C. The detection limit was also lower when the assay was performed at 37 degrees C with a minimum detection level of 2.4 log CFU/ml compared to 3.4 log CFU/ml for 25 degrees C. The assay was used to determine thermal inactivation using T4 phages heated at 70 degrees C for 0 to 30 min, and phage concentrations were determined using the bioluminescence assay and a standard plaque assay. There was no significant difference between the two enumeration methods (P>0.01). This study suggests the bioluminescence-based assay can be used as an alternative for quantitatively monitoring phage infectivity, instead of conventional standard plaque assays. PMID:19628012

Kim, S; Schuler, B; Terekhov, A; Auer, J; Mauer, L J; Perry, L; Applegate, B

2009-07-21

341

Properties of Two Marine Bacteriophages.  

National Technical Information Service (NTIS)

Various properties have been determined for two bacteriophages, NCMB 384 and 385, and their host, NCMB 397, a Cytophaga sp., isolated from the marine environment. The purified bacteriophages have been subjected to serological analysis, results of which in...

P. K. Chen R. V. Citarella O. Salazar R. R. Colwell

1965-01-01

342

A new lysozyme from the eastern oyster ( Crassostrea virginica ) indicates adaptive evolution of i -type lysozymes  

Microsoft Academic Search

.  A new lysozyme (cv-lysozyme 2) with a MALDI molecular mass of 12 984.6 Da was purified from crystalline styles and digestive\\u000a glands of eastern oysters (Crassostrea virginica) and its cDNA sequenced. Quantitative real time RT-PCR detected cv-lysozyme 2 gene expression primarily in digestive gland\\u000a tissues, and in situ hybridization located cv-lysozyme 2 gene expression in basophil cells of digestive tubules. Cv-lysozyme 2

Q.-G. Xue; N. Itoh; K. L. Schey; Y.-L. Li; R. K. Cooper; J. F. La Peyre

2007-01-01

343

BACTERIOPHAGE T4 MULTIPLICATION IN A GLUCOSE-LIMITED ESCHERICHIA COLI BIOFILM. (R825503)  

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

344

Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase  

Microsoft Academic Search

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with

Craig Tuerk; Larry Gold

1990-01-01

345

THERMAL STABILITY AND FOLDING KINETICS OF LYSOZYME  

Technology Transfer Automated Retrieval System (TEKTRAN)

A number of researchers reported linear correlations between lysozyme denaturation temperatures and the folding rate in the presence of different organic solvents and various levels of pH and ionic strength. This work is focused on determining the kinetics of lysozyme folding in the presence of amy...

346

Purification, characterization and comparison of reptile lysozymes.  

PubMed

Cation exchange column chromatography and gel filtration chromatography were used to purify four reptile lysozymes from egg white: SSTL A and SSTL B from soft shelled turtle (Trionyx sinensis), ASTL from Asiatic soft shelled turtle (Amyda cartilagenea) and GSTL from green sea turtle (Chelonia mydas). The molecular masses of the purified reptile lysozymes were estimated to be 14 kDa by SDS-PAGE. Enzyme activity of the four lysozymes could be confirmed by gel zymograms and showed charge differences on native-PAGE. SSTL A, SSTL B and ASTL had sharp pH optima of about pH 6.0, which contrasts with that of GSTL, which showed dual pH optima at about pH 6.0 and pH 8.0. The activities of the reptile lysozymes rapidly decreased within 30 min of incubation at 90 degrees C except for ASTL, which was more stable. Partial N-terminal amino acid sequencing and peptide mapping strongly suggested that the enzymes were C-type lysozymes. Interestingly, the mature SSTL lysozymes show an extra Gly residue at the N-terminus, which was previously found in soft-shelled turtle lysozyme. The reptile lysozymes showed lytic activity against several species of bacteria, such as Micrococcus luteus and Vibrio cholerae, but showed only weak activity to Pseudomonas aeruginosa and lacked activity towards Aeromonas hydrophila. PMID:16549391

Thammasirirak, Sompong; Ponkham, Pornpimol; Preecharram, Sutthidech; Khanchanuan, Rathakarn; Phonyothee, Phalakorn; Daduang, Sakda; Srisomsap, Chantragan; Araki, Tomohiro; Svasti, Jisnuson

2006-03-06

347

Stationary phase-like properties of the bacteriophage ? Rex exclusion phenotype  

Microsoft Academic Search

The rex genes of bacteriophage u were found to protect lysogenic Escherichia coli K host cells against killing by phage T4 rII, when compared in parallel to isogenic Rexm lysogens and nonlysogens. This protective effect was abrogated upon mutation of the host stationary-phase sigma factor RpoS. Rex+ lysogens infected by T4 rII contracted, formed aggregates and shed flagella, thus resembling

R. A. Slavcev; S. Hayes

2003-01-01

348

Bacteriophages and their Genomes  

PubMed Central

Bacteriophages occupy a unique position in biology, representing an absolute majority of all organisms in the biosphere. Because their genomes are relatively small, elucidating the genetic diversity of the phage population, deciphering their origins, and identifying the evolutionary mechanisms that shape the population would seem readily feasible. And yet the pace of phage genome characterization has slowed over the past three years, reflecting in part a need to transition from sequencing known and well-characterized bacteriophages to the isolation and comparative analysis of new isolates. The current state of bacteriophage genomics shows that the genetic diversity of the population is very high, that phages have been actively evolving for billions of years with active engagement of horizontal genetic exchange, and that their genomes are consequently pervasively mosaic in their architectures. But we have barely scratched the surface and the next years of phage genome exploration promise to be especially revealing.

Hatfull, Graham F.; Hendrix, Roger W.

2011-01-01

349

MOLECULAR CHARACTERIZATION OF LYSOZYME TYPE II GENE IN RAINBOW TROUT.  

Technology Transfer Automated Retrieval System (TEKTRAN)

Rainbow trout (Oncorhynchus mykiss) has two types of lysozyme. Type II lysozyme differs from type I by only one amino acid, but only type II lysozyme has significant bactericidal activity. Due to this novel antibacterial property, lysozyme II appears to be a candidate gene for potentially enhancing ...

350

Inhibition of lysozyme by taurine dibromamine.  

PubMed

Hypobromous acid (HOBr) is a powerful oxidant produced by stimulated neutrophils and eosinophils. Taurine, a non-protein amino acid present in high amounts in the leukocytes, reacts instantaneously with HOBr leading to their haloamine derivative taurine dibromamine (Tau-NBr2). Lysozyme is a bactericidal enzyme also present in leukocytes and in secretory fluids. The inhibition of lysozyme is a pathway for bacterial proliferation in inflammatory sites. Here, we investigated the inhibition of the enzymatic activity of lysozyme when it was submitted to oxidation by Tau-NBr2. We found that the oxidation of lysozyme by Tau-NBr2 decreased its enzymatic activity in 80%, which was significant higher compared to the effect of its precursor HOBr (30%). The study and comparison of Tau-NBr2 and HOBr regarding the alterations provoked in the intrinsic fluorescence, synchronous fluorescence, resonance light scattering and near and far-UV circular dichroism spectra of lysozyme and oxidized lysozyme revealed that tryptophan residues in the active site of the protein were the main target for Tau-NBr2 and could explain its efficacy as inhibitor of lysozyme enzymatic activity. This property of Tau-NBr2 may have pathological significance, since it can be easily produced in the inflammatory sites. PMID:23590281

Petronio, M S; Ximenes, V F

2013-11-01

351

Problem-Solving Test: RNA and Protein Synthesis in Bacteriophage-Infected "E. coli" Cells  

ERIC Educational Resources Information Center

|The classic experiment presented in this problem-solving test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in "Escherichia coli" cells infected with bacteriophage T4. The work described in this test led to one of the most seminal discoveries of early molecular biology: it dealt a…

Szeberenyi, Jozsef

2008-01-01

352

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis  

Microsoft Academic Search

In E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages ?, P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165\\/70). In the other type DNA synthesis continues at

Erich Lanka; Heinz Schuster

1970-01-01

353

New insights into the possible role of bacteriophages in host defense and disease  

Microsoft Academic Search

BACKGROUND: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics – resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells. PRESENTATION OF THE HYPOTHESIS: At least some phages (e.g., T4 coliphage)

Andrzej Gorski; Krystyna Dabrowska; Kinga Switala-Jele?; Maria Nowaczyk; Beata Weber-Dabrowska; Janusz Boratynski; Joanna Wietrzyk; Adam Opolski

2003-01-01

354

Stability and in vitro DNA packaging of bacteriophages: effects of dextrans, sugars, and polyols  

SciTech Connect

Attempts were made to increase the efficiency of infectious particle formation during the in vitro assembly of bacteriophage T7 from procapsids and DNA. It was found that dextrans and some smaller, related compounds (sucrose and sorbitol) increase this efficiency by a factor of 8 to 50. Dextrans also inhibited elevated temperature-induced emptying of DNA from bacteriophages T7, P22, and T4, suggesting that the stimulation of assembly is caused, at least in part, by the stabilization of packaged DNA in capsids. The data indicated that the sugars and polyols can slow DNA emptying from bacteriophages at elevated temperature whether they permeate the bacteriophage capsid or not. In contrast, the data suggested that permeation of some particle, probably a capsid, results in inhibition of in vitro T7 assembly.

Serwer, P. (The Univ. of Texas Health Science Center, San Antonio); Masker, W.E.; Allen, J.L.

1983-02-01

355

Several new bacteriophage T4 genes, mapped by sequencing deletion endpoints between genes 56 (dCTPase) and dda (a DNA-dependent ATPase-helicase) modulate transcription 1 Published in conjunction with A Wisconsin Gathering Honoring Waclaw Szybalski on the occasion of his 75th year and 20years of Editorship-in-Chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA. 1  

Microsoft Academic Search

We have analyzed DNA of wild-type T4 and of 13 independent large viable deletions isolated by Homyk and Weil (Virology 61 (1974) 505–523) and by Little (Virology 53 (1973) 47–59), by sequencing, cloning, and expression studies. The deletions can be explained by illegitimate recombination between short (4- to 15-bp) ectopic repeats. In four deletions, adjacent regions are partially homologous, and

Gisela Mosig; Nancy E. Colowick; Bradley C. Pietz

1998-01-01

356

Structural and Mechanistic Studies of Pesticin, a Bacterial Homolog of Phage Lysozymes*  

PubMed Central

Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme.

Patzer, Silke I.; Albrecht, Reinhard; Braun, Volkmar; Zeth, Kornelius

2012-01-01

357

Structural and mechanistic studies of pesticin, a bacterial homolog of phage lysozymes.  

PubMed

Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme. PMID:22593569

Patzer, Silke I; Albrecht, Reinhard; Braun, Volkmar; Zeth, Kornelius

2012-05-16

358

Crystal Structure of the Phage T4 Recombinase UvsX and Its Functional Interaction with the T4 SF2 Helicase UvsW  

SciTech Connect

Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.

Gajewski, Stefan; Webb, Michael R.; Galkin, Vitold; Egelman, Edward H.; Kreuzer, Kenneth N.; White, Stephen W. (Duke); (UV); (SJCH)

2012-07-11

359

Method for Produce Sanitation using Bacteriophages.  

National Technical Information Service (NTIS)

A method for produce sanitation using bacteriophages is disclosed. According to one embodiment of the present invention, the method includes the steps of (1) providing at least one bacteriophage; and (2) applying the bacteriophage to the produce. The prod...

A. Sulakvelidze J. G. Morris Z. Alavidze G. R. Pasternack T. C. Brown

2003-01-01

360

A Comparative Study of Anthrax Bacteriophages.  

National Technical Information Service (NTIS)

A study of the morphology of negative colonies of various anthrax bacteriophages showed that according to morphology the negative colonies of anthrax bacteriophages included all the known types described in other bacteriophages. On the basis of the study ...

E. N. Levina V. R. Arkhipova

1968-01-01

361

Division M: Bacteriophage  

NSDL National Science Digital Library

This website from the American Society for Microbiology provides current information on Bacteriophages ('Phages' for short), the tiny, microscopic viruses whose hosts are bacterial cells. The site is organized into five sections: general information, resources for teachers, images (micrographs, diagrams, etc.), upcoming meetings and events, and related links. Uncluttered but full of information, the site is an excellent resource for educators and students.

2007-07-23

362

BACTERIOPHAGE THERAPY AND CAMPYLOBACTER  

Technology Transfer Automated Retrieval System (TEKTRAN)

The book chapter reports efforts to exploit Campylobacter-specific bacteriophages to reduce the numbers of Campylobacter jejuni and C. coli colonizing poultry and contaminating poultry meat products. Controlling campylobacters in poultry represents one of the greatest challenges to the agriculture a...

363

Femtosecond laser processing of lysozyme crystals  

Microsoft Academic Search

We have developed a novel processing technique for lysozyme crystals using femtosecond laser ablation. This procedure enables us to make problematic protein crystals suitable for X-ray diffraction measurements to determine the three-dimensional (3D) protein structure.

M. Kashii; H. Kitano; Y. Hosokawa; H. Adachi; Y. Mori; T. Sasaki; H. Yoshikawa

2005-01-01

364

Sperm Specific Lysozyme-Like Proteins.  

National Technical Information Service (NTIS)

The present invention is directed to a family of testis specific proteins (SLLPs) that share high sequence identity to lysozyme-C proteins. The application encompasses compositions comprising the SLLP proteins, antibodies specific for the SLLP polypeptide...

A. Mandal J. C. Herr L. C. Digilio M. B. Herrero

2004-01-01

365

Purification of lysozyme by multistage affinity filtration  

Microsoft Academic Search

A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified

L. He; Y. Sun

2002-01-01

366

Studies with an antibody to rat lysozyme  

PubMed Central

An antibody to rat lysozyme has been prepared and its activity and specificity studied by inhibition and double diffusion techniques. By means of this antibody together with fluorescent anti-rabbit globulin, the distribution of lysozyme in rat and mouse polymorphs and macrophages has been examined and some changes which follow phagocytosis demonstrated. ImagesFIGS. 5-7FIGS. 8-13FIG. 14FIGS. 2-4

Glynn, A. A.; Parkman, R.

1964-01-01

367

Secondary structure of T4 gene 33 protein. Fourier transform infrared and circular dichroic spectroscopic studies.  

PubMed

The secondary structure of bacteriophage T4 gene 33 protein (gp33) has been quantitatively examined by using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy were used to quantitate the spectral information from the amide I bands. The relative areas of these component bands indicate 21% alpha-helix, 25% beta-sheet, 34% turn, 12% random coil and 8% other undefined structures in gp33. An analysis of the CD spectrum of gp33 at the same pH and temperature revealed 19% alpha-helix, 25% beta-sheet, 13% turn and 43% random coil structures. The possible reasons for the discrepancies in estimates of the contributions to the secondary structure from turns and random coils are discussed. PMID:9184943

Shao, W; Kearns, D R; Sanders, G M

1997-04-01

368

Analysis of the DNA translocation and unwinding activities of T4 phage helicases  

PubMed Central

Helicases are an important class of enzymes involved in DNA and RNA metabolism that couple the energy of ATP hydrolysis to unwind duplex DNA and RNA structures. Understanding the mechanism of helicase action is vital due to their involvement in various biological processes such as DNA replication, repair and recombination. Furthermore, the duplex DNA unwinding property of this class of enzymes is closely related to their single-stranded DNA translocation. Hence the study of its translocation properties is essential to understanding helicase activity. Here we review the methods that are employed to analyze the DNA translocation and unwinding activities of the bacteriophage T4 UvsW and Dda helicases. These methods have been successfully employed to study the functions of helicases from large superfamilies.

Perumal, Senthil K.; Raney, Kevin D.; Benkovic, Stephen J.

2010-01-01

369

Kinetics of single DNA molecule denaturation by T4 Gene 32 protein  

NASA Astrophysics Data System (ADS)

Bacteriophage T4 gene 32 protein (32 protein) specifically binds single-stranded DNA, a property essential for its role in DNA replication, recombination, and repair. Although on a thermodynamic basis, single-stranded DNA binding proteins should lower the thermal melting temperature of double-stranded DNA (dsDNA), 32 protein does not. Using single molecule force spectroscopy, we show for the first time that 32 protein is capable of slowly destabilizing natural dsDNA. Direct measurements of single DNA molecule denaturation and renaturation kinetics in the presence of 32 protein and its proteolytic fragments reveal three types of kinetic behavior, attributable to specific protein structural domains, which regulate 32 protein's helix-destabilizing capabilities. This regulation is potentially biologically significant because uncontrolled helix-destabilization would be lethal to the cell. We also obtain equilibrium measurements of the DNA helix-coil transition free energy in the presence of these proteins for the first time.

Pant, Kiran; Karpel, Richard L.; Williams, Mark C.

2003-03-01

370

Bacteriophage in polar inland waters  

Microsoft Academic Search

Bacteriophages are found wherever microbial life is present and play a significant role in aquatic ecosystems. They mediate\\u000a microbial abundance, production, respiration, diversity, genetic transfer, nutrient cycling and particle size distribution.\\u000a Most studies of bacteriophage ecology have been undertaken at temperate latitudes. Data on bacteriophages in polar inland\\u000a waters are scant but the indications are that they play an active

Christin Säwström; John Lisle; Alexandre M. Anesio; John C. Priscu; Johanna Laybourn-Parry

2008-01-01

371

Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae  

SciTech Connect

A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity.

Valerie, K.; Fronko, G.; Henderson, E.E.; de Riel, J.K.

1986-10-01

372

Mutational Analysis of the mRNA Operator for T4 DNA Polymerase  

PubMed Central

Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C->A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A->U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

Andrake, M. D.; Karam, J. D.

1991-01-01

373

Periplasmic domains define holin-antiholin interactions in t4 lysis inhibition.  

PubMed

Bacteriophage T4 effects host lysis with a holin, T, and an endolysin, E. T and E accumulate in the membrane and cytoplasm, respectively, throughout the period of late gene expression. At an allele-specific time, T triggers to disrupt the membrane, allowing E to enter the periplasm and attack the peptidoglycan. T triggering can be blocked by secondary infections, leading to the state of lysis inhibition (LIN). LIN requires the T4 antiholin, RI, and is sensitive to the addition of energy poisons. T is unusual among holins in having a large C-terminal periplasmic domain. The rI gene encodes a polypeptide of 97 residues, of which 72 are predicted to be a periplasmic domain. Here, we show that the periplasmic domain of RI is necessary and sufficient to block T-mediated lysis. Moreover, when overexpressed, the periplasmic domain of T (T(CTD)) was found to abolish LIN in T4 infections and to convert wild-type (wt) T4 plaques from small and fuzzy edged to the classic "r" large, sharp-edged plaque morphology. Although RI could be detected in whole cells, attempts to monitor it during subcellular fractionation were unsuccessful, presumably because RI is a highly unstable protein. However, fusing green fluorescence protein (GFP) to the N terminus of RI created a more stable chimera that could be demonstrated to form complexes with wild-type T(CTD) and also with its LIN-defective T75I variant. These results suggest that the function of the unusual periplasmic domain of T is to transduce environmental information for the real-time control of lysis timing. PMID:16166524

Tran, Tram Anh T; Struck, Douglas K; Young, Ry

2005-10-01

374

The amino acid sequence of wood duck lysozyme.  

PubMed

The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme. PMID:10052146

Araki, T; Torikata, T

1999-01-01

375

Guards of the great wall: bacterial lysozyme inhibitors.  

PubMed

Peptidoglycan is the major structural component of the bacterial cell wall. It provides resistance against turgor and its cleavage by hydrolases such as lysozymes results in bacteriolysis. Most, if not all, animals produce lysozymes as key effectors of their innate immune system. Recently, highly specific bacterial proteinaceous lysozyme inhibitors against the three major animal lysozyme families have been discovered in bacteria, and these may represent a bacterial answer to animal lysozymes. Here, we will review their properties and phylogenetic distribution, present their structure and molecular interaction mechanism with lysozyme, and discuss their possible biological functions and potential applications. PMID:22840966

Callewaert, Lien; Van Herreweghe, Joris M; Vanderkelen, Lise; Leysen, Seppe; Voet, Arnout; Michiels, Chris W

2012-07-27

376

Bacteriophage-Based Biosensors  

Microsoft Academic Search

\\u000a Bacteriophages, or phages for short, are viruses that infect bacteria and are considered the main regulators of microbial\\u000a balance on Earth. These viruses are extremely specific, and their long-term survivability and ability to reproduce quickly\\u000a in suitable hosts play a major role in the preservation of a dynamic equilibrium amid the diverse variety of bacterial species\\u000a in the Earth’s ecosystem.

Mohammed Zourob; Steven Ripp

377

T-4G Simulator and T-4 Ground Training Devices in USAF Undergraduate Pilot Training.  

ERIC Educational Resources Information Center

|The objective of the project was to investigate the utility of using an A/F37A-T4G T-37 flight simulator within the context of Air Force undergraduate pilot training. Twenty-one subjects, selected from three undergraduate pilot training classes, were given contact flight training in a TP4G/EPT simulator before going to T-37 aircraft for further…

Woodruff, Robert R.; Smith, James F.

378

The Structure of the Phage T4 DNA Packaging Motor Suggests a Mechanism Dependent on Electrostatic Forces  

SciTech Connect

Viral genomes are packaged into procapsids by powerful molecular motors. We report the crystal structure of the DNA packaging motor protein, gene product 17 (gp17), in bacteriophage T4. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. We show that another function of the C-terminal domain is to translocate the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a tensed state. A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 shows that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a relaxed state. These structures suggest a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors.

Sun, Siyang; Kondabagil, Kiran; Draper, Bonnie; Alam, Tanfis I.; Bowman, Valorie D.; Zhang, Zhihong; Hegde, Shylaja; Fokine, Andrei; Rossmann, Michael G.; Rao, Venigalla B. (CUA); (Purdue)

2009-06-30

379

T4-Like Phage Bp7, a Potential Antimicrobial Agent for Controlling Drug-Resistant Escherichia coli in Chickens.  

PubMed

Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4, 62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the "T4-like" genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens. PMID:23835183

Zhang, Can; Li, Wenli; Liu, Wenhua; Zou, Ling; Yan, Chen; Lu, Kai; Ren, Huiying

2013-07-08

380

Enhanced antimicrobial activity of engineered human lysozyme.  

PubMed

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted toward the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high-throughput screen was implemented to functionally interrogate combinatorial libraries of charge-engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge-engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing. PMID:20604527

Scanlon, Thomas C; Teneback, Charlotte C; Gill, Avinash; Bement, Jenna L; Weiner, Joshua A; Lamppa, John W; Leclair, Laurie W; Griswold, Karl E

2010-09-17

381

Enhanced antimicrobial activity of engineered human lysozyme  

PubMed Central

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted towards the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high throughput screen was implemented to functionally interrogate combinatorial libraries of charge engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing.

Scanlon, Thomas C.; Teneback, Charlotte C.; Gill, Avinash; Bement, Jenna L.; Weiner, Joshua A.; Lamppa, John W.; Leclair, Laurie W.; Griswold, Karl E.

2010-01-01

382

Lysozyme Catalyzes the Formation of Antimicrobial Silver Nanoparticles (POSTPRINT).  

National Technical Information Service (NTIS)

Hen egg white lysozyme acted as the sole reducing agent and catalyzed the formation of silver nanoparticles in the presence of light. Stable silver colloids formed after mixing lysozyme and silver acetate in methanol and the resulting nanoparticles were c...

D. M. Eby G. R. Johnson K. E. Farrington N. M. Schaeublin S. M. Hussain

2009-01-01

383

Lysozyme activity in the plaice ( Pleuronectes platessa L.)  

Microsoft Academic Search

Zusammenfassung Die Untersuchung der Lysozymverteilung im Gewebe des Knochenfischs,Pleuronectes platessa L., ergab, dass das Plasma-Lysozym von Leukozyten abstammt, das Hautschleim-Lysozym dagegen von spezifischen Epidermiszellen.

Thelma C. Fletcher; Ann White

1973-01-01

384

Bacteriophage 5' untranslated regions for control of plastid transgene expression.  

PubMed

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation. PMID:23053542

Yang, Huijun; Gray, Benjamin N; Ahner, Beth A; Hanson, Maureen R

2012-09-30

385

Cloning and characterization of the tiger shrimp lysozyme  

Microsoft Academic Search

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated\\u000a from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of\\u000a 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two

Ye Xing; Gao Feng-Ying; Zheng Qing-Mei; Bai Jun-Jie; Wang Huan; Lao Hai-Hua; Jian Qing

2009-01-01

386

Molecular Evolution of Vertebrate Goose-Type Lysozyme Genes  

Microsoft Academic Search

We have found that mammalian genomes contain two lysozyme g genes. To better understand the function of the lysozyme g genes we have examined the evolution of this small gene family. The lysozyme g gene structure has been largely conserved during vertebrate evolution, except at the 5' end of the gene, which varies in number of exons. The expression pattern

David M. Irwin; Zhiyuan M. Gong

2003-01-01

387

The cytochemical localization of lysozyme in Paneth cell granules  

Microsoft Academic Search

Synopsis  The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue

Y. Ghoos; G. Vantrappen

1971-01-01

388

Escherichia Coli Mutations That Prevent the Action of the T4 Unf/Alc Protein Map in an RNA Polymerase Gene  

PubMed Central

Bacteriophage T4 has the substituted base hydroxymethylcytosine in its DNA and presumably shuts off host transcription by specifically blocking transcription of cytosine-containing DNA. When T4 incorporates cytosine into its own DNA, the shutoff mechanism is directed back at T4, blocking its late gene expression and phage production. Mutations which permit T4 multiplication with cytosine DNA should be in genes required for host shutoff. The only such mutations characterized thus far have been in the phage unf/alc gene. The product of this gene is also required for the unfolding of the host nucleoid after infection, hence its dual name unf/alc. As part of our investigation of the mechanism of action of unf/alc, we have isolated Escherichia coli mutants which propagate cytosine T4 even if the phage are genotypically alc(+). These same E. coli mutants are delayed in the T4-induced unfolding of their nucleoid, lending strong support to the conclusion that blocking transcription and unfolding the host nucleoid are but different manifestations of the same activity. We have mapped two of the mutations, called paf mutations for prevent alc function. They both map at about 90 min, probably in the rpoB gene encoding a subunit of RNA polymerase. From the behavior of Paf mutants, we hypothesize that the unf/alc gene product of T4 interacts somehow with the host RNA polymerase to block transcription of cytosine DNA and unfold the host nucleoid.

Snyder, L.; Jorissen, L.

1988-01-01

389

Removal of particle-associated bacteriophages by dual-media filtration at different filter cycle stages and impacts on subsequent UV disinfection  

Microsoft Academic Search

This bench-scale study investigated the passage of particle-associated bacteriophage through a dual-media (anthracite-sand) filter over a complete filter cycle and the effect on subsequent ultraviolet (UV) disinfection. Two model viruses, bacteriophages MS2 and T4, were considered. The water matrix was de-chlorinated tap water with either kaolin or Aldrich® humic acid (AHA) added and coagulated with alum to form floc before

Michael R. Templeton; Robert C. Andrews; Ron Hofmann

2007-01-01

390

T4 phages against Escherichia coli diarrhea: Potential and problems  

Microsoft Academic Search

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and

Emmanuel Denou; Anne Bruttin; Caroline Barretto; Catherine Ngom-Bru; Harald Brüssow; Sophie Zuber

2009-01-01

391

Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection  

Technology Transfer Automated Retrieval System (TEKTRAN)

The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

392

Lysozyme Resistance in Streptococcus suis Is Highly Variable and Multifactorial  

PubMed Central

Background Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a variety of body fluids and immune cells. Lysozyme acts as a peptidoglycan degrading enzyme causing bacterial lysis. Several pathogens have developed mechanisms to evade lysozyme-mediated killing. In the present study we compared the lysozyme sensitivity of various S. suis isolates and investigated the molecular basis of lysozyme resistance for this pathogen. Results The lysozyme minimal inhibitory concentrations of a wide panel of S. suis isolates varied between 0.3 to 10 mg/ml. By inactivating the oatA gene in a serotype 2 and a serotype 9 strain, we showed that OatA-mediated peptidoglycan modification partly contributes to lysozyme resistance. Furthermore, inactivation of the murMN operon provided evidence that additional peptidoglycan crosslinking is not involved in lysozyme resistance in S. suis. Besides a targeted approach, we also used an unbiased approach for identifying factors involved in lysozyme resistance. Based on whole genome comparisons of a lysozyme sensitive strain and selected lysozyme resistant derivatives, we detected several single nucleotide polymorphisms (SNPs) that were correlated with the lysozyme resistance trait. Two SNPs caused defects in protein expression of an autolysin and a capsule sugar transferase. Analysis of specific isogenic mutants, confirmed the involvement of autolysin activity and capsule structures in lysozyme resistance of S. suis. Conclusions This study shows that lysozyme resistance levels are highly variable among S. suis isolates and serotypes. Furthermore, the results show that lysozyme resistance in S. suis can involve different mechanisms including OatA-mediated peptidolycan modification, autolysin activity and capsule production.

Wichgers Schreur, Paul J.; van Weeghel, Christian; Rebel, Johanna M. J.; Smits, Mari A.; van Putten, Jos P. M.; Smith, Hilde E.

2012-01-01

393

Lysozyme Deficiency-An Inherited Disorder of Rabbits  

PubMed Central

A genetic disorder of rabbits consisting of a deficiency of the enzyme lysozyme is characterized. The condition appears to be inherited as an autosomal recessive trait. Most of the tissues of lysozyme-deficient rabbits including bone marrow, liver, lung. spleen and bone had levels of lysozyme which were 1% or less of the levels in the corresponding tissues of normal rabbits when measured with the lysoplate method. Levels of lysozyme in the kidney and serum were 6% of controls, but the thymus of the lysozyme-deficient rabbits had normal levels of the enzyme. All leukocytes of the lysozyme-deficient rabbits were negative for lysozyme when examined by a histobacterial technic. No morphologic lesions could be detected in any of the tissues of the lysozyme-deficient rabbits. Although several species of animals have been reported to be lysozyme deficient, this appears to be the first report of lysozyme deficiency occurring as a mutant condition. It is suggested that these mutant rabbits may be useful as a resource for experiments designed to delineate the biologic role of lysozyme. ImagesFigs 5-8Fig 9Fig 1Fig 2Fig 3Fig 4

Prieur, David J.; Olson, Harry M.; Young, David M.

1974-01-01

394

T4 phages against Escherichia coli diarrhea: potential and problems.  

PubMed

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and colon and lower titers in the small intestine, but were not detected in the blood, liver or spleen. No adverse effects were observed after one-month exposure to phage nor were serum anti-T4 antibodies detected. T4 phages belonging to the same subgroup showed closely related genomes that differed by 12 (phage JS10 vs. JS98 reference) to 17 (phage JSE vs. RB49 reference) insertion/deletions mostly representing single small ORFs. Bioinformatic analysis did not reveal undesired genes in the T4 genomes. Sequence variability was seen over the tail fibre genes, but the variability did not correlate with phage host range. The investigated T4 phages were not only species- but also strain-specific, necessitating the use of phage cocktails consisting of 10 and 16 T4 phage isolates to cover half to two thirds of E. coli strains representing the five main pathotypes isolated from diarrhea patients. PMID:19339031

Denou, Emmanuel; Bruttin, Anne; Barretto, Caroline; Ngom-Bru, Catherine; Brüssow, Harald; Zuber, Sophie

2009-04-01

395

Comparative Genomics of the T4Like Escherichia coli Phage JS98: Implications for the Evolution of T4 Phages  

Microsoft Academic Search

About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the

Sandra Chibani-Chennoufi; Carlos Canchaya; Anne Bruttin; Harald Brussow

2004-01-01

396

INDEPENDENT FUNCTIONS OF VIRAL PROTEIN AND NUCLEIC ACID IN GROWTH OF BACTERIOPHAGE  

Microsoft Academic Search

The work of Doermaml (1948), Doermann and Dissosway (1949), and Anderson and Doermann (1952) has shown that bacteriophages T2, T3, and T4 multiply in the bacterial cell in a non-infective form. The same is true of the phage carried by certain lysogenic bacteria (Lwoff and Gutmann, 1950). Little else is known about the vegetative phase of these viruses. The experi-

A. D. Hershey; MARTHA CHASE

1952-01-01

397

HIV1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages  

Microsoft Academic Search

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Qß, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on

Ruth Levitz; Karl Drlica; Ellen Murphy

1994-01-01

398

Phenotypic characterization and genomic analysis of the Shigella sonnei bacteriophage SP18  

Microsoft Academic Search

A novel bacteriophage that infects Shigella sonnei was isolated from the Gap River in Korea, and its phenotypic and genomic characteristics were investigated. The virus, called\\u000a SP18, showed morphology characteristic of the family Myoviridae, and phylogenetic analysis of major capsid gene (gp23) sequences classified it as a T4-like phage. Based on host spectrum\\u000a analysis, it is lytic to S. sonnei,

Kyoung-Ho Kim; Ho-Won Chang; Young-Do Nam; Seong Woon Roh; Jin-Woo Bae

2010-01-01

399

Enzymatic Oligomerization of Bacteriophage P22 DNA and of Linear Simian Virus 40 DNA  

Microsoft Academic Search

Linear double-stranded molecules of the circularly permuted and terminally redundant DNA of Salmonella bacteriophage P22 have been converted to oligomeric products in the presence of polynucleotide ligase coded for by the coliphage T4. The reaction has been monitored by sucrose density-gradient centrifugation and electron microscopy. It goes slowly and gives yields of 30-40%. The products are mainly dimers and trimers,

Vittorio Sgaramella

1972-01-01

400

Factors influencing recombinant human lysozyme extraction and cation exchange adsorption.  

PubMed

Human lysozyme has numerous potential therapeutic applications to a broad spectrum of human diseases. This glycosidic enzyme is present in tears, saliva, nasal secretions, and milk--sources not amendable for commercial development. Recently, a high expression level of recombinant human lysozyme (0.5% dry weight) was achieved in transgenic rice seed. This paper evaluates the effects of pH and ionic strength on rice protein and lysozyme extractability, as well as their interactions with the strong cation-exchange resin, SP-Sepharose FF. The extraction conditions that maximized lysozyme yield and the ratio of extracted human lysozyme to native rice protein were not optimal for lysozyme adsorption. The conditions that gave the highest extracted lysozyme to native protein ratio were pH 4.5 and 100 mM NaCl in 50 mM sodium acetate buffer. At pH 4.5, salt concentrations above 100 mM NaCl reduced the lysozyme-to-protein ratio. The best conditions for lysozyme adsorption were pH 4.5 and 50 mM sodium acetate buffer. Lysozyme extraction and subsequent adsorption at pH 4.5 and 50 mM NaCl was an acceptable compromise between lysozyme extractability, adsorption, and purity. The primary recovery of human lysozyme from pH 6 extracts, irrespective of ionic strength, was inferior to that using pH 4.5 with unacceptably low saturation capacities and lysozyme purity. High purity was achieved with a single chromatography step by adjusting the pH 4.5 extract to pH 6 before adsorption. The disadvantage of this approach was the drastically lower saturation capacity compared to adsorption at pH 4.5. PMID:16739958

Wilken, Lisa R; Nikolov, Zivko L

401

Influence of bacteriophage preparations on intracellular killing of bacteria by human phagocytes in vitro.  

PubMed

Bacteriophages are viruses that infect bacteria. It was shown that bacteriophage therapy is an effective method of combating bacterial infections, including infections caused by antibiotic-resistant bacterial strains. One of the main obstacles to widespread use of phage preparations is limited knowledge regarding the influence of bacteriophages on human organisms. In our study, we evaluated whether application of phage preparations impair bactericidal activities of human phagocytes (granulocytes and monocytes). In our study, we used preparations of phages T2 and T4 specific to Escherichia coli and A3 phage specific to Staphylococcus aureus. We found that bacteriophage preparations do not influence intracellular killing of bacteria by human phagocytes. The effect is irrespective of phage preparation type (lysate, purified phage preparation), phage titer of the preparation, and whether bacteria phagocytosed by phagocyte cells are sensitive or insensitive to phage (bacteriophages homologous and heterologous to bacteria). Although the results of our study are preliminary, they support previous data indicating safety of therapeutic application of phages. PMID:23458442

Kurzepa-Skaradzinska, Aneta; Lusiak-Szelachowska, Marzanna; Skaradzinski, Grzegorz; Jonczyk-Matysiak, Ewa; Weber-Dabrowska, Beata; Zaczek, Maciej; Maj, Tomasz; Slawek, Anna; Rymowicz, Waldemar; Klak, Marlena; Miedzybrodzki, Ryszard; Gorski, Andrzej

2013-03-04

402

Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium.  

PubMed

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This study investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4, and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45% reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium- adhered bacteria. PMID:21140149

Sillankorva, Sanna; Oliveira, Dulce; Moura, Alexandra; Henriques, Mariana; Faustino, Alberta; Nicolau, Ana; Azeredo, Joana

2010-12-08

403

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli.  

PubMed

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 x 10(3)cfu/mL of E. coli in the sample within 2h with high accuracy (CV=1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells. PMID:20561957

Minikh, O; Tolba, M; Brovko, L Y; Griffiths, M W

2010-06-01

404

Recombinant Expression and Purification of T4 Phage Hoc, Soc, gp23, gp24 Proteins in Native Conformations with Stability Studies  

PubMed Central

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.

Miernikiewicz, Paulina; Owczarek, Barbara; Piotrowicz, Agnieszka; Boczkowska, Barbara; Rzewucka, Kamila; Figura, Grzegorz; Letarov, Andrey; Kulikov, Eugene; Kopciuch, Agnieszka; Switala-Jelen, Kinga; Oslizlo, Anna; Hodyra, Katarzyna; Gubernator, Jerzy; Dabrowska, Krystyna

2012-01-01

405

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.  

PubMed

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro. PMID:22808021

Miernikiewicz, Paulina; Owczarek, Barbara; Piotrowicz, Agnieszka; Boczkowska, Barbara; Rzewucka, Kamila; Figura, Grzegorz; Letarov, Andrey; Kulikov, Eugene; Kopciuch, Agnieszka; Swita?a-Jele?, Kinga; O?liz?o, Anna; Hodyra, Katarzyna; Gubernator, Jerzy; D?browska, Krystyna

2012-07-13

406

Thyroid hormones in the elderly sick: "T4 euthyroidism".  

PubMed Central

Thyroid function and serum levels of triiodothyronine (T3) and thyroxine (T4) were investigated in 79 euthyroid geriatric patients. Of the 59 inpatients and 20 outpatients 35 (59%) and 2, respectively, had low T3 levels. In contrast, 7 (12%) and 6 (30%), respectively, had raised T4 levels. Two further patients were excluded from the study because of raised levels of thyroid-stimulating hormone. Thyroxine-binding globulin was greatly increased in both groups of patients, but low serum albumin levels were present in 31 (39%). Despite these changes free T3 and T4 indices closely followed total T3 and T4 levels. The difference between the two groups of patients did not correlate with body weight, diagnostic categories, age, drug treatment, or duration of stay in hospital.

Burrows, A W; Shakespear, R A; Hesch, R D; Cooper, E; Aickin, C M; Burke, C W

1975-01-01

407

Colorimetric and fluorometric dual-readout sensor for lysozyme.  

PubMed

A novel, highly sensitive and selective dual-readout sensor (colorimetric and fluorometric) for the detection of lysozyme was proposed. The fluorescence of triazolylcoumarin molecules was quenched by Au nanoparticles (AuNPs) initially through the fluorescence resonance energy transfer (FRET), after the addition of lysozyme, the stronger binding of lysozyme onto the surfaces of AuNPs made triazolylcoumarin molecules remove from the AuNPs surface and led to the recovery of the fluorescence of triazolylcoumarin molecules, and accompanied by the discernable color change of the solution from red to purple. The lowest detectable concentration for lysozyme was 50 ng mL(-1) by the naked eye, and the limit of detection (LOD) was 23 ng mL(-1) by fluorescence measurements. In addition, satisfactory results for lysozyme detection in hen egg white were confirmed in the study. Moreover, the presented sensor provides a reliable option to determine lysozyme with high sensitivity and selectivity. PMID:23978821

Zheng, Hanye; Qiu, Suyan; Xu, Kefeng; Luo, Linguang; Song, Yibiao; Lin, Zhenyu; Guo, Longhua; Qiu, Bin; Chen, Guonan

2013-09-30

408

Hydration of lysozyme studied by Raman spectroscopy.  

PubMed

Hydration plays a fundamental role in maintaining the three-dimensional structure and function of proteins. In this study, Raman spectroscopy was used to probe the hydration induced structural changes at various sites of lysozyme under isothermal conditions in the range of water contents from 0 to 44 wt %. Raman hydration curves were constructed from detailed analysis of marker bands. Transition inflection points (w(m)) and onsets determined from the hydration curves have shown that structural changes start at 7-10 and end at about 35 wt % water. The onset of structural changes coincides with the onset of the broad glass transition earlier observed in this system. The increase of ?-helix content starts at very low concentrations of water with w(m) = 12 wt %. Monitoring the development of importance for enzymatic action hydrophobic clusters has revealed wm = 15 wt % and completion of the process at 25 wt %. The parameters of 621 cm(-1) (Phe) and 1448 cm(-1) (CH2 bending) modes were found to be sensitive to hydration, suggesting changes in organization of water molecules near the protein surface. The native structure of lysozyme was achieved at 35 wt % water where its content is high enough for filling the space between lysozyme molecules. PMID:23557185

Kocherbitov, Vitaly; Latynis, Jekaterina; Misiunas, Audrius; Barauskas, Justas; Niaura, Gediminas

2013-04-22

409

Characterization of endolysin from a Salmonella Typhimurium-infecting bacteriophage SPN1S.  

PubMed

The full genome sequence of bacteriophage SPN1S, which infects Salmonella, contains genes that encode homologues of holin, endolysin and Rz/Rz1-like accessory proteins, which are 4 phage lysis proteins. The ability of these proteins to lyse Escherichia coli cells when overexpressed was evaluated. In contrast to other endolysins, the expression of endolysin and Rz/Rz1-like proteins was sufficient to cause lysis. The endolysin was tagged with oligohistidine at the N-terminus and purified by affinity chromatography. The endolysin has a lysozyme-like superfamily domain, and its activity was much stronger than that of lysozyme from chicken egg white. We used the chelating agent, ethylenediaminetetraacetic acid (EDTA), to increase outer membrane permeability, and it greatly enhanced the lytic activity of SPN1S endolysin. The antimicrobial activity of endolysin was stable over broad pH and temperature ranges and was active from pH 7.0 to 10.5 and from 25 °C to 45 °C. The SPN1S endolysin could kill most of the tested Gram-negative strains, but the Gram-positive strains were resistant. SPN1S endolysin, like lysozyme, cleaves the glycosidic bond of peptidoglycan. These results suggested that SPN1S endolysin has potential as a therapeutic agent against Gram-negative bacteria. PMID:22289622

Lim, Jeong-A; Shin, Hakdong; Kang, Dong-Hyun; Ryu, Sangryeol

2012-01-18

410

Regulation of the functional activity of lysozyme by alkylhydroxybenzenes  

Microsoft Academic Search

In our study, we investigated the capacity of alkylhydroxybenzenes (AHB), which are microbial anabiosis autoinducers, for\\u000a alteration of the enzymatic activity of the hen egg-white lysozyme, as well as the efficiency of hydrolysis of specific (peptidoglycan)\\u000a and nonspecific (chitin) substrates catalyzed by lysozyme. AHB homologues (C7-AHB and C12-AHB), which differ in their hydrophobicity\\u000a and effects in their interaction with lysozyme,

A. S. Petrovskii; D. G. Deryabin; N. G. Loiko; N. A. Mikhailenko; T. G. Kobzeva; P. A. Kanaev; Yu. A. Nikolaev; Yu. F. Krupyanskii; A. N. Kozlova; G. I. El’-Registan

2009-01-01

411

The associative nature of adenylyl transfer catalyzed by T4 DNA ligase  

PubMed Central

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD+ and assistance of a divalent metal cofactor Mg2+. Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover 31P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase–nucleotide complex is triggered in situ by the photo release of caged Mg2+, and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4–5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.

Cherepanov, Alexey V.; Doroshenko, Elena V.; Matysik, Jorg; de Vries, Simon; de Groot, Huub J. M.

2008-01-01

412

Bacterial 'immunity' against bacteriophages  

PubMed Central

Vertebrate animals possess multiple anti-pathogen defenses. Individual mechanisms usually are differentiated into those that are immunologically adaptive vs. more “primitive” anti-pathogen phenomena described as innate responses. Here I frame defenses used by bacteria against bacteriophages as analogous to these animal immune functions. Included are numerous anti-phage defenses in addition to the adaptive immunity associated with CRISPR/cas systems. As these other anti-pathogen mechanisms are non-adaptive they can be described as making up an innate bacterial immunity. This exercise was undertaken in light of the recent excitement over the discovery that CRISPR/cas systems can serve, as noted, as a form of bacterial adaptive immunity. The broader goal, however, is to gain novel insight into bacterial defenses against phages by fitting these mechanisms into considerations of how multicellular organisms also defend themselves against pathogens. This commentary can be viewed in addition as a bid toward integrating these numerous bacterial anti-phage defenses into a more unified immunology.

2012-01-01

413

[Bacteriophages from micrococci (author's transl)].  

PubMed

Using 14 indicator strains we investigated the possibilities of isolation of bacteriophages from micrococci (two strains of M. luteus, three strains of M. varians and one strain of M. roseus). Three phages were released after mitomycin C-induction, four phages after UV-rays-induction. These seven strains seem to be the first known bacteriophages, which were released from naturally lysogenic micrococci. Induction experiments with beta-propiolacton and dimethylsulfate as well as tests of spontaneous lysogeny produced only negative results. PMID:1179882

Peters, G; Pulverer, G

1975-07-01

414

Isolation of Yersinia ruckeri Bacteriophages  

PubMed Central

Eight bacteriophages effective against Yersinia ruckeri, the enteric redmouth disease bacterium, were isolated. Phage YerA41, a tailed icosahedral virus isolated from sewage enrichments, lysed 34 of 35 strains of Y. ruckeri serovar I, but was inactive against 15 strains belonging to three other serological groups. Six other phages lysed strains of serovars II, V, and I?, a subgroup of serovar I. YerL62, a phage obtained by mitomycin C induction, was specific for one of three serovar V strains. These bacteriophages, particularly YerA41, have potential value for fish disease diagnostic work. Images

Stevenson, R. M. W.; Airdrie, D. W.

1984-01-01

415

T4 lymphopenia in patients with active pulmonary tuberculosis.  

PubMed Central

The numbers of cells bearing the T3 (pan-T cell), the T4 (putative helper/inducer cells), the T8 (putative suppressor/cytotoxic cells) and B cell phenotypic markers were counted in venous blood samples from 26 newly diagnosed pulmonary tuberculosis patients and 29 healthy controls from East Java. The absolute T cell count was lower in the patients and T4 cells were fewer in patients (mean 748/mm3) than in controls (mean 1,043/mm3), but there were no significant differences in total T8 cell and B cell counts between patients and controls. The T4:T8 ratio was not disturbed in many patients, but it was less than 1.6 in 11 of 26 patients and in only three of 29 controls: this ratio was less than 1.2 (the lower limit of 'normal') in six patients but no controls. The intensity of the T4 lymphopenia was unrelated to the extent of the lesion seen radiologically or the size of the skin test reaction to PPD. Levels of interferon-alpha were not elevated in the serum of any of the patients or controls. It is suggested that the T4 lymphopenia was a reaction to the mycobacterial infection and not a manifestation of underlying secondary (acquired) immune deficiency.

Beck, J S; Potts, R C; Kardjito, T; Grange, J M

1985-01-01

416

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage ?29 tail  

PubMed Central

The small bacteriophage ?29 must penetrate the ?250-? thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage ?29 is noncontractile and ?380 ? long. A 1.8-? resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the ?29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13? mutants with the ?29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.

Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.; Bowman, Valorie D.; Anderson, Dwight L.; Rossmann, Michael G.

2008-01-01

417

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage [psi]29 tail  

SciTech Connect

The small bacteriophage {phi}29 must penetrate the {approx}250-{angstrom} thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage {phi}29 is noncontractile and {approx}380 {angstrom} long. A 1.8-{angstrom} resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the {phi}29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13{sup -} mutants with the {phi}29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.

Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.; Bowman, Valorie D.; Anderson, Dwight L.; Rossmann, Michael G. (Purdue); (UMM)

2009-08-28

418