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PubMed1992

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin). Refinement of native and mutant proteins.

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.

Amino Acid Sequence↗

PubMed2001

Typing and antimicrobial susceptibilities of methicillin resistant Staphylococcus aureus (MRSA) strains isolated in a hospital in Korea.

Methicillin-resistant Staphylococcus aureus (MRSA) strains may cause serious nosocomial infections, including pneumonia and septicemia. The rate of methicillin-resistance among S. aureus isolates in Korea is over 50%. In this study, 90 MRSA isolates from Kyung Hee University Hospital were characterized employing bacteriophage typing, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. Eighty percent of the strains could be phage-typed. The largest group or 40% of the strains belonged to lyso group III, followed by 32% of the isolates which produced a reaction with regional additional phages. Phage type 83A was most frequently encountered, followed by phage type D11. PFGE patterns confirmed the presence of two major clusters, which comprise the isolates belonging to lyso group III and the strains that were typable with regional additional phages. The latter group also contained a number of strains that were nontypable with bacteriophages. The resistance rates to ciprofloxacin, erythromycin, tetracycline, gentamicin and clindamycin were over 94%. Strains with intermediate resistance to vancomycin strains or resistance to mupirocin were not found. In conclusion, this study demonstrates that the results of phage typing are confirmed and supplemented by PFGE data.

Bacteriophage Typing↗

PubMed1984

Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4.

A strain of Escherichia coli, CP 790302, severely restricts the growth of wild-type bacteriophage T4. In broth culture, most infections of single cells are abortive, although a few infected cells exhibit reduced burst sizes. In contrast, bacteriophage T4 mutants impaired in the ability to modify valyl-tRNA synthetase develop normally on this strain. Biochemical evidence indicates that the phage-modified valyl-tRNA synthetase in CP 790302 is different from that previously described. Although the enzyme is able to support normal protein synthesis, a disproportionate amount of phage structural protein (serum blocking power) fails to mature into particles of the appropriate density. The results with host strain CP 790302 are consistent with either a gratuitous inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA synthetase might normally play in viral assembly.

Amino Acyl-tRNA Synthetases↗

PubMed2006

Sequence and comparative genomic analysis of lactococcal bacteriophages jj50, 712 and P008: evolutionary insights into the 936 phage species.

The complete genome sequences of three lactococcal 936-type bacteriophages, 712, jj50 and P008, were determined. Comparative genomic analysis of these phages with the previously sequenced 936-type phages, sk1 and bIL170, reveals a strict conservation of the overall genetic organization of this geographically diverse phage group. Genetic divergence was mainly observed in the early expressed region of the phage genomes, where a number of deletions, exchanges and insertions appear to have occurred. These genetic differences may be responsible for the observed differential sensitivity to the lactococcal DNA injection blocking protein, Sie(2009), and the abortive infection system, AbiA.

Bacterial Proteins↗

PubMed1981

Ultrastructure of bacteriophages used for typing of Staphylococcus aureus.

This paper reports the results of a study on the morphology, size and homogeneity of the bacteriophages used for typing Staphylococcus aureus. Fifty-five bacteriophages were studied. Preparations for the electron microscope were made directly from lysates or from purified and concentrated bacteriophages, and were contrasted with neutralized phosphotungstic acid (KPT) and examined under an electron microscope at x 40 000 microscopic magnification. The studied bacteriophages differed with regard to size and shape. The bacteriophages were classified into two morphological groups: BI and BIII. In the BI group, there morphological types were distinguished: B14, B15 and B16. Only one morphological type was distinguished in the BIII group, designated by the symbol BIII2. Only lysates of 42C, 70 and D bacteriophages contained virions of two morphological types: BI6 an BIII2. Until now, it is not clear whether the bacteriophages are a mixture of two different virions, or whether they represent variation in the size of their heads. Of the 55 bacteriophages studied, 25 were examined by other investigators. The results showed that of the 25 bacteriophages 5 belonged to different morphological types. It was concluded that the sets of standard bacteriophages should be checked by means of the electron microscope.

Bacteriophage Typing↗

PubMed2003

Emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infection in Queensland, Australia.

OBJECTIVES: To investigate the incidence and epidemiology of non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) infection in south-east Queensland, Australia. STUDY DESIGN: A retrospective survey was done of hospital records of all patients who had non-multiresistant MRSA isolated at Ipswich Hospital (a 250-bed general hospital, 40 km south-west of Brisbane, Queensland, Australia) between March 2000 and June 2001. Laboratory typing of these isolates was done with antibiogram, pulsed-field gel electrophoresis, bacteriophage typing and coagulase gene typing. RESULTS: There were 44 infections caused by nmMRSA. Seventeen infections (39%) occurred in patients from the south-west Pacific Islands (predominantly Samoa, Tonga and New Zealand). Laboratory typing showed that the isolates in Pacific Islanders were Pacific Island strains, and 16/17 of these infections were community acquired. Twenty-three infections (52%) occurred in Caucasians. Eleven of the isolates from Caucasians (48%) were a new predominantly community-acquired strain that we have termed the 'R' pulsotype, nine (39%) were Pacific Island strains, and three (13%) were health care institution-associated strains. Four infections occurred in patients who were not Caucasians or Pacific Islanders. Overall, 34 of all 44 infections (77%) were community acquired. CONCLUSIONS: Non-multiresistant MRSA infection, relatively frequently observed in Pacific Islanders in south-east Queensland, is now a risk for Caucasians as well, and is usually community acquired. Clinicians should consider taking microbiological specimens for culture and antimicrobial susceptibility testing in patients with suspected staphylococcal infections who are not responding to empirical therapy with beta-lactam antibiotics.

Adult↗

PubMed1978

Evidence for a near UV-induced photoproduct of 5-hydroxymethylcytosine in bacteriophage T4 that can be recognized by endonuclease V.

Non-photoreactivable endonuclease V-sensitive sites have been detected in the DNA of wild type bacteriophage T4 irradiated with near UV light (320 nm). Such sites were not detected in the DNA of (a) wild type T4 irradiated with far UV (254 nm) or (B) in T4 mutants in which non-glucosylated 5-hydroxy-methylcytosine (5HMC) or cytosine replaces glucosylated 5HMC normally present in T4, irradiated with 320 nm or 254 nm light. Although the non-photoreactivable sites accounted for approximately 50% of the endonuclease V-sensitive sites in the DNA of glucosylated T4 irradiated with near UV, there was very little difference in the sensitivities of T4 containing glucosylated 5HMC, non-glucosylated 5HMC and cytosine to near UV (313 nm). We propose that the photoproduct responsible for the non-photoreactivable, but endonuclease V-sensitive, sites in glucosylated DNA is formed from glucosylated 5HMC and that a similar photoproduct is formed from non-glucosylated 5HMC or cytosine in the appropriate phage strains. We further propose that the glucosylated 5HMC photoproduct is non-photoreactivable whereas the cytosine and non-glucosylated 5HMC photoproducts are photoreactivable and are therefore possibly cyclobutane dimers.

5-Methylcytosine↗

PubMed2006

Effective plasmid pX3 transduction in Lactobacillus delbrueckii by bacteriophage LL-H.

High-frequency plasmid transductions in Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus strains mediated by pac-type bacteriophages were observed and further investigated. The frequency of plasmid transduction by phages LL-H and LL-S attained levels of from 0.10 to about 1 with plasmid p X 3, but only about 2 x 10(-2) with plasmid pJK650. Infection of L. delbrueckii subsp. lactis strain LKT(pX3) or ATCC 15808(pX3) with phage LL-H resulted in intensive concatemerization of plasmid pX3, and most progeny phage particles contained concatemers of plasmid DNA instead of phage LL-H DNA. The synthesis of phage LL-H DNA was depressed. No evident homology or recombination was observed between phage LL-H DNA and plasmid pX3. The unusually high frequency of plasmid pX3 transduction by phage LL-H could be considered to result from specific interaction(s) between a particular phage and plasmid. These interactions may include pX3-mediated blockage of phage LL-H DNA replication and effective use of a particular pac-like site located about 1 kb from BglII in the smaller NdeI-BglII fragment of plasmid pX3. Phage LL-H together with plasmid vector pX3 could be used as effective plasmid transduction tools for genetic engineering of L. delbrueckii subsp. lactis and subsp. bulgaricus strains.

Bacteriophages↗

PubMed1990

Solution structure of bacteriophage T4D and icosahedral capsid geometry visualized in freeze-fractured, deep-etched replicas.

The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.

Capsid↗

PubMed1987

[Transfecting activity of Pseudomonas aeruginosa bacteriophage SM].

Factors affecting the efficiency of transfection of Ps. aeruginosa PAO1 cells by the temperate SM bacteriophage DNA have been determined. The efficiency of transfection by DNA preparations isolated from the wild type bacteriophage SMc+ or its thermoinducible mutant SM cts6 is practically the same. The frequency of transfection is (7-9) X 10(4) of infectious centers per mkg of transfecting DNA. Variability in the frequencies of transfection has been registered depending of the infection conditions or on the transfer of the Ps. aeruginosa PAO1 recipient strain population into the competence phase. The efficiency of transfection is increased by the addition of Ca2+ or Mg2+ ions affecting the adsorption and absorbtion of phage DNA by the recipient cells. Optimal concentrations of the bivalent metal ions are 0.15M CaCl2 and 0.2M MgCl2. The results obtained have been used for optimizing the conditions of Ps. aeruginosa PAO1 transfection by SM bacteriophage DNA.

Bacteriophages↗

PubMed1984

Epidemiology of colitis induced by Clostridium difficile in hamsters: application of a bacteriophage and bacteriocin typing system.

The epidemiology of colitis induced by Clostridium difficile in hamsters was studied with a new bacteriophage and bacteriocin typing system. Fatal enterocolitis was induced by administration of N-formimidoyl thienamycin. Environmental cultures were obtained repeatedly throughout the experiments. Thirteen percent of 90 healthy hamsters were already colonized with C difficile on arrival from the supplier. Mortality from enterocolitis after antibiotic administration was 75% and was not diminished by use of a laminar-flow facility. The same uncommon bacteriocin type (83/1309/2329) of toxigenic C difficile that colonized hamsters on arrival was recovered from the cecal contents of all hamsters dying with enterocolitis and from most environmental isolates. Previously uncolonized , antibiotic-treated hamsters placed into cages where animals had died from enterocolitis also developed enterocolitis with the same bacteriocin type (83/1309/2329), an outcome suggesting acquisition of C difficile from the environment.

Animals↗

PubMed2003

Shigella dysenteriae type 1-specific bacteriophage from environmental waters in Bangladesh.

Shigella dysenteriae type 1 is the causative agent of the most severe form of bacillary dysentery, which occurs as epidemics in many developing countries. We isolated a bacteriophage from surface water samples from Bangladesh that specifically lyses strains of S. dysenteriae type 1. This phage, designated SF-9, belongs to the Podoviridae family and has a 41-kb double-stranded DNA genome. Further screening of water samples for the prevalence of the phage revealed 9 of 71 (12.6%) water samples which were positive for the phage. These water samples were also positive in PCR assays for one or more S. dysenteriae type 1-specific genes, including ipaBCD and stx1, and live S. dysenteriae type 1 was isolated from three phage-positive samples. The results of this study suggest that phage SF-9 may have epidemiological applications in tracing the presence of S. dysenteriae type 1 in environmental waters.

Bacterial Proteins↗

PubMed2002

Trade-offs and coexistence in microbial microcosms.

Trade-offs among the abilities of organisms to respond to different environmental factors are often assumed to play a major role in the coexistence of species. There has been extensive theoretical study of the role of such trade-offs in ecological communities but it has proven difficult to study such trade-offs experimentally. Microorganisms are ideal model systems with which to experimentally study the causes and consequences of ecological trade-offs. In model communities of E. coli B and T-type bacteriophage, a trade-off in E. coli between resistance to bacteriophage and competitive ability is often observed. This trade-off can allow the coexistence of different ecological types of E. coli. The magnitude of this trade-off affects, in predictable ways, the structure, dynamics and response to environmental change of these communities. Genetic factors, environmental factors, and gene-by-environment interactions determine the magnitude of this trade-off. Environmental control of the magnitude of trade-offs represents one avenue by which environmental change can alter community properties such as invasability, stability and coexistence.

Coliphages↗

PubMed1980

A mutant of E. coli that restricts growth of bacteriophage T4 at elevated temperatures.

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

Capsid↗

PubMed1984

Recognition of single-stranded DNA by the bacteriophage T4-induced type II topoisomerase.

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.

Amino Acid Sequence↗

PubMed1976

Renaturation of bacteriophage lambda DNA. Determination of the optimal renaturation conditions using a single-strand-specific DNase and alkaline-sucrose-gradient assay system.

Reannealed hybrid molecules of wild-type bacteriophage lambda DNA were prepared in aqueous solutions of formamide at a variety of NaCl concentrations at both room temperature ( 22 degrees C) and 37 degrees C. Treatment of the hybrid DNA molecules with the single-strand-specific nuclease S1 from Aspergillus oryzae followed by alkaline sucrose gradient sedimentation was used to monitor the extent and fidelity of hybridization. The optimal renaturation conditions at room temperature were found to be: 50% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 mug DNA/ml. Optimal conditions at 37 degrees C were: 32% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 mug DNA/ml. Under these conditions approximately 85-90% of the input single-stranded DNA (molecular weight 1.5 X 10(7)) was rendered S1-nuclease-resistant within 8 h at room temperature and 5 h at 37 degrees C. Neither Mg2+ nor spermidine appeared to have an effect on either the extent or fidelity of duplex formation. Experiments performed with excess enzyme and with lambda/lambda imm 434 heteroduplex hybrids suggested that the hybrid that the hybrid DNA molecules formed under optimal conditions contained no, or only short (less than 1%), mismatched regions.

Binding Sites↗

PubMed1975

Miniphage-a class of satellite phage to M13.

Satellite or defective bacteriophage particles can appear in extensively recycled stocks of coliphage M13. These particles, herein known as miniphage, replicate using the wild type bacteriophage as a helper. Their physical properties (u.v. spectra, sedimentation of DNA and bacterophage, electrophoretic moblitiy) are described and a method for the isolation of specific satellite bacteriophage is presented.

Amino Acids↗

PubMed1986

Bradyrhizobium japonicum Serocluster 123 and Diversity among Member Isolates.

Diversity was examined within a group of 79 isolates of Bradyrhizobium japonicum reactive to fluorescent antibodies (FAs) prepared against B. japonicum USDA 123. Analyses were by means of cross-adsorbed FAs, bacteriophage typing, and endonuclease restriction digest patterns. Serogroups 127 and 129 shared antigenic determinants with serogroup 123 but not with each other. Bacteriophage and DNA digest patterns reflected more common features between serogroups 123 and 127 than between 123 and 129. Serogroups 129 and 122 showed FA cross-reactivity. The term serocluster was proposed to reflect interrelationships observed among the serogroups.

Journal Article↗