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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)



Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths.  


Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths. PMID:2062826

Bell, J A; Wilson, K P; Zhang, X J; Faber, H R; Nicholson, H; Matthews, B W



Bacteriophage T4 Genome†  

PubMed Central

Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

Miller, Eric S.; Kutter, Elizabeth; Mosig, Gisela; Arisaka, Fumio; Kunisawa, Takashi; Ruger, Wolfgang



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




T4 bacteriophage as a phage display platform.  


Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems. PMID:24828789

Gamkrelidze, Mariam; D?browska, Krystyna



Lessons from the lysozyme of phage T4  

PubMed Central

An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (Tm) for any point mutant is 5.1°C for the mutant Ser 117 ? Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 Å that apparently has an unfavorable van der Waals contact. Increases in Tm of more than 3–4°C for point mutants are rare, whereas several different types of destabilizing substitutions decrease Tm by 20°C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of “large-to-small” mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C-terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically-engineered dimers. Among the 43 space groups, P212121 and P21 were observed most frequently, consistent with the prediction of Wukovitz and Yeates.

Baase, Walter A; Liu, Lijun; Tronrud, Dale E; Matthews, Brian W



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



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.



A Genetic Screen for Mutations That Increase the Thermal Stability of Phage T4 Lysozyme  

Microsoft Academic Search

A method has been developed to screen for mutants of phage T4 lysozyme that are more stable than the wild-type enzyme. Using an assay that detects lysozyme activity on Petri plates [Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, A., Terzaghi, E. & Inouye, M. (1966) Cold Spring Harbor Symp. Quant. Biol. 31, 77-84], protein synthesized during the formation

Tom Alber; Joan A. Wozniak



A Promiscuous DNA Packaging Machine from Bacteriophage T4  

PubMed Central

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%–25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.

Zhang, Zhihong; Kottadiel, Vishal I.; Vafabakhsh, Reza; Dai, Li; Chemla, Yann R.; Ha, Taekjip; Rao, Venigalla B.



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


Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56, denA and denB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA with EcoRI have been cloned using the vector plasmid pCR1. Clones containing T4 DNA were identified by hybridization with radioactive early and late T4 RNA. A simple marker rescue technique is described for the genetic identification of the cloned T4 fragments. Some of the T4-hybrid plasmids which contain entire T4 genes can complement temperature sensitive and amber mutants of T4. PMID:927440

Mattson, T; Van Houwe, G; Bolle, A; Selzer, G; Epstein, R



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


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



Phylogenetic diversity of T4-like bacteriophages in Lake Baikal, East Siberia.  


Among the tailed phages, the myoviruses, those with contractile tails, are widespread and diverse. An important component of the Myoviridae family is the genus 'T4-like viruses'. The present study was aimed at elucidating the molecular diversity of T4-type bacteriophages in Lake Baikal by partial sequencing of g23 genes of T4-type bacteriophages. Our study revealed that the g23 gene sequences investigated were highly diverse and different from those of T4-like bacteriophages and from g23 clones obtained from different environments. Phylogenetic analysis showed that all g23 fragments from Lake Baikal, except for the one sequence, were more closely related to marine T4 cyanophages and to previously described subgroups of uncultured T4 phages from marine and rice field environments. PMID:20579103

Butina, Tatyana Vladimirovna; Belykh, Olga I; Maksimenko, Svetlana Yu; Belikov, Sergey I



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.



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.



Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.  

PubMed Central

New mutants of bacteriophage T4 that overproduce the enzyme dihydrofolate reductase were investigated. Unlike previously described overproducers of this enzyme (Johnson and Hall, 1974), these mutants did not overproduce deoxycytidylate deaminase. Overproduction of dihydrofolate reductase by the new mutants occurred because enzymatic activity continued to increase for a longer period of time in cells infected by the mutants than in cells infected by wild-type phage. This continued increase occurred even in the presence of rifampin, indicating that the overproduction is probably due to a post-transcriptional event. Both these new overproducers and the previously described overproducers were studied by using polyacrylamide gel electrophoresis. The two types of overproducers appeared to be very different. The previously described overproducers showed a delay and/or reduction in the synthesis of several proteins that normally started to be made 4 to 6 min after infection. Several proteins could be seen to be overproduced on the gels. The new overproducers did not show the delay in the synthesis of some proteins and only overproduced a few proteins. The new gene defined by the new overproducers is between the gene coding for thymidine kinase and the gene coding for lysozyme. Images

Chace, K V; Hall, D H



T4 phage lysozyme: a protein designed for understanding tryptophan photophysics  

NASA Astrophysics Data System (ADS)

Bacteriophage T4 lysozyme in its wild type form contains three tryptophan residues (at sequence postions 126, 138 and 158). These three residues are in rather different environments in the protein: 126 and 158 are near the protein surface while residue 138 is more buried. T4 lysozyme has been genetically engineered to prepare all possible variants in which one or more of the tryptophan residues have been replaced by tyrosine. The available data supports the hypothesis that this substitution has, at most, a very minor effect on the structure of the protein. The three species with single tryptophan residues have been investigated in detail. The surface location of residue 126 compared to the buried location of residue 138 is reflected in the difference in collisional quenching observed with added potassium iodide. It is found that the spectral and radiative properties of the three proteins are very similar but that their radiationless decay properties are quite distinct. This is apparently due to short-range collisional quenching by neighboring side chains. Comparison with solution quenching measurements permits the identification of the specific quenching groups involved for each tryptophan residue and provides a semi-quantitative rationale for the radiationless decay rate. This collisional quenching interpretation is supported by mutational effects on fluorescence quantum yield. This simple picture of the behavior of these single-tryptophan proteins is clearly revealed in this particular case because of the unambiguous choice of collisional quenching groups. The time dependence of the fluorescence decay of each of these single-tryptophan proteins is quite complex. Several methods of analysis are presented and discussed in terms of their underlying physical basis. Internal collisional quenching, as suggested from the comparative studies, is expected to lead to non-exponential behavior. This is consistent with the observed time dependence. Analysis of the temporal nature of the fluorescence as a function of emission wavelength is also revealing. Such data can be used to test discrete component, distribution and relaxation models of the time decay. It is found, in agreement with previous studies for other proteins, that the average lifetime for the emission increases with increasing emission wavelength. Analysis of the overall emission wavelength dependence of the time dependent data in a global sense based on a discrete population model shows acceptable agreement with the data in only one of the three cases. Application of several continuous distribution models to this data at each emission wavelength reveals that as the emission is moved to the red, a negative component appears in the distribution of decay components. This is a characteristic feature of relaxation behavior resulting in emission from kinetic species that are not present at the time of excitation. This negative preexponential character is not revealed by discrete component analyses since these do not have sufficient flexibility to describe the underlying complexity of the relaxing distribution. Finally, examination of the three proteins containing two tryptophan residues indicates that there is energy transfer between these residues in these cases and in the wild type protein. The order of energy transfer is in accord with the variation of the magnitude of the ratio k2/R6 controlling the efficiency of Forster energy transfer.

Hudson, Bruce S.; Harris, Dan



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.



T4-lysozyme fusion for the production of human formyl peptide receptors for structural determination.  


T4-lysozyme (T4L) fusion was introduced in the intracellular loop of a G protein-coupled receptor (GPCR) of human formyl peptide receptor 3 (FPR3), and the ability of T4L fusion to be used in the production of human FPR3 for structural determination was evaluated in this work. The T4L variant of human FPR3 termed FPR3-T4L was expressed in stable tetracycline-inducible HEK293 cells. A systematic detergent screening showed that fos-choline-14 was the optimal detergent to solubilize and subsequently purify FPR3-T4L from HEK293 cells. Immunoaffinity purification in combination with gel filtration was employed to purify the T4L-fused receptor to high homogeneity. The final yield of the human FPR3-T4L monomer from 2 g of cells was 0.2 mg. Circular dichroism spectroscopy indicated that the receptor adopted a correct secondary structure after purification, while ligand binding measurement indicated that the receptor was functional. Thus, the presence of T4L fusion did not evidently disturb the expression in HEK293 cells, proper folding, and functionality of human FPR3. Our study of evaluating T4L fusion for the recombinant production of human formyl peptide receptor would facilitate ongoing efforts in the structural characterization of GPCRs. PMID:24407945

Wang, Xiaoqiang; Cui, Ying; Wang, Jiqian



On the origin of the polar order of T4 lysozyme on planar model surfaces.  


Site directed spin labeling is used to investigate the origin of the macroscopic alignment of T4 lysozyme vectorially tethered to planar biomimetic surfaces. T4 lysozyme was adsorbed to a quartz-supported dioleoylphosphatidylcholine (DOPC) bilayer by selective binding of the histidine-tagged protein to functionalized headgroups (1,2-dioleoyl-sn-glycero-3-[[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl], DOGS NTA) of the bilayer. This results in a polar oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance (EPR) spectra. In order to reveal the mechanism of the protein alignment, the influence of protein coverage on the order of the molecules was addressed. Along the lines described previously for a full monolayer (Jacobsen, et al. Biophys. J. 2005, 88, 4351), the polar orientation of the molecules was inferred from an analysis of the EPR line shape using the stochastic Liouville equation (SLE) approach developed by Freed and co-workers. The simulations reveal that the orientation of the protein is strongly determined by lateral protein-protein interactions. In comparison to the lipid bilayer, a fusion protein of T4 lysozyme (T4L) with Annexin XII was investigated, where the two-dimensional crystallization of Annexin XII on a dioleoylphosphatidylserine (DOPS) bilayer provides a surface layer of regularly anchored T4L molecules. For this system, it is found that the interaction between T4L and Annexin plays a more important role for understanding the structure in the adsorbed state. PMID:18171040

Jacobsen, Kerstin; Risse, Thomas



The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.  

PubMed Central

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.

Sjoberg, B M; Hahne, S; Mathews, C Z; Mathews, C K; Rand, K N; Gait, M J



Characterization of New Regulatory Mutants of Bacteriophage T4  

PubMed Central

Plating techniques which eliminate T4 plaque formation on Escherichia coli by folate analogue inhibition of dihydrofolate (FH2) reductase (EC allowed the isolation of folate analogue-resistant (far) mutants of T4. One class of far mutants overproduces the phage-induced FH2 reductase. Deoxycytidylate deaminase (EC, thymidine kinase (EC, and deoxycytidine triphosphatase (EC are also overproduced by 20 min after infection at 37 C. The overproduction of FH2 reductase by these far mutants is not affected by the absence of DNA synthesis. Other types of mutations that affect the synthesis of early enzymes cause overproduction in the absence of DNA synthesis of some of the above enzymes but not of FH2 reductase. Therefore, overproducing far mutants apparently have mutations in previously undescribed genes controlling the expression of the T4 genome. Three of four mutants under study map near gene 56, and one maps near gene 52. All of these mutants show delays in DNA synthesis, phage production, and lysis and appear to show decreased levels of RNA synthesis based on the cumulative incorporation of uridine.

Johnson, James R.; Hall, Dwight H.



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

PubMed Central

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.



A role for two DNA helicases in the replication of T4 bacteriophage DNA.  


The T4 bacteriophage gene 41 protein is the highly processive DNA helicase of the T4 primosome, a central part of the protein machinery that moves the T4 DNA replication fork. The T4 gene 59 protein accelerates the loading of 41 protein onto DNA covered with 32 protein (the T4 single strand binding protein), and it makes the 41 protein DNA helicase activity rapidly available to catalyze replication fork movement through a DNA double helix (Barry, J., and Alberts, B.M. (1994) J. Biol. Chem. 269, 33049-33062). With the aid of the 59 protein, we show that the T4 primosome (the T4 gene 41 and 61 proteins) can move rapidly through a promoter-bound RNA polymerase molecule that would otherwise stop replication fork movement. A second, very different DNA helicase, the T4 dda protein, provides an alternative pathway for replication past this DNA-bound RNA polymerase (Bedinger, P., Hochstrasser, M., Jongeneel, C. V., and Alberts, B.M. (1983) Cell 34, 115-123). Combined with other data, these in vitro experiments allow us to propose a model that explains why either the 59 protein or the dda protein, but not both, are required to begin efficient DNA replication inside the T4 bacteriophage-infected cell. PMID:7806534

Barry, J; Alberts, B



Hotspot Sites for Acridine-Induced Frameshift Mutations in Bacteriophage T4 Correspond to Sites of Action of the T4 Type II Topoisomerase.  

National Technical Information Service (NTIS)

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activi...

L. S. Ripley J. S. Dubins J. G. deBoer D. M. DeMarini A. M. Bogerd



Allelic or NonAllelic? Complementation Studies with Bacteriophage T4 rII Mutations  

Microsoft Academic Search

An easy-to-perform exercise to demonstrate complementation is presented. Complementation tests are used to determine whether mutations affecting the same phenotype are within the same gene (allelic) or in different genes (non-allelic). In this exercise, mutations in the rII locus of bacteriophage T4 are studied. Host E. coli bacteria are co-infected with different T4 mutant phage. If two mutations are in

Susan J. Karcher


Effects of the bacteriophage T4 dda protein on DNA synthesis catalyzed by purified T4 replication proteins.  


The T4 bacteriophage dda protein is a DNA-dependent ATPase and DNA helicase that is the product of an apparently nonessential T4 gene. We have examined its effects on in vitro DNA synthesis catalyzed by a purified, multienzyme T4 DNA replication system. When DNA synthesis is catalyzed by the T4 DNA polymerase on a single-stranded DNA template, the addition of the dda protein is without effect whether or not other replication proteins are present. In contrast, on a double-stranded DNA template, where a mixture of the DNA polymerase, its accessory proteins, and the gene 32 protein is required, the dda protein greatly stimulates DNA synthesis. The dda protein exerts this effect by speeding up the rate of replication fork movement; in this respect, it acts identically with the other DNA helicase in the T4 replication system, the T4 gene 41 protein. However, whereas a 41 protein molecule remains bound to the same replication fork for a prolonged period, the dda protein seems to be continually dissociating from the replication fork and rebinding to it as the fork moves. Some gene 32 protein is required to observe DNA synthesis on a double-stranded DNA template, even in the presence of the dda protein. However, there is a direct competition between this helix-destabilizing protein and the dda protein for binding to single-stranded DNA, causing the rate of replication fork movement to decrease at a high ratio of gene 32 protein to dda protein. As shown elsewhere, the dda protein becomes absolutely required for in vitro DNA synthesis when E. coli RNA polymerase molecules are bound to the DNA template, because these molecules otherwise stop fork movement (Bedinger, P., Hochstrasser, M., Jongeneel, C.V., and Alberts, B. M. (1983) Cell 34, 115-123). PMID:6092352

Jongeneel, C V; Bedinger, P; Alberts, B M



Assembly and infection process of bacteriophage T4  

NASA Astrophysics Data System (ADS)

Bacterophage T4 consists of three parts, namely, a head, a tail, and six tail fibers, each of which is assembled along an independent pathway and then joined. In contrast to simple plant viruses such as tobacco mosaic virus, disassembly and reassembly of the virion is not possible. This is due mainly to the fact that the assembly involves not only irreversible steps such as cleavage of covalent bonds of some constituent proteins, but also that it requires a scaffold and involves the inner membrane of the host cell. Another unique feature of the assembly as a biological nanomachine is the involvement of specific protein devices such as a ``ruler molecule,'' which determines the length of the tail, an ATP-driven DNA packaging protein complex, and phage-encoded molecular chaperones. Recent structural biological studies of the phage started to unveil the molecular mechanics of structural transformation of the tail upon infection.

Arisaka, Fumio



Inactivation of Bacteriophage T4 by Ethyl Methanesulfonate: Influence of Host and Viral Genotypes  

PubMed Central

Inactivation of bacteriophage T4 by ethyl methanesulfonate (EMS) is a complex process which depends critically upon the conditions of treatment and upon both the viral and the host genotypes. EMS-inactivated particles are capable of multiplicity and cross-reactivation, indicating the need for caution in using EMS in certain types of mutation studies. The pyrimidine dimer excision systems of the phage and the host do not affect the EMS sensitivity of T4, but the T4x+y+ system does. Mutational defects in the deoxyribonucleic acid (DNA) ligase and the DNA polymerase systems both of the virus and of its host also affect viral EMS sensitivity.

Ray, Usharanjan; Bartenstein, Lynne; Drake, John W.



Control of promoter utilization by bacteriophage T4-induced modification of RNA polymerase alpha subunit.  

PubMed Central

After infection of Escherichia coli cells, bacteriophage T4 induces several changes in the host DNA-dependent RNA polymerase. A well-characterized chemical change is a two-step ADP-ribosylation of the enzyme's alpha subunit (1). In order to investigate the effect of this change on RNA polymerase transcriptional properties in an in vitro system, we have reconstituted the enzyme from separated individual subunits which were obtained from normal or T4-modified RNA polymerases. It is demonstrated that the enzymes containing T4-modified alpha differ from the enzymes with normal alpha in two respects: (i) their overall activity on T4 DNA is reduced and (ii) they fail to utilize certain T4 promotors while efficiently utilizing other promoters. Among the promoters which are switched off by alpha modification are the two promoters of the D region and one of the two promoters of the T4 tRNA gene cluster. The differential effect of alpha modification on the expression of the tRNA and the D regions in vitro correlates with the previously established pattern of their transcription in vivo. It is suggested that the T4-induced ADP-ribosylation of RNA polymerase alpha subunit is involved in the shutoff of the early bacteriophage genes at the late stage of phage development. Images

Goldfarb, A; Palm, P



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.



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



Bacteriophage T4 baseplate components. III. Location and properties of the bacteriophage structural thymidylate synthetase.  

PubMed Central

Two T4D thymidylate synthetase (td) temperature-sensitive mutants have been isolated and characterized. Both mutants produce heat-labile phage particles. This observation supports the view that this viral-induced protein is a phage structural component. Further, antiserum to td has been shown to block a specific step in tail plate morphogenesis. The results indicated that the td protein is largely covered by the T4D tail plate gene 11 protein. Since the phageinduced dihydrofolate reductase (dfr) also is partially covered by the gene 11 protein, it appears that td was adjacent to the tail plate dfr. This location has been confirmed by constructing a T4D mutant which is dfrtstdts and showing that these two tail plate constituents interact and give altered physical properties to the phage particles produced. A structural relationship for the tail plate folate, dfr, and td has been reported.

Kozloff, L M; Crosby, L K; Lute, M



Investigations of the interactions of saccharides with the lysozyme from bacteriophage lambda.  


The bacteriophage lambda R gene has been isolated into an Escherichia coli expression system and the R gene product, a lysozyme, has been overexpressed and purified to homogeneity using an efficient purification procedure. A turbidimetric assay utilizing chloroform-treated E. coli cells has been optimized to assess the bacteriolytic activity of the purified enzyme. Using this assay, oligomers of beta (1 --> 4) N-acetyl-D-glucosamine at high concentrations were shown to inhibit lysozyme but were not cleaved by the enzyme. Differential scanning calorimetry revealed that the thermal denaturation of lysozyme was found to increase in the presence of (GlcNAc)3 and (GlcNAc)5. The lysozyme was also expressed in an E. coli strain auxotrophic for methionine, allowing for the incorporation of [methyl-13C]methionine into the enzyme. An alteration of the [1H-13C]HMQC NMR spectra of the labelled enzyme was observed in the presence of (GlcNAc)5. Commercially available nitrophenyl glycosides did not act as substrates for lambda lysozyme. The results indicate that lambda lysozyme has specific interactions with oligosaccharides of N-acetylglucosamine, but is incapable of hydrolyzing these sugars. The relevance of the structure of peptidoglycan to the activity of lambda lysozyme is discussed. PMID:7873585

Duewel, H S; Daub, E; Honek, J F



Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants  

PubMed Central

The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering.

Lopez, Carlos J.; Yang, Zhongyu; Altenbach, Christian; Hubbell, Wayne L.



Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants.  


The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering. PMID:24167295

López, Carlos J; Yang, Zhongyu; Altenbach, Christian; Hubbell, Wayne L



Bacteriophage T4 baseplate components. I. Binding and location of the folic acid.  

PubMed Central

Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.

Kozloff, L M; Lute, M; Crosby, L K



In Vitro Complementation as an Assay for New Proteins Required for Bacteriophage T4 DNA Replication: Purification of the Complex Specified by T4 Genes 44 and 62  

PubMed Central

We have developed an in vitro complementation assay for six T4 bacteriophage gene products believed to be components of the T4 DNA replication apparatus. This assay is based upon the fact that DNA synthesis in an infected cell lysate that lacks a given gene product is specifically stimulated by addition of the missing product. By the use of such an assay, two proteins that appear to be the products of T4 genes 44 and 62 have been purified to electrophoretic homogeneity as a single complex of the two polypeptide chains.

Barry, Jack; Alberts, Bruce



Structural role of the polyglutamate portion of the folate found in T4D bacteriophage baseplate.  

PubMed Central

Three types of reagents were used to determine the structural role and location of the polyglutamate portion of the Escherichia coli T4D bacteriophage baseplate dihydropteroyl hexaglutamate. These reagents were examined for their effect in vitro on some of the final steps in phage baseplate morphogenesis. The reagents were (i) a series of oligopeptides composed solely of glutamic acid residues but with various chemical linkages and chain lengths; (ii) a homogeneous preparation of carboxypeptidase G1, an exopeptidase that hydrolyzes carboxyl-terminal glutamates (or aspartates) from simple oligopeptides, including the gamma-glutamyl bonds on folyl polyglutamates as well as the bond between the carboxyl group of the p-aminobenzoyl moiety and the amino group of the first glutamic acid residue of folic acid; and (iii) antisera prepared against a polyglutamate hapten. All three types of reagent markedly inhibited the attachment of the phage long tail fibers to the baseplate. Other steps in baseplate assembly such as the addition of T4D gene 11 or gene 12 products were not affected by any of these reagents. These results indicate that the polyglutamate portion of the folate is located near the attachment site on the bacteriophage baseplate for the long tail fibers.

Kozloff, L M; Crosby, L K; Baugh, C M



Focused genetic recombination of bacteriophage t4 initiated by double-strand breaks.  

PubMed Central

A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCDelta strain of bacteriophage T4 was developed. The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene. Under segC(+) conditions, the ets1 behaves as a recombination hotspot. Crosses of the ets1 against rII markers located to the left and to the right of ets1 gave similar results, thus demonstrating the equal and symmetrical initiation of recombination by either part of the broken chromosome. Frequency/distance relationships were studied in a series of two- and three-factor crosses with other rIIB and rIIA mutants (all segC(+)) separated from ets1 by 12-2100 bp. The observed relationships were readily interpretable in terms of the modified splice/patch coupling model. The advantages of this localized or focused recombination over that distributed along the chromosome, as a model for studying the recombination-replication pathway in T4 in vivo, are discussed.

Shcherbakov, Victor; Granovsky, Igor; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Pyatkov, Konstantin; Shlyapnikov, Michael; Shubina, Olga



A Multifrequency Electron Spin Resonance Study of the Dynamics of Spin Labeled T4 Lysozyme  

PubMed Central

An extensive set of ESR spectra was obtained over a wide range of frequencies (9, 95, 170 and 240 GHz) and temperatures (2 to 32°C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model: the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature, and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2, are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.

Zhang, Ziwei; Fleissner, Mark R.; Tipikin, Dmitriy; Liang, Zhichun; Moscicki, Jozef K.; Earle, Keith; Hubbell, Wayne



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


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



Genetic Analysis of Bacteriophage P22 Lysozyme Structure  

PubMed Central

The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains. Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30°; an additional seven were defective at 40°. Revertants were isolated on the ``missuppressing'' hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants. It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host. Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13. Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype.

Rennell, D.; Poteete, A. R.



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



Marine T4-type bacteriophages, a ubiquitous component of the dark matter of the biosphere  

NASA Astrophysics Data System (ADS)

Tailed bacteriophages are the most abundant biological entities in marine environments. However, most of these marine phages are uncharacterized because few of their hosts have been cultivated. To learn more about such phages, we designed a set of degenerate PCR primers for phage T4 g23, which encodes the major capsid protein in all of the T4-type phages, an important family of the tailed phage. These primers were used to amplify g23-related sequences from diverse marine environments (fjords and bays of British Columbia, the eastern Gulf of Mexico, and the western Arctic Ocean) revealing a remarkable level of molecular diversity, which in some cases was correlated with morphological variation of the virions. Phylogenetic analysis showed that although some of these sequences were closely related to well studied subgroups of the T4-type phage, such as the T-evens, the majority of them belong to five previously uncharacterized subgroups. These data indicate that the host range of T4-type phages is much broader than previously imagined and that the laboratory isolate T4 belongs to a phage family that is extraordinarily widespread and diverse in the biosphere. Author contributions: J.F., F.T., and H.M.K. designed research; J.F. and F.T. performed research; C.A.S. contributed new reagents/analytic tools; J.F., C.A.S., and H.M.K. analyzed data; and J.F., C.A.S., and H.M.K. wrote the paper.This paper was submitted directly (Track II) to the PNAS office.Data deposition: The nucleotide sequences of g23 reported in this paper have been deposited in the GenBank database (accession nos. DQ105858-DQ105942).

Filée, Jonathan; Tétart, Françoise; Suttle, Curtis A.; Krisch, H. M.



Double-strand break repair in tandem repeats during bacteriophage T4 infection.  

PubMed Central

Recombinational repair of double-strand breaks in tandemly repeated sequences often results in the loss of one or more copies of the repeat. The single-strand annealing (SSA) model for repair has been proposed to account for this nonconservative recombination. In this study we present a plasmid-based physical assay that measures SSA during bacteriophage T4 infection and apply this assay to the genetic analysis of break repair. SSA occurs readily in broken plasmid DNA and is independent of the strand exchange protein UvsX and its accessory factor UvsY. We use the unique features of T4 DNA metabolism to examine the link between SSA repair and DNA replication and demonstrate directly that the DNA polymerase and the major replicative helicase of the phage are not required for SSA repair. We also show that the Escherichia coli RecBCD enzyme can mediate the degradation of broken DNA during early, but not late, times of infection. Finally, we consider the status of broken ends during the course of the infection and propose a model for SSA during T4 infections.

Tomso, D J; Kreuzer, K N



SP62, a Viable Mutant of Bacteriophage T4D Defective in Regulation of Phage Enzyme Synthesis  

PubMed Central

SP62 is a mutant of bacteriophage T4D that was discovered because it produces fewer phage than the wild type in the presence of 5-fluorodeoxyuridine. In the absence of phage DNA synthesis, SP62 solubilizes host DNA slower than normal; this may explain the sensitivity to 5-fluorodeoxyuridine. In Escherichia coli B at 37 C in the absence of drugs, SP62 makes DNA at a normal rate and the kinetics of appearance of phage are nearly normal. Under the same conditions, SP62 produces T4 lysozyme (gene e) at a normal rate until 20 min, but then produces it at twice the normal rate until at least 60 min. It has long been known that, when T4 DNA synthesis is blocked (DNA? state) in an otherwise normal infection, the synthesis of a number of early enzymes continues beyond the shutoff time of about 12 min seen in the DNA+ state, but still stops at about 20 min. We have termed the 12-min shutoff event S1 and the 20-min shutoff event S2. We show here that, in the DNA+ state, SP62 makes four early enzymes normally, i.e., S1 occurs. However, in the DNA? state (where S1 is missing), SP62 continues to make dCTPase (gene 56), dCMP hydroxymethylase (gene 42), and deoxynucleotide kinase (gene 1) for at least an hour; this results in production of up to 13 times the normal level of dCTPase at 60 min after infection, or 6 times the DNA? level. We conclude that SP62 is defective in the second shutoff mechanism, S2, for these three enzymes. In contrast, SP62 causes premature cessation of dTMP synthetase production in the DNA? state; the result is a twofold underproduction of dTMP synthetase. Autoradiograms of pulse-labeled proteins separated by slab-gel electrophoresis in the presence of sodium dodecyl sulfate show that a number of other T4 early proteins, including the products of genes 45, 46, and rIIA, are synthesized longer than normal by SP62 in the DNA? state. Few late proteins are made in the DNA? state, but in autoradiograms examining the DNA+ state there is little or no effect of the SP62 mutation on the synthesis of T4 late or early proteins. Circumstantial evidence is presented favoring a role for the gene of SP62 in translation of certain mRNAs. At very high temperatures (above 43 C) in the absence of drugs, phage production, but not DNA synthesis, is much reduced in SP62 infections relative to wild-type T4 infections; this temperature sensitivity is greater on E. coli CR63 than on E. coli B. This property has facilitated recognition of the SP62 genotype and aided in complementation testing and genetic mapping. A later publication will provide evidence that SP62 defines a new T4 gene named regA, which maps between genes 43 and 62. Images

Wiberg, John S.; Mendelsohn, Steven; Warner, Virginia; Hercules, Kathleen; Aldrich, Cynthia; Munro, Judith L.



Nucleotide sequence and revised map location of the arn gene from bacteriophage T4.  


Non-glucosylated (Glu-) T-even phage DNAs are restricted by Escherichia coli RgIA and RgIB endonucleases with different specificities. RgIB endonuclease activity is strongly inhibited by anti-restriction endonuclease (Arn) encoded by the bacteriophage T4 genome. The nucleotide sequence of the arn gene encoding Arn was determined. The product of the cloned arn gene was overexpressed by the T7 RNA polymerase/promoter system, and its molecular size is consistent with that predicted from the open reading frame of the arn gene. The arn gene is located between the asiA gene and motA gene in the region of 161,300-161,578 nucleotides. PMID:9387160

Kim, B C; Kim, K; Park, E H; Lim, C J



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.



Evidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor.  


How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle. PMID:24937091

Migliori, Amy D; Keller, Nicholas; Alam, Tanfis I; Mahalingam, Marthandan; Rao, Venigalla B; Arya, Gaurav; Smith, Douglas E



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



The 3'-5' proofreading exonuclease of bacteriophage T4 DNA polymerase is stimulated by other T4 DNA replication proteins.  


The bacteriophage T4 DNA polymerase has an intrinsic 3'-5' proofreading exonuclease activity that plays a central role in determining the fidelity of T4 DNA replication. In order to monitor this activity, we have measured the rate at which the polymerase decreases the size of a double-stranded DNA substrate in the absence of deoxyribonucleoside triphosphates. With this assay, we find that the addition of the polymerase accessory proteins, 45 protein and 44/62 protein, increases the rate at which the polymerase-associated exonuclease digests the DNA substrate 3- to 4-fold. This stimulation requires the continuous hydrolysis of ATP catalyzed by the accessory protein complex. When added alone, the T4 helix-destabilizing protein, 32 protein, inhibits the exonuclease rate at high concentrations (greater than 100 micrograms/ml), while stimulating about 3-fold at low concentrations. The 32 protein and the accessory proteins together increase the exonuclease rate 8- to 10-fold above that found for the polymerase alone. The bacteriophage T7 DNA polymerase displays a similar 3'-5' exonuclease activity, but this exonuclease is not stimulated by any of the T4 replication proteins. It therefore appears that specific protein-protein interactions are involved. PMID:6604051

Bedinger, P; Alberts, B M



The Roles of the Bacteriophage T4 r Genes in Lysis Inhibition and Fine-Structure Genetics: A New Perspective  

Microsoft Academic Search

Seldom has the study of a set of genes contributed more to our understanding of molecular genetics than has the characterization of the rapid-lysis genes of bacteriophage T4. For example, T4 rII mutants were used to define gene structure and mutagen effects at the molecular level and to help unravel the genetic code. The large-plaque morphology of these mutants reflects

Patrick Paddison; Stephen T. Abedon; Holly Kloos Dressman; Katherine Gailbreath; Julia Tracy; Eric Mosser; James Neitzel; Burton Guttman; Elizabeth Kutter


Membrane Interaction of the Portal Protein gp20 of Bacteriophage T4  

PubMed Central

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.

Quinten, Tobias A.



Membrane interaction of the portal protein gp20 of bacteriophage T4.  


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



Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers.  


Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A. M., Nossal, N. G., and Griffith, J. D. (2003) J. Biol. Chem. 278, 21276-21285). Here we probe the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. 6% of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. Under fixation conditions that extend the lagging strand, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein remains on the fork after loading the helicase and is present on molecules with extensive replication. PMID:17105722

Nossal, Nancy G; Makhov, Alexander M; Chastain, Paul D; Jones, Charles E; Griffith, Jack D



Properties of the T4 bacteriophage DNA replication apparatus: the T4 dda DNA helicase is required to pass a bound RNA polymerase molecule.  


The interaction of DNA replication forks with both stationary and transcribing RNA polymerase molecules has been examined in vitro, using the multienzyme T4 bacteriophage DNA replication system and purified E. coli RNA polymerase. We have found that a single stationary RNA polymerase molecule can block the movement of the T4 replication fork when bound to a promoter on a double-stranded fd DNA template. When transcription is allowed (in the same direction as replication), the replication fork appears to follow the moving RNA polymerase molecule at the relatively slow rate of transcription. The barriers to fork movement formed by E. coli RNA polymerase are eliminated by the addition of small amounts of a purified T4-encoded DNA helicase, the product of the dda gene. We find that replication complexes containing the dda protein cause stationary RNA polymerase molecules to dissociate from the DNA. PMID:6136341

Bedinger, P; Hochstrasser, M; Jongeneel, C V; Alberts, B M



Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor  

Microsoft Academic Search

Helicases are molecular motor enzymes that unwind and translocate nucleic acids. One of the central questions regarding helicase activity is whether the process of coupling ATP hydrolysis to DNA unwinding requires an oligomeric form of the enzyme. We have applied a pre-steady-state kinetics approach to address this question with the bacteriophage T4 Dda helicase. If a helicase can function as

Bindu Nanduri; Alicia K. Byrd; Robert L. Eoff; Alan J. Tackett; Kevin D. Raney



In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.  

PubMed Central

Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons.

Hulen, C; Legault-Demare, J



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


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



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.



Biochemical characterization of bacteriophage T4 Mre11-Rad50 complex.  


The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair. PMID:21081488

Herdendorf, Timothy J; Albrecht, Dustin W; Benkovic, Stephen J; Nelson, Scott W



Biochemical Characterization of Bacteriophage T4 Mre11-Rad50 Complex*  

PubMed Central

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn2+-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3? to 5? dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3? ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg2+ as a cofactor for its nuclease activity. Additionally, the Mg2+-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3? ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.

Herdendorf, Timothy J.; Albrecht, Dustin W.; Benkovic, Stephen J.; Nelson, Scott W.



Using transposon Tn5 insertions to sequence bacteriophage T4 gene 11.  


A simple and rapid method of creating an overlapping set of deletions in cloned DNA in preparation for sequencing has been developed. The method is based on a positive selection for Tn5 transposition into the cloned DNA fragment on a high-copy-number filamid, resolution of potential filamid dimers by filamentous phage infection, and the use of Tn5 both as a "portable" restriction enzyme site for in vitro DNA deletion and a "portable" sequencing primer binding site to initiate DNA sequencing reactions using a custom primer complementary to the outside ends of IS50. This new method has been utilized to sequence bacteriophage T4 gene 11, encoding the T4 baseplate protein gp11. The coding sequence of gene 11 is 657 bp in length, and predicts a primary structure of 219 amino acids that agrees well with the biochemical data previously obtained. DNA sequence around gene 11 suggests that the expression of genes 10, 11, and 12 of phage T4 are translationally coupled. Plasmids carrying deletions generated using this method have been used to map genetically five amber alleles of gene 11. These amber alleles were sequenced to confirm the proposed reading frame. The five amber alleles actually represent two different mutational changes at either codon 206 or 207, changing these adjacent glutamine codons to amber. The position of these amber alleles lends support to earlier studies identifying the carboxyl terminus of gp11 as essential in the interaction of P11 with baseplate protein P10 (Plishker and Berget, 1984). PMID:2548819

Barrett, B K; Berget, P B



Level of specific prereplicative mRNA's during bacteriophage T4 regA-, 43- and T4 43- infection of Escherichia coli B.  

PubMed Central

The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation. Images

Trimble, R B; Maley



Folding and Function of a T4 Lysozyme Containing 10 Consecutive Alanines Illustrate the Redundancy of Information in an Amino Acid Sequence  

NASA Astrophysics Data System (ADS)

Single and multiple Xaa -> Ala substitutions were constructed in the ?-helix comprising residues 39-50 in bacteriophage T4 lysozyme. The variant with alanines at 10 consecutive positions (A40-49) folds normally and has activity essentially the same as wild type, although it is less stable. The crystal structure of this polyalanine mutant displays no significant change in the main-chain atoms of the helix when compared with the wild-type structure. The individual substitutions of the solvent-exposed residues Asn-40, Ser-44, and Glu-45 with alanine tend to increase the thermostability of the protein, whereas replacements of the buried or partially buried residues Lys-43 and Leu-46 are destabilizing. The melting temperature of the lysozyme in which Lys-43 and Leu-46 are retained and positions 40, 44, 45, 47, and 48 are substituted with alanine (i.e., A40-42/44-45/47-49) is increased by 3.1^circC relative to wild type at pH 3.0, but reduced by 1.6^circC at pH 6.7. In the case of the charged amino acids Glu-45 and Lys-48, the changes in melting temperature indicate that the putative salt bridge between these two residues contributes essentially nothing to the stability of the protein. The results clearly demonstrate that there is considerable redundancy in the sequence information in the polypeptide chain; not every amino acid is essential for folding. Also, further evidence is provided that the replacement of fully solvent-exposed residues within ?-helices with alanines may be a general way to increase protein stability. The general approach may permit a simplification of the protein folding problem by retaining only amino acids proven to be essential for folding and replacing the remainder with alanine.

Heinz, Dirk W.; Baase, Walt A.; Matthews, Brian W.



Genetic analysis of the bacteriophage T4-encoded cochaperonin Gp31.  

PubMed Central

Previous genetic and biochemical analyses have established that the bacteriophage T4-encoded Gp31 is a cochaperonin that interacts with Escherichia coli's GroEL to ensure the timely and accurate folding of Gp23, the bacteriophage-encoded major capsid protein. The heptameric Gp31 cochaperonin, like the E. coli GroES cochaperonin, interacts with GroEL primarily through its unstructured mobile loop segment. Upon binding to GroEL, the mobile loop adopts a structured, beta-hairpin turn. In this article, we present extensive genetic data that strongly substantiate and extend these biochemical studies. These studies begin with the isolation of mutations in gene 31 based on the ability to plaque on groEL44 mutant bacteria, whose mutant product interacts weakly with Gp31. Our genetic system is unique because it also allows for the direct selection of revertants of such gene 31 mutations, based on their ability to plaque on groEL515 mutant bacteria. Interestingly, all of these revertants are pseudorevertants because the original 31 mutation is maintained. In addition, we show that the classical tsA70 mutation in gene 31 changes a conserved hydrophobic residue in the mobile loop to a hydrophilic one. Pseudorevertants of tsA70, which enable growth at the restrictive temperatures, acquire the same mutation previously shown to allow plaque formation on groEL44 mutant bacteria. Our genetic analyses highlight the crucial importance of all three highly conserved hydrophobic residues of the mobile loop of Gp31 in the productive interaction with GroEL.

Richardson, A; Georgopoulos, C



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.



Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies  

PubMed Central

Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5?-O-(3-thiotriphosphate) (MgATP?S) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41·DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the primosome on the lagging strand of DNA at a replication fork.

Zhang, Zhiquan; Spiering, Michelle M.; Trakselis, Michael A.; Ishmael, Faoud T.; Xi, Jun; Benkovic, Stephen J.; Hammes, Gordon G.



Nucleotide sequences involved in bacteriophage T4 gene 32 translational self-regulation.  

PubMed Central

We have determined the nucleotide sequence of a cloned segment of the bacteriophage T4D chromosome, which contains the regulatory sequences and the structural gene (gene 32) for the single-stranded DNA binding protein (gp32). The amino acid sequence predicted by translation of the structural gene agrees well with that published for gp32 [Williams, K. R., Lo-Presti, M. B., Setoguchi, M. & Konigsberg, W. H. (1980) Proc. Natl. Acad. Sci. USA 77, 4614-4617]. To localize the nucleotide sequence involved in translational self-regulation of gene 32, we have constructed a series of plasmids in which gene 32 is fused to an amino-terminal deletion mutant of the beta-galactosidase gene of Escherichia coli. Expression of a beta-galactosidase fusion protein that contains only the first seven amino acids of gp32 is still repressed by gp32. The ribosomal binding site of gene 32 is flanked by a repetitive A+T-rich sequence. Preferential and cooperative binding of gp32 to this region of its mRNA could inhibit translation initiation and, thus, would account for the autoregulation. Images

Krisch, H M; Allet, B



Coordination of DNA ends during double-strand-break repair in bacteriophage T4.  

PubMed Central

The extensive chromosome replication (ECR) model of double-strand-break repair (DSBR) proposes that each end of a double-strand break (DSB) is repaired independently by initiating extensive semiconservative DNA replication after strand invasion into homologous template DNA. In contrast, several other DSBR models propose that the two ends of a break are repaired in a coordinated manner using a single repair template with only limited DNA synthesis. We have developed plasmid and chromosomal recombinational repair assays to assess coordination of the broken ends during DSBR in bacteriophage T4. Results from the plasmid assay demonstrate that the two ends of a DSB can be repaired independently using homologous regions on two different plasmids and that extensive replication is triggered in the process. These findings are consistent with the ECR model of DSBR. However, results from the chromosomal assay imply that the two ends of a DSB utilize the same homologous repair template even when many potential templates are present, suggesting coordination of the broken ends during chromosomal repair. This result is consistent with several coordinated models of DSBR, including a modified version of the ECR model.

Stohr, Bradley A; Kreuzer, Kenneth N



T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by 'Dickeya solani'.  


The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics. PMID:22413005

Adriaenssens, Evelien M; Van Vaerenbergh, Johan; Vandenheuvel, Dieter; Dunon, Vincent; Ceyssens, Pieter-Jan; De Proft, Maurice; Kropinski, Andrew M; Noben, Jean-Paul; Maes, Martine; Lavigne, Rob



Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase  

Microsoft Academic Search

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non- sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used

Alan J. Tackett; David R. Corey; Kevin D. Rane



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



Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus uv-specific endonucleases  

SciTech Connect

A comparison was made of the activity of the uv-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultraviolet light-irradiated DNA substrates of defined sequence. The two enzyms cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.

Gordon, L.K.; Haseltine, W.A.



Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing  

PubMed Central

Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb3+ ions and the selective 2?-hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb3+ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem–loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.

Todd, Gabrielle C.; Walter, Nils G.



Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor.  


Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging. PMID:16258174

Mitchell, Michael S; Rao, Venigalla B



Distance measurements by fluorescence energy homotransfer: evaluation in T4 lysozyme and correlation with dipolar coupling between spin labels.  


We demonstrate the feasibility and practical limitations of using steady-state anisotropy to determine distances from fluorescence homotransfer in the context of a protein of known crystal structure. Eight double mutants of T4 lysozyme spanning the distance range between 20 A and 50 A were labeled with a methanethiosulfonate derivative of fluorescein. The measured distances in liquid solution are in agreement with those determined from dipolar coupling between spin labels in the frozen state. They can be interpreted in the context of the crystal structure after accounting for the probe linking arm. Overall, the results establish the necessary calibration for this spectroscopic ruler. The measurement of similar distance trends using independent probes sets the stage for the complementary use of homotransfer and dipolar coupling in the determination of static structures and detection of conformational changes. PMID:17142264

Zou, Ping; Surendhran, Kavitha; Mchaourab, Hassane S



Diversity of the major capsid genes (g23) of T4-like bacteriophages in the eutrophic Lake Kotokel in East Siberia, Russia.  


Numerous studies revealed high diversity of T4-like bacteriophages in various environments, but so far, little is known about T4-like virus diversity in freshwater bodies, particularly in eutrophic lakes. The present study was aimed at elucidating molecular diversity of T4-like bacteriophages in eutrophic Lake Kotokel located near Lake Baikal by partial sequencing of the major capsid genes (g23) of T4-like bacteriophages. The majority of g23 fragments from Lake Kotokel were most similar to those from freshwater lakes and paddy fields. Despite the proximity and direct water connection between Lake Kotokel and Lake Baikal, g23 sequence assemblages from two lakes were different. UniFrac analysis showed that uncultured T4-like viruses from Lake Kotokel tended to cluster with those from the distant lake of the same trophic status. This fact suggested that the trophic conditions affected the formation of viral populations, particularly of T4-like viruses, in freshwater environments. PMID:23539063

Butina, Tatyana V; Belykh, Olga I; Potapov, Sergey A; Sorokovikova, Ekaterina G



Bacteriophage t4 nanoparticles as materials in sensor applications: variables that influence their organization and assembly on surfaces.  


Bacteriophage T4 nanoparticles possess characteristics that make them ideal candidates as materials for sensors, particularly as sensor probes. Their surface can be modified, either through genetic engineering or direct chemical conjugation to display functional moieties such as antibodies or other proteins to recognize a specific target. However, in order for T4 nanoparticles to be utilized as a sensor probe, it is necessary to understand and control the variables that determine their assembly and organization on a surface. The aim of this work is to discuss some of variables that we have identified as influencing the behavior of T4 nanoparticles on surfaces. The effect of pH, ionic strength, substrate characteristics, nanoparticle concentration and charge was addressed qualitatively using atomic force microscopy (AFM). PMID:22454586

Archer, Marie J; Liu, Jinny L



Orthogonal Spin Labeling and Gd(III)-Nitroxide Distance Measurements on Bacteriophage T4-Lysozyme  

PubMed Central

We present the first example of chemo-selective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a Gadolinium (III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity.

Garbuio, Luca; Bordignon, Enrica; Brooks, Evan K.; Hubbell, Wayne L.; Jeschke, Gunnar; Yulikov, Maxim



Effective inhibition of lytic development of bacteriophages ?, P1 and T4 by starvation of their host, Escherichia coli  

PubMed Central

Background Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. Results Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, ?, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. Conclusion Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

Los, Marcin; Golec, Piotr; Los, Joanna M; Weglewska-Jurkiewicz, Anna; Czyz, Agata; Wegrzyn, Alicja; Wegrzyn, Grzegorz; Neubauer, Peter



Structural Refinement from Restrained-Ensemble Simulations Based on EPR/DEER Data: Application to T4 Lysozyme  

PubMed Central

DEER (Double Electron Electron Resonance) is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of distance histograms between pairs of nitroxide spin-labels linked to a protein in a native-like solution environment. However, exploiting the huge amount of information provided by ESR/DEER histograms to refine structural models is extremely challenging. In this study, a restrained ensemble (RE) molecular dynamics (MD) simulation methodology is developed to address this issue. In RE simulation, the spin-spin distance distribution histograms calculated from a multiple-copy MD simulation are enforced, via a global ensemble-based energy restraint, to match those obtained from ESR/DEER experiments. The RE simulation is applied to 51 ESR/DEER distance histogram data from spin-labels inserted at 37 different positions in T4 lysozyme (T4L). The rotamer population distribution along the five dihedral angles connecting the nitroxide ring to the protein backbone is determined and shown to be consistent with available information from X-ray crystallography. For the purpose of structural refinement, the concept of a simplified nitroxide dummy spin-label is designed and parameterized on the basis of these all-atom RE simulations with explicit solvent. It is demonstrated that RE simulations with the dummy nitroxide spin-labels imposing the ESR/DEER experimental distance distribution data are able to systematically correct and refine a series of distorted T4L structures, while simple harmonic distance restraints are unsuccessful. This computationally efficient approach allows experimental restraints from DEER experiments to be incorporated into RE simulations for efficient structural refinement.

Islam, Shahidul M.; Stein, Richard A.; Mchaourab, Hassane; Roux, Benoit



Structural refinement from restrained-ensemble simulations based on EPR/DEER data: application to T4 lysozyme.  


DEER (double electron-electron resonance) is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of distance histograms between pairs of nitroxide spin-labels linked to a protein in a native-like solution environment. However, exploiting the huge amount of information provided by ESR/DEER histograms to refine structural models is extremely challenging. In this study, a restrained ensemble (RE) molecular dynamics (MD) simulation methodology is developed to address this issue. In RE simulation, the spin-spin distance distribution histograms calculated from a multiple-copy MD simulation are enforced, via a global ensemble-based energy restraint, to match those obtained from ESR/DEER experiments. The RE simulation is applied to 51 ESR/DEER distance histogram data from spin-labels inserted at 37 different positions in T4 lysozyme (T4L). The rotamer population distribution along the five dihedral angles connecting the nitroxide ring to the protein backbone is determined and shown to be consistent with available information from X-ray crystallography. For the purpose of structural refinement, the concept of a simplified nitroxide dummy spin-label is designed and parametrized on the basis of these all-atom RE simulations with explicit solvent. It is demonstrated that RE simulations with the dummy nitroxide spin-labels imposing the ESR/DEER experimental distance distribution data are able to systematically correct and refine a series of distorted T4L structures, while simple harmonic distance restraints are unsuccessful. This computationally efficient approach allows experimental restraints from DEER experiments to be incorporated into RE simulations for efficient structural refinement. PMID:23510103

Islam, Shahidul M; Stein, Richard A; McHaourab, Hassane S; Roux, Benoît



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



Complete Genome Sequence of the Broad-Host-Range Vibriophage KVP40: Comparative Genomics of a T4-Related Bacteriophage  

PubMed Central

The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined. The genome sequence is 244,835 bp, with an overall G+C content of 42.6%. It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators. Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp. While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4. At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity). The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40. There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes. KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns. KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified. There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40. From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages. Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved.

Miller, Eric S.; Heidelberg, John F.; Eisen, Jonathan A.; Nelson, William C.; Durkin, A. Scott; Ciecko, Ann; Feldblyum, Tamara V.; White, Owen; Paulsen, Ian T.; Nierman, William C.; Lee, Jong; Szczypinski, Bridget; Fraser, Claire M.




Microsoft Academic Search

Ever since lysozyme was discovered by Fleming in 1922, this protein has emerged as a model for investigations on protein structure and function. Over the years, several high-resolution structures have yielded a wealth of structural data on this protein. Extensive studies on folding of lysozyme have shown how different regions of this protein dynamically interact with one another. Data is

Rajaram Swaminathan; Vijay Kumar Ravi; Satish Kumar; Mattaparthi Venkata Satish Kumar; Nividh Chandra



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.



Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase. Processivity and orientation to the methylation target.  


We carried out steady state and pre-steady state (burst) kinetic analyses of the bacteriophage T4 Dam DNA-(N(6)-adenine)-methyltransferase (MTase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to Ade in oligonucleotide duplexes containing one or two specific GATC sites with different combinations of methylated and unmodified targets. We compared the results for ligated 40-mer duplexes with those of the mixtures of the two unligated duplexes used to generate the 40-mers. The salient results are as follows: (i) T4 Dam MTase modifies 40-mer duplexes in a processive fashion. (ii) During processive movement, T4 Dam rapidly exchanges product S-adenosyl-l-homocysteine (AdoHcy) for substrate AdoMet without dissociating from the DNA duplex. (iii) T4 Dam processivity is consistent with an ordered bi-bi mechanism AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. However, in contrast to the steady state, here DNA(Me) upward arrow signifies departure from a methylated site GMTC upward arrow without physically dissociating from the DNA. (iv) Following methyl transfer at one site and linear diffusion to a hemimethylated site, a reconstituted T4 Dam-AdoMet complex rapidly reorients itself to the (productive) unmethylated strand. T4 Dam-AdoHcy cannot reorient at an enzymatically created GMTC site. (v) The inhibition potential of fully methylated sites 5'-GMTC/5'-GMTC is much lower for a long DNA molecule compared with short single-site duplexes. PMID:12501249

Zinoviev, Victor V; Evdokimov, Alexey A; Malygin, Ernst G; Schlagman, Samuel L; Hattman, Stanley



Nucleotide sequence determination of bacteriophage T4 glycine transfer ribonucleic acid  

PubMed Central

The nucleotide sequence of a T4 tRNA with an anticodon for glycine has been determined using 32P-labeled material from T4-infected cultures of Escherichia coli. The sequence is: pGCGGAUAUCGUAUAAUGmGDAUUACCUCAGACUUCCAA?CUGAUGAUGUGAGT?CGAUUCUCAUUAUCCGCUCCA-OH. The 74 nucleotide sequence can be arranged in the classic cloverleaf pattern for tRNAs. The anticodon of T4 tRNAGly is UCC with a possible modification of the U. The tRNA molecule would thus be expected to recognize the glycine codons GGG and GGA. Comparative analysis of tRNAsGly from T2 and T6 indicate that their sequences are identical with that from T4. Images

Stahl, Stephen; Paddock, Gary V.; Abelson, John



An ORFan No More: The Bacteriophage T4 39.2 Gene Product, NwgI, Modulates GroEL Chaperone Function  

PubMed Central

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.

Ang, Debbie; Georgopoulos, Costa



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))



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


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



Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.  


Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications. PMID:14654700

Blondal, Thorarinn; Hjorleifsdottir, Sigridur H; Fridjonsson, Olafur F; Aevarsson, Arnthor; Skirnisdottir, Sigurlaug; Hermannsdottir, Anna Gudny; Hreggvidsson, Gudmundur O; Smith, Albert Vernon; Kristjansson, Jakob K



The Mutagenic Effect of D2O on Bacteriophage T4  

Microsoft Academic Search

E. coli and T4 phage were grown in MI media containing various ; concentrations of Dâ0. Since the final phage titer attainable and the ; growth rate of the bacteria decreases sharply when the concentration of Dâ0 ; exceeds 50%, a more thorough investigation was carried out to test the mutaagenic ; effect of 50% Dâ0. It was found that

Michael Konrad



Synthesis of functional bacteriophage T4-delayed early mRNA in the absence of protein synthesis.  

PubMed Central

When Escherichia coli B207 is grown either aerobically or under limited aerobic conditions, pretreated with chloramphenicol to block protein synthesis, and then infected with bacteriophage T4, the phage RNA which accumulates, termed "immediately early" (IE), contains the transcripts of a limited number of prereplicative genes. Among the transcripts which accumulate is the mRNA which serves as a template for deoxycytidylate hydroxymethylase (HMase) synthesis. Among the prereplicative gene transcripts which do not accumulate under these conditions are deoxycytidine triphosphatase (dCTPase), alpha-glucosyl transferase (alphg-gt), and deoxynucleotide kinase (kinase); these genes have been termed "delayed early" (DE). In contrast, when protein synthesis is inhibited by depleting aerobically grown E. coli B207 of K+, both IE and DE T4 RNA accumulate, but these transcripts do not contain functional HMase, dCTPase, alpha-gt, or kinase mRNA's. However, if E. coli is grown under conditions of limited aeration and then depleted of K+ prior to T4 infection, the T4 RNA which accumulates contains both IE and DE transcripts and functional HMase, dCTPase, and alpha-gt mRNA's. Functional kinase mRNA does not accumulate under these conditions. The results of these experiments indicate that the synthesis of functional DE RNA in the absence of simultaneous protein synthesis, depends on the physiological condition of the cells and the way in which protein synthesis is inhibited. In addition, data is presented which suggests that extensive transcription of DE genes in the absence of protein synthesis results in the inhibition of transcription of certain IE genes.

Morse, J W; Cohen, P S



Repetitive lagging strand DNA synthesis by the bacteriophage T4 replisome.  


Our studies on the T4 replisome build on the seminal work from the Alberts laboratory. They discovered essentially all the proteins that constitute the T4 replisome, isolated them, and measured their enzymatic activities. Ultimately, in brilliant experiments they reconstituted in vitro a functioning replisome and in the absence of structural information created a mosaic as to how such a machine might be assembled. Their consideration of the problem of continuous leading strand synthesis opposing discontinuous lagging strand synthesis led to their imaginative proposal of the trombone model, an illustration that graces all textbooks of biochemistry. Our subsequent work deepens their findings through experiments that focus on defining the kinetics, structural elements, and protein-protein contacts essential for replisome assembly and function. In this highlight we address when Okazaki primer synthesis is initiated and how the primer is captured by a recycling lagging strand polymerase--problems that the Alberts laboratory likewise found mysterious and significant for all replisomes. PMID:18931782

Spiering, Michelle M; Nelson, Scott W; Benkovic, Stephen J



Intragenic Complementation byGene42AmberMutations of Bacteriophage T4  

Microsoft Academic Search

were shownbyinvivoandinvitro experiments toparticipate inbothpositive and negative intragenic complementation. Thisarguesthatincomplete polypeptide chains canparticipate insubunit interaction. ChiuandGreenberg (4)haveobserved intra- genic complementation intsx tsmixedinfec- tions byusing tsmutants defective ingene42of phage T4,thegenethat specifies dCMPhydrox- ymethylase (13). Inthepresent study, am xts andam x am+ complementation tests were carried outwithgene42mutants bothinvivo andinvitro, andpositive aswellasnegative complementation reactions wereobserved. Theseassays weredonetotest whether incom- pletepolypeptide chains canparticipate in




Photodynamic inactivation and mutagenesis by angelicin (isopsoralen) or thiopyronin (methylene red) in wild-type and repair-deficient strains of bacteriophage T4  

SciTech Connect

Photodynamic inactivation of bacteriophage T4 particles, mediated by either angelicin or thiopyronin, is enhanced by defects in the T4 uvsW-uvsX-uvsY postreplication repair system but not by a defect in the denV pyrimidine-dimer-excision system. There was no evidence for functional interactions between the two repair systems. As observed previously with 8-methoxypsoralen, photodynamic mutagenesis with angelicin is abolished by defects in the uvsW-uvsX-uvsY system.

Drake, J.W.



Functional analysis of the highly antigenic outer capsid protein, Hoc, a virus decoration protein from T4-like bacteriophages.  


Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the centre of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modelling 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 beta-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. PMID:20497329

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



Transcriptional Regulation of T4 Bacteriophage-Specific Enzymes Synthesized In Vitro  

PubMed Central

In contrast to dihydrofolate reductase and four other phage-specific enzymes, the initiation of deoxynucleotide kinase is essentially prevented if rifampin is added to a culture of Escherichia coli B cells within 1.5 min after infection with T4. Deoxynucleotide kinase thus belongs to a group of so-called delayed-early enzymes that is not initiated from an immediate-early promoter site. We prepared crude extracts from infected cells in a manner designed to maintain the integrity of the complexes of native, endogenous T4 DNA with bacterial structural and enzymatic units concerned with RNA synthesis. The initiation of the synthesis of the mRNA for dihydrofolate reductase, an example of an immediate-early enzyme, and deoxynucleotide kinase, a special type of delayed-early enzyme, was studied with these extracts prepared from cells infected in the absence or presence of chloramphenicol. Initiation of transcription of the dihydrofolate reductase gene is immediate when programmed by extracts made either from cells treated with chloramphenicol prior to infection (CM extracts) or from cells 3 min into the normal infection cycle (3-min extracts). However, initiation of transcription of the deoxynucleotide kinase gene programmed by CM extracts is delayed 2 min relative to the immediate initiation of transcription of the deoxynucleotide kinase gene programmed by 3-min extracts. These experiments duplicated in vitro effects of the antibiotics on the synthesis of phage-specific mRNA previously noted only in vivo.

Cohen, Paul S.; Natale, Peter J.; Buchanan, John M.



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 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



The Mechanism of Inactivation of T4 Bacteriophage by Tritium Decay  

PubMed Central

Coliphage T4 was used as a model system to study the mechanism of biological inactivation produced by tritium decay. Experimentally, tritiated precursors were incorporated into phage DNA (thymidine-3H) or into phage protein (3H-amino acids). The ratio of killing efficiencies for decays originating in phage DNA to those originating in phage protein was 2.6. Inactivation by decays from labeled amino acids was assumed to occur exclusively from ?-particle irradiation of phage DNA. If decays originating in DNA are due solely to irradiation of DNA, then the killing efficiencies reflect the energy transfer paths in phage DNA for decays originating in phage DNA and in the protein coat. The energy transfer paths were determined for the two cases with the help of a computer and found to be very nearly equal to the experimentally determined ratio (2.6). The killing efficiencies for decays originating in phage DNA were 0.12 and for decays originating in protein 0.046.

Funk, Fred; Person, Stanley; Bockrath, Richard C.



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


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



Response of the bacteriophage T4 replisome to non-coding lesions and regression of a stalled replication fork  

PubMed Central

DNA is constantly damaged by endogenous and exogenous agents. The resulting DNA lesions have the potential to halt the progression of the replisome, possibly leading to replication fork collapse. Here, we examine the effect of a non-coding DNA lesion in either the leading or lagging strand template on the bacteriophage T4 replisome. A damaged base in the lagging strand template does not affect the progression of the replication fork. Instead, the stalled lagging strand polymerase recycles from the lesion and initiates synthesis of the new Okazaki fragment upstream from the damaged base. In contrast, when the replisome encounters a blocking lesion in the leading strand template, the replication fork only travels approximately 1 kb beyond the point of the DNA lesion before complete replication fork collapse. The primosome and lagging strand polymerase remain active during this period and an Okazaki fragment is synthesized beyond the point of the leading strand lesion. There is no evidence for a new priming event on the leading strand template. Instead, the DNA structure that is produced by the stalled replication fork is a substrate for the DNA repair helicase, UvsW. UvsW catalyzes the regression of a stalled replication fork into a “chicken foot” structure that has been postulated to be an intermediate in an error-free lesion bypass pathway.

Nelson, Scott W.; Benkovic, Stephen J.



Sequence and characterization of the bacteriophage T4 comC alpha gene product, a possible transcription antitermination factor.  

PubMed Central

We have sequenced a 1,340-bp region of the bacteriophage T4 DNA spanning the comC alpha gene, a gene which has been implicated in transcription antitermination. We show that comC alpha, identified unambiguously by sequencing several missense and nonsense mutations within the gene, codes for an acidic polypeptide of 141 residues, with a predicted molecular weight of 16,680. We have identified its product on one- and two-dimensional gel systems and found that it migrates abnormally as a protein with a molecular weight of 22,000. One of the missense mutations (comC alpha 803) is a glycine-to-arginine change, and the resulting protein exhibits a substantially faster electrophoretic mobility. The ComC alpha protein appears immediately after infection. Its rate of synthesis is maximum around 2 to 3 min postinfection (at 37 degrees C) and then starts to decrease slowly. Some residual biosynthesis is still detectable during the late period of phage development. Images

Sanson, B; Uzan, M



The Structure of Gene Product 6 of Bacteriophage T4, the Hinge-Pin of the Baseplate  

SciTech Connect

The baseplate of bacteriophage T4 is a multicomponent protein complex, which controls phage attachment to the host. It assembles from six wedges and a central hub. During infection the baseplate undergoes a large conformational change from a dome-shaped to a flat, star-shaped structure. We report the crystal structure of the C-terminal half of gene product (gp) 6 and investigate its motion with respect to the other proteins during the baseplate rearrangement. Six gp6 dimers interdigitate, forming a ring that maintains the integrity of the baseplate in both conformations. One baseplate wedge contains an N-terminal dimer of gp6, whereas neighboring wedges are tied together through the C-terminal dimer of gp6. The dimeric interactions are preserved throughout the rearrangement of the baseplate. However, the hinge angle between the N- and C-terminal parts of gp6 changes by {approx}15{sup o}, accounting for a 10 {angstrom} radial increase in the diameter of the gp6 ring.

Aksyuk, Anastasia A.; Leiman, Petr G.; Shneider, Mikhail M.; Mesyanzhinov, Vadim V.; Rossmann, Michael G.; (SOIBC); (Purdue)



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


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



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


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



Cryoelectron Microscopy Analysis of Small Heat Shock Protein 16.5 (Hsp16.5) Complexes with T4 Lysozyme Reveals the Structural Basis of Multimode Binding*  

PubMed Central

Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the ?-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human ?B-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.

Shi, Jian; Koteiche, Hanane A.; McDonald, Ezelle T.; Fox, Tara L.; Stewart, Phoebe L.; Mchaourab, Hassane S.



Testing homology modeling on mutant proteins: predicting structural and thermodynamic effects in the Ala98-->Val mutants of T4 lysozyme.  


Backgound. Current approaches to homology modeling predict how amino acid substitutions will alter a protein's structure, primarily by modeling sidechain conformations upon essentially immobile backbone frameworks. However, recent crystal structures of T4 lysozyme mutants reveal significant shifts of the mainchain and other potentially serious problems for sidechain rotamer-based modeling. This paper evaluates the accuracy of structural and thermodynamic predictions from two common sidechain modeling approaches to measure errors caused by the fixed-backbone approximation. Results. Tested on a series of T4 lysozyme mutants, this sidechain rotamer library approach did not handle mainchain shifts well, correctly predicting the sidechain conformations of only two of six mutants. By contrast, allowing sidechains to move more flexibly appeared to compensate for the rigidity of the mainchain and gave reasonably accurate coordinate predictions (rms errors of 0.5-1.0 Å for each mutated sidechain), better on average than 90% of possible conformations. The calculated packing energies correlated well with experimental stabilities (r2=0.81) and correctly captured the cooperative interactions of several neighboring mutations. Conclusion. Mutant modeling can be relatively accurate despite the fixed-backbone approximation. Mainchain shifts (0.2-0.5 Å) cause increased sidechain coordinate errors of 0.1-0.8 Å, torsional errors of 10-30°, and exaggerated strain energy for overpacked mutants, compared with the same calculations performed with the correct mutant backbones. PMID:9162134




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



Selective inhibition by methoxyamine of the apurinic\\/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4  

Microsoft Academic Search

The UV endonucleases from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic\\/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. The authors have explored the potential use

Michel Liuzzi; Michael Weinfeld; Malcolm C. Paterson



Bacteriophage T4 can produce progeny virions in extremely slowly growing Escherichia coli host: comparison of a mathematical model with the experimental data.  


Development of bacteriophage T4 depends on the physiological state of its host cell. Based on previous studies performed under laboratory conditions with different media determining various growth rates of Escherichia coli, a mathematical model was developed which suggested that phage T4 development cannot proceed efficiently in bacteria growing with a doubling time longer than 160 min. Contrary to this prediction, using a chemostat culture system allowing for culturing E. coli at different growth rates without changes in the medium composition, we found that T4 can yield progeny in host cells growing with a doubling time as long as 21 h. Our results indicate that the actual limiting growth rate of the host culture for the development of phage T4 is about 0.033 h(-1) , corresponding to the doubling time of about 21 h. PMID:24386916

Golec, Piotr; Karczewska-Golec, Joanna; ?o?, Marcin; W?grzyn, Grzegorz



Role of gene 59 of bacteriophage T4 in repair of UV-irradiated and alkylated DNA in vivo.  

PubMed Central

Nonsense mutants in gene 59 (amC5, amHL628) were used to study the role of this gene in the repair of UV-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to UV irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after UV irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to UV irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that UV repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B (su minus), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of UV lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules.

Wu, R; Wu, J L; Yeh, Y C



Cpl-7, a lysozyme encoded by a pneumococcal bacteriophage with a novel cell wall-binding motif.  


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. PMID:20720016

Bustamante, Noemí; Campillo, Nuria E; García, Ernesto; Gallego, Cristina; Pera, Benet; Diakun, Gregory P; Sáiz, José Luis; García, Pedro; Díaz, J Fernando; Menéndez, Margarita



Control of Gene Function in Bacteriophage T4 IV. Post-Transcriptional Shutoff of Expression of Early Genes  

PubMed Central

The selective and sequential shutoff of synthesis of early T4 proteins in bacteria infected with DNA-negative mutants is under the active control of one or more T4-induced proteins. Selective shutoff of synthesis of early T4 proteins is accompanied by a selective degradation of distinct species of T4 mRNA. We present circumstantial evidence that selective degradation of mRNA is the cause, and not the consequence, of selective termination of expression of early T4 genes. The mutation sp62 inactivates the shutoff mechanism and prevents the selective degradation of distinct species of T4 mRNA. Images

Sauerbier, Walter; Hercules, Kathleen



Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6.  

PubMed Central

In bacteriophage T4, there is a strong tendency for genes that encode interacting proteins to be clustered on the chromosome. There is 1.6 kb of DNA between the DNA helicase (gene 41) and the DNA primase (gene 61) genes of this virus. The DNA sequence of this region suggests that it contains five genes, designated as open reading frames (ORFs) 61.1 to 61.5, predicted to encode proteins ranging in size from 5.94 to 22.88 kDa. Are these ORFs actually genes? As one test, we compared the DNA sequence of this region in bacteriophages T2, T4, and T6 and found that ORFs 61.1, 61.3, 61.4, and 61.5 are highly conserved among the three closely related viruses. In contrast, ORF 61.2 is conserved between phages T4 and T6 yet is absent from phage T2, where it is replaced by another ORF, T2 ORF 61.2, which is not found in the T4 and T6 genomes. As a second, independent test for coding sequences, we calculated the codon base position preferences for all ORFs in this region that could encode proteins that contain at least 30 amino acids. Both the T4/T6 and T2 versions of ORF 61.2, as well as the other ORFs, have codon base position preferences that are indistinguishable from those of known T4 genes (coefficients of 0.81 to 0.94); the six other possible ORFs of at least 90 bp in this region are ruled out as genes by this test (coefficients less than zero). Thus, both evolutionary conservation and codon usage patterns lead us to conclude that ORFs 61.1 to 61.5 represent important protein-coding sequences for this family of bacteriophages. Because they are located between the genes that encode the two interacting proteins of the T4 primosome (DNA helicase plus DNA primase), one or more may function in DNA replication by modulating primosome function. Images

Selick, H E; Stormo, G D; Dyson, R L; Alberts, B M



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



Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid 1  

PubMed Central

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine (3H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the 3H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4. Images

Hercules, Kitty; Munro, Judith L.; Mendelsohn, Steven; Wiberg, John S.



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


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



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.



Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains  

PubMed Central

RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain—a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.

Ho, C. Kiong; Shuman, Stewart



Assembly of Human Immunodeficiency Virus (HIV) Antigens on Bacteriophage T4: a Novel In Vitro Approach To Construct Multicomponent HIV Vaccines  

PubMed Central

Bacteriophage T4 capsid is an elongated icosahedron decorated with 155 copies of Hoc, a nonessential highly antigenic outer capsid protein. One Hoc monomer is present in the center of each major capsid protein (gp23*) hexon. We describe an in vitro assembly system which allows display of HIV antigens, p24-gag, Nef, and an engineered gp41 C-peptide trimer, on phage T4 capsid surface through Hoc-capsid interactions. In-frame fusions were constructed by splicing the human immunodeficiency virus (HIV) genes to the 5? or 3? end of the Hoc gene. The Hoc fusion proteins were expressed, purified, and displayed on hoc? phage particles in a defined in vitro system. Single or multiple antigens were efficiently displayed, leading to saturation of all available capsid binding sites. The displayed p24 was highly immunogenic in mice in the absence of any external adjuvant, eliciting strong p24-specific antibodies, as well as Th1 and Th2 cellular responses with a bias toward the Th2 response. The phage T4 system offers new direction and insights for HIV vaccine development with the potential to increase the breadth of both cellular and humoral immune responses.

Sathaliyawala, Taheri; Rao, Mangala; Maclean, Danielle M.; Birx, Deborah L.; Alving, Carl R.; Rao, Venigalla B.



Assembly of human immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel in vitro approach to construct multicomponent HIV vaccines.  


Bacteriophage T4 capsid is an elongated icosahedron decorated with 155 copies of Hoc, a nonessential highly antigenic outer capsid protein. One Hoc monomer is present in the center of each major capsid protein (gp23*) hexon. We describe an in vitro assembly system which allows display of HIV antigens, p24-gag, Nef, and an engineered gp41 C-peptide trimer, on phage T4 capsid surface through Hoc-capsid interactions. In-frame fusions were constructed by splicing the human immunodeficiency virus (HIV) genes to the 5' or 3' end of the Hoc gene. The Hoc fusion proteins were expressed, purified, and displayed on hoc(-) phage particles in a defined in vitro system. Single or multiple antigens were efficiently displayed, leading to saturation of all available capsid binding sites. The displayed p24 was highly immunogenic in mice in the absence of any external adjuvant, eliciting strong p24-specific antibodies, as well as Th1 and Th2 cellular responses with a bias toward the Th2 response. The phage T4 system offers new direction and insights for HIV vaccine development with the potential to increase the breadth of both cellular and humoral immune responses. PMID:16840347

Sathaliyawala, Taheri; Rao, Mangala; Maclean, Danielle M; Birx, Deborah L; Alving, Carl R; Rao, Venigalla B



Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not.  

PubMed Central

Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.

Doan, P L; Belanger, K G; Kreuzer, K N



Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not.  


Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms. PMID:11238396

Doan, P L; Belanger, K G; Kreuzer, K N



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

PubMed Central

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 105 to 106 PFU. We also describe a lysogenic system for possible use in microbially enhanced oil recovery techniques.

Chang, Philip L.; Yen, Teh Fu



Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme  

NASA Astrophysics Data System (ADS)

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, 1 s 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.



Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex.  


The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections. PMID:23979587

Almond, Joshua R; Stohr, Bradley A; Panigrahi, Anil K; Albrecht, Dustin W; Nelson, Scott W; Kreuzer, Kenneth N



Complete genome sequence and characterization of a broad-host range T4-like bacteriophage phiAS5 infecting Aeromonas salmonicida subsp. salmonicida.  


In this study, we report one lytic Myoviridae bacteriophage (phage) infecting Aeromonas salmonicida subsp. salmonicida. The phage (named as phiAS5) was isolated from environmental river waters in Korea, and showed broad infectivity to other bacterial species in the family Aeromonadaceae as well as antibiotic-resistant A. salmonicida subsp. salmonicida strains. The biological properties and complete genome of phiAS5 were simultaneously investigated. The complete genome of phiAS5 composed of linear double-stranded DNA of 225,268 bp with G+C content of 43.0%, and encoded 343 putative ORFs, 69 putative promoters, 33 transcriptional terminator regions and 24 tRNA-encoding genes. A high degree of similarity to other T4-like Aeromonas phage was found in most ORFs of phiAS5. Therefore, the genome of phiAS5 was further compared with T4 phage and the closest relative, Aeromonas phage Aeh1, and the result demonstrated that it could be classified as a new member of the T4-like group. The bacteriolytic activity of phiAS5 against A. salmonicida subsp. salmonicida was evaluated at different doses of multiplicity of infection using one each of virulent strain that possesses the ascV gene and multi-drug resistant strain, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, phiAS5 may have the potential for reducing the impacts of virulent or antibiotic-resistant A. salmonicida subsp. salmonicida in aquaculture and may also advance our understanding of the biodiversity of T4-like Aeromonas phages. PMID:22226819

Kim, Ji Hyung; Son, Jee Soo; Choi, Yun Jaie; Choresca, Casiano H; Shin, Sang Phil; Han, Jee Eun; Jun, Jin Woo; Park, Se Chang



Molecular Architecture of Bacteriophage T4 Capsid: Vertex Structure and Bimodal Binding of the Stabilizing Accessory Protein, Soc  

Microsoft Academic Search

T4 encodes two dispensable proteins that bind to the outer surface of the mature capsid. Soc (9 kDa) stabilizes the capsid against extremes of alkaline pH and temperature, but Hoc (40 kDa) has no perceptible effect. Both proteins have been developed as display platforms. Their positions on the hexagonal surface lattice of gp23*, the major capsid protein, were previously defined

Kenji Iwasaki; Benes L. Trus; Paul T. Wingfield; Naiqian Cheng; Gregorina Campusano; Venigalla B. Rao; Alasdair C. Steven



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



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.



Complete genomic sequence of a T4-like bacteriophage, phiAS4, infecting Aeromonas salmonicida subsp. salmonicida.  


A newly identified virulent phage (named phiAS4) infecting Aeromonas salmonicida subsp. salmonicida was isolated from river water in Korea. Morphological analysis of phiAS4 by transmission electron microscopy revealed that it belonged to the family Myoviridae. The genome of phiAS4 comprised a linear double-stranded DNA of 163,875 bp with a G + C content of 41.3%, and genomic analysis revealed 271 putative ORFs, 67 putative promoters, 25 putative terminator regions, and 16 tRNA-encoding genes. Most of the ORFs of phiAS4 showed a high degree of similarity to those of Aeromonas phage 25, which belongs to the T4-like group. Moreover, the comparison of the genome of phiAS4 with those of its relatives demonstrated that phage phiAS4 is closely related to members of the T4-like group and can be classified as a new member of the T4-like phages infecting bacteria of the family Aeromonadaceae. PMID:22116495

Kim, J H; Son, J S; Choi, Y J; Choresca, C H; Shin, S P; Han, J E; Jun, J W; Park, S C



RNA Initiation with Dinucleoside Monophosphates during Transcription of Bacteriophage T4 DNA with RNA Polymerase of Escherichia coli  

PubMed Central

The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: (i) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. (ii) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5?-guanosyl dinucleoside monophosphates. (iii) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. (iv) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. (v) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 “early” type. (vi) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of 3H-labeled stimulatory dinucleoside monophosphates support the above conclusions.

Hoffman, David J.; Niyogi, Salil K.



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



Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4  

SciTech Connect

The UV endonucleases from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. The authors have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV-treated, (/sup 3/H)thymine-labeled poly(dA) x poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical-(5 kJ/m/sup 2/, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. The data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. The results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.

Liuzzi, M.; Weinfeld, M.; Paterson, M.C.



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

PubMed Central

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 loss of helicase activity and 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.

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



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


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



Sequence analysis of bacteriophage T4 DNA packaging/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases  

PubMed Central

Phage DNA packaging is believed to be driven by a rotary device coupled to an ATPase ‘motor’. Recent evidence suggests that the phage DNA packaging motor is one of the strongest force-generating molecular motors reported to date. However, the ATPase center that is responsible for generating this force is unknown. In order to identify the DNA translocating ATPase, the sequences of the packaging/terminase genes of coliphages T4 and RB49 and vibriophages KVP40 and KVP20 have been analyzed. Alignment of the terminase polypeptide sequences revealed a number of functional signatures in the terminase genes 16 and 17. Most importantly, the data provide compelling evidence for an ATPase catalytic center in the N-terminal half of the large terminase subunit gp17. An analogous ATPase domain consisting of conserved functional signatures is also identified in the large terminase subunit of other bacteriophages and herpesviruses. Interestingly, the putative terminase ATPase domain exhibits some of the common features found in the ATPase domain of DEAD box helicases. Residues that would be critical for ATPase catalysis and its coupling to DNA packaging are identified. Com binatorial mutagenesis shows that the predicted threonine residues in the putative ATPase coupling motif are indeed critical for function.

Mitchell, Michael S.; Matsuzaki, Shigenobu; Imai, Shosuke; Rao, Venigalla B.



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


TiO(2)-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO(2), CuO and hybrid CuO/TiO(2) prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO(2) prepared by a sol-gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol-gel preparations. However, these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO(2) which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some self-cleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO(2)/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries. PMID:18317747

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



Elasticity theory for self-assembled protein lattices with application to the martensitic phase transition in bacteriophage T4 tail sheath  

NASA Astrophysics Data System (ADS)

We propose an elasticity theory for one- and two-dimensional arrays of globular proteins for which the free energy is affected by relative position and relative rotation between neighboring molecules. The kinematics of such assemblies is described, the conditions of compatibility are found, a form of the free energy is given, and formulas for applied forces and moments are developed. It is shown that fully relaxed states of sheets consist of helically deformed sheets which themselves are composed of helical chains of molecules in rational directions. We apply the theory to the fascinating contractile deformation that occurs in the tail sheath of the virus bacteriophage T4, which aids its invasion of its bacterial host. Using electron density maps of extended and contracted sheaths, we approximate the domains of each molecule by ellipsoids and then evaluate our formulas for the position and orientation of each molecule. We show that, with the resulting kinematic description, the configurations of extended and contracted tail sheaths are generated by a simple formula. We proposed a constrained version of the theory based on measurements on extended and contracted sheath. Following a suggestion of Pauling [Discuss. Faraday Soc. 13, 170 (1953)], we develop a simple model of the molecular interaction. The resulting free energy is found to have a double-well structure. Certain simple deformations are studied (tension, torsion inflation); the theory predicts a first-order Poynting effect and some unexpected relations among moduli. Finally, the force of penetration is given, and a possibly interesting program of epitaxial growth and patterning of such sheets is suggested.

Falk, Wayne; James, Richard D.



Bacteriophage T4 MotA activator and the ?-flap tip of RNA polymerase target the same set of ?70 carboxyl-terminal residues.  


Sigma factors, the specificity subunits of RNA polymerase, are involved in interactions with promoter DNA, the core subunits of RNA polymerase, and transcription factors. The bacteriophage T4-encoded activator, MotA, is one such factor, which engages the C terminus of the Escherichia coli housekeeping sigma factor, ?(70). MotA functions in concert with a phage-encoded co-activator, AsiA, as a molecular switch. This process, termed sigma appropriation, inhibits host transcription while activating transcription from a class of phage promoters. Previous work has demonstrated that MotA contacts the C terminus of ?(70), H5, a region that is normally bound within RNA polymerase by its interaction with the ?-flap tip. To identify the specific ?(70) residues responsible for interacting with MotA and the ?-flap tip, we generated single substitutions throughout the C terminus of ?(70). We find that MotA targets H5 residues that are normally engaged by the ?-flap. In two-hybrid assays, the interaction of ?(70) with either the ?-flap tip or MotA is impaired by alanine substitutions at residues Leu-607, Arg-608, Phe-610, Leu-611, and Asp-613. Transcription assays identify Phe-610 and Leu-611 as the key residues for MotA/AsiA-dependent transcription. Phe-610 is a crucial residue in the H5/?-flap tip interaction using promoter clearance assays with RNA polymerase alone. Our results show how the actions of small transcriptional factors on a defined local region of RNA polymerase can fundamentally change the specificity of polymerase. PMID:21911499

Bonocora, Richard P; Decker, Phillip K; Glass, Stephanie; Knipling, Leslie; Hinton, Deborah M



Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid.  


Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology. PMID:17544446

Li, Qin; Shivachandra, Sathish B; Zhang, Zhihong; Rao, Venigalla B



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



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:



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



Effect of DNA-negative and maturation-defective conditions on accumulation of functional messengers for T4 bacteriophage-specific dihydrofolate reductase and deoxynucleoside monophosphate kinase.  

PubMed Central

Messengers for T4 phage-specific deoxynucleoside monophosphate kinase overaccumulated in nonpermissive hosts infected with amber-defective viruses that displayed either the DNA-negative or maturation-defective phenotype. Under both conditions, however, transcription of functional messengers for dihydrofolate reductase followed essentially normal kinetics.

Witmer, H



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


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 (?10(11) 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. PMID:21687531

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



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



Expression of bacteriophage phiEa1h lysozyme in Escherichia coli and its activity in growth inhibition of Erwinia amylovora.  


A 3.3 kb fragment from Erwinia amylovora phage Ea1h in plasmid pJH94 was previously characterized and found to contain an exopolysaccharide depolymerase (dpo) gene and two additional ORFs encoding 178 and 119 amino acids. ORF178 (lyz) and ORF119 (hol) were found to overlap by 19 bp and they resembled genes encoding lysozymes and holins. In nucleotide sequence alignments, lyz had structurally conserved regions with residues important for lysozyme function. The lyz gene was cloned into an expression vector and expressed in Escherichia coli. Active lysozyme was detected only when E. coli cells with the lyz gene and a kanamycin-resistance cassette were grown in the presence of kanamycin. Growth of Erw. amylovora was inhibited after addition of enzyme exceeding a threshold for lysozyme to target cells. When immature pears were soaked in lysates of induced cells, symptoms such as ooze formation and necrosis were retarded or inhibited after inoculation with Erw. amylovora. PMID:15289567

Kim, Won-Sik; Salm, Heike; Geider, Klaus



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



Mutations of the Bacteriophage T4 Type II DNA Topoisomerase That Alter Sensitivity to Antitumor Agent 4'-(9-Acridinylamino)methanesulfon- m-anisidide and an Antibacterial Quinolone1  

Microsoft Academic Search

Various antitumor and antibacterial agents target type II DNA topoi- somerases, stabilizing a cleaved DNA reaction intermediate and thereby converting topoisomerase into a cellular poison. Two 4'-<9-acridinylami- no)methanesulfon-m-anisidide (m-AMSA 1-resistant hacteriophage T4 to- poisomerases have previously been characterized biochemically, and we have now determined the sequence of the causative mutations. In one case, a mutation (K457K) in a conserved domain

Catherine H. Freudenreich; Christine Chang; Kenneth N. Kreuzer


Differential Bacteriophage Mortality on Exposure to Copper ?  

PubMed Central

Many studies report that copper can be used to control microbial growth, including that of viruses. We determined the rates of copper-mediated inactivation for a wide range of bacteriophages. We used two methods to test the effect of copper on bacteriophage survival. One method involved placing small volumes of bacteriophage lysate on copper and stainless steel coupons. Following exposure, metal coupons were rinsed with lysogeny broth, and the resulting fluid was serially diluted and plated on agar with the corresponding bacterial host. The second method involved adding copper sulfate (CuSO4) to bacteriophage lysates to a final concentration of 5 mM. Aliquots were removed from the mixture, serially diluted, and plated with the appropriate bacterial host. Significant mortality was observed among the double-stranded RNA (dsRNA) bacteriophages ?6 and ?8, the single-stranded RNA (ssRNA) bacteriophage PP7, the ssDNA bacteriophage ?X174, and the dsDNA bacteriophage PM2. However, the dsDNA bacteriophages PRD1, T4, and ? were relatively unaffected by copper. Interestingly, lipid-containing bacteriophages were most susceptible to copper toxicity. In addition, in the first experimental method, the pattern of bacteriophage ?6 survival over time showed a plateau in mortality after lysates dried out. This finding suggests that copper's effect on bacteriophage is mediated by the presence of water.

Li, Jinyu; Dennehy, John J.



Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems.  

PubMed Central

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.

Wiggins, B A; Alexander, M



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).



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


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



Antiviral effect of cationic compounds on bacteriophages  

PubMed Central

The antiviral activity of several cationic compounds – cetyltrimethylammonium bromide (CTAB), chitosan, nisin, and lysozyme – was investigated on the bacteriophage c2 (DNA head and non-contractile tail) infecting Lactococcus strains and the bacteriophage MS2 (F-specific RNA) infecting E. coli. Firstly, these activities were evaluated in a phosphate buffer pH 7 – 10 mM. The CTAB had a virucidal effect on the Lactococcus bacteriophages, but not on the MS2. After 1 min of contact with 0.125 mM CTAB, the c2 population was reduced from 6 to 1.5 log(pfu)/mL and completely deactivated at 1 mM. On the contrary, chitosan inhibited the MS2 more than it did the bacteriophages c2. No antiviral effect was observed for the nisin or the lysozyme on bacteriophages after 1 min of treatment. A 1 and 2.5 log reduction was respectively observed for nisin and lysozyme when the treatment time increased (5 or 10 min). These results showed that the antiviral effect depended both on the virus and structure of the antimicrobial compounds. The antiviral activity of these compounds was also evaluated in different physico-chemical conditions and in complex matrices. The antiviral activity of CTAB was impaired in acid pH and with an increase of the ionic strength. These results might be explained by the electrostatic interactions between cationic compounds and negatively charged particles such as bacteriophages or other compounds in a matrix. Milk proved to be protective suggesting the components of food could interfere with antimicrobial compounds.

Ly-Chatain, Mai H.; Moussaoui, Saliha; Vera, Annabelle; Rigobello, Veronique; Demarigny, Yann



Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid  

PubMed Central

Evidence was presented to support the hypothesis that long single strands appearing at late times (15 min after infection) are produced as a result of recombination and not as a continuous elongation during the replication process. The production of long strands does not depend on the multiplicity of infection, and the first long strands appear at the time when 20 to 50 phage equivalent units of deoxyribonucleic (DNA) are synthesized, and not earlier. The addition of chloramphenicol at 5 min, which prevents molecular recombination but allows replication of DNA, prevents the formation of long, single strands. Chloramphenicol added between 8 and 10 min after infection, a time at which molecular recombination is fully expressed and covalent repair of recombinant molecules is allowed, does not prevent formation of long single strands. Cutting of single-strand DNA with a limited amount of endonuclease I allows confirmation that the fast-sedimenting characteristic of intracellular denatured DNA is caused primarily by the length of the strands, and not by the formation of aggregates. The computer simulation of two recombination models indicates the feasibility of random breakage and rejoining of molecules in generating long concatenates.

Miller, Robert C.; Kozinski, Andrzej W.; Litwin, Samuel



t4 Workshop Report  

PubMed Central

Summary In October 2010, a group of experts met as part of the transatlantic think tank for toxicology (t4) to exchange ideas about the current status and future of safety testing of nanomaterials. At present, there is no widely accepted path forward to assure appropriate and effective hazard identification for engineered nanomaterials. The group discussed needs for characterization of nanomaterials and identified testing protocols that incorporate the use of innovative alternative whole models such as zebrafish or C. elegans, as well as in vitro or alternative methods to examine specific functional pathways and modes of action. The group proposed elements of a potential testing scheme for nanomaterials that works towards an integrated testing strategy, incorporating the goals of the NRC report Toxicity Testing in the 21st Century: A Vision and a Strategy by focusing on pathways of toxic response, and utilizing an evidence-based strategy for developing the knowledge base for safety assessment. Finally, the group recommended that a reliable, open, curated database be developed that interfaces with existing databases to enable sharing of information.

Silbergeld, Ellen K.; Contreras, Elizabeth Q.; Hartung, Thomas; Hirsch, Cordula; Hogberg, Helena; Jachak, Ashish C.; Jordan, William; Landsiedel, Robert; Morris, Jeffery; Patri, Anil; Pounds, Joel G.; de Vizcaya Ruiz, Andrea; Shvedova, Anna; Tanguay, Robert; Tatarazako, Norihasa; van Vliet, Erwin; Walker, Nigel J.; Wiesner, Mark; Wilcox, Neil; Zurlo, Joanne



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.



Lytic bacteriophages  

PubMed Central

Foodborne illnesses resulting from the consumption of produce commodities contaminated with enteric pathogens continue to be a significant public health issue. Lytic bacteriophages may provide an effective and natural intervention to reduce bacterial pathogens on fresh and fresh-cut produce commodities. The use of multi-phage cocktails specific for a single pathogen has been most frequently assessed on produce commodities to minimize the development of bacteriophage insensitive mutants (BIM) in target pathogen populations. Regulatory approval for the use of several lytic phage products specific for bacterial pathogens such as Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in foods and on food processing surfaces has been granted by various agencies in the US and other countries, possibly allowing for the more widespread use of bacteriophages in the decontamination of fresh and minimally processed produce. Research studies have shown lytic bacteriophages specific for E. coli O157:H7, Salmonella spp. and Listeria monocytogenes have been effective in reducing pathogen populations on leafy greens, sprouts and tomatoes.

Sharma, Manan



Bacteriophage Genomics  

PubMed Central

The last three years have seen an escalation in the number of sequenced bacteriophage genomes with more than five hundred now in the NCBI phage database, representing a more than three-fold increase from 2005. These span at least 70 different bacterial hosts, with two-thirds of the sequenced genomes of phages representing only eight bacterial hosts. Three key features emerge from the comparative analysis of these genomes. First, they span a very high degree of genetic diversity, suggesting early evolutionary origins. Secondly, the genome architectures are mosaic, reflecting an unusually high degree of horizontal genetic exchange in their evolution. Thirdly, phage genomes contain a very high proportion of novel genetic sequences of unknown function, and likely represent the largest reservoir of unexplored genes. With an estimated 1031 bacterial and archael viruses in the in the biosphere, our view of the virosphere will draw into sharper focus as further bacteriophage genomes are characterized.

Hatfull, Graham F.



Bacteriophage prehistory  

PubMed Central

We identified 30 actual or presumptive “bacteriophage” references dating between the years 1895 and 1917 and have further explored one of the oldest: Hankin's 1896 study of a bactericidal action associated with the waters of the Ganges and Jumna rivers in India. As Hankin's work took place approximately 20 years prior to the actual discovery of bacteriophages, no claims were made as to a possible phage nature of the phenomenon. Here we suggest that it may be imprudent to assume nevertheless that it represents an early observation of phagemediated bactericidal activity. Our principal argument is that the antibacterial aspect of these river waters was able to retain full potency following “heating” for one-half hour in hermetically sealed tubes, where heating in “open” tubes resulted in loss of antibacterial activity. We also suggest that environmental phage counts would have had to have been unusually high—greater than 106/ml impacting a single host strain—to achieve the rates of bacterial loss that Hankin observed.

Thomas-Abedon, Cameron; Thomas, Anne; Mazure, Hubert



Template reporter bacteriophage platform and multiple bacterial detection assays based thereon  

NASA Technical Reports Server (NTRS)

The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

Goodridge, Lawrence (Inventor)



Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose.  


The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution. The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound. Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled. This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase. Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present. The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid. PMID:11341831

Leung, A K; Duewel, H S; Honek, J F; Berghuis, A M



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.



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)



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



Synergistic Interactions of T4 Early Proteins Concerned with Their Binding to DNA  

PubMed Central

Under appropriate conditions, 19 distinct early proteins produced by bacteriophage T4 bind DNA with various affinities. They include products of genes that have been implicated in T4 DNA metabolism, such as DNA negative, DNA arrest, and DNA delay genes, and others whose roles have not yet been defined. The identifiable DNA-binding proteins are the products of genes 43, rIIA, 46, 30, 39, 52, ?gt, 32, and internal protein III. In some cases, protein-protein interactions are involved in the formation of the DNA-protein complexes. Images

Huang, Wai Mun; Buchanan, John M.



Microneedle-mediated transdermal bacteriophage delivery.  


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



Optical activity of human lysozyme.  


The ultraviolet circular dichroism spectra of human lysozyme are presented. Effects of pH and added inhibitor (N-acetyl-D-glucosamine) were examined and the results were compared with similar measurements of hen egg-white lysozyme. The near-ultraviolet CD spectral bands are substantially different in the human and hen egg-white enzymes. In addition to marked dissimilarities in the spectral interval 260-300 nm, an unusual CD band occurs at an anomalous wavelength (313 nm) in human lysozyme. The pH dependence of the latter suggests a possible interaction, absent in hen egg-white lysozyme, between a tryptophan and a tyrosine residue. Analysis of the spectra furthermore suggests lesser net rotational strengths of tryptophan bands in hen egg-white lysozyme than in human lysozyme, although the latter has one less tryptophan residue. The relationship between the CD spectra and the sequence differences of the proteins is discussed, as well as the CD spectra (published by others) of a closely related protein, bovine alpha-lactalbumin. Contributions of cystine residues to the spectra are examined in the light of possible differences in chirality of one of the four disulfide bridges.The far-ultraviolet CD spectra of human and egg-white lysozyme are quite similar, though not identical. In view of the pronounced differences in side-chain optical activity, and of the effect of pH variation on the far-ultraviolet CD spectrum of human lysozyme, it is likely that at least part of the observed difference in spectra is due to nonpeptide optical activity, and that the proteins have a secondary structure in common. PMID:5276753

Halper, J P; Latovitzki, N; Bernstein, H; Beychok, S



Optical Activity of Human Lysozyme  

Microsoft Academic Search

The ultraviolet circular dichroism spectra of human lysozyme are presented. Effects of pH and added inhibitor (N-acetyl-D-glucosamine) were examined and the results were compared with similar measurements of hen egg-white lysozyme. The near-ultraviolet CD spectral bands are substantially different in the human and hen egg-white enzymes. In addition to marked dissimilarities in the spectral interval 260-300 nm, an unusual CD

J. P. Halper; N. Latovitzki; H. Bernstein; S. Beychok



Protein determinants of phage T4 lysis inhibition  

PubMed Central

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.

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



Bacteriophage Interference in Bacillus subtilis 168  

PubMed Central

Strains of Bacillus subtilis lysogenic for temperate bacteriophage SPO2 inhibit the development of bacteriophage ?1. After infection by bacteriophage ?1, DNA and RNA synthesis in the lysogenic host terminates, culminating in cell death. Bacteriophage SPO2 also prevents the production of bacteriophage ?105. Mechanisms for these two types of bacteriophage interference are discussed.

Yasbin, R. E.; Ganesan, A. T.; Young, F. E.



Bacteriophage of Halobacterium salinarium  

Microsoft Academic Search

Halobacterium salinarium is a member of the Halobacteria, a group of obligate, extremely halophilic organisms requiring at least 15% NaCl in their growth media. No bacteriophage has previously been found in association with members of this group.

Terje Torsvik; Ian D. Dundas




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.



The distribution of impurities in lysozyme crystals  

NASA Astrophysics Data System (ADS)

The effective distribution coefficient, K Ieff, of turkey egg white lysozyme into host hen egg white lysozyme crystals was measured for varying levels of relative supersaturation, impurity concentrations and temperatures. Turkey egg white lysozyme served as a test impurity of high homology to hen egg white lysozyme. K Ieff increased with increasing host protein concentration and temperature, consistent with a kinetically controlled incorporation process. The principles of slow crystal growth rate to limit impurity incorporation were applied to a commercial source of lysozyme with concomitant improvement in purity of re-crystallized protein.

Schutt, Kelly J.; Gosavi, Rajendrakumar A.; White, Donald B.; Schall, Constance A.



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



Salivary Lysozyme and Prevalent Hypertension  

PubMed Central

Although the etiology of essential hypertension is not clearly understood, endothelial dysfunction from chronic infection and/or impaired glucose metabolism may be involved. We hypothesized that salivary lysozyme, a marker for oral infection and hyperglycemia, might display a significant relationship with hypertension, an early stage of cardiovascular disease. Logistic regression analyses of the Kuopio Oral Health and Heart Study demonstrated that persons with higher lysozyme levels were more likely to have hypertension, after adjustment for age, gender, smoking, BMI, diabetes, the ratio of total cholesterol to HDL cholesterol, and C-reactive protein. The exposure to increasing quartiles of lysozyme was associated with adjusted Odds Ratios for the outcome, hypertension, 1.00 (referent), 1.25, 1.42, and 2.56 (linear trend p < 0.003). When we restricted the sample to the individuals without heart disease (N = 250), we observed a non-significant trend for increasing odds. Our hypothesis—"high salivary lysozyme levels are associated with the odds of hypertension"—was confirmed.

Qvarnstrom, M.; Janket, S.; Jones, J.A.; Nuutinen, P.; Baird, A.E.; Nunn, M.E.; Van Dyke, T.E.; Meurman, J.H.



Self-Association of Gene-32 Protein of Bacteriophage T4  

PubMed Central

The self-association of gene-32 protein has been studied by sedimentation equilibrium centrifugation and polyacrylamide gel electrophoresis, in order to better understand its role in DNA replication and genetic recombination. The monomer molecular weight of gene-32 protein is 38,000 in guanidine hydrochloride and 34,000 in sodium dodecyl sulfate, in agreement with the results of Alberts and coworkers. Stable dimers of gene-32 protein occur under various conditions, among which are high ionic strength and pH 10. The occurrence of stable dimers under some conditions and higher aggregates under others indicates there are two types of protein-protein interactions occurring in gene-32 protein self-association. The association that occurs above about 0.1 mg/ml concentration of protein produces at least decamers. A model for the DNA replication fork is postulated that requires the two different interactions that occur in gene-32 protein aggregation. In the model, gene-32 protein holds the two strands of the DNA duplex in a conformation that prevents their reannealing and, therefore, facilitates replication and recombination. Images

Carroll, Robert B.; Neet, Kenneth E.; Goldthwait, David A.



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



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.

M., American S.



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



CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4  

PubMed Central

Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine.

Church, George M.



Recombination in bacteriophage P2: recA dependent enhancement by ultraviolet irradiation and by transfection with mixed DNA dimers  

Microsoft Academic Search

Bacteriophage P2 is known for its exceptionally low rate of spontaneous (non-integrative) recombination, which however may be stimulated by ultraviolet irradiation of the phage. We show here that ligated dimers, made in vitro from mixtures of DNAs of two P2 mutants, upon transfection of lysozyme-spheroplasts give origin to recombinants at high frequency. While spontaneous P2 recombination occurs independently of the

T. Hudnik-Plevnik; G. Bertani



The Bacillus subtilis Extracytoplasmic Function ? Factor ?V Is Induced by Lysozyme and Provides Resistance to Lysozyme?†  

PubMed Central

Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative ? factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative ? factors are the extracytoplasmic function (ECF) ? factors. In this paper, we demonstrate that the expression of the ECF ? factor ?V in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that ?V is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon ?V. Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for ?V-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF ? factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF ? factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF ? factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.

Ho, Theresa D.; Hastie, Jessica L.; Intile, Peter J.; Ellermeier, Craig D.



Morphogenesis of the T4 tail and tail fibers  

PubMed Central

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.



Structural aberrations in T-even bacteriophage. IX. Effect of mixed infection on the production of giant bacteriophage.  

PubMed Central

To date, the production of T-even bacteriophage with giant heads has been achieved in two ways: (i) by use of canavanine-arginine treatment of Escherichia coli B cultures infected by wild-type bacteriophage (Cummings and Bolin, Bacteriol. Rev. 40:314-359, 1976; Cummings et al., Virology 54:245-261, 1973), which give a size distribution of giants that is phage specific (Cummings et al., Virology 54:245-261, 1973); and (ii) by infection with certain missense mutants of T4D gene 23 (Doermann et al., J. Virol. 12:374-385, 1973; ICN-UCLA Symposium on Molecular Biology, p. 243-285, 1973) or temperature-sensitive mutants of gene 24 (Aebi et al., J. Supramol. Struct. 2:253-275, 1974; Biljenga et al., J. Mol. Biol. 103:469-498, 1976). We now report the effect of mixed infection with several mutants of T4D on both the production and the size of giant bacteriophage. We found that gene 24 mutant is a critical partner for the production of giants. Infection using T4.24 mutants together with either T4.23 mutants, T4B+ or T6+ led to the formation of giants with heads 10- to 14-fold longer than normal-length heads. Infection with amber 24-bypass 24 double mutants of T4D led to the production of giants when gene 23 mutant was used to co-infect. Addition of canavanine to the co-infected cultures could alter the size distribution of giants, depending on which phage were used to coinfect. Gene 22 mutants had a modifying effect on these results. In the absence of canavanine co-infection with gene 22 mutants prevented the production of giants, and in the presence of canavanine giants of 1.5 to 5 head lengths were found. We have interpreted these results to mean that critical concentrations of gene products 22, 23, and 24 interact to control head length in T-even bacteriophage. Images

Cummings, D J; Chapman, V A; DeLong, S S



Fluorescence Studies of Lysozyme Nucleation  

NASA Technical Reports Server (NTRS)

Fluorescence is one of the most powerful tools available for the study of macromolecules. For example, fluorescence can be used to study self association through methods such as anisotropy (the rotational rate of the molecule in solution), quenching (the accessibility of a bound probe to the bulk solution), and resonance energy transfer (measurement of the distance between two species). Fluorescence can also be used to study the local environment of the probe molecules, and the changes in that environment which accompany crystal nucleation and growth. However fluorescent techniques have been very much underutilized in macromolecular growth studies. One major advantage is that the fluorescent species generally must be at low concentration, typically ca 10-5 to 10-6 M. Thus one can study a very wide range of solution conditions, ranging from very high to very low protein concentration, he latter of which are not readily accessible to scattering techniques. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme (CEWL). Fluorescent probes have been attached to two different sites, ASP 101 and the N-terrninal amine, with a sought for use in different lines of study. Preliminary resonance energy transfer studies have been -carried out using pyrene acetic acid (Ex 340 mn, Em 376 nm) lysozyme as a donor and cascade blue (Ex 377 run, Em 423 nm) labeled lysozyme as an acceptor. The emission of both the pyrene and cascade blue probes was followed as a function of the salt protein concentrations. The data show an increase in cascade blue and a concomitant decrease in the pyrene fluorescence as either the salt or protein concentrations are increased, suggesting that the two species are approaching each other close enough for resonance energy transfer to occur. This data can be analyzed to measure the distance between the probe molecules and, knowing their locations on the protein molecule their distances from and orientations with respect to each other. The results of these and other studies will be discussed.

Pusey, Marc L.; Smith, Lori



Bacteriophages of Listeria  

Microsoft Academic Search

Bacteriophages have been shown to influence the evolution of their host and, in several cases, have a major effect on pathogenicity\\u000a and\\/or virulence of bacterial pathogens. Several mechanisms allow phages to change the biology and associated phenotypes of\\u000a their host. This chapter aims at explaining these mechanisms in the context of Listeria evolution and pathogenesis, using examples from other pathogens.

Steven Hagens; Martin J. Loessner


Enzymatic characterization of a lysin encoded by bacteriophage EL  

PubMed Central

The bacteriophage EL is a virus that specifically attacks the human pathogen Pseudomonas aeruginosa. This phage carries a large genome that encodes for its own chaperonin which presumably facilitates the proper folding of phage proteins independently of the host chaperonin system. EL also encodes a lysin enzyme, a critical component of the lytic cycle that is responsible for digesting the peptidoglycan layer of the host cell wall. Previously, this lysin was believed to be a substrate of the chaperonin encoded by phage EL. In order to characterize the activity of the EL lysin, and to determine whether lysin activity is contingent on chaperonin-mediated folding, a series of peptidoglycan hydrolysis activity assays were performed. Results indicate that the EL-encoded lysin has similar enzymatic activity to that of the Gallus gallus lysozyme and that the EL lysin folds into a functional enzyme in the absence of phage chaperonin and should not be considered a substrate.

Tafoya, Diana A.; Hildenbrand, Zacariah L.; Herrera, Nadia; Molugu, Sudheer K.; Mesyanzhinov, Vadim V.; Miroshnikov, Konstantin A.; Bernal, Ricardo A.



Origin activation requires both replicative and accessory helicases during T4 infection  

PubMed Central

Summary The bacteriophage T4 has served as an in vitro model for the study of DNA replication for several decades, yet less is known about this process during infection. Recent work has shown that viral DNA synthesis is initiated from at least five origins of replication distributed across the 172 kb chromosome, but continued synthesis is dependent on recombination. Two proteins are predicted to facilitate loading of the hexameric 41 helicase at the origins, the Dda accessory helicase and the 59 loading protein. Using a real time, genome-wide assay to monitor replication during infections, it is shown here that dda mutant viruses no longer preferentially initiate synthesis near the origins, implying that the Dda accessory helicase has a fundamental role in origin selection and activation. In contrast, at least two origins function efficiently without 59 loading protein, indicating that other factors load 41 helicase at these loci. Hence, normal T4 replication includes two mechanistically distinct classes of origins, one requiring 59 helicase loader, and a second that does not. Since both mechanisms require an additional factor, repEB, for sustained activation, normal T4 origin function appears to include at least three common elements, origin selection and initial activation, replisome loading, and persistence.

Brister, J. Rodney



Inhibition of lysozyme by taurine dibromamine.  


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

Petrônio, M S; Ximenes, V F



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)



Crystal structure of the phage T4 recombinase UvsX and its functional interaction with the T4 SF2 helicase UvsW  

PubMed Central

Summary 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 resembles that of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed EM 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 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.



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



Lysozyme pattern formation in evaporating drops.  


Liquid droplets containing suspended particles deposited on a solid, flat surface generally form ringlike structures due to the redistribution of solute during evaporation (the "coffee ring effect"). The forms of the deposited patterns depend on interactions between solute(s), solvent, and substrate. In this study, deposition patterns from droplets of a simplified model biological fluid (DI water + lysozyme) are examined by scanning probe and optical microscopy. The overall lysozyme residue morphology is complex (with both a perimeter "rim" and undulating interior) but varies little with concentration. However, the final packing of lysozyme molecules is strongly dependent on initial concentration. PMID:22339498

Gorr, Heather Meloy; Zueger, Joshua M; Barnard, John A



Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.  


Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E



Replication of phase fd RF with fd gene 2 protein and phage T4 enzymes.  


Bacteriophage fd replicative form DNA with a nick in the viral strand serves as a template for DNa replication with purified bacteriophage T4 enzymes. As anticipated from previous in vitro studies carried out with this system (Morris, C. F., Sinha, N. K., and Alberts, B. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4800-4804), DNA is synthesized by a rolling circle mechanism. We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made. The products are mostly unit length linear single strands, indicating that the circularization step normally catalyzed by gene 2 protein subsequent to its site-specific cleavage of an fd DNA strand occurs only inefficiently in this system. The gene 2 protein reduces the level of DNA synthesis by 2-fold at low concentrations, even though it only cleaves the DNA products efficiently at higher levels of the enzyme. This indicates that there are at least two different effects of the fd gene 2 protein in processing of viral fd DNA. PMID:7016862

Meyer, T F; Bäumel, I; Geider, K; Bedinger, P



Secondary structure of T4 gene 33 protein. Fourier transform infrared and circular dichroic spectroscopic studies.  


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



A New Family of Lysozyme Inhibitors Contributing to Lysozyme Tolerance in Gram-Negative Bacteria  

Microsoft Academic Search

Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel

Lien Callewaert; Abram Aertsen; Daphne Deckers; Kristof G. A. Vanoirbeek; Lise Vanderkelen; Joris M. Van Herreweghe; Barbara Masschalck; Dorothy Nakimbugwe; Johan Robben; Chris W. Michiels



The SPO1-related bacteriophages  

Microsoft Academic Search

A large and diverse group of bacteriophages has been termed ‘SPO1-like viruses’. To date, molecular data and genome sequences\\u000a are available for Bacillus phage SPO1 and eight related phages infecting members of other bacterial genera. Many additional bacteriophages have been\\u000a described as SPO1-related, but very few data are available for most of them. We present an overview of putative ‘SPO1-like

Jochen Klumpp; Rob Lavigne; Martin J. Loessner; Hans-Wolfgang Ackermann



Crystallogenesis of adenosine A(2A) receptor-T4 lysozyme fusion protein: a practical route for the structure.  


G-protein-coupled receptors (GPCRs) represent a major class of receptors through which a number of signals ranging from photons to large glycoprotein hormones are recognized. Human genome encodes about 800 GPCRs, yet very little structural information is available on this class of receptors. Structural studies provide a wealth of information about not only the activation mechanism of the receptor but also the crucial information about the ligand-binding pocket which could lead to the development of subtype-specific ligands. The crystal structure of human adenosine A(2A) receptor was solved in complex with a high-affinity antagonist ZM241385 at 2.6Å resolution. Here, we describe the methods that were undertaken to solve the fusion protein structure. PMID:23332700

Ashok, Yashwanth; Nanekar, Rahul T; Jaakola, Veli-Pekka



T4-like phage Bp7, a potential antimicrobial agent for controlling drug-resistant Escherichia coli in chickens.  


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



Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient  

NASA Technical Reports Server (NTRS)

Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A or B impurities added were 0.76, 0.38 and 0.1 milligram per millimeter while HEWL concentration were 20, 30 and 40 milligram per milliliter. The crystals grown in 18 experiments for each impurity were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K = 2.15 plus or minus 0.13 for A and K = 3.42 plus or minus 0.25 for B. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that impurity adsorption and incorporation rate is proportional to the impurity concentration and that the growth rate is proportional to the crystallizing protein in solution. With the kinetic coefficient for crystallization, beta = 5.10(exp -7) centimeters per second, the frequency at which an impurity molecule near the growing interface irreversibly joins a molecular site on the crystal was found to be 3 1 per second, much higher than the average frequency for crystal molecules. For best quality protein crystals it is better to have low microheterogeneous protein impurity concentration and high supers aturation.

Thomas, B. R.; Chernov, A. A.



DNA-Directed Synthesis In Vitro of T4 Phage-Specific Enzymes  

PubMed Central

The synthesis of deoxynucleotide kinase (EC 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 ?-glucosyl transferase (EC shows that potassium ions are required for maximal enzyme production. Examination of the RNA-directed system indicates that potassium ions are more effective than ammonium ion in the translation of messenger RNA for the formation of the biologically active proteins studied. From a comparison of the magnitude of the effect of ions on both the DNA- and RNA-directed systems, we conclude that potassium ions may also have a marked stimulatory effect on transcription of the deoxynucleotide kinase gene of T4 DNA. The time required for messenger initiation and completion and the size distribution of messenger RNA formed in vitro were also examined.

Natale, Peter J.; Buchanan, John M.



Study of lysozyme resistance in Rhodococcus equi.  


Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses. PMID:24170270

Hébert, Laurent; Bidaud, Pauline; Goux, Didier; Benachour, Abdellah; Laugier, Claire; Petry, Sandrine



X-ray topography of a lysozyme crystal  

SciTech Connect

X-ray topography methods were employed to identify defects in lysozyme crystals. White-beam and monochromatic topographs of lysozyme crystals obtained at the National Synchrotron Light Source are presented.

Stojanoff,V.; Siddons, D.



Bacteriophages that infect Bacillus bacteria (Anthrax)  

US Patent & Trademark Office Database

The invention provides bacteriophages that infect Bacillus bacteria, including Bacillus anthracis, and compositions containing the bacteriophages. The invention also provides methods for using the bacteriophages of the invention to prevent and treat infection of an organism by Bacillus bacteria. Methods and materials to decontaminate a surface or an organism that is contaminated with Bacillus bacteria or Bacillus spores is also provided.



Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.  


Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented. PMID:9449356

Thuman-Commike, P A; Greene, B; Malinski, J A; King, J; Chiu, W



Coacervates of lysozyme and ?-casein.  


Complexes are formed when positively charged lysozyme (LYZ) is mixed with negatively charged caseins. Adding ?-casein (BCN) to LYZ leads to flocculation even at low addition levels. Titrating LYZ into BCN shows that complexes are formed up to a critical composition (x=[LYZ]/([LYZ]+[BCN]). The formation of these complex coacervates increases asymptotically toward the molar charge equivalent ratio (xcrit), where the size of the complexes also seems to grow asymptotically. At xcrit, insoluble precipitates of charge-neutral complexes are formed. The precipitates can be re-dispersed by adding NaCl. The value of xcrit shifts to higher values on the LYZ side with increasing salt concentration and pH. Increasing the pH, de-protonates the BCN and protonates the LYZ, and therefore, charge neutrality will shift toward the LYZ side. xcrit increases linearly from 0.2 at no salt to 0.5 at 0.5M NaCl. It ends abruptly at a salt concentration of 0.5M after which a clear mixed solution remains. Away from the charge equivalent ratio, it seems that the buildup of charges limits the complex size. A simple scaling law to predict the size of the complex is proposed. By assuming that surface charge density is constant or can reach only a maximum value, it follows that scattering intensity is proportional to |(1-x/xcrit)|(-3) where x is the mole fraction of one protein and xcrit the value of the mole fraction at the charge equivalent ratio. Both scattering intensity and particle size seem to obey this simple assumption. For BCN-LYZ, the buildup occurs only at the LYZside in contrast to lactoferrin which forms stable complexes on either side of xcrit. The reason that the complexes are formed at the BCN side only may be due to the small size of LYZ, which induces a bending energy in the BCN on adsorption. PMID:23511012

Anema, Skelte G; de Kruif, C G Kees



Ultrasound and Hypersound Speeds in Lysozyme Solutions  

NASA Astrophysics Data System (ADS)

Ultrasound velocimetry and Brillouin spectroscopy provide information on the compressibility of proteins and the surrounding hydration layer. Employing both techniques we investigate the sound speeds at GHz (hypersound) and MHz (ultrasound) frequencies in lysozyme solutions (250 mg / ml, pH 7) and pure water over the temperature range from 275 K to 335 K. Compared to water the Brillouin peaks in the lysozyme solutions are shifted by about 400 MHz towards higher frequencies. This shift reflects the change in sound speed and is attributed to the influence of the compressibility of the protein and bound water in the hydration shell. In addition, we measure a dispersion of the sound velocity in the lysozyme solution. The higher sound speed at GHz frequencies, as measured by Brillouin scattering, may indicate additional relaxation processes as compared to pure bulk water, where no sound dispersion between ultrasound speed and hypersound speed is observed.

Schulte, Alfons; Pruner, Christian; Wilhelm, Emmerich; Asenbaum, Augustinus



Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance  

Microsoft Academic Search

Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783

Laurent Hebert; Pascal Courtin; Riccardo Torelli; Maurizio Sanguinetti; Marie-Pierre Chapot-Chartier; Yanick Auffray; Abdellah Benachour



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.



[Bacteriophages from micrococci (author's transl)].  


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



Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49  

PubMed Central

RB49 is a virulent bacteriophage that infects Escherichia coli. Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage. To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the ?170-kb genome. Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis. However, when translated, about 70% of them encoded proteins with homology to T4 proteins. Among these sequences were the numerous components of the virion and the phage DNA replication apparatus. Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome. The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication. This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues. Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences. The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies. For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters. These results indicate that RB49 and T4 have diverged substantially from their last common ancestor. The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes.

Desplats, Carine; Dez, Christophe; Tetart, Francoise; Eleaume, Heidy; Krisch, H. M.



Protonation favors aggregation of lysozyme with SDS.  


Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (?11). Negatively charged SDS was used to 'antagonize' positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as far-UV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0-1.0 and maximum aggregation was observed at pH 1.0. The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation. PMID:24647567

Khan, Javed M; Chaturvedi, Sumit K; Rahman, Shah K; Ishtikhar, Mohd; Qadeer, Atiyatul; Ahmad, Ejaz; Khan, Rizwan H



Bacteriophages: antibacterials with a future?  

Microsoft Academic Search

The hypothesis as to whether a benign species of bacteria could kill a virulent kind has to this point been untested.Recently it was shown that in the macrophage, bacteriophages, when properly introduced through a nonvirulent microbe, had a killing rate for virulent AIDS Mycobacterium tuberculosis and Mycobacterium avium far in excess of modern day antibiotics.The study in effect brought a

Lawrence Broxmeyer



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.



Lysozymes and lysozyme-like proteins from the fall armyworm, Spodoptera frugiperda.  


Lysozyme is an important component of the insect non-specific immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. By searching an EST database from the fall armyworm, Spodoptera frugiperda (Negre et al., 2006), we identified five sequences encoding proteins of the lysozyme family. The deduced protein sequences corresponded to three classical c-type lysozymes Sf-Lys1, Sf-Lys2 and Sf-Lys3, and two lysozyme-like proteins, Sf-LLP1 and Sf-LLP2. Sf-Lys1 was purified from the hemolymph of Escherichia coli-challenged S. frugiperda larvae. The mature protein had a molecular mass of 13.975 Da with an isoelectric point of 8.77 and showed 98.3% and 96.7% identity with lysozymes from Spodoptera litura and Spodoptera exigua, respectively. As the other insect lysozymes, Sf-Lys1 was active against gram positive bacteria such as Micrococcus luteus but also induced a slight permeabilization of the inner membrane of E. coli. Genes encoding these five Sf-Lys or Sf-LLPs were differentially up-regulated in three immune-competent tissues (hemocytes, fat body and gut) after challenges with non-pathogenic bacteria, E. coli and M. luteus, or entomopathogenic bacterium, Photorhabdus luminescens. Sf-Lys1 and Sf-Lys2 were mainly induced in fat body in the presence of E. coli or P. luminescens. Sf-Lys3, which had an acidic isoelectric point, was found to be the most up-regulated of all five Sf-Lys or Sf-LLPs in hemocytes and gut after challenge with P. luminescens. More molecular data are now available to investigate differences in physiological functions of these different members of the lysozyme superfamily. PMID:19828200

Chapelle, Michael; Girard, Pierre-Alain; Cousserans, François; Volkoff, Nathalie-Anne; Duvic, Bernard



Classification of Myoviridae bacteriophages using protein sequence similarity  

PubMed Central

Background We advocate unifying classical and genomic classification of bacteriophages by integration of proteomic data and physicochemical parameters. Our previous application of this approach to the entirely sequenced members of the Podoviridae fully supported the current phage classification of the International Committee on Taxonomy of Viruses (ICTV). It appears that horizontal gene transfer generally does not totally obliterate evolutionary relationships between phages. Results CoreGenes/CoreExtractor proteome comparison techniques applied to 102 Myoviridae suggest the establishment of three subfamilies (Peduovirinae, Teequatrovirinae, the Spounavirinae) and eight new independent genera (Bcep781, BcepMu, FelixO1, HAP1, Bzx1, PB1, phiCD119, and phiKZ-like viruses). The Peduovirinae subfamily, derived from the P2-related phages, is composed of two distinct genera: the "P2-like viruses", and the "HP1-like viruses". At present, the more complex Teequatrovirinae subfamily has two genera, the "T4-like" and "KVP40-like viruses". In the genus "T4-like viruses" proper, four groups sharing >70% proteins are distinguished: T4-type, 44RR-type, RB43-type, and RB49-type viruses. The Spounavirinae contain the "SPO1-"and "Twort-like viruses." Conclusion The hierarchical clustering of these groupings provide biologically significant subdivisions, which are consistent with our previous analysis of the Podoviridae.



Bacteriophage polysaccharide depolymerases and biomedical applications.  


Polysaccharide depolymerase, a polysaccharide hydrolase encoded by bacteriophages (or 'phages'), can specifically degrade the macromolecule carbohydrates of the host bacterial envelope. This enzyme assists the bacteriophage in adsorbing, invading, and disintegrating the host bacteria. Polysaccharide depolymerase activity continues even within biofilms. This effectiveness means phages are promising candidates for novel antibiotic scaffolds. A comprehensive compendium of bacteriophage polysaccharide depolymerases has been compiled, together with their potential biomedical applications, such as novel antibiotics, adjuvants for antibiotics, bacterial biofilm disruptants, and diagnostic kits. PMID:24352884

Yan, Jianlong; Mao, Jiaoxiao; Xie, Jianping



Preparation of lysozyme molecularly imprinted polymers and purification of lysozyme from egg white.  


Molecular imprinting as a promising and facile separation technique has received much attention because of its high selectivity for target molecules. In this study, lysozyme molecularly imprinted polymers (Lys-MIPs) were successfully prepared by the entrapment method with lysozyme as the template molecule, acrylamide as the functional monomer and N,N-methylenebisacrylamide as the cross-linker. The removal of the template lysozyme from the molecularly imprinted polymers was investigated in detail by two methods. The synthesized Lys-MIPs were characterized by scanning electron microscopy and Fourier transform-infrared, and the adsorption capacity, selectivity and reproducibility of the Lys-MIPs were also evaluated. The maximum adsorption capacity reached 94.8 mg/g, which is twice that of nonmolecularly imprinted polymers, and satisfactory selectivity and reproducibility were achieved. Using the Lys-MIP column, lysozyme could be separated completely from egg white, with purity close to 100% and mass recovery of 98.2%. This illustrated that the synthesized Lys-MIPs had high specific recognition and selectivity to the template lysozyme when they were applied to a mixture of protein standards and a real sample. PMID:24861763

Wang, Xuejiao; Dong, Shaohua; Bai, Quan



Glassy dynamics and enzymatic activity of lysozyme.  


There has been much interest in the analogies between dynamic processes in proteins and in other complex systems such as viscous liquids and glasses. We have investigated the dynamics of protons along chains of hydrogen-bonded water molecules adsorbed on the surface of the globular protein lysozyme. The hydration dependence of the dielectric relaxation time is fitted by a modified Vogel-Fulcher-Tamman equation, in which the variable temperature has been replaced with hydration. We find that the relaxation time diverges at a singular hydration that coincides with the critical water content required to trigger lysozyme enzymatic activity. This surprising correlation suggests a direct coupling between protein function and glasslike behavior, with possible implications for the turnover number of the enzyme. PMID:11735996

Pizzitutti, F; Bruni, F



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.



Genome and Proteome of Campylobacter jejuni Bacteriophage NCTC 12673?†  

PubMed Central

Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.

Kropinski, Andrew M.; Arutyunov, Denis; Foss, Mary; Cunningham, Anna; Ding, Wen; Singh, Amit; Pavlov, Andrey R.; Henry, Matthew; Evoy, Stephane; Kelly, John; Szymanski, Christine M.



Arrangement of coding and intervening sequences of chicken lysozyme gene.  

PubMed Central

Hybrid phages that contain chicken lysozyme gene sequences have been isolated from a chicken DNA library. Two overlapping clones covering a region of 22 kilobase pairs around this gene have been studied by restriction mapping. Southern hybridization, and electron microscopic analysis of hybrids between lysozyme mRNA and the cloned cellular DNA. Three intervening sequences interrupt the lysozyme structural gene. The cellular gene is at least 3.9 kilobases long, about 6 times the length of the structural gene. Images

Lindenmaier, W; Nguyen-Huu, M C; Lurz, R; Stratmann, M; Blin, N; Wurtz, T; Hauser, H J; Sippel, A E; Schutz, G



Thyroid Hormones, T3 and T4, in the Brain  

PubMed Central

Thyroid hormones (THs) are essential for fetal and post-natal nervous system development and also play an important role in the maintenance of adult brain function. Of the two major THs, T4 (3,5,3?,5?-tetraiodo-l-thyronine) is classically viewed as an pro-hormone that must be converted to T3 (3,5,3?-tri-iodo-l-thyronine) via tissue-level deiodinases for biological activity. THs primarily mediate their effects by binding to thyroid hormone receptor (TR) isoforms, predominantly TR?1 and TR?1, which are expressed in different tissues and exhibit distinctive roles in endocrinology. Notably, the ability to respond to T4 and to T3 differs for the two TR isoforms, with TR?1 generally more responsive to T4 than TR?1. TR?1 is also the most abundantly expressed TR isoform in the brain, encompassing 70–80% of all TR expression in this tissue. Conversion of T4 into T3 via deiodinase 2 in astrocytes has been classically viewed as critical for generating local T3 for neurons. However, deiodinase-deficient mice do not exhibit obvious defectives in brain development or function. Considering that TR?1 is well-established as the predominant isoform in brain, and that TR?1 responds to both T3 and T4, we suggest T4 may play a more active role in brain physiology than has been previously accepted.

Schroeder, Amy C.; Privalsky, Martin L.



Recombinant bacteriophage lysins as antibacterials  

PubMed Central

With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential.

Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim



Evaluation of oriented lysozyme immobilized with monoclonal antibody  

NASA Astrophysics Data System (ADS)

The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.

Aoyagi, Satoka; Okada, Keigo; Shigyo, Ayako; Man, Naoki; Karen, Akiya



Low-temperature T4-like coliphages vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20  

Microsoft Academic Search

Bacteriophages vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20, showing an unusual low-temperature plating profile and producing\\u000a constantly growing plaques, were isolated from aquatic environments of Lithuania. Although vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20\\u000a resembled phage T4 both in their genome size and in their major structural protein (gp23) pattern, physiological properties\\u000a of all three phages tested differed significantly from those of T4. With an optimum

Laura KalinieneVytautas Klausa; Vytautas Klausa; Lidija Truncaite



Disruption of the bacteriophage T4 Mre11 dimer interface reveals a two-state mechanism for exonuclease activity.  


The Mre11-Rad50 (MR) complex is a central player in DNA repair and is implicated in the processing of DNA ends caused by double strand breaks. Recent crystal structures of the MR complex suggest that several conformational rearrangements occur during its ATP hydrolysis cycle. A comparison of the Mre11 dimer interface from these structures suggests that the interface is dynamic in nature and may adopt several different arrangements. To probe the functional significance of the Mre11 dimer interface, we have generated and characterized a dimer disruption Mre11 mutant (L101D-Mre11). Although L101D-Mre11 binds to Rad50 and dsDNA with affinity comparable with the wild-type enzyme, it does not activate the ATP hydrolysis activity of Rad50, suggesting that the allosteric communication between Mre11 and Rad50 has been interrupted. Additionally, the dsDNA exonuclease activity of the L101D-MR complex has been reduced by 10-fold under conditions where processive exonuclease activity is required. However, we unexpectedly found that under steady state conditions, the nuclease activity of the L101D-MR complex is significantly greater than that of the wild-type complex. Based on steady state and single-turnover nuclease assays, we have assigned the rate-determining step of the steady state nuclease reaction to be the productive assembly of the complex at the dsDNA end. Together, our data suggest that the Mre11 dimer interface adopts at least two different states during the exonuclease reaction. PMID:22798142

Albrecht, Dustin W; Herdendorf, Timothy J; Nelson, Scott W



An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Bacteriophage T4 Replication Fork In Vivo  

Microsoft Academic Search

Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and\\/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and




Properties of r Mutants of Bacteriophage T4 Photodynamically Induced in the Presence of Thiopyronin and Psoralen  

PubMed Central

About 4 × 10?4r mutants were induced per lethal hit, a frequency characteristic of weak mutagens. Collections of mutants produced in the presence of either dye were indistinguishable in most of their properties. The rII mutants differed sharply from spontaneous mutants in their mutational spectra (fine scale map distribution) and their reversion responses to specific mutagens. Few or none of the induced mutants were induced to revert with proflavine (sign mutants; reading frame shift mutants). A majority were induced to revert with base analogues (base pair substitution mutants), and about half of these also responded to the hydroxymethylcytosine-specific agent hydroxylamine. A large minority of the mutants reverted spontaneously but failed to respond either to proflavine or to base analogues. We believe these mutants, as well as some of the mutants which did respond to base analogues, to be transversions (base pair substitutions which reverse the purine-pyrimidine orientation).

Drake, John W.; McGuire, Janice



Control of synthesis of mRNA's for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase.  

PubMed Central

A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.

Witmer, H; Baros, A; Ende, D; Dosmar, M



Realignment of the Genetic Map of the Terminus of the rIIB Cistron of Bacteriophage T4  

PubMed Central

Duplication end-point mapping in the rIIB cistron indicates that the order of the B8-B10b segments is the inverse of that presented in Benzer's (1961) genetic maps. This finding is supported by two- and three-factor crosses and the phenotypes of rII deletions extending into the D region.

Parma, David H.; Dill, Marsha; Slocum, Mary K.



Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family.  


Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features. PMID:9914527

Ito, Y; Yoshikawa, A; Hotani, T; Fukuda, S; Sugimura, K; Imoto, T



Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae  

PubMed Central

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ?CPV4 and ?ZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ?CP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ?CP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage ?29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

Volozhantsev, Nikolay V.; Oakley, Brian B.; Morales, Cesar A.; Verevkin, Vladimir V.; Bannov, Vasily A.; Krasilnikova, Valentina M.; Popova, Anastasia V.; Zhilenkov, Eugeni L.; Garrish, Johnna K.; Schegg, Kathleen M.; Woolsey, Rebekah; Quilici, David R.; Line, J. Eric; Hiett, Kelli L.; Siragusa, Gregory R.; Svetoch, Edward A.; Seal, Bruce S.



Bacteria screening, viability, and confirmation assays using bacteriophage-impedimetric/loop-mediated isothermal amplification dual-response biosensors.  


Here, we integrate two complementary detection strategies for the identification and quantification of Escherichia coli based on bacteriophage T4 as a natural bioreceptor for living bacteria cells. The first approach involves screening and viability assays, employing bacteriophage as the recognition element in label-free electrochemical impedance spectroscopy. The complementary approach is a confirmation by loop-mediated isothermal amplification (LAMP) to amplify specifically the E. coli Tuf gene after lysis of the bound E. coli cells, followed by detection using linear sweep voltammetry. Bacteriphage T4 was cross-linked, in the presence of 1,4-phenylene diisothiocyanate, on a cysteamine-modified gold electrode. The impedimetric biosensor exhibits specific and reproducible detection with sensitivity over the concentration range of 10(3)-10(9) cfu/mL, while the linear response of the LAMP approach was determined to be 10(2)-10(7) cfu/mL. The limit of detection (LOD) of 8 × 10(2) cfu/mL in less than 15 min and 10(2) cfu/mL within a response time of 40 min were achieved for the impedimetric and LAMP method, respectively. This work provides evidence that integration of the T4-bacteriophage-modified biosensor and LAMP can achieve screening, viability, and confirmation in less than 1 h. PMID:23510137

Tlili, Chaker; Sokullu, Esen; Safavieh, Mohammadali; Tolba, Mona; Ahmed, Minhaz Uddin; Zourob, Mohammed




PubMed Central

Markedly increased quantities of lysozyme have been found in the serum and urine (ranging to 2.6 g per day) of ten consecutive cases of monocytic and monomyelocytic leukemia. The enzyme has been isolated from the urine of several cases and physicochemically and immunochemically characterized. It is apparently identical to the lysozyme of normal tears, saliva, leukocytes, and serum, but structurally different from the lysozyme of hen's egg white. The activity of the human enzyme assayed with M. lysodeikticus organisms is 3 to 12 times greater than egg white lysozyme at equivalent concentrations. An agar plate method has been developed for quantitating lysozyme activity in small samples (approximately 25 µl) of serum, urine, or other biological fluids. The range and reproducibility of this method were found to be superior to previously available lysozyme assay procedures. Present evidence indicates that lysozyme is the principal, if not the sole, product of the proliferating monocytes in monocytic and monomyelocytic leukemia, and quantitation of serum and urine lysozyme should be a useful diagnostic procedure for these leukemias.

Osserman, Elliott F.; Lawlor, Dolores P.



Lysozyme loading and release from hydrogels carrying pendant phosphate groups.  


To develop a polymeric matrix for efficiently loading cationic biomolecules, polyelectrolyte hydrogels carrying pendant phosphate groups were synthesized by copolymerizing 2-methacryloyloxyethyl dihydrogen phosphate with N-isopropylacrylamide and N, N1-methylene-bis-acrylamide. The phosphate-carrying monomer yielded anionic hydrogels, which formed ionic complexes with the cationic protein, lysozyme. It was shown that the amount of complexed lysozyme reached 2.1 g g-1 dry gel, corresponding to 1.3 x 10(-3) mol phosphate group per gram lysozyme, when 40 mol% of phosphate-carrying monomer was incorporated in a hydrogel. When the hydrogel complexed with lysozyme was placed in deionized water and various KCl solutions, of varying concentrations of up to 0.5 M KCl, no lysozyme was released in deionized water, while increasing amounts of lysozyme were released as the KCl concentration increased. This confirmed that lysozyme was loaded in the hydrogel through electrostatic interactions. It was further found that the complexed lysozyme retained its enzymatic activity after being released from the hydrogel. These results suggest the use of this system for the controlled release of cationic protein drugs. PMID:9505202

Nakamae, K; Nizuka, T; Miyata, T; Furukawa, M; Nishino, T; Kato, K; Inoue, T; Hoffman, A S; Kanzaki, Y



Diversification and adaptive sequence evolution of Caenorhabditis lysozymes (Nematoda: Rhabditidae)  

Microsoft Academic Search

BACKGROUND: Lysozymes are important model enzymes in biomedical research with a ubiquitous taxonomic distribution ranging from phages up to plants and animals. Their main function appears to be defence against pathogens, although some of them have also been implicated in digestion. Whereas most organisms have only few lysozyme genes, nematodes of the genus Caenorhabditis possess a surprisingly large repertoire of

Hinrich Schulenburg; Claudia Boehnisch



Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida)  

Microsoft Academic Search

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes – four times, in comparison to the

Marta J. Fio?ka; Miros?aw P. Zagaja; Monika Hu?as-Stasiak; Jerzy Wielbo


Continuing adventures in lysozyme crystal growth. [in earth laboratory experiments  

NASA Technical Reports Server (NTRS)

Results obtained on the crystal nucleation and growth of lysozyme are presented. Special attention is given to the anion-protein binding, the nucleation kinetics, the mechanisms of aggregation, and the conditions that promote or inhibit lysozyme (110)-face growth rate. The emerging theory that is currently being used for data interpretation and for designing new experimental approaches is outlined.

Pusey, Marc L.



Tetragonal Lysozyme, From Monomer to Crystal  

NASA Technical Reports Server (NTRS)

The data now leads us to a comprehensive model for the process by which tetragonal lysozyme crystals are nucleated and subsequently grow. Lysozyme is typically desolubilized by addition of ionic salts. The salt anions bind to basic and other sites on the protein and promote protein-protein interactions, i.e., initiate the nucleation self assembly process. Formation of protein-protein interactions occurs at the expense of the protein-anion interactions, with the anions being released to the solution. The association follows a defined pattern, forming the "head to side" interactions of the crystal 4(3) helix. The presence of the high salt also promotes hydrophobic interactions between the protein molecules, further tightening their interaction. The solute assembly process persists after crystal nucleation, and the 4(3) helical structures form the subsequent growth units. AFM measurements show that the growth units follow the dimensions of these helices, and that those on the surface are more compact about the c-axis than in the bulk crystal, with adjacent helices riot being in contact. This further supports the role of hydrophobic interactions, as the surface is still in contact with the bulk solution. Once buried within the crystal the protein:salt ratio radically changes and the hydrophobic interactions relax to those measured crystallographically. Thus the crystal growth process recapitulates the initial stages of the nucleation process, and the two seamlessly merge. Experimental evidence, based upon face growth rate, AFM, and fluorescence energy transfer data, for a postulated model of the nucleation of tetragonal lysozyme crystals and how it transitions into crystal growth will be presented.

Pusey, Marc L.; Curreri, Peter A. (Technical Monitor)



Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144  

SciTech Connect

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.; Mesyanzhinov, Vadim V.; Rossmann, Michael G. (SOIBC); (Purdue)



Determination of monomer concentrations in crystallizing lysozyme solutions  

NASA Technical Reports Server (NTRS)

We have developed a non-optical technique for the study of aggregation in lysozyme and other protein solutions. By monitoring the rate at which lysozyme traverses a semipermeable membrane it was possible to quantitate the degree of aggregation in supersaturated solutions. Using this technique, we have measured the concentration of monomers and larger aggregates in under- and oversaturated lysozyme solutions, and in the presence of crystals, at pH 4.0 and 3 percent NaCl (0.1M NaAc). Comparison of these concentration profiles with (110) face growth rate data supports the theory that tetragonal lysozyme crystals grow by addition of preformed aggregates and not by monomer addition. The data suggest that a considerable population of aggregates larger than dimers are present at lysozyme concentrations above 22 mg/ml. Determination of dimer concentrations, and equilibrium constants for subsequent aggregation levels, are currently underway.

Wilson, L. J.; Pusey, Marc L.



Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections.  


There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme's electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose. PMID:24637705

Griswold, Karl E; Bement, Jenna L; Teneback, Charlotte C; Scanlon, Thomas C; Wargo, Matthew J; Leclair, Laurie W



Deformation behavior of aluminum alloy 6111-T4  

Microsoft Academic Search

Although aluminum alloys have found increasing usage in the automotive industry, their lower tensile elongations as compared with the low carbon steels they replace has raised concern about their lower formability. Lower formability imposes design and economic constraints on the automakers. The cause behind this lower elongation is the primary focus of this research. The specific alloy studied is 6111-T4

Carol Tseng



T-4G Methodology: Undergraduate Pilot Training T-37 Phase.  

ERIC Educational Resources Information Center

The report's brief introduction describes the application of T-4G methodology to the T-37 instrument phase of undergraduate pilot training. The methodology is characterized by instruction in trainers, proficiency advancement, a highly structured syllabus, the training manager concept, early exposure to instrument training, and hands-on training.…

Woodruff, Robert R.; And Others


Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium1  

PubMed Central

There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as nonfoodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria.

Seal, Bruce S.



Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida).  


In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm. PMID:22019387

Fio?ka, Marta J; Zagaja, Miros?aw P; Hu?as-Stasiak, Monika; Wielbo, Jerzy



Expression and prognostic significance of lysozyme in male breast cancer  

PubMed Central

Background Lysozyme, one of the major protein components of human milk that is also synthesized by a significant percentage of breast carcinomas, is associated with lesions that have a favorable outcome in female breast cancer. Here we evaluate the expression and prognostic value of lysozyme in male breast cancer (MBC). Methods Lysozyme expression was examined by immunohistochemical methods in a series of 60 MBC tissue sections and in 15 patients with gynecomastia. Staining was quantified using the HSCORE (histological score) system, which considers both the intensity and the percentage of cells staining at each intensity. Prognostic value of lysozyme was retrospectively evaluated by multivariate analysis taking into account conventional prognostic factors. Results Lysozyme immunostaining was negative in all cases of gynecomastia. A total of 27 of 60 MBC sections (45%) stained positively for this protein, but there were clear differences among them with regard to the intensity and percentage of stained cells. Statistical analysis showed that lysozyme HSCORE values in relation to age, tumor size, nodal status, histological grade, estrogen receptor status, metastasis and histological type did not increase the statistical significance. Univariate analysis confirmed that both nodal involvement and lysozyme values were significant predictors of short-term relapse-free survival. Multivariate analysis, according to Cox's regression model, also showed that nodal status and lysozyme levels were significant independent indicators of short-term relapse-free survival. Conclusion Tumor expression of lysozyme is associated with lesions that have an unfavorable outcome in male breast cancer. This milk protein may be a new prognostic factor in patients with breast cancer.

Serra, Carlos; Vizoso, Francisco; Alonso, Lorena; Rodriguez, Juan C; Gonzalez, Luis O; Fernandez, Maria; Lamelas, Maria L; Sanchez, Luis M; Garcia-Muniz, Jose L; Baltasar, Aniceto; Medrano, Justo



Increased inflammation in lysozyme M-deficient mice in response to Micrococcus luteusand its peptidoglycan  

Microsoft Academic Search

genic mice deficient in lysozyme M after challenge by the normally nonpathogenic and highly lysozyme-sensitive bacterium Micrococcus luteus. Despite partial com- pensation by newly expressed lysozyme P in macrophages, lysozyme M-deficient mice developed much more severe le- sions than wild-type mice. The tissue injury was due to the failure of lysozyme M-deficient mice to inactivate peptidogly- can, resulting in an

Tomas Ganz; Victoria Gabayan; Hsiang-I Liao; Lide Liu; Ami Oren; Thomas Graf; Alexander M. Cole



Nucleation and Growth According to Lysozyme  

NASA Technical Reports Server (NTRS)

How does one take a molecule, strongly asymmetric in both shape and charge distribution, and assemble it into a crystal? We propose a model for the nucleation and crystal growth process for tetragonal lysozyme that may be very germane to other monomeric proteins. The first species formed is postulated to be a dimer. Through repeating associations involving the same intermolecular interactions this becomes the 4(sub 3) helix, that in turn serves as the basic unit for nucleation and crystal growth. High salt attenuates surface charges while promoting hydrophobic interactions. Symmetry facilitates helix self-association. Assembly stability is enhanced when a four helix structure is obtained, with each bound to two neighbors. Only two unique interactions are required. The first are those for helix formation, where the dominant interaction is the intermolecular bridging anion. The second is the anti-parallel side-by-side helix-helix interaction, guided by alternating pairs of symmetry related salt bridges along each side. At this stage all eight unique positions of the P4(sub 3)2(sub 1)2(sub 1) unit cell are filled. From the above, the process is one of a) attenuating the most strongly interacting groups, such that b) the molecules begin to self-associate in defined patterns, so that c) symmetry is obtained, which d) propagates as a growing crystal. Simple and conceptually obvious in hindsight, this tells much about what we are empirically doing when we crystallize macromolecules. By adjusting the solution parameters we are empirically balancing the intermolecular interactions, preferentially attenuating the dominant strong (for lysozyme the charged groups) while strengthening the lesser strong (hydrophobic) interactions. Lysozyme is atypical in the breadth of its crystallization conditions; many proteins only crystallize under narrowly defined conditions, pointing to the criticality of the empirical balancing process. Lack of a singularly defined association pathway leads to formation of multiple species, i.e., amorphous precipitation. Weak interactions, such as hydrogen bonds, are promiscuous, serving to strengthen rather than define specific interactions. Participation in an interaction sequesters that surface from subsequent interactions, and we expect the strongest bonds to form first. When two molecules self associate the resulting species will have an axis of symmetry. Subsequent interactions between two associated species having equivalent interactions will also have symmetry. Only a few unique sets of interactions are required to give any of the commonly found space groups for monomeric proteins. This model and what it suggests will be discussed.

Pusey, Marc L.; Curreri, Peter A. (Technical Monitor)



Fractal properties of lysozyme: A neutron scattering study  

NASA Astrophysics Data System (ADS)

The spatial structure and dynamics of hen egg white lysozyme have been investigated by small-angle and inelastic neutron scattering. Analysis of the results was carried using the fractal approach, which allowed determination of the fractal and fracton dimensions of lysozyme, i.e., consideration of the protein structure and dynamics by using a unified approach. Small-angle neutron scattering studies of thermal denaturation of lysozyme have revealed changes in the fractal dimension in the vicinity of the thermal denaturation temperature that reflect changes in the spatial organization of protein.

Lushnikov, S. G.; Svanidze, A. V.; Gvasaliya, S. N.; Torok, G.; Rosta, L.; Sashin, I. L.



40 CFR 721.10337 - Copper, iodotris(triphenylphosphine)-, (T-4)-.  

Code of Federal Regulations, 2013 CFR

...Copper, iodotris(triphenylphosphine)-, (T-4)-. 721.10337 Section 721.10337...Copper, iodotris(triphenylphosphine)-, (T-4)-. (a) Chemical substance and...copper, iodotris(triphenylphosphine)-, (T-4)- (PMN P-04-6; CAS No....



Tubular Heads in Bacteriophages from Lactic Streptococci  

PubMed Central

Tubular bacteriophage heads were observed in the lysate of two phages from Streptococcus lactis obtained from single plaques without mutagenesis. The frequency of appearance of the tubular heads was 2.5 and 16%. Images

Chopin, Marie-Christine; Rousseau, Micheline



Isolation of Bacteriophages Active Against Neisseria Meningitidis.  

National Technical Information Service (NTIS)

Five distinct bacteriophages have been isolated from strains of Neisseria meningitidis. Filtrates with titers of .0001 to .000001 were produced with a modified Swanstrom and Adams semisolid agar procedure, employing Eugonbroth with added agar and an incub...

S. G. Cary D. H. Hunter



Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride  

SciTech Connect

Recent studies have outlined the use of eutectic solution of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without the solvent crystallization. The eutectic point in (H2O)R(LiCl) system corresponds to R 7.3, and it is of interest to investigate whether less concentrated aqueous solutions of LiCl could be employed in low-temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low-temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins.

Mamontov, Eugene [ORNL] [ORNL; O'Neill, Hugh Michael [ORNL] [ORNL



Mutagenic Specificity of a Novel T4 DNA Polymerase Mutant  

PubMed Central

The in vivo mutational specificity of a novel T4 DNA polymerase mutator mutant, tsM19, was determined. Two genetic tester systems were used to characterize the mutant. Results of our studies indicate that tsM19 promotes transition and transversion mutagenesis and, possibly, frameshift mutagenesis. Central G:C base pairs in runs of three or more consecutive G:C base pairs may be target sites for tsM19-induced transitions.

Reha-Krantz, Linda J.; Liesner, Evelyn M.



Bacteriophages: New Tools for Safer Food?  

Microsoft Academic Search

:  The recent FDA approval of Listeria-specific bacteriophage preparations for food preservation has opened the door to new applications\\u000a of these natural bacterial killers. Bacteriophages are viruses that only infect and lyse bacterial cells and are harmless\\u000a to mammalians. There is now a renewed interest to use the ability of these viruses to kill pathogenic bacteria for biotechnological\\u000a purposes. We will

E. Strauch; J. A. Hammerl; S. Hertwig



Arthrobacter globiformis and its bacteriophage in soil  

NASA Technical Reports Server (NTRS)

An attempt was made to correlate bacteriophages for Arthrobacter globiformis with soils containing that bacterium. The phages were not detected unless the soil was nutritionally amended (with glucose or sucrose) and incubated for several days. Phage was continuously produced after amendment without the addition of host Arthrobacter. These results indicate that the bacteriophage is present in a masked state and that the bacteria are present in an insensitive form which becomes sensitive after addition of nutrient.

Casida, L. E., Jr.; Liu, K.-C.



Taking Bacteriophage Therapy Seriously: A Moral Argument  

PubMed Central

The excessive and improper use of antibiotics has led to an increasing incidence of bacterial resistance. In Europe the yearly number of infections caused by multidrug resistant bacteria is more than 400.000, each year resulting in 25.000 attributable deaths. Few new antibiotics are in the pipeline of the pharmaceutical industry. Early in the 20th century, bacteriophages were described as entities that can control bacterial populations. Although bacteriophage therapy was developed and practiced in Europe and the former Soviet republics, the use of bacteriophages in clinical setting was neglected in Western Europe since the introduction of traditional antibiotics. Given the worldwide antibiotic crisis there is now a growing interest in making bacteriophage therapy available for use in modern western medicine. Despite the growing interest, access to bacteriophage therapy remains highly problematic. In this paper, we argue that the current state of affairs is morally unacceptable and that all stakeholders (pharmaceutical industry, competent authorities, lawmakers, regulators, and politicians) have the moral duty and the shared responsibility towards making bacteriophage therapy urgently available for all patients in need.

Verbeken, Gilbert; Huys, Isabelle; Jennes, Serge; Chanishvili, Nina; Gorski, Andrzej; De Vos, Daniel



21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2010 CFR serum, plasma, leukocytes, and urine. Lysozyme measurements are used in the diagnosis and treatment of monocytic leukemia and kidney disease. (b) Classification. Class I (general controls). The device is exempt from the...



21 CFR 862.1490 - Lysozyme (muramidase) test system.  

Code of Federal Regulations, 2010 CFR serum, plasma, leukocytes, and urine. Lysozyme measurements are used in the diagnosis and treatment of monocytic leukemia and kidney disease. (b) Classification. Class I (general controls). The device is exempt from the...



Fabrication of polypyrrole nano-arrays in lysozyme single crystals  

NASA Astrophysics Data System (ADS)

A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals.A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals. Electronic supplementary information (ESI) available: Optical microscopy, SEM, TEM images, FTIR spectra and tables, conductivity plot. Experimental methods. See DOI: 10.1039/c2nr32413j

England, Matt W.; Lambert, Elizabeth M.; Li, Mei; Turyanska, Lyudmila; Patil, Avinash J.; Mann, Stephen



Immobilization of lysozyme on polyvinylalcohol films for active packaging applications.  


A new technique for the immobilization of lysozyme onto the surface of polyvinylalcohol films is presented. The active compound was sprayed along with a suitable bonding agent onto the surface of the cross-linked polymeric matrix. Active compound release tests determined the amount of lysozyme immobilized on the film surface. With the use of Micrococcus lysodeikticus, the antimicrobial activity of the films was determined and the results correlated with the amount of immobilized lysozyme. This new technique was effective for immobilizing the enzyme, and the developed films were active against the test microorganism. Results were compared with those obtained with a different immobilizing technique, in which the active compound was bound into the bulk of the polymeric film. As expected, the surface-immobilized lysozyme films have a higher antimicrobial activity than bulk-bound films. PMID:16629031

Conte, A; Buonocore, G G; Bevilacqua, A; Sinigaglia, M; Del Nobile, M A



Effects of Oxygen on Irradiated Solid Egg-White Lysozyme.  

National Technical Information Service (NTIS)

Chromatographically homogeneous egg-white lysozyme has been subjected to gamma radiation in vacuo and in the presence of oxygen. Soluble active and inactive fractions from these samples have been isolated by salt precipitation and chromatographic steps. S...

B. M. Tolbert C. O. Stevens G. R. Bergstrom



Location of Bromide Ions in Tetragonal Lysozyme Crystals  

NASA Technical Reports Server (NTRS)

Anions have been shown to play a dominant role in the crystallization of chicken egg white lysozyme from salt solutions. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystal grown in bromide and chloride solutions. Five possible anion binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four of these sites corresponded to four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed.

Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.



T4 genes in the marine ecosystem: studies of the T4-like cyanophages and their role in marine ecology  

PubMed Central

From genomic sequencing it has become apparent that the marine cyanomyoviruses capable of infecting strains of unicellular cyanobacteria assigned to the genera Synechococcus and Prochlorococcus are not only morphologically similar to T4, but are also genetically related, typically sharing some 40-48 genes. The large majority of these common genes are the same in all marine cyanomyoviruses so far characterized. Given the fundamental physiological differences between marine unicellular cyanobacteria and heterotrophic hosts of T4-like phages it is not surprising that the study of cyanomyoviruses has revealed novel and fascinating facets of the phage-host relationship. One of the most interesting features of the marine cyanomyoviruses is their possession of a number of genes that are clearly of host origin such as those involved in photosynthesis, like the psbA gene that encodes a core component of the photosystem II reaction centre. Other host-derived genes encode enzymes involved in carbon metabolism, phosphate acquisition and ppGpp metabolism. The impact of these host-derived genes on phage fitness has still largely to be assessed and represents one of the most important topics in the study of this group of T4-like phages in the laboratory. However, these phages are also of considerable environmental significance by virtue of their impact on key contributors to oceanic primary production and the true extent and nature of this impact has still to be accurately assessed.



Study of ethanol-lysozyme interactions using neutron diffraction  

Microsoft Academic Search

Single-crystal neutron diffraction has been used to observe the interactions between deuterated ethanol (CD3CD2OH) and lysozyme in triclinic crystals of hen egg white lysozyme soaked in 25% (v\\/v) ethanol solutions. A total of 6047 observed reflections to a resolution of 2 A were used, and 13 possible ethanol sites were identified. The three highest occupied sites are close to locations

M. S. Lehmann; S. A. Mason; G. J. McIntyre



Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function  

Microsoft Academic Search

A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13?700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and

Rosana Regel; Sergio R Matioli; Walter R Terra



Lysozyme detection on aptamer functionalized graphene-coated SPR interfaces.  


The paper reports on a surface plasmon resonance (SPR)-based approach for the sensitive and selective detection of lysozyme. The SPR sensor consists of a 50 nm gold film coated with a thin film of reduced graphene oxide (rGO) functionalized with anti-lysozyme DNA aptamer. The SPR chip coating with rGO matrix was achieved through electrophoretic deposition of graphene oxide (GO) at 150 V. Electrophoretic deposition resulted in partial reduction of GO to rGO with a thickness depending on the deposition time. For very short time pulses of 20 s, the resulting rGO film had a thickness of several nanometers and was appropriate for SPR sensing. The utility of the graphene-based SPR sensor for the selective and sensitive detection of proteins was demonstrated using lysozyme as model protein. Functionalization of rGO matrix with anti-lysozyme DNA aptamer through ?-stacking interactions allowed selective SPR detection of lysozyme. The graphene-based SPR biosensor provides a means for the label-free, concentration-dependent and selective detection of lysozymes with a detection limit of 0.5 nM. PMID:23871871

Subramanian, Palaniappan; Lesniewski, Adam; Kaminska, Izabela; Vlandas, Alexis; Vasilescu, Alina; Niedziolka-Jonsson, Joanna; Pichonat, Emmanuelle; Happy, Henri; Boukherroub, Rabah; Szunerits, Sabine



Interplay between the mechanics of bacteriophage fibers and the strength of virus-host links  

NASA Astrophysics Data System (ADS)

Viral fibers play a central role in many virus infection mechanisms since they recognize the corresponding host and establish a mechanical link to its surface. Specifically, bacteriophages have to anchor to bacteria through the fibers surrounding the tail before starting the viral DNA translocation into the host. The protein gene product (gp) 37 from bacteriophage T4 long tail fibers forms a fibrous parallel homotrimer located at the distal end of the long tail fibers. Biochemical data indicate that, at least, three of these fibers are required for initial host cell interaction but do not reveal why three and no other numbers are required. By using atomic force microscopy, we obtained high-resolution images of gp37 fibers adsorbed on a mica substrate in buffer conditions and probed their local mechanical properties. Our experiments of radial indentation at the nanometer scale provided a radial stiffness of ˜0.08 N/m and a breaking force of ˜120 pN. In addition, we performed finite element analysis and determined a Young's modulus of ˜20 MPa. From these mechanical parameters, we hypothesize that three viral fibers provide enough mechanical strength to prevent a T4 virus from being detached from the bacteria by the viral particle Brownian motion, delivering a biophysical justification for the previous biochemical data.

Ares, P.; Garcia-Doval, C.; Llauró, A.; Gómez-Herrero, J.; van Raaij, M. J.; de Pablo, P. J.



Structures of monoclinic lysozyme iodide at 1.6 A and of triclinic lysozyme nitrate at 1.1 A.  


Hen egg-white lysozyme is one of the most thoroughly studied of enzymes and has been the subject of study by many methods, including X-ray crystallography. The present work extends the X-ray crystallography to higher resolution, includes the positions of the anions, and examines the contacts of the neighbors in greater detail. Data were collected at room temperature on a Rigaku R-axis area detector with rotating-anode X-ray generator to 1.6 A resolution for monoclinic lysozyme iodide at pH 4.0, to 1.8 A for monoclinic lysozyme iodide at pH 8.0, and to 1.1 A resolution for triclinic lysozyme nitrate at pH 4.5. The structures have been refined by SHELX93 with the expected number of anion sites being accounted for. Two regions of the protein have been found to be variable: residues 65-75 and 99-104. Except for 65-75 and 99-104, lysozyme is a very stable molecule with the crystal forms being held together by the electrostatic contacts of the anions and by layers of water molecules. The anion positions can be described as paired half sites, each half being contributed by a different lysozyme molecule. The many different crystal forms of lysozyme may be due to different combinations of the many such half sites on the surface. A hypothesis is presented for lysozyme in the different crystal forms and which may be extended to behavior in solution. Suggestions for future crystallographic research are proposed, involving anions of different shape and charge. PMID:9757091

Steinrauf, L K



Complete Genome Sequence of Bacillus thuringiensis Bacteriophage BMBtp2.  


Bacillus thuringiensis is an insect pathogen which has been widely used for biocontrol. During B. thuringiensis fermentation, lysogenic bacteriophages cause severe losses of yield. Here, we announce the complete genome sequence of a bacteriophage, BMBtp2, which is induced from B. thuringiensis strain YBT-1765, which may be helpful to clarify the mechanism involved in bacteriophage contamination. PMID:23405296

Dong, Zhaoxia; Peng, Donghai; Wang, Yueying; Zhu, Lei; Ruan, Lifang; Sun, Ming



Assessment of drinking water quality using indicator bacteria and bacteriophages  

Microsoft Academic Search

Bacterial indicators and bacteriophages suggested as potential indicators of water quality were determined by public laboratories in water from springs, household water wells, and rural and metropolitan water supplies in north-eastern Spain. Indicator bacteria were detected more frequently than bacteriophages in springs, household water wells and rural water supplies. In contrast, positive bacteriophage detections were more numerous than those of

Ana Audicana; Mercedes Cancer



Selective Antibacterial Properties of Lysozyme for Oral Microorganisms  

PubMed Central

The antibacterial properties of lysozyme were investigated with oral microorganisms representing the seven serotypes (a through g) of Streptococcus mutans, Veillonella alcalescens, and the virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14. Growth of bacteria in defined medium was monitored spectrophotometrically after the addition of various amounts (25 ?g to 5 mg/ml) of enzyme. No growth inhibition of V. alcalescens was observed. Inhibition of A. viscosus T14(V) and A. viscosus T14(AV) occurred with 160 ?g of lysozyme per ml. Of the S. mutans cultures tested, the serotype a and b strains were inhibited with as little as 25 ?g of enzyme per ml, whereas e and f strains were most resistant to the bacteriostatic activity of lysozyme. The presence of dl-threonine or sucrose in growth medium did not significantly affect the results. A lysoplate assay was developed to rapidly survey the bacterial cultures for their susceptibility to the lytic ability of the enzyme. Lysis, as a measure of a zone of clearing in agarose plates, occurred for all microorganisms in the presence of lysozyme after the subsequent addition of NaCl or detergent. The bactericidal activity of lysozyme was determined on S. mutans BHT and S. mutans LM-7 by the pour plate technique. Preincubation of S. mutans LM-7 with as much as 1 mg of enzyme for 90 min did not affect viability or growth, whereas preincubation of S. mutans BHT with 1 mg of lysozyme resulted in no recoverable colony-forming units. An antigen containing extract of S. mutans LM-7 blocked the growth inhibitory property of lysozyme. Human lysozyme was a more effective antibacterial factor than hen egg white lysozyme. Total growth inhibition of S. mutans BHT was effected with 40 ?g of human enzyme, and as little as 10 ?g of human enzyme inhibited growth for greater than 20 h. The data presented indicate that different mechanisms may be responsible for the bacteriostatic, lytic, and bactericidal properties of the enzyme and that lysozyme is a selective but effective antibacterial factor for oral microorganisms. ImagesFig. 3

Iacono, Vincent J.; MacKay, Bruce J.; DiRienzo, Sharon; Pollock, Jerry J.



Low-temperature T4-like coliphages vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20.  


Bacteriophages vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20, showing an unusual low-temperature plating profile and producing constantly growing plaques, were isolated from aquatic environments of Lithuania. Although vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20 resembled phage T4 both in their genome size and in their major structural protein (gp23) pattern, physiological properties of all three phages tested differed significantly from those of T4. With an optimum temperature for plating around 24 degrees C and a high efficiency of plating in the range 7-30 degrees C, bacteriophages vB_EcoM-VR7 and vB_EcoM-VR20 failed to plate at 37 degrees C, whereas phage vB_EcoM-VR5 could not be plated at 40 degrees C. Sequence analysis of diagnostic g23 PCR products revealed that g23 of vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20 differed from the corresponding T4 g23 DNA sequence by 21, 21 and 20%, respectively. PMID:20361343

Kaliniene, Laura; Klausa, Vytautas; Truncaite, Lidija



Characterization of Temperate Bacteriophages of Bacillus subtilis by the Restriction Endonuclease EcoRI: Evidence for Three Different Temperate Bacteriophages  

PubMed Central

Temperate bacteriophages of Bacillus subtilis were characterized according to host range and digestion of the bacteriophage genome by endonuclease EcoRI. The three bacteriophages, ?3T, SPO2, and ?105, were all heteroimmune, and the DNA digests showed dissimilar patterns by agarose-ethidium bromide gel electrophoresis. Images

Wilson, G. A.; Williams, M. T.; Baney, H. W.; Young, F. E.



Lysis of Grouped and Ungrouped Streptococci by Lysozyme 1  

PubMed Central

Thirty strains of streptococci were tested for lysis with lysozyme, and 29 of these could be lysed by the following method: (i) suspension of the cells to a Klett reading of 200 units (no. 42 filter) in 0.01 m tris(hydroxymethyl)aminomethane buffer, pH 8.2, after washing twice with the buffer; (ii) addition of lysozyme to a final concentration of 250 ?g/ml with incubation for 60 min at 37 C; (iii) addition of sodium lauryl sulfate (SLS) to a final concentration of 0.2% and incubation up to an additional 15 min at 37 C. Significant lysis was obtained only after the addition of SLS. (Strains of groups A, E, and G were treated with trypsin at a concentration of 200 ?g/ml for 2 hr at 37 C before exposure to lysozyme.) These parameters for optimal lysis of streptococci by lysozyme were established by testing the group D Streptococcus faecalis strain 31 which lyses readily with lysozyme and the group H strain Challis which is less susceptible to the action of the enzyme. Viability of S. faecalis decreased 96% after 3 min of exposure to 250 ?g of lysozyme per ml, whereas the more resistant strain Challis retained 27% of the initial viability after the same period. After 60 min, there was almost total loss of viability in each case. Variations of three methods of lysing streptococci with lysozyme were compared with respect to the decrease in turbidity and the release of protein and deoxyribonucleic acid (DNA) effected by each variation. The method presented in this paper allowed the greatest release of these cytoplasmic constituents from S. faecalis and strain Challis. Transformation experiments using DNA obtained from strain Challis (streptomycinresistant) by this method showed that the DNA released is biologically active.

Coleman, S. E.; van de Rijn, Ivo; Bleiweis, A. S.



Lytic sensitivity of Actinobacillus actinomycetemcomitans Y4 to lysozyme.  

PubMed Central

The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be an important mechanism by which lysozyme participates in the regulation of this suspected periodontal pathogen. Images

Iacono, V J; Boldt, P R; MacKay, B J; Cho, M I; Pollock, J J



Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY  

PubMed Central

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.

Pant, Kiran; Shokri, Leila; Karpel, Richard L.; Morrical, Scott W.; Williams, Mark C.



A new lysozyme from the eastern oyster, Crassostrea virginica, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion  

PubMed Central

Background Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense but also modified in some instances for digestion. The biochemical and evolutionary changes between these different functional forms has been well-studied in the c-type lysozymes of vertebrates, but less so in the i-type lysozymes prevalent in most invertebrate animals. Some bivalve molluscs possess both defensive and digestive lysozymes. Results We report a third lysozyme from the oyster Crassostrea virginica, cv-lysozyme 3. The chemical properties of cv-lysozyme 3 (including molecular weight, isoelectric point, basic amino acid residue number, and predicted protease cutting sites) suggest it represents a transitional form between lysozymes used for digestion and immunity. The cv-lysozyme 3 protein inhibited the growth of bacteria (consistent with a defensive function), but semi-quantitative RT-PCR suggested the gene was expressed mainly in digestive glands. Purified cv-lysozyme 3 expressed maximum muramidase activity within a range of pH (7.0 and 8.0) and ionic strength (I = 0.005-0.01) unfavorable for either cv-lysozyme 1 or cv-lysozyme 2 activities. The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands. Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion. Conclusion The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms.



Bacteriophage protein-protein interactions.  


Bacteriophages T7, ?, P22, and P2/P4 (from Escherichia coli), as well as ?29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages ? and T7. For example, the ?55 proteins encoded by the T7 genome are connected by ?43 interactions with another ?15 between the phage and its host. The chapter compiles published interactions for the well-studied phages ? (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ?29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage ? and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter



My Life with Bacteriophage ?29  

PubMed Central

This article is a survey of my scientific work over 52 years. During my postdoctoral stay in Severo Ochoa's laboratory, I determined the direction of reading of the genetic message, and I discovered two proteins that I showed to be involved in the initiation of protein synthesis. The work I have done in Spain with bacteriophage ?29 for 45 years has been very rewarding. I can say that I was lucky because I did not expect that ?29 would give so many interesting results, but I worked hard, with a lot of dedication and enthusiasm, and I was there when the luck arrived. I would like to emphasize our work on the control of ?29 DNA transcription and, in particular, the finding for the first time of a protein covalently linked to the 5?-ends of ?29 DNA that we later showed to be the primer for the initiation of phage DNA replication. Very relevant was the discovery of the ?29 DNA polymerase, with its properties of extremely high processivity and strand displacement capacity, together with its high fidelity. The ?29 DNA polymerase has become an ideal enzyme for DNA amplification, both rolling-circle and whole-genome linear amplification. I am also very proud of the many brilliant students and collaborators with whom I have worked over the years and who have become excellent scientists. This Reflections article is not intended to be the end of my scientific career. I expect to work for many years to come.

Salas, Margarita



Bacteriophage typing of Listeria species.  

PubMed Central

A bacteriophage typing scheme for differentiating Listeria isolates from dairy products and various other foodstuffs was developed. Sixteen selected phages isolated from both environmental sources and lysogenic strains were used for typing and, according to their lytic spectra, divided into four groups. Thus far, 41 distinct patterns of lysis were seen when this set was used in typing 57 defined reference strains, representing all five confirmed species and 16 serotypes in addition to 454 Listeria isolates of primarily foodborne origin. Overall, typability was 84.5%; i.e., a strain was lysed by at least one phage at 100x routine test dilution. Strains belonging to serovar 3 were mostly resistant to lysis by the phages employed. The results were highly reproducible, as determined in retyping trials several weeks later. Some phages isolated from environmental sources showed a wider lytic spectrum than did those isolated from lysogenic strains. In accordance with this, the phages were found in different clusters within a computer-generated linkage map. Species specificity and serovar specificity of the lytic reaction were not found. None of the phages was able to lyse strains of Listeria grayi, Listeria murrayi or Jonesia denitrificans. This phage typing system may provide important information for a means of recognizing and eliminating sources of contamination by Listeria spp. within dairy plant equipment.

Loessner, M J; Busse, M



Bacteriophages: antibacterials with a future?  


The hypothesis as to whether a benign species of bacteria could kill a virulent kind has to this point been untested. Recently it was shown that in the macrophage, bacteriophages, when properly introduced through a nonvirulent microbe, had a killing rate for virulent AIDS Mycobacterium tuberculosis and Mycobacterium avium far in excess of modern day antibiotics. The study in effect brought a natural phenomena, lysogeny, whereby one bacterial colony kills another thru phage weaponry, to bear in the conquest of hard-to-kill, antibiotic resistant pathogens. This killing occurred intracellularly, within the white blood cell using Mycobacterium smegmatis, a benign bacterial species found generally in smegma secretions from human genitalia as well as soil, dust and water, and first identified in 1884. The subsequent treatment of M. avium-infected, as well as M. tuberculosis-infected RAW 264.7 macrophages, with M. smegmatis transiently infected with TM4 resulted in a unexpectedly large time- and titer-dependent reduction in the number of viable intracellular bacilli. In addition, the M. smegmatis vacuole harboring TM4 fused with the M. avium vacuole in macrophages. These results suggested a potentially novel concept to kill intracellular pathogenic bacteria and warrant future development. PMID:15142642

Broxmeyer, Lawrence



Inducible bacteriophages from ruminal bacteria.  


The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria. PMID:2504111

Klieve, A V; Hudman, J F; Bauchop, T



Bacteriophage-Based Pathogen Detection  

NASA Astrophysics Data System (ADS)

Considered the most abundant organism on Earth, at a population approaching 1031, bacteriophage, or phage for short, mediate interactions with myriad bacterial hosts that has for decades been exploited in phage typing schemes for signature identification of clinical, food-borne, and water-borne pathogens. With over 5,000 phage being morphologically characterized and grouped as to susceptible host, there exists an enormous cache of bacterial-specific sensors that has more recently been incorporated into novel bio-recognition assays with heightened sensitivity, specificity, and speed. These assays take many forms, ranging from straightforward visualization of labeled phage as they attach to their specific bacterial hosts to reporter phage that genetically deposit trackable signals within their bacterial hosts to the detection of progeny phage or other uniquely identifiable elements released from infected host cells. A comprehensive review of these and other phage-based detection assays, as directed towards the detection and monitoring of bacterial pathogens, will be provided in this chapter.

Ripp, Steven


HyShot-T4 Supersonic Combustion Experiments  

NASA Technical Reports Server (NTRS)

A series of experiments were initiated to investigate the operation of a two-dimensional, hypersonic, airbreathing engine (scramjet) inclined at angles of attack to the freestream. The experiments were undertaken to obtain data for use in the Hyshot flight test program. Experiments on the Hyshot scramjet were under taken in the T4 shock tunnel. Experiments were made at a nominal total enthalpy of 3.0MJkg (exp -1) using a nozzle that produced flows with a Mach number of approximately 6.5. The conditions produced correspond to flight at Mach 7.6 at an altitude range of 35.7-21.4km. A summary of the flow conditions is included. The scramjet was tested at 0, plus 2, plus 4, minus 2 and minus 4 degrees angle of attack. Experiments were also undertaken at 2 and 4 degrees angle of skew.

Paull, A.; Frost, M.; Alesi, H.



Bacteriophages with the Ability to Degrade Uropathogenic Escherichia Coli Biofilms  

PubMed Central

Escherichia coli-associated urinary tract infections (UTIs) are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC) have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages) as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12) which were able to lyse 80.5% of a subset (42) of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages’ genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2–12 h of incubation.

Chibeu, Andrew; Lingohr, Erika J.; Masson, Luke; Manges, Amee; Harel, Josee; Ackermann, Hans-W.; Kropinski, Andrew M.; Boerlin, Patrick



Effects of Purification on the Crystallization of Lysozyme  

NASA Technical Reports Server (NTRS)

We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20 C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal- orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

Ewing, Felecia L.; Forsythe, Elizabeth L.; Van Der Woerd, Mark; Pusey, Marc L.



Sensitization by Ethylenediaminetetraacetate of Clostridium perfringens Type A Spores to Germination by Lysozyme1  

PubMed Central

Clostridium perfringens spores (three strains) were normally resistant to germination by lysozyme. In the absence of disulfide bond-breaking reagents, tetrasodium ethylenediaminetetraacetate rapidly sensitized the spores to lysozyme.

Adams, D. M.



Insights into the structure-function relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1.  


The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile. PMID:18667432

Monterroso, Begoña; Sáiz, José Luis; García, Pedro; García, José Luis; Menéndez, Margarita



Studies on tetragonal lysozyme crystal growth rates.  


A computer-controlled apparatus able to simultaneously follow the face growth rate of up to 40 crystals was developed. This apparatus was used to investigate the effects of solution pH on the (110) and (101) face growth rates of tetragonal lysozyme. Growth rates were measured at pH 4.0, 4.4, 4.8 and 5.2, in 0.1 M sodium acetate buffer with 5% NaCl, 295 K. Initial crystal sizes ranged from 10 to 40 microm. Plots of log supersaturation ratio (either C/C(sat) or C/C(sat) - 1) versus log(growth rate) are not linear, typically having a slope of approximately 8 at the lowest growth rates determined (10(-6) microm s(-1)), which falls off to a slope of approximately 2 at the highest growth rates (10(-2) microm s(-1)) measured. Ratios of C/C(sat) ranged from 4 to >20. The data show that lower solubility solutions require higher supersaturation ratios for equivalent growth rates. Data for the growth rate of the (101) face at pH 4.0 were widely scattered, especially at lower supersaturation ratios. Time-lapse video of crystals at low supersaturations shows that initially only the (110) faces grow, leaving 'notches' at the (110)-(110) corners. These corners then fill in and macro-steps appear on the (101) faces which rapidly move inward in the form of an octagon, restoring the crystal to a 'normal' appearance. This phenomenon has been observed for tetragonal crystals grown in either still or flowing solutions. Flowing solutions at lower supersaturations also gave cases where the corners did not fill in, with the (110) faces continuing to grow out until growth ceased. PMID:15299425

Forsythe, E; Ewing, F; Pusey, M



Modeling Tetragonal Lysozyme Crystal Growth Rates  

NASA Technical Reports Server (NTRS)

Tetragonal lysozyme 110 face crystal growth rates, measured over 5 orders of magnitude in range, can be described using a model where growth occurs by 2D nucleation on the crystal surface for solution supersaturations of c/c(sub eq) less than or equal to 7 +/- 2. Based upon the model, the step energy per unit length, beta was estimated to be approx. 5.3 +/- 0.4 x 10(exp -7) erg/mol-cm, which for a step height of 56 A corresponds to barrier of approx. 7 +/- 1 k(sub B)T at 300 K. For supersaturations of c/c(sub eq) > 8, the model emphasizing crystal growth by 2D nucleation not only could not predict, but also consistently overestimated, the highest observable crystal growth rates. Kinetic roughening is hypothesized to occur at a cross-over supersaturation of c/c(sub eq) > 8, where crystal growth is postulated to occur by a different process such as adsorption. Under this assumption, all growth rate data indicated that a kinetic roughening transition and subsequent crystal growth by adsorption for all solution conditions, varying in buffer pH, temperature and precipitant concentration, occurs for c/c(sub eq)(T, pH, NaCl) in the range between 5 and 10, with an energy barrier for adsorption estimated to be approx. 20 k(sub B)T at 300 K. Based upon these and other estimates, we determined the size of the critical surface nucleate, at the crossover supersaturation and higher concentrations, to range from 4 to 10 molecules.

Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, Marc L.



Analysis of Monomer Aggregation and Crystal Growth Rates of Lysozyme  

NASA Technical Reports Server (NTRS)

This project was originally conceived to analyze the extensive data of tetragonal lysozyme crystal growth rates collected at NASA/MSFC by Dr. Marc L. Pusey's research group. At that time the lack of analysis of the growth rates was hindering progress in understanding the growth mechanism of tetragonal lysozyme and other protein crystals. After the project was initiated our initial analysis revealed unexpected complexities in the growth rate behavior. This resulted in an expansion in the scope of the project to include a comprehensive investigation of the growth mechanisms of tetragonal lysozyme crystals. A discussion of this research is included as well a list of presentations and publications resulting from the research. This project contributed significantly toward the education of several students and fostered extensive collaborations between investigators.

Nadarajah, Arunan



Protist-Type Lysozymes of the Nematode Caenorhabditis elegans Contribute to Resistance against Pathogenic Bacillus thuringiensis  

Microsoft Academic Search

Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role

Claudia Boehnisch; Daniel Wong; Michael Habig; Kerstin Isermann; Nicolaas K. Michiels; Thomas Roeder; Robin C. May; Hinrich Schulenburg



Crystal structures of K33 mutant hen lysozymes with enhanced activities.  


Using random mutagenesis, we previously obtained K33N mutant lysozyme that showed a large lytic halo on the plate coating Micrococcus luteus. In order to examine the effects of mutation of K33N on enzyme activity, we prepared K33N and K33A mutant lysozymes from yeast. It was found that the activities of both the mutant lysozymes were higher than those of the wild-type lysozyme based on the results of the activity measurements against M. luteus (lytic activity) and glycol chitin. Moreover, 3D structures of K33N and K33A mutant lysozyme were solved by X-ray crystallographic analyses. The side chain of K33 in the wild-type lysozyme hydrogen bonded with N37 involved in the substrate-binding region, and the orientation of the side chain of N37 in K33 mutant lysozymes were different in the wild-type lysozyme. These results suggest that the enhancement of activity in K33N mutant lysozyme was due to an alteration in the orientation of the side chain of N37. On the other hand, K33N lysozyme was less stable than the wild-type lysozyme. Lysozyme may sacrifice its enzyme activity to acquire the conformational stability at position 33. PMID:18776207

Goto, Takashi; Ohkuri, Takatoshi; Shioi, Seijiro; Abe, Yoshito; Imoto, Taiji; Ueda, Tadashi



Bacteriophage control of foodborne bacteriat.  


Bacteriophages are measurable components of the natural microflora in the food production continuum from the farm to the retail outlet. Phages are remarkably stable in these environments and are readily recovered from soil, sewage, water, farm and processing plant effluents, feces, and retail foods. Purified high-titer phage lysates have been used for the species-specific control of bacteria during the pre- and postharvest phases of food production and storage. For example, the inhibition of the phytopathogens Erwinia amylovara and Xanthomonas campestris has reduced the incidence of diseases such as fire blight in apples and bacterial spot of tomato and peaches. Research on preslaughter treatment of food animals has demonstrated phage control of salmonellosis in chickens, enteropathogenic Escherichia coli infections in calves, piglets, and lambs, and E. coli O157:H7 shedding by beef cattle. Phages have also been applied to control the growth of pathogens such as Listeria monocytogenes, Salmonella, and Campylobacter jejuni in a variety of refrigerated foods such as fruit, dairy products, poultry, and red meats. Phage control of spoilage bacteria (e.g., Pseudomonas spp. and Brochothrix thermosphacta) in raw chilled meats can result in a significant extension of storage life. Phage biocontrol strategies for food preservation have the advantages of being self-perpetuating, highly discriminatory, natural, and cost-effective. Some of the drawbacks of biopreservation with phages are a limited host range, the requirement for threshold numbers of the bacterial targets, phage-resistant mutants, and the potential for the transduction of undesirable characteristics from one bacterial strain to another. Most research to date has involved experimentally infected plants and animals or artificially inoculated foods. This technology must be transferred to the field and to commercial environments to assess the possibility of controlling natural contaminants under more realistic production and processing conditions. PMID:15895751

Greer, G Gordon



Complete amino acid sequence of three reptile lysozymes.  


To study the structure and function of reptile lysozymes, we have reported their purification, and in this study we have established the amino acid sequence of three egg white lysozymes in soft-shelled turtle eggs (SSTL A and SSTL B from Trionyx sinensis, ASTL from Amyda cartilaginea) by using the rapid peptide mapping method. The established amino acid sequence of SSTL A, SSTL B, and ASTL showed substitutions of 43, 42, and 44 residues respectively when compared with the HEWL (hen egg white lysozyme) sequence. In these reptile lysozymes, SSTL A had one substitution compared with SSTL B (Gly126Asp) and had an N-terminal extra Gly and 11 substitutions compared with ASTL. SSTL B had an N-terminal extra Gly and 10 residues different from ASTL. The sequence of SSTL B was identical to soft-shelled turtle lysozyme from STL (Trionyx sinensis japonicus). The Ile residue at position 93 of ASTL is the first report in all C-type lysozymes. Furthermore, amino acid substitutions (Phe34His, Arg45Tyr, Thr47Arg, and Arg114Tyr) were also found at subsites E and F when compared with HEWL. The time course using N-acetylglucosamine pentamer as a substrate exhibited a reduction of the rate constant of glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution for amino acids mentioned above for substrate binding at subsites E and F. Interestingly, the variable binding free energy values occurred on ASTL, may be contributed from substitutions at outside of subsites E and F. PMID:19747569

Ponkham, Pornpimol; Daduang, Sakda; Kitimasak, Wachira; Krittanai, Chartchai; Chokchaichamnankit, Daranee; Srisomsap, Chantragan; Svasti, Jisnuson; Kawamura, Shunsuke; Araki, Tomohiro; Thammasirirak, Sompong



Spectrophotometric studies on the interaction between (-)-epigallocatechin gallate and lysozyme  

NASA Astrophysics Data System (ADS)

Various reported antibacterial activities of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea prompted us to study its binding with lysozyme. This has been investigated by fluorescence, circular dichroism (CD) and protein-ligand docking. The binding parameters were determined using a modified Stern-Volmer equation. The thermodynamic parameters are indicative of an initial hydrophobic association. The complex is, however, held together predominantly by van der Waals interactions and hydrogen bonding. CD studies do not indicate any significant changes in the secondary structure of lysozyme. Docking studies revealed that specific interactions are observed with residues Trp 62 and Trp 63.

Ghosh, Kalyan Sundar; Sahoo, Bijaya Ketan; Dasgupta, Swagata



Phase transition in tetragonal hen egg-white lysozyme crystals  

NASA Astrophysics Data System (ADS)

Lattice dynamics of tetragonal hen egg-white lysozyme crystals was studied by Brillouin light scattering and differential ac-nanocalorimetry with controlled relative humidity from 298 to 330 K. Temperature dependences of fast quasitransverse phonon velocity and integral intensity were found to exhibit anomalies in the vicinity of 306 K. An anomaly in the temperature behavior of heat capacity was also observed near this temperature. It is concluded that the anomalies point to a structural phase transition. Possible mechanisms of phase transition in lysozyme crystals are discussed.

Svanidze, A. V.; Huth, H.; Lushnikov, S. G.; Kojima, Seiji; Schick, C.



Enteric Bacteriophages in Saint Louis Bay, Mississippi.  

National Technical Information Service (NTIS)

Samples from four stations in Saint Louis Bay, Mississippi were examined monthly for the presence of enteric bacteriophages over the period from July 1971 to March 1972. The samples were of surface and bottom water, bottom sediment, and surface and gut of...

J. O. Graves



Immobilization of bacteriophages on modified silica particles  

Microsoft Academic Search

Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties,

Rebecca Cademartiri; Hany Anany; Isabelle Gross; Rahul Bhayani; Mansel Griffiths; Michael A. Brook



Long-Circulating Bacteriophage as Antibacterial Agents  

Microsoft Academic Search

The increased prevalence of multidrugresistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the

Carl R. Merril; Biswajit Biswas; Richard Carlton; Nicole C. Jensen; G. Joseph Creed; Steve Zullo; Sankar Adhya



76 FR 16285 - Food Additives Permitted for Direct Addition to Food for Human Consumption; Bacteriophage...  

Federal Register 2010, 2011, 2012, 2013

...Human Consumption; Bacteriophage Preparation AGENCY...for the use of a bacteriophage preparation as an...antimicrobial agent against Listeria monocytogenes on...approving the use of a Listeria-specific bacteriophage preparation...



Effect of secondary structure on the interactions of peptide T4 LYS (11-36) in mixtures of aqueous sodium chloride and 2,2,2,-Trifluoroethanol  

SciTech Connect

The potential of mean force for protein-protein interactions is key to the development of a statistical-mechanical model for salt-induced protein precipitation and crystallization, and for understanding certain disease states, including cataract formation and {beta}-amyloid pathology in Alzheimer's disease. Fluorescence anisotropy provides a method for quantitative characterization of intermolecular interactions due to reversible association. Monomer-dimer equilibria for the peptide T4 LYS(11-36) were studied by fluorescence anisotropy. This peptide, derived from the {beta}-sheet region of the T4 lysozyme molecule, has the potential to form amyloid fibrils. 2,2,2-trifluoroethanol (TFE) induces a change in peptide secondary structure, and was used in aqueous solutions at concentrations from 0 to 50% (v/v) at 25 and 37 C to examine the role of peptide conformation on peptide-peptide interactions. The association constant for dimerization increased with rising TFE concentration and with falling temperature. The peptide-peptide potential of mean force was computed from these association constants. Circular-dichroism measurements showed that the secondary structure of the peptide plays an important role in these strong attractive interactions due to intermolecular hydrogen-bond formation and hydrophobic interactions.

Anderson, Camille O.; Spiegelberg, Susanne; Prausnitz, John M.; Blanch, Harvey W.



40 CFR 721.1880 - Borate(1-), tris(acetato-.kappa.O)hydro-, sodium, (T-4)-.  

Code of Federal Regulations, 2013 CFR

...acetato-.kappa.O)hydro-, sodium, (T-4)-. 721.1880 Section 721.1880...acetato-.kappa.O)hydro-, sodium, (T-4)-. (a) Chemical substance and...acetato-.kappa.O)hydro-, sodium, (T-4)- (PMN P-00-0922; CAS...



Sequences of two highly divergent canine type c lysozymes: implications for the evolutionary origins of the lysozyme/alpha-lactalbumin superfamily.  


The amino acid sequences of two canine lysozymes, from milk and spleen, have been elucidated by direct sequence analyses of the purified proteins and fragments generated from them. The two enzymes are highly divergent, differing from each other by 45% in sequence, but each is closely similar to lysozymes previously obtained from other mammalian species. The milk lysozyme is similar in sequence to equine and donkey milk lysozymes (83% identity) and, like these enzymes, contains a bound Ca2+ ion while the spleen enzyme is most similar in sequence to the majority of previously studied mammalian and avian lysozymes (80 to 83% identity) and, based on its sequence, does not contain a Ca(2+)-binding site. This demonstrates that Ca(2+)-binding lysozymes are expressed in at least two mammalian orders, the carnivores and perissodactyls, as well as confirming that the genes for the Ca(2+)-binding and conventional lysozymes are paralogous. The latter point was further confirmed by the isolation and partial sequence analysis of a conventional lysozyme from equine spleen. The relationships of these new lysozyme sequences to those of other lysozymes and their homologues, the alpha-lactalbumins, were analyzed using different molecular phylogeny algorithms, producing a new model for the evolutionary origins of the superfamily. The most significant conclusion to be drawn from this model is that Ca(2+)-binding activity was an ancient feature of this protein superfamily which was lost during the evolutionary development of the conventional lysozymes. It also supports a previous suggestion that the alpha-lactalbumins and lysozymes diverged at a time earlier than the divergence of the fishes and tetrapods. PMID:8080284

Grobler, J A; Rao, K R; Pervaiz, S; Brew, K



[Friedrich Mauz: T4 assessor and military psychiatrist].  


Friedrich Mauz is one of the medical perpetrators of the second tier whose biography is difficult to comprehend. Autobiographies from three different political systems exist - Weimar Republic, the Third Reich, and postwar Germany in which he constantly reinvented himself. While after 1933 he suddenly emphasized his participation in the civil war turmoil during the early period of the Weimar Republic and his patriotism, he then depicted himself after 1945 as an apolitical person characterized by Württemberg pietism who inwardly rejected the Nazi State but had found himself prepared to accept "all sorts of humiliating concessions." He claimed that he had always remained true to his scientific code of conduct and had distanced himself from psychiatric genetics. In point of fact, Mauz was among those exonerated in the denazification trial in 1946 and was able to pursue his career in the Federal Republic of Germany. However, if the sources are read against the grain, a different picture emerges. Mauz's career stalled in the 1930s, not because he had been politically offensive, but because his scientific work was flimsy and considered lacking originality, particularly since he had chosen constitution research and psychotherapy as his main fields of interest, which were overshadowed by research in genetic psychiatry in the 1930s. Mauz tendered his services to the Nazi policy of genetic health, served as a medical assessor in proceedings based on the "Law for the Prevention of Genetically Diseased Offspring," permitted himself to be recruited for the T4 program as a medical expert, even participated in the deliberations on a future "Law on Euthanasia," and as a consulting psychiatrist for the German Armed Forces contributed to military medicine. PMID:22399061

Silberzahn-Jandt, G; Schmuhl, H-W



Functional Characterization of a c-type Lysozyme from Indian Shrimp Fenneropenaeus indicus.  


Lysozyme gene from Fenneropenaeus indicus was cloned, expressed in Escherichia coli and characterized. The cDNA consists of 477 base pairs and encodes amino acid sequence of 159 residues. F. indicus lysozyme had high identity (98 %) with Fenneropenaeus merguiensis and Fenneropenaeus chinensis and exhibits low to moderate identities with lysozymes of other invertebrates and vertebrates. This lysozyme is presumed to be chicken types as it possesses two catalytic and eight cysteine residues that are conserved across c-type lysozymes and a c-terminal extension, which is a characteristic of lysozymes from marine invertebrates. Further, the antimicrobial properties of the recombinant lysozyme from F. indicus were determined in comparison with recombinant hen egg white lysozyme. This exhibited high activity against a Gram-negative pathogenic bacterium Salmonella typhimurium and two fungal strains Pichia pastoris and Saccharomyces cerevisiae in turbidimetric assay. Distribution of lysozyme gene and protein in tissues of shrimps infected with white spot syndrome virus revealed that the high levels of lysozyme are correlated with low and high viral load in abdominal muscle and tail, respectively. In conclusion, lysozyme from F. indicus has a broad spectrum of antimicrobial properties, which once again emphasizes its role in shrimp innate immune response. PMID:24676722

Karthik, Viswanathan; Kamalakannan, Vijayan; Thomas, Ancy; Sudheer, Naduvilamuriparambu Saidumuhammed; Singh, Issac S Bright; Narayanan, Rangarajan Badri



Electrostatic self-assembly between biological polymers & macroions: Interactions of F-actin & DNA with lysozyme  

NASA Astrophysics Data System (ADS)

The pathological self-assembly of polyelectrolytes such as DNA and F-actin with cationic antimicrobial proteins such as lysozyme may have significant clinical consequences in Cystic Fibrosis (CF) lung infections. Wild-type lysozyme is a compact, cationic, globular protein which carries a net charge of +9e at neutral pH. Our Small Angle X-ray Scattering (SAXS) experiments on F-actin-lysozyme complexes indicate that the wild-type lysozyme close packs into 1-D columns between hexagonally organized F-actin filaments. We will present SAXS results of the interactions of F-actin and DNA with genetically engineered lysozyme mutants that carry a reduced charge of +5e. We have also used fluorescence microscopy to investigate the morphologies and sizes of such bundles induced with divalent cations, wild-type lysozyme, and mutant lysozymes.

Sanders, Lori K.; Matthews, Brian W.; Wong, Gerard C. L.



Kinetic Roughening Transition and Energetics of Tetragonal Lysozyme Crystal Growth  

NASA Technical Reports Server (NTRS)

Interpretation of lysozyme crystal growth rates using well-established physical theories enabled the discovery of a phenomenon possibly indicative of kinetic roughening. For example, lysozyme crystals grown above a critical supersaturation sigma, (where supersaturation sigma = ln c/c(sub eq), c = the protein concentration and c(sub eq) = the solubility concentration) exhibit microscopically rough surfaces due to the continuous addition of growth units anywhere on the surface of a crystal. The rate of crystal growth, V(sub c), for the continuous growth process is determined by the continuous flux of macromolecules onto a unit area of the crystal surface, a, from a distance, xi, per unit time due to diffusion, and a probability of attachment onto the crystal surface, expressed. Based upon models applied, the energetics of lysozyme crystal growth was determined. The magnitudes of the energy barriers of crystal growth for both the (110) and (101) faces of tetragonal lysozyme crystals are compared. Finally, evidence supportive of the kinetic roughening hypothesis is presented.

Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, Marc L.



Chronic neutrophilic leukaemia with enlarged lymph nodes and lysozyme deficiency  

PubMed Central

A further case of chronic neutrophilic leukaemia is reported and compared to fourteen previously reported cases. The presence of enlarged lymph nodes as the first clinical sign and the existence of a relative lysozyme deficiency of the granulocytes were striking features. Images

Feremans, Walter; Marcelis, LUC; Ardichvili, Daniel



Locations of Bromide Ions in Tetragonal Lysozyme Crystals  

NASA Technical Reports Server (NTRS)

Anions have been shown to play a dominant role in the crystallization of chicken egg-white lysozyme from salt solutions. Previous studies employing X-ray crystallography have found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystals grown in bromide and chloride solutions. Five possible anion-binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four further sites were found which corresponded to the four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion-binding sites in lysozyme remain unchanged even when different anions and different crystal forms of lysozyme are employed.

Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.



Radiation-Produced Aggregation and Inactivation in Egg White Lysozyme.  

National Technical Information Service (NTIS)

Chromatographically homogeneous egg white lysozyme has been subjected under reduced pressure to 0.67-m.e.v. gamma-rays. At 37% destruction of enzymic activity, three inactive aggregates and one partially active fraction have been isolated by salt precipit...

C. O. Stevens H. E. Sauberlich G. R. Bergstrom



The Multiple Nature of Crystalline Egg-White Lysozyme.  

National Technical Information Service (NTIS)

Seven components were demonstrated chromatographically in crystalline preparations of egg-white lysozyme. This was effected with a chromatographic procedure employing BioRex 70 columns eluted with 0.2 M potassium phosphate buffer, pH 7.09. Six components ...

C. O. Stevens G. R. Bergstrom



Science Study Aids 6: Lysozyme - The Cooperative Enzyme.  

ERIC Educational Resources Information Center

This publication is the sixth of a series of seven supplementary investigative materials for use in secondary science classes providing up-to-date research-related investigations. This unit is structured for grade levels 10 through 12. It is concerned with the crystallization of an enzyme, lysozyme, from egg white. The first part of this guide…

Boeschen, John; Alderton, Gordon


Muramidase (lysozyme) in Crohn's disease and in ulcerative colitis  

Microsoft Academic Search

Estimation of lysozyme (LZM) activity in the serum was suggested as a valuable test to distinguish between Crohn's disease and ulcerative colitis. Subsequently several reports either supported or denied the original observation. Selection of patients and methodological differences were suggested as an explanation for the controversy. We estimated serum LZM in a large group of patients using the lysoplate method

W. Pruzanski; N. Marcon; C. Ottaway; E. Prokipchuk



Quantitative tear lysozyme assay: a new technique for transporting specimens.  

PubMed Central

We have developed a method for assaying the concentration of tear lysozyme using eluates of tear fluid collected on filter paper discs. Specimens can be stored and transported to remote laboratories for assay. We have shown that the 'indirect' or eluate method gives statistically comparable results to the 'direct' method using fresh, neat tear fluid. Images

Seal, D V; Mackie, I A; Coakes, R L; Farooqi, B



Does Warming a Lysozyme Solution Cook Ones Data?  

NASA Technical Reports Server (NTRS)

Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.0 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubility are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to the initiation of the crystallization process. We have now measured the kinetics of this process and investigated its reversibility. An aliquot of a stock protein solution is held at a given temperature, and at periodic intervals used to set up batch crystallization experiments. The batch solutions were incubated at 20 C until macroscopic crystals were obtained, at which point the number of crystals in each well were counted. The transition effects increased with temperature, slowly falling off at 30 C with a half time (time to approx. 1/2 the t = 0 number of crystals) of approx. 5 hours, and an estimated half time of approx. 0.5 hours at 43 C. Further, the process was not reversible by simple cooling. After holding a lysozyme solution at 37 C (prior to addition of precipitant) for 16 hours, then cooling and holding it at 4 C, no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Thus every thermal excursion above the phase transition point results in a further decrease in the nucleation rate of that solution, the extent being a function of the time and temperature. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. We have previously shown that putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. We may be able to use this differential behavior to elucidate how flow affects tile lysozyme crystal growth process.

Pusey, Marc; Burke, Michael; Judge, Russell



Two Modes of In Vivo Transcription for Genes 43 and 45 of Phage T4  

PubMed Central

Sensitivities of the expression of early genes of phage T4 to UV light were determined at various stages of intracellular development of T4 wild type, a DNA-negative mutant (T4 DO), and T4 tsG1, (a mutant that exhibits delayed expression of some T4 early genes). Whereas the sensitivities of some genes in the T4 wild type and T4 DO remain constant, genes 43 and 45 exhibit greatly reduced sensitivities several minutes after the onset of phage development. Since UV sensitivities are a measure of the distance of a gene from its promotor, these observations indicate a switch from distal, “immediate early” promotors to proximal, “delayed early” promotors for genes 43 and 45. In the tsG1 mutant this decrease in UV sensitivities of genes 43 and 45 does not occur at 42 C, suggesting that at high temperature this mutant does not utilize the delayed early promotors. Images

Hercules, Kathleen; Sauerbier, Walter



Functional relationship between bacteriophages G4 and phi X174.  

PubMed Central

Mutants of bacteriophage G4 were isolated and characterized, and their mutations were mapped. They constitute six different genes, namely, A, B, E, F, G, and H. The functional relationship with bacteriophage phi X174 was determined by complementation experiments using amber mutants of phi X and amber mutants of G4. Bacteriophage phi X was able to use the products of G4 genes E, F, G, and H. In bacteriophage G4, however, only the phi X gene H product was functional. Images

Borrias, W E; Hagenaar, M; Van Den Brekel, R; Kuhlemeijer, C; Weisbeek, P J



The prognosis of tumors with only microscopic skin involvement without clinical T4b signs is significantly better than T4b tumors in breast carcinoma.  


The number of studies forming a base for tumor (T)-node (N)-metastasis (M) classification by comparing T4b tumors with only histological skin involvement in breast carcinoma is limited and results are contradictory. In this study, the survival of patients with T4b tumor and patients whose tumor had only microscopic skin involvement without clinical T4b signs were compared. The file records of 101 patients with T4b tumor (group A) and 79 patients whose tumor had only microscopic skin involvement (group B) were reviewed. The endpoint was disease recurrence. For the whole series, disease-free survival (DFS) of group B patients was significantly better compared with group A patients treated with either adjuvant (p<0.001) or neoadjuvant (p<0.001) therapies. When patients were subgrouped according to tumor size, DFS of group B patients was significantly better than group A patients receiving either adjuvant or neoadjuvant therapy for all tumor size subgroups of ?3, >3, ?5, and >5cm. Presence of T4b clinical signs had independent prognostic value in multivariate Cox analysis. In conclusion, tumors with only histological skin involvement without clinical T4b signs should be classified as T1-T3 according to their size instead of T4 as stated in the TNM classification. PMID:21129094

Duraker, Nüvit; Bat?, Bak?r; Caynak, Zeynep Civelek; Demir, Davut; Kurtulu?, Idris



Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7*  

PubMed Central

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

Dauden, Maria I.; Martin-Benito, Jaime; Sanchez-Ferrero, Juan C.; Pulido-Cid, Mar; Valpuesta, Jose M.; Carrascosa, Jose L.



Large terminase conformational change induced by connector binding in bacteriophage T7.  


During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7. PMID:23632014

Daudén, María I; Martín-Benito, Jaime; Sánchez-Ferrero, Juan C; Pulido-Cid, Mar; Valpuesta, José M; Carrascosa, José L



Targeted filamentous bacteriophages as therapeutic agents.  


Bacteriophages (phages) have been used for therapy of bacterial infections, for genetic research, as tools for the discovery of specific target binding proteins and for vaccine development. The aim of this article is to present advances in genetic and chemical engineering of filamentous bacteriophages that facilitated their application for therapeutic purposes. We review studies where phages were applied for in vivo imaging, as gene delivery vehicles and as drug carriers. Target specificity is based on peptides or proteins displayed on the phage coat. The cargo may be a packaged gene incorporated into the phage genome for gene delivery applications, or imaging agents or cytotoxic drugs chemically conjugated at high density onto the phage coat. We believe that the combination of those separately developed methodologies would result in clinical applications of phage-based therapeutics. PMID:18318653

Yacoby, Iftach; Benhar, Itai



The structural peptidoglycan hydrolase gp181 of bacteriophage phiKZ.  


Gp181 (2237 amino acids) of Pseudomonas aeruginosa bacteriophage phiKZ (Myoviridae) is a structural virion protein, which bears a peptidoglycan hydrolase domain near its C-terminus. This protein is supposed to degrade the peptidoglycan locally during the infection process. Nine deletional mutants allowed delineation of the peptidoglycan hydrolase domain between amino acids 1880-2042 (gp181M8) and analysis of its biochemical properties. Gp181M8 tolerates a high ionic strength (>320mM) and is less sensitive to long thermal treatments compared to the similar phiKZ endolysin. Gp181M8 lysed all tested outer membrane-permeabilized Gram-negative species. The C-terminal distal end (amino acids 2043-2237) enhances the specific activity of gp181M8 threefold, resulting in a twelve times higher activity than commercial hen egg white lysozyme. These biochemical properties suggest that this novel peptidoglycan hydrolase domain may be suitable for enzybiotic applications. PMID:18671939

Briers, Yves; Miroshnikov, Konstantin; Chertkov, Oleg; Nekrasov, Alexei; Mesyanzhinov, Vadim; Volckaert, Guido; Lavigne, Rob



??????????????????? ?????????????????????????????????????????????? Isolation of Bacteriophages Specific to Enteric Bacteria  

Microsoft Academic Search

Bacteria in family Enterobacteriaceae are pathogenic to human and animals. They often contaminate food and water. In this study four bacteriophages specific to Shigella dysenteriae DMS2137, Salmonella Typhi DMS5784, Escherichia coli ATCC25922 and Enterobacter aerogenes were isolated from sewage water in Ubon Ratchathani, Thailand, using double-layer plaque technique. All of them showed specificity only to their hosts. These results indicated

Kanjana Nukdee


Drugs against superbugs: private lessons from bacteriophages.  


Bacterial genomics has provided a plethora of potential targets for antibacterial drug discovery, however, success in the hunt for new antibiotics will hinge on selecting targets with the highest potential. A recent paper by Liu and coworkers describes a new approach to target selection that uncovers strategies used by bacteriophage to disable bacteria. The method uses key phage proteins to identify and validate vulnerable targets and exploits them further in the identification of new antibacterial leads. PMID:15331220

Brown, Eric D



[T4 tracheo-bronchial carcinoma: clinical evaluation of 48 sleeve pneumonectomies].  


The aim of the study was to verify the indications for surgery in T4 tracheo-bronchial carcinomas. Forty-eight tracheal-sleeve pneumonectomies for T4 bronchogenic carcinoma were performed in our unit from 1986 to 2003. The patients were 42 males and 6 females. A postero-lateral thoracotomy was preferred (46 right, 2 left). Bronchial reimplantation was performed additionally (tracheal-sleeve lobectomy) in 2 patients on the right side. The morbidity was 25% and the mortality 6.2% (1 acute respiratory distress syndrome, 1 myocardial infarction, 1 anastomotic fistula). Twenty-three cases were sT4N2M0, 14 sT4N1M0, and 11 sT4N0M0. The sT4N2M0 and sT4N1M0 cases were not associated with more than 3 year survival, despite adjuvant therapies; sT4N0M0 squamous cell carcinomas, on the other hand, had > 40% 10-year survival with no adjuvant therapy. Associated prosthetic replacement of the superior vena cava neither affected the risk nor improved the prognosis. Surgery for T4 tracheo-bronchial carcinoma appears feasible for well differentiated sT4N0 squamous cell carcinomas; at more advanced stages this procedure is no more than a dangerous form of palliation. PMID:15771031

Politi, Leonardo; Scanagatta, Paolo; Salani, Anna; Andreani, Matteo; Montinaro, Flavio; Vassallo, Miguel; Crisci, Clemente



Enzyme characterisation and gene expression profiling of Atlantic salmon chicken- and goose-type lysozymes.  


Lysozymes represent important innate immune components against bacteria. In this study, Atlantic salmon (Salmo salar) goose (g-) and chicken (c-) types of lysozyme were subjected to protein characterisations and tissue expression analyses. Specific bacterial protein inhibitors of g- and c-type lysozymes were employed to discriminate between respective enzyme activities. Blood, gills and liver contained activities exclusive for the g-type lysozyme. Only haematopoietic organs (head kidney and spleen) contained enzyme activities of both g- and c-lysozyme enzymes and c-type activity was not found outside these organs. Gene transcript levels proportional to enzyme activity levels were detected for the g-type lysozyme but not for the c-type. In vitro studies revealed significant induction of c-type gene expression and enzyme activity in macrophages after incubation with lipopolysaccharide (LPS) while expression of the g-type lysozyme gene was unaffected. The activity of purified native c-type enzyme was profoundly reduced by divalent cations and displayed low tolerance to monovalent cations, while the native g-type lysozyme was stimulated by monovalent cations and tolerated low concentrations of divalent cations. Activities of both enzymes increased with temperature elevations up to 60°C. The native g-type lysozyme responses to temperature in particular are in apparent conflict to the ones for the recombinant salmon g-lysozyme. Our results imply separate expression regulations and different functions of c- and g-type lysozymes in salmon. LPS-induced expression of c-type lysozyme and broad constitutive tissue distribution of g-type lysozyme in salmon is different from findings in other studied fish species. PMID:23396098

Myrnes, Bjørnar; Seppola, Marit; Johansen, Audny; Overbø, Kersti; Callewaert, Lien; Vanderkelen, Lise; Michiels, Chris W; Nilsen, Inge W



Interaction of T4 UvsW helicase and single-stranded DNA binding protein gp32 through its carboxy-terminal acidic tail.  


Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA (ssDNA) binding protein gp32 is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW-catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy-terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions. PMID:23732982

Perumal, Senthil K; Nelson, Scott W; Benkovic, Stephen J



Bacteriophage P70: Unique Morphology and Unrelatedness to Other Listeria Bacteriophages  

PubMed Central

Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages.

Schmuki, Martina M.; Erne, Doris; Loessner, Martin J.



Bacteriophage P70: unique morphology and unrelatedness to other Listeria bacteriophages.  


Listeria monocytogenes is an important food-borne pathogen, and its bacteriophages find many uses in detection and biocontrol of its host. The novel broad-host-range virulent phage P70 has a unique morphology with an elongated capsid. Its genome sequence was determined by a hybrid sequencing strategy employing Sanger and PacBio techniques. The P70 genome contains 67,170 bp and 119 open reading frames (ORFs). Our analyses suggest that P70 represents an archetype of virus unrelated to other known Listeria bacteriophages. PMID:22993158

Schmuki, Martina M; Erne, Doris; Loessner, Martin J; Klumpp, Jochen



Bacteriophages and the control of Erwinia carotovora subsp. carotovora  

Microsoft Academic Search

Fourteen bacteriophages of Erwinia carotovora subsp. carotovora, the causal agent of bacterial soft rot, were isolated from samples of fertilizer solutions taken from a greenhouse producing calla lily (Zantedeschia spp.). To avoid a single-host selection bias, a mixture of four bacterial hosts was used to enrich bacteriophages. Molecular characterization of the phages with restriction endonuclease digestions indicated that two distinct

M. Ravensdale; T. J. Blom; J. A. Gracia-Garza; A. M. Svircev; R. J. Smith



21 CFR 172.785 - Listeria-specific bacteriophage preparation.  

Code of Federal Regulations, 2013 CFR

...Additives § 172.785 Listeria -specific bacteriophage...antimicrobial agent specific for Listeria monocytogenes (L...activity) bacteriophages (phages) specific against L...genomic sequence of the phages. (4) L. monocytogenes...entitled âLMP-102TM Listeria monocytogenes...



21 CFR 172.785 - Listeria-specific bacteriophage preparation.  

Code of Federal Regulations, 2010 CFR

...Additives § 172.785 Listeria -specific bacteriophage...antimicrobial agent specific for Listeria monocytogenes (L...activity) bacteriophages (phages) specific against L...genomic sequence of the phages. (4) L. monocytogenes...entitled âLMP-102TM Listeria monocytogenes...



21 CFR 172.785 - Listeria-specific bacteriophage preparation.  

Code of Federal Regulations, 2010 CFR

...Additives § 172.785 Listeria -specific bacteriophage...antimicrobial agent specific for Listeria monocytogenes (L...activity) bacteriophages (phages) specific against L...genomic sequence of the phages. (4) L. monocytogenes...entitled âLMP-102TM Listeria monocytogenes...



Genetically engineered acidophilic heterotrophic bacteria by bacteriophage transduction  

Microsoft Academic Search

A bacteriophage capable of infecting acidophilic heterotrophic bacteria and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the

T. E. Ward; D. F. Bruhn; D. F. Bulmer



DNA origami structures directly assembled from intact bacteriophages.  


Keeping it simple: a purified DNA single strand derived from bacteriophage M13 is typically used as scaffold material for DNA origami. This requires time-consuming purification procedures to attain single-stranded DNA. The direct use of intact bacteriophage particles as a scaffold source for DNA origami assemblies is reported. PMID:24532395

Nickels, Philipp C; Ke, Yonggang; Jungmann, Ralf; Smith, David M; Leichsenring, Marc; Shih, William M; Liedl, Tim; Högberg, Björn



Do we need still more trials on T-4 and T-3 combination therapy in hypothyroidism?  

Microsoft Academic Search

Approximately 10% of hypothyroid patients are dissatisfied with the outcome of levothyroxine replacement. It is unlikely that slight over- or under-treatment with thyroxine (T-4) explains remaining complaints Meta-analysis of randomized clinical trials shows no advantage of T-4\\/tri-iodothyronine (T-3) combination therapy over T-4 monotherapy However, each of these trials can be criticized, and none is perfect: most of them failed to

Wilmar M Wiersinga



Locations of Halide Ions in Tetragonal Lysozyme Crystals  

NASA Technical Reports Server (NTRS)

Anions play an important role in the crystallization of lysozyme, and are known to bind to the crystalline protein. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. Studies using other approaches have reported more chloride ion binding sites, but their locations were not known. Knowing the precise location of these anions is also useful in determining the correct electrostatic fields surrounding the protein. In the first part of this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from the lysozyme crystals grown in bromide and chloride solutions under identical conditions. The anion locations were then obtained from standard crystallographic methods and five possible anion binding sites were found in this manner. The sole chloride ion binding site found in previous studies was confirmed. The remaining four sites were new ones for tetragonal lysozyme crystals. However, three of these new sites and the previously found one corresponded to the four unique binding sites found for nitrate ions in monoclinic crystals. This suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed. It is unlikely that there are many more anions in the tetragonal lysozyme crystal structure. Assuming osmotic equilibrium it can be shown that there are at most three more anions in the crystal channels. Some of the new anion binding sites found in this study were, as expected, in pockets containing basic residues. However, some of them were near neutral, but polar, residues. Thus, the study also showed the importance of uncharged, but polar groups, on the protein surface in determining its electrostatic field. This was important for the second part of this study where the electrostatic field surrounding the protein was accurately determined. This was achieved by solving the linearized version of the Poisson-Boltzmann equation for the protein in solution. The solution was computed employing the commercial code Delphi which uses a finite difference technique. This has recently become available as a module in the general protein visualization code Insight II. Partial charges were assigned to the polar groups of lysozyme for the calculations done here. The calculations showed the complexity of the electrostatic field surrounding the protein. Although most of the region near the protein surface had a positive field strength, the active site cleft was negatively charged and this was projected a considerable distance. This might explain the occurrence of "head-to-side" interactions in the formation of lysozyme aggregates in solution. Pockets of high positive field strength were also found in the vicinity of the anion locations obtained from the crystallographic part of this study, confirming the validity of these calculations. This study clearly shows not only the importance of determining the counterion locations in protein crystals and the electrostatic fields surrounding the protein, but also the advantage of performing them together.

Lim, Kap; Adimurthy, Ganapathi; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.



Mouse lysozyme M gene: isolation, characterization, and expression studies.  

PubMed Central

We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels. Images

Cross, M; Mangelsdorf, I; Wedel, A; Renkawitz, R



Actin - Lysozyme Interactions in Model Cystic Fibrosis Sputum  

NASA Astrophysics Data System (ADS)

Cystic fibrosis sputum is a complex fluid consisting of mucin (a glycoprotein), lysozyme (a cationic polypeptide), water, salt, as well as a high concentration of a number of anionic biological polyelectrolytes such as DNA and F-actin. The interactions governing these components are poorly understood, but may have important clinical consequences. For example, the formation of these biological polyelectrolytes into ordered gel phases may contribute significantly to the observed high viscosity of CF sputum. In this work, a number of model systems containing actin, lysozyme, and KCl were created to simulate CF sputum in vitro. These model systems were studied using small angle x-ray scattering and confocal fluorescence microscopy. Preliminary results will be presented. This work was supported by NSF DMR-0071761, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

Sanders, Lori; Slimmer, Scott; Angelini, Thomas; Wong, Gerard C. L.



Tunable supramolecular hydrogel for in situ encapsulation and sustained release of bioactive lysozyme.  


To develop new matrices for the entrapment and sustained release of bioactive lysozyme, a series of supramolecular hydrogels based on ?-cyclodextrin (?-CD) and water-soluble poly(?-caprolactone)-poly(ethylene glycol) block copolymer (PCL-b-PEG) were prepared in the presence of chicken egg lysozyme. Different from commonly used polymeric microspheres and chemically crosslinked hydrogels for lysozyme encapsulation, such hydrogel matrices could be formed under mild conditions without high temperature and the use of chemical emulsifiers or crosslinkers. Their gelation rate, mechanical strength and shear viscosity as well as the release behavior for the encapsulated lysozyme could be tuned easily by the change of ?-CD or PCL-b-PEG amount. For the encapsulated lysozyme, its conformation and biological activity could be well maintained when compared to native lysozyme. For the resultant supramolecular hydrogels, they were also confirmed to have a good biocompatibility by MTT assay using mice skin fibroblast (L929). PMID:21536304

Ma, Dong; Zhang, Li-Ming; Xie, Xi; Liu, Tao; Xie, Min-Qiang



Concurrent chemoradiotherapy for T4 patients with hypopharyngeal and laryngeal squamous cell carcinomas  

Microsoft Academic Search

ObjectivesConcurrent chemoradiotherapy (CCR) was given for the previously untreated T4 hypopharyngeal and laryngeal squamous cell carcinoma patients and the response and survival rates were evaluated.

Goshi Nishimura; Mamoru Tsukuda; Choichi Horiuchi; Kenichi Satake; Takafumi Yoshida; Junichi Nagao; Mariko Kawakami; Norio Kondo; Yasuhiro Arai; Takahide Taguchi; Hideki Matsuda; Yasukazu Mikami



Acoustic absorption and thixotropic structure in lysozyme solution  

Microsoft Academic Search

Summary  The acoustic-absorption coefficient is measured in a 1% w\\/w lysozyme solution, at frequencies ranging from 5 to 50 MHz, as\\u000a a function of time. It is shown that a relaxation process due to diffusional motion of clusters exists. Such a process is\\u000a gradually quenched, as the interacting clusters become fixed in a thixotropic solidlike structure. The obtained results are\\u000a discussed

R. Giordano; F. Mallamace; F. Wanderlingh



Solid-phase competitive luminescence immunoassay for lysozyme in faeces.  


We have developed a new simple solid-phase luminescence immunoassay (LIA) for the determination of faecal lysozyme. The assay utilises a polyclonal capture antibody coated to polystyrene beads and acridinium ester-labelled human lysozyme as tracer. Samples are incubated with polystyrene beads and tracer overnight at 4 degrees C. After a thorough washing step, emitted light is measured by an automated luminometer for 2 seconds. The standard curve uses five standards ranging from 0.025 to 6.4 mg/l. The method has a sensitivity of 0.02 mg/l. Dilution recoveries for three samples were 88, 104 and 108%. Intraassay coefficients of variation (CV, n = 24) were 10.1% and 11.7% for a healthy control and a patient sample; interassay CV (n = 16) were 6.7% and 13.1% for the same healthy control but another patient sample. The normal range of faecal lysozyme in 80 healthy controls was found to be 0.02-1 mg/l (97.5 percentile) with a median of 0.28 mg/l. Fifty-three patients with Crohn's disease had faecal lysozyme values ranging from 0.16 to 100.7 mg/l with a median of 1.75 mg/l, and 30 patients with ulcerative colitis showed levels between 0.09 and 118 mg/l with a median of 1.11 mg/l. The assay has proved useful for differentiating healthy individuals from those with inflammatory bowel disease and might be a valuable tool for diagnosing or evaluating inflammatory bowel disease. PMID:8542654

Gao, P; John, M R; Schmidt-Gayk, H; Arndt, B; Scheida, M; Theuer, D



Structural Basis of Protein Oxidation Resistance: A Lysozyme Study  

PubMed Central

Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD) simulations, we find the protein parts with increased root-mean-square deviation (RMSD) to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation.

Girod, Marion; Enjalbert, Quentin; Brunet, Claire; Antoine, Rodolphe; Lemoine, Jerome; Lukac, Iva; Radman, Miroslav; Krisko, Anita; Dugourd, Philippe



Magnetic molecularly imprinted nanoparticles for recognition of lysozyme.  


Molecular imprinting is an attractive technique for preparing mimics of natural, biological receptors. Nevertheless, the imprinting of macromolecule remains a challenge due to their bulkiness and sensitivity to denaturation. In this work, we presented a method for preparing multifunctional lysozyme-imprinted nanoparticles (magnetic susceptibility, molecular recognition and environmental response). The magnetic susceptibility was imparted through the successful encapsulation of Fe3O4 nanoparticles. Selective lysozyme recognition depended on molecularly imprinted film. Moreover, it was also a hydrophilic stimuli-responsive polymer, which could undergo a reversible change of imprinted cavity in response to a small change in the environmental conditions. Thus, magnetic molecularly imprinted nanoparticles had high adsorption capacity (0.11 mg mg(-1)), controlled selectivity and direct magnetic separation (22.1 emicro g(-1)) in crude samples. After preconcentration and purification with magnetic MIPs nanoparticles, a sensitive chemiluminescence method was developed for determination of lysozyme in human serum samples. The results indicated that the spiked recoveries were changed from 92.5 to 113.7%, and the RSD was lower than 11.8%. PMID:20829022

Jing, Tao; Du, Hairong; Dai, Qing; Xia, Huan; Niu, Jiwei; Hao, Qiaolin; Mei, Surong; Zhou, Yikai



Design and structural analysis of an engineered thermostable chicken lysozyme.  

PubMed Central

A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (delta Tm approximately +10.5 degrees C) and chemical (delta Cm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 degrees C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mol-1 greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with delta Tm = 3.3 degrees C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.

Shih, P.; Kirsch, J. F.



Modeling the electrophoresis of lysozyme. II. Inclusion of ion relaxation.  

PubMed Central

In this work, boundary element methods are used to model the electrophoretic mobility of lysozyme over the pH range 2-6. The model treats the protein as a rigid body of arbitrary shape and charge distribution derived from the crystal structure. Extending earlier studies, the present work treats the equilibrium electrostatic potential at the level of the full Poisson-Boltzmann (PB) equation and accounts for ion relaxation. This is achieved by solving simultaneously the Poisson, ion transport, and Navier-Stokes equations by an iterative boundary element procedure. Treating the equilibrium electrostatics at the level of the full rather than the linear PB equation, but leaving relaxation out, does improve agreement between experimental and simulated mobilities, including ion relaxation improves it even more. The effects of nonlinear electrostatics and ion relaxation are greatest at low pH, where the net charge on lysozyme is greatest. In the absence of relaxation, a linear dependence of mobility and average polyion surface potential, (lambda zero)s, is observed, and the mobility is well described by the equation [formula: see text] where epsilon 0 is the dielectric constant of the solvent, and eta is the solvent viscosity. This breaks down, however, when ion relaxation is included and the mobility is less than predicted by the above equation. Whether or not ion relaxation is included, the mobility is found to be fairly insensitive to the charge distribution within the lysozyme model or the internal dielectric constant.

Allison, S A; Potter, M; McCammon, J A



Influence of Hydration on the Dynamics of Lysozyme  

PubMed Central

Quasielastic neutron and light-scattering techniques along with molecular dynamics simulations were employed to study the influence of hydration on the internal dynamics of lysozyme. We identified three major relaxation processes that contribute to the observed dynamics in the picosecond to nanosecond time range: 1), fluctuations of methyl groups; 2), fast picosecond relaxation; and 3), a slow relaxation process. A low-temperature onset of anharmonicity at T ? 100 K is ascribed to methyl-group dynamics that is not sensitive to hydration level. The increase of hydration level seems to first increase the fast relaxation process and then activate the slow relaxation process at h ? 0.2. The quasielastic scattering intensity associated with the slow process increases sharply with an increase of hydration to above h ? 0.2. Activation of the slow process is responsible for the dynamical transition at T ? 200 K. The dependence of the slow process on hydration correlates with the hydration dependence of the enzymatic activity of lysozyme, whereas the dependence of the fast process seems to correlate with the hydration dependence of hydrogen exchange of lysozyme.

Roh, J. H.; Curtis, J. E.; Azzam, S.; Novikov, V. N.; Peral, I.; Chowdhuri, Z.; Gregory, R. B.; Sokolov, A. P.



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

Microsoft Academic Search

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

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



Analysis of Two Lysozyme Genes and Antimicrobial Functions of Their Recombinant Proteins in Asian Seabass  

PubMed Central

Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu50 and Asp67) and a “GSTDYGIFQINS” motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu71, Asp84, Asp95) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

Fu, Gui Hong; Bai, Zhi Yi; Xia, Jun Hong; Liu, Feng; Liu, Peng; Yue, Gen Hua



[Properties and clinical application of quasi-liquid form of lysozyme].  


The course of transendoscopic quasi-liquid form of lysozyme treatment of the relapse of peptic ulcer was microbiologically and clinically substantiated. A high antimicrobial and sorption activity of the quasi-liquid form of lysozyme with respect to the microflora isolated from the mucous membrane of the stomach and the duodenum was shown. The transendoscopic introduction of this form of lysozyme significantly reduces the time of the cicatrization of ulcers and the stay of patients in the hospital. PMID:11550551

Strelets, E V; Egorova, E N; Chernin, V V; Chervinets, V M; Bazlov, S N



An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white  

Microsoft Academic Search

This article describes an integrated process for simultaneous purification of lysozyme, ovalbumin, and ovomucoid from hen\\u000a egg white. The crude egg white extract was passed through a cation exchanger Streamline™ SP and the bound lysozyme was eluted with 5% ammonium carbonate, pH 9.0, containing 1 M NaCl after elution of avidin. This partially purified lysozyme was further purified 639-fold on

Ipsita Roy; M. V. S. Rao; Munishwar N. Gupta



Antibacterial activity of hen egg white lysozyme against Listeria monocytogenes Scott A in foods.  


Egg white lysozyme killed or prevented growth of Listeria monocytogenes Scott A in several foods. Lysozyme was more active in vegetables than in animal-derived foods that we tested. For maximum activity in certain foods, EDTA was required in addition to lysozyme. Lysozyme with EDTA effectively killed inoculated populations of 10(4) L. monocytogenes per g in fresh corn, fresh green beans, shredded cabbage, shredded lettuce, and carrots during storage at 5 degrees C. Control incubations without lysozyme supported growth of L. monocytogenes to 10(6) to 10(7)/g. Lysozyme had less activity in animal-derived foods, including fresh pork sausage (bratwurst) and Camembert cheese. In bratwurst, lysozyme with EDTA prevented L. monocytogenes from growing for 2 to 3 weeks but did not kill significant numbers of cells and did not prevent eventual growth. The control sausages not containing lysozyme supported rapid and heavy growth, which indicated that lysozyme was bacteriostatic for 2 to 3 weeks in fresh pork sausage. We also prepared Camembert cheese containing 10(4) L. monocytogenes cells per g and investigated the changes during ripening in cheeses supplemented with lysozyme and EDTA. Cheeses with lysozyme by itself or together with EDTA reduced the L. monocytogenes population by approximately 10-fold over the first 3 to 4 weeks of ripening. In the same period, the control cheese wheels without added lysozyme with and without chelator slowly started to grown and eventually reached 10(6) to 10(7) CFU/g after 55 days of ripening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2494938

Hughey, V L; Wilger, P A; Johnson, E A



Accelerated study on lysozyme deposition on poly(HEMA) contact lenses.  


A technique was developed to accelerate lysozyme deposition on poly(HEMA) contact lenses and measure the amounts of the deposited lysozyme. This technique was for evaluation of bendazac lysine solution, a contact lens cleaning and wetting solution. Effect of temperature on lysozyme deposition on poly(HEMA) contact lenses was examined. Five temperatures ranging from 25 degrees C to 90 degrees C were chosen to examine the temperature effect. The amounts of lysozyme deposited on poly(HEMA) contact lenses at 25 C and 60 C were 0.27 microg/lens and 0.61 microg/lens, respectively. The amount increased sharply to 23 microg/lens at 70 degrees C with the maximum of 31 microg/lens at 90 degrees C. Kinetics of lysozyme deposition on poly(HEMA) contact lenses was examined at 80 degrees C. Lysozyme deposition increased sharply during the first 2 h and reached a plateau after 2 h. Effectiveness of various cleaning procedures was examined using bendazac lysine solution. When the contact lenses were washed without rubbing with fingers, the bendazac lysine reduced the amount of deposited lysozyme by more than 40% from 18.3 microg/lens to 10.6 microg/lens. The effect of bendazac lysine was most prominent when the contact lenses were shaken during storage in the presence of lysozyme in solution. If the contact lenses were cleaned by rubbing with fingers, the effect of bendazac lysine solution on the prevention of lysozyme deposition was negligible. PMID:9870756

Kidane, A; Szabocsik, J M; Park, K



Conjugation and modeled structure/function analysis of lysozyme on glycine esterified cotton cellulose-fibers.  


The antimicrobial activity of lysozyme covalently bound to glycine-derivatized cotton cellulose was assessed in a 96-well format. Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction. The attachment to cotton fibers was made through both a single glycine and a glycine dipeptide esterified to cotton cellulose. Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples. The antibacterial activity of the lysozyme-conjugated cotton cellulose against Bacillus subtilis was assessed as a suspension of pulverized cotton fibers in microtiter wells. Inhibition of B. subtilis growth was observed to be optimal within a range of 0.14-0.3 mM (equivalent to 4-20 mg of lysozyme-bound cotton/mL) of lysozyme. Enhancement of activity over soluble lysozyme may result from the solid-phase protection afforded by the cellulose linkage of the glycoprotein against proteolytic lysis. Computational models of lysozyme based on its crystal structure attached through aspartate, glutamate, and COOH-terminal residues to cellopentaose-(3) Gly-O-6-glycyl-glycine ester were constructed. The models demonstrate no steric constraints to the active-site cleft from the glycine-conjugated cellulose chain when lysozyme is bound at the carboxylates of Asp-87, Glu-7, Asp-119, Asp-18, and COOH-terminal Leu-129. The more robust antibacterial activity of the enzyme when bonded to cotton fibers suggests good potential for biologically active enzymes on cotton-based fabrics. PMID:10898567

Edwards, J V; Sethumadhavan, K; Ullah, A H



Structure, Magnetism, and Thermodynamics of the Novel Rare Earth-Based Ln5T4 Intermetallics  

Microsoft Academic Search

After approximately 30 years of dormancy, the binary, ternary, and multicomponent intermetallic compounds of rare earth metals (R) with the group 14 elements (T) at the R 5 T 4 stoichiometry have become a goldmine for materials science, condensed matter physics, and solid-state chemistry. In addition to providing numerous opportunities to clarify elusive structure-property relationships, the R 5 T 4

V. K. Pecharsky; K. A. Jr. Gschneidner



[Normal values of T3-index and of thyroxine (T4) (author's transl)].  


The normal values of T3-index and of thyroxine (T4) have been evaluated comparing a binomial distribution to frequential curve of all the values obtained. The findings are: T3-index = 89 divided by 112; T4 = 3.7 divided by 15.3 microgram/100 cm3 of serum. PMID:461849

Di Pietrantonj, F; Fabrizzi, G; Proietti, A



Bacteriophage diversity in the North Sea.  


In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10(4) to 10(7) particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the gamma subdivision of the class Proteobacteria. PMID:9797256

Wichels, A; Biel, S S; Gelderblom, H R; Brinkhoff, T; Muyzer, G; Schütt, C



Bacteriophages as pathogens and immune modulators?  


While Shiga toxins (Stx) are key determinants of enterohemorrhagic Escherichia coli (EHEC) pathophysiology in humans, their dissemination to target organs following gastrointestinal EHEC infection is still poorly understood. Most types of Stx target cells with globotriaosylceramide (Gb3) receptors, which are expressed on endothelial cells. According to current theory, Stx is trafficked on the surface of peripheral blood cells, and transfer of toxin from these trafficking cells to endothelial cells results in microvascular damage to target organs, including the kidneys and brain. Inside the cell, Stx inhibits protein synthesis, resulting in cell death. Host "repair" responses can lead to microthrombus formation, erythrocyte damage, and reduced oxygen supply, potentially resulting in organ failure. A recent study [L. V. Bentancor et al., mBio 4(5):e00501-13, 2013, doi:10.1128/mBio.00501-13] indicates that another mechanism for Stx "dissemination" needs to be considered. Bentancor et al. demonstrated that high-pressure injection of a plasmid encoding the "prokaryotic" Stx2 sequence into mice can lead to mortality, with pathology indicative of Stx activity and antibody responses to Stx. While the plasmid levels and injection methodology were extreme, the study indicates that these sequences are potentially taken up into eukaryotic cells, transcribed, and translated, producing active Stx. Stx genes are present on integrated bacteriophage genomes in EHEC, and Stx-encoding phages are released following bacterial lysis in the gastrointestinal tract. We therefore need to consider whether bacteriophage sequences can be expressed in eukaryotic cells, what the wider implications are for our understanding of many "bacterial" diseases, and the possibility of developing novel interventions that target bacteriophages. PMID:24222490

Lengeling, A; Mahajan, A; Gally, D L



M13 Bacteriophage Based Protein Sensors  

NASA Astrophysics Data System (ADS)

Despite significant progress in biotechnology and biosensing, early detection and disease diagnosis remains a critical issue for improving patient survival rates and well-being. Many of the typical detection schemes currently used possess issues such as low sensitivity and accuracy and are also time consuming to run and expensive. In addition, multiplexed detection remains difficult to achieve. Therefore, developing advanced approaches for reliable, simple, quantitative analysis of multiple markers in solution that also are highly sensitive are still in demand. In recent years, much of the research has primarily focused on improving two key components of biosensors: the bio-recognition agent (bio-receptor) and the transducer. Particular bio-receptors that have been used include antibodies, aptamers, molecular imprinted polymers, and small affinity peptides. In terms of transducing agents, nanomaterials have been considered as attractive candidates due to their inherent nanoscale size, durability and unique chemical and physical properties. The key focus of this thesis is the design of a protein detection and identification system that is based on chemically engineered M13 bacteriophage coupled with nanomaterials. The first chapter provides an introduction of biosensors and M13 bacteriophage in general, where the advantages of each are provided. In chapter 2, an efficient and enzyme-free sensor is demonstrated from modified M13 bacteriophage to generate highly sensitive colorimetric signals from gold nanocrystals. In chapter 3, DNA conjugated M13 were used to enable facile and rapid detection of antigens in solution that also provides modalities for identification. Lastly, high DNA loadings per phage was achieved via hydrozone chemistry and these were applied in conjunction with Raman active DNA-gold/silver core/shell nanoparticles toward highly sensitive SERS sensing.

Lee, Ju Hun


Frequency of ultraviolet radiation-induced mutation at the hprt locus in repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4.  


The frequency of spontaneous and ultraviolet radiation (UVR)-induced mutation at the hprt locus was determined in control and denV-transfected, repair-proficient murine fibroblasts. Control cells removed an average of 25% of pyrimidine dimers induced by exposure to 150 J/m2 UVR from an FS40 sunlamp within 24 h; under the same conditions of induction and repair, denV-transfected cells removed an average of 71% of pyrimidine dimers. Control cells were somewhat more resistant than denV-transfected cells to killing by UVR. The average frequency of spontaneous mutation at the hprt locus for control and denV-transfected cells was 3 and 15 6-thioguanine (6-TG)-resistant colonies per 10(6) surviving cells, respectively; there was no statistically significant difference between control and denV-transfected cells. However, after exposure to 75 or 150 J/m2 UVR, denV-transfected cells had a significantly lower frequency of mutation to 6-TG resistance. After exposure to a fluence of 75 J/m2, the average frequency of UVR-induced mutation at the hprt locus was 166 mutant colonies per 10(6) surviving cells for control cells and 92 mutant colonies for denV-transfected cells; after 150 J/m2, control cells had 205 6-TG-resistant colonies per 10(6) cells, while denV-transfected cells had 61 mutant colonies. These results demonstrate that UVR-induced pyrimidine dimers are mutagenic photoproducts in mammalian cells. PMID:8234481

Kusewitt, D F; Budge, C L; Anderson, M M; Ryan, S L; Ley, R D



AtlasT4SS: A curated database for type IV secretion systems  

PubMed Central

Background The type IV secretion system (T4SS) can be classified as a large family of macromolecule transporter systems, divided into three recognized sub-families, according to the well-known functions. The major sub-family is the conjugation system, which allows transfer of genetic material, such as a nucleoprotein, via cell contact among bacteria. Also, the conjugation system can transfer genetic material from bacteria to eukaryotic cells; such is the case with the T-DNA transfer of Agrobacterium tumefaciens to host plant cells. The system of effector protein transport constitutes the second sub-family, and the third one corresponds to the DNA uptake/release system. Genome analyses have revealed numerous T4SS in Bacteria and Archaea. The purpose of this work was to organize, classify, and integrate the T4SS data into a single database, called AtlasT4SS - the first public database devoted exclusively to this prokaryotic secretion system. Description The AtlasT4SS is a manual curated database that describes a large number of proteins related to the type IV secretion system reported so far in Gram-negative and Gram-positive bacteria, as well as in Archaea. The database was created using the RDBMS MySQL and the Catalyst Framework based in the Perl programming language and using the Model-View-Controller (MVC) design pattern for Web. The current version holds a comprehensive collection of 1,617 T4SS proteins from 58 Bacteria (49 Gram-negative and 9 Gram-Positive), one Archaea and 11 plasmids. By applying the bi-directional best hit (BBH) relationship in pairwise genome comparison, it was possible to obtain a core set of 134 clusters of orthologous genes encoding T4SS proteins. Conclusions In our database we present one way of classifying orthologous groups of T4SSs in a hierarchical classification scheme with three levels. The first level comprises four classes that are based on the organization of genetic determinants, shared homologies, and evolutionary relationships: (i) F-T4SS, (ii) P-T4SS, (iii) I-T4SS, and (iv) GI-T4SS. The second level designates a specific well-known protein families otherwise an uncharacterized protein family. Finally, in the third level, each protein of an ortholog cluster is classified according to its involvement in a specific cellular process. AtlasT4SS database is open access and is available at



Bacteriophages as biocontrol agents of food pathogens.  


Bacteriophages have long been recognized for their potential as biotherapeutic agents. The recent approval for the use of phages of Listeria monocytogenes for food safety purposes has increased the impetus of phage research to uncover phage-mediated applications with activity against other food pathogens. Areas of emerging and growing significance, such as predictive modelling and genomics, have shown their potential and impact on the development of new technologies to combat food pathogens. This review will highlight recent advances in the research of phages that target food pathogens and that promote their use in biosanitation, while it will also discuss its limitations. PMID:21115341

Mahony, Jennifer; McAuliffe, Olivia; Ross, R Paul; van Sinderen, Douwe



Modified lysozymes as novel broad spectrum natural antimicrobial agents in foods.  


In recent years much attention and interest have been directed toward application of natural antimicrobial agents in foods. Some naturally occurring proteins such as lactoperoxidase, lactoferrin, and lysozyme have received considerable attention and are being considered as potential antimicrobial agents in foods. Lysozyme kills bacteria by hydrolyzing the peptidoglycan layer of the cell wall of certain bacterial species, hence its application as a natural antimicrobial agent has been suggested. However, limitations in the action of lysozyme against only Gram-positive bacteria have prompted scientists to extend the antimicrobial effects of lysozyme by several types of chemical modifications. During the last 2 decades extensive research has been directed toward modification of lysozyme in order to improve its antimicrobial properties. This review will report on the latest information available on lysozyme modifications and examine the applicability of the modified lysozymes in controlling growth of Gram-positive and Gram-negative bacteria in foods. The results of modifications of lysozyme using its conjugation with different small molecule, polysaccharides, as well as modifications using proteolytic enzymes will be reviewed. These types of modifications have not only increased the functional properties of lysozyme (such as solubility and heat stability) but also extended the antimicrobial activity of lysozyme. Many examples will be given to show that modification can decrease the count of Gram-negative bacteria in bacterial culture and in foods by as much as 5 log CFU/mL and in some cases essentially eliminated Escherichia coli. In conclusion this review demonstrates that modified lysozymes are excellent natural food preservatives, which can be used in food industry. PMID:24837015

Aminlari, Ladan; Mohammadi Hashemi, Marjan; Aminlari, Mahmoud



The tertiary structure of an i-type lysozyme isolated from the common orient clam (Meretrix lusoria).  


To evaluate the structure-function relationships of invertebrate lysozymes, a new invertebrate-type (i-type) lysozyme was isolated from the common orient clam (Meretrix lusoria) and the tertiary structure of this enzyme was determined. Comparison of the tertiary structure of this enzyme with those of chicken and Venerupi philippinarum lysozymes revealed that the location of the side chain of the second catalytic residue, an aspartic acid, and the N-acetylglucosamine trimer bound at subsites A-C were different. Furthermore, the amino acid electrostatically interacting with Asp30 in V. philippinarum lysozyme, Lys108, was substituted by Gly in M. lusoria lysozyme and no other possible amino acid that could contribute to this interaction was found in M. lusoria lysozyme. It therefore seems that the substitutions of the amino acids at the interface of the V. philippinarum lysozyme dimer are likely to change the oligomeric state of the M. lusoria lysozyme. PMID:24192349

Kuwano, Yuko; Yoneda, Kazunari; Kawaguchi, Yuya; Araki, Tomohiro



Bacteriophages and their role in food safety.  


The interest for natural antimicrobial compounds has increased due to alterations in consumer positions towards the use of chemical preservatives in foodstuff and food processing surfaces. Bacteriophages fit in the class of natural antimicrobial and their effectiveness in controlling bacterial pathogens in agro-food industry has led to the development of different phage products already approved by USFDA and USDA. The majority of these products are to be used in farm animals or animal products such as carcasses, meats and also in agricultural and horticultural products. Treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases and ultimately promote safe environments in animal and plant food production, processing, and handling. This is an overview of recent work carried out with phages as tools to promote food safety, starting with a general introduction describing the prevalence of foodborne pathogens and bacteriophages and a more detailed discussion on the use of phage therapy to prevent and treat experimentally induced infections of animals against the most common foodborne pathogens, the use of phages as biocontrol agents in foods, and also their use as biosanitizers of food contact surfaces. PMID:23316235

Sillankorva, Sanna M; Oliveira, Hugo; Azeredo, Joana



Immobilization of bacteriophages on modified silica particles.  


Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties, for example, biotin or cellulose binding domains; procedures that are both time and money intensive. We report that morphologically different bacteriophages, active against a variety of food-borne bacteria: Escherichia coli; Salmonella enterica; Listeria monocytogenes; and Shigella boydii, will effectively physisorb to silica particles, prepared by silica surface modification with poly(ethylene glycol), carboxylic acid groups, or amines. The phages remain infective to their host bacteria while adsorbed on the surface of the silica particles. The number of infective phage bound to the silica is enhanced by the presence of ionic surfaces, with greater surface charge - to a maximum - correlating with greater concentration of adsorbed phage. Above the maximum charge concentration, the number of active phage drops. PMID:19945158

Cademartiri, Rebecca; Anany, Hany; Gross, Isabelle; Bhayani, Rahul; Griffiths, Mansel; Brook, Michael A



Bacteriophage Infecting the Myxobacterium Chondrococcus columnaris  

PubMed Central

Kingsbury, David T. (University of Washington, Seattle), and Erling J. Ordal. Bacteriophage infecting the myxobacterium Chondrococcus columnaris. J. Bacteriol. 91:1327–1332. 1966.—During a series of screening experiments, seven bacteriophages which infect the pathogenic myxobacterium Chondrococcus columnaris were isolated. Of these, one was chosen for detailed study. This phage has a wide host range among strains of C. columnaris, but does not infect other myxobacterial species tested. Morphologically, this phage resembles coliphage T2, though it is smaller. It has a head diameter of 600 A, a tail length of 1,000 A, and a tail width of 200 A. The head is attached to the tail by a well-defined neck. The turbid plaques produced by this phage are similar in appearance to those produced by coliphage ?, and average 1 mm in diameter. The phage has a latent period of 100 min, a rise period of an additional 90 min, and a burst size of 23. Calcium ions at a concentration of 0.004 m are required for adsorption. This requirement cannot be met by substitution of magnesium ions. A purified preparation of 2 × 1012 phage particles was extracted with phenol, and the nucleic acid was identified as deoxyribonucleic acid (DNA). Base ratios of the phage DNA and the DNA of two propagating strains were similar. Streptomycin at a concentration of 70 ?g/ml inhibits phage infection at an early stage, probably by inhibiting injection of the phage DNA. Images

Kingsbury, David T.; Ordal, Erling J.



Understanding bacteriophage specificity in natural microbial communities.  


Studying the coevolutionary dynamics between bacteria and the bacteriophage viruses that infect them is critical to understanding both microbial diversity and ecosystem functioning. Phages can play a key role in shaping bacterial population dynamics and can significantly alter both intra- and inter-specific competition among bacterial hosts. Predicting how phages might influence community stability and apparent competition, however, requires an understanding of how bacteria-phage interaction networks evolve as a function of host diversity and community dynamics. Here, we first review the progress that has been made in understanding phage specificity, including the use of experimental evolution, we then introduce a new dataset on natural bacteriophages collected from the phyllosphere of horse chestnut trees, and finally we highlight that bacterial sensitivity to phage is rarely a binary trait and that this variation should be taken into account and reported. We emphasize that there is currently insufficient evidence to make broad generalizations about phage host range in natural populations, the limits of phage adaptation to novel hosts, or the implications of phage specificity in shaping microbial communities. However, the combination of experimental and genomic approaches with the study of natural communities will allow new insight to the evolution and impact of phage specificity within complex bacterial communities. PMID:23478639

Koskella, Britt; Meaden, Sean



Enteroviruses and Bacteriophages in Bathing Waters  

PubMed Central

A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters. A number of bacterial indicators and bacteriophages were also tested. Cultivable enteroviruses were detected in 55% of the samples, most of which complied with bacteriological criteria. In contrast, viral genomes were only detected in 20% of the samples by reverse transcription-PCR. Somatic coliphages outnumbered all other indicators. F-specific RNA phages were detected in only 15% of the samples, whereas phages infecting Bacteroides thetaiotaomicron were detected in 70% of samples. A numerical relationship between the numbers of enteroviruses and the numbers of enterococci and somatic coliphages was observed. In situ inactivation experiments showed that viruses persisted significantly longer than the bacterial indicators. Only somatic coliphages and bacteriophages infecting Bacteroides persisted longer than the viruses. These results explain the numbers of enteroviruses and indicators in bathing waters attending the numbers usually found in sewage in the area. Somatic coliphages show a very good potential to predict the risk of viruses being present in bathing waters.

Moce-Llivina, Laura; Lucena, Francisco; Jofre, Juan



Recombinant Destabilase-Lysozyme: Synthesis de novo in E. coli and Action Mechanism of the Enzyme Expressed in Spodoptera frugiperda  

Microsoft Academic Search

Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families. In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli. Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial,

L. L. Zavalova; I. P. Baskova; E. V. Barsova; E. V. Snezhkov; S. B. Akopov; S. A. Lopatin



Genomic characterization, phylogeny and gene regulation of g-type lysozyme in sole ( Solea senegalensis)  

Microsoft Academic Search

The g-type lysozyme is a key protein of the innate immune system to fight bacterial infections. In this study we cloned and characterized the gene encoding for g-type lysozyme in Senegalese sole (Solea senegalensis). The deduced amino acid sequence comprised 195 residues containing the three conserved catalytic residues and two cysteines. A BAC analysis revealed that the gene is structured

Marian Ponce; Emilio Salas-Leiton; Ana Garcia-Cegarra; Anais Boglino; Olivier Coste; Carlos Infante; Enric Gisbert; Laureana Rebordinos; Manuel Manchado


[The influence of occupational exposure to manganese on levels of serum lysozyme].  


The serum lysozyme level of workers engaged in the production of iron-manganese alloys was observed. The lysozyme level was statistically significantly increased compared with the control group. There was no evidence with age and years of work. PMID:8834628

Misiewicz, A; Jele?, B; Radwan, K; Karmoli?ski, M; Dziewit, T



Synergy of Histone-Derived Peptides of Coho Salmon with Lysozyme and Flounder Pleurocidin  

Microsoft Academic Search

Recent research has identified endogenous cationic antimicrobial peptides as important factors in the innate immunity of many organisms, including fish. It is known that antimicrobial activity, as well as lysozyme activ- ity, can be induced in coho salmon (Oncorhynchus kisutch) mucus after exposure of the fish to infectious agents. Since lysozyme alone does not have antimicrobial activity against Vibrio anguillarum




Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles  

Microsoft Academic Search

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined

E. H. W. Pap; M. C. Houbiers; J. S. Santema; A. van Hoek; A. J. W. G. Visser



Three in one: Identification, expression and enzymatic activity of lysozymes in amphioxus.  


The lysozymes identified so far in animals belong to the g-type, c-type, and i-type. Vertebrate animals possess only the former two types, i.e., g- and c-types, while all the three types have been reported in invertebrates. Here we demonstrate that (1) three cDNAs that encode g-, c-, and i-type lysozymes, respectively, were identified in a single species of the amphioxus Branchiostoma japonicum; (2) all the 3-type genes displayed distinct tissue-specific expression pattern; (3) recombinant g-, c-, and i-type lysozymes all exhibited enzymatic activities; and (4) native g-, c-, and i-type lysozymes were identified in the different tissues of amphioxus. Collectively, these results suggest the presence of all the 3-type lysozymes in a single animal species, first such data ever reported. The presence of biologically active i-type lysozyme in amphioxus also suggests that i-type lysozyme gene is retained at least in Protochordata, contrasting to the previous proposal that i-type lysozyme gene has been lost in a common ancestor of all chordates. PMID:24968076

Xu, Na; Pan, Junli; Liu, Shousheng; Xue, Qinggang; Zhang, Shicui



Lysozyme microencapsulation within biodegradable PLGA microspheres: urea effect on protein release and stability.  


Lysozyme was encapsulated within biodegradable poly(D, L-lactide-co-glycolide) microspheres by a double emulsion solvent evaporation method for studying its release mechanism associated with protein stability problems. When urea, a protein unfolding agent, was added into the incubation medium lysozyme release rate from the microspheres increased with the increase in urea concentration. The enhanced lysozyme release was attributed to the suppression of protein aggregation, to the facilitated diffusion of unfolded lysozyme by an efficient reptile motion of unfolded protein molecules through porous channels in microspheres, and to the largely decreased extent of nonspecific protein adsorption onto the enlarged surface area of degrading polymer microspheres in the presence of urea. Encapsulating lysozyme in an unfolded form within PLGA microspheres was attempted by using urea as an excipient. This new urea-based formulation exhibited a more sustained lysozyme release profile than the control formulation, and released lysozyme from the microspheres showed a much less amount of lysozyme dimer population while maintaining a correct conformation after refolding in the incubation medium. This study provides new insights for the formulation of protein encapsulated PLGA microspheres. PMID:10992231

Nam, Y S; Song, S H; Choi, J Y; Park, T G



Effect of lysozyme on plasma membranes of liver and heart cells: lectin binding assay.  


Lectin binding assay was applied to polyacrylamide slices to study the effects of lysozyme on carbohydrate profile of plasma membranes from liver and heart tissues. The investigations were carried out on polyacrylamide slices immobilized with wheat germ agglutinin or Concanavalin A. It was shown that lysozyme changes the carbohydrate profile of plasma membranes of liver and heart cells. PMID:17317226

Mikaelyan, M V; Poghosyan, G G; Stepanyan, I E; Gasparyan, V K



The Action of Lysozyme on Heat-killed Gram-positive Microorganisms  

Microsoft Academic Search

SUMMARY: The change in the Gram staining reaction which occurs when heat- killed Gram-positive Clostridium welchii and Staphylococcus albus are incubated with lysozyme is due to the removal of the ribonucleic acid component of the Gram complex, and is brought about by the hydrolysis of certain sugar linkages in polysaccharides located at the cell surface. Lysozyme, the enzyme in egg-white

M. Webb



Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio  

Microsoft Academic Search

The purpose of this study was to quantify the gene expression of lysozyme, an important antibacterial protein produced by shrimp hemocytes, within tissues of Litopenaeus vannamei Boone in response to a pathogen challenge. We quantified lysozyme transcripts with a real-time PCR method and used these data, along with total hemocyte counts, to infer patterns of hemocyte trafficking during the immune

Erin J. Burge; Daniel J. Madigan; Louis E. Burnett; Karen G. Burnett