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At least 1,063 records · Page 59Linked to original sources
PubMed1978

Synthesis of phage M13 coat protein and its assembly into membranes in vitro.

The coat protein (gene 8 product) of coliphage M1O is an integral protein of the host cell membrane at all stages of virus infection. This protein, when made in a cell-free reaction, has been shown by others to have an additional NH2-terminal peptide region and is referred to as "procoat." It is initially not membrane-bound but, upon exposure to Escherichia coli membrane vesicles or to liposomes prepared from E. coli lipids, it assembles into the bilayer in an integral fashion. Much of this protein is shown to be exposed on the inner surface of the liposome. We suggest that refolding of procoat as it encounters the bilayer is sufficient to transport large segments of the peptide chain through the apolar hydrocarbon core.

Cell-Free System↗
PubMed1978

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts.

The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [(3)H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The (3)H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg(2+)-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.

Cell Line↗
PubMed1979

Translational and post-translational cleavage of M13 procoat protein: extracts of both the cytoplasmic and outer membranes of Escherichia coli contain leader peptidase activity.

The coat protein of coliphage M13 is an integral protein of the host cytoplasmic membrane at all stages of the infectious cycle. Both in in vivo and DNA-directed in vitro synthesis, it is initially made with an NH2-terminal "leader peptide" of 23 amino acids and is termed procoat. We now report that leader peptidase, and activity which removes the leader peptide and converts procoat to coat, is found in both the inner (cytoplasmic) and outer membrane of Escherichia coli. However, only cytoplasmic membranes will catalyze cleavage of procoat in the absence of detergent. Leader peptidase will cleave procoat either during translation or after protein synthesis is complete.

Cell Membrane↗
PubMed1979

Photoreactive labeling of M13 coat protein in model membranes by use of a glycolipid probe.

Coliphage M13 coat protein in synthetic bilayer membranes was labeled by use of 12-(4-azide-2-nitrophenoxy)stearoyl[1-14C]glucosamine, a photoreactive glycolipid probe that spontaneously inserts into membranes. In this model system, the probe preferentially labeled the proteins over the lipids. Experiments designed to test the probe's restriction to integral membrane proteins revealed that extrinsic proteins as well as external peptide fragments of integral membrane proteins were not accessible to the photogenerated nitrene on the fatty acid chain. Only integral membrane peptides were labeled by the membrane-bound probe. These results indicate that protein labeling can be effected specifically from within the hydrocarbon milieu of a model membrane system.

Affinity Labels↗
PubMed1979

Soluble precursor of an integral membrane protein: synthesis of procoat protein in Escherichia coli infected with bacteriophage M13.

Prior to virus assembly, the major coat protein of coliphage M13 is an integral protein of the host cytoplasmic membrane. Coat protein synthesized in vitro is initially made with an NH2-terminal "leader peptide" of 23 amino acids and is termed "procoat." We now report that procoat is a biosynthetic precursor of coat protein in vivo. Conversion of procoat to coat occurs within 30 sec in cells infected with wild-type virus. This proteolytic processing is delayed in cells infected by M13 mutants (in genes 1, 5, or 7) that are defective in virus assembly. Pulse--chase experiments in combination with subcellular fractionation show that procoat is synthesized in a soluble form in the cytoplasm and is then incorporated into the cytoplasmic membrane, where it is converted to coat protein. This finding is supported by the observation that procoat is synthesized exclusively by polysomes that are not membrane bound. These results are interpreted in terms of the "membrane-triggered folding" hypothesis of membrane protein assembly.

Bacterial Proteins↗
PubMed1980

Antiviral response elicited by a completely synthetic antigen with built-in adjuvanticity.

In a previous study we demonstrated that antiviral response against the coliphage MS-2 can be elicited by immunization with a synthetic antigen consisting of a conjugate (P2-A -- L) of a synthetic fragment (P2) of the virus coat protein attached to a synthetic polymeric carrier. The antiviral response was induced when the antigen was administered in complete Freund's adjuvant or when it was administered in incomplete adjuvant, provided that a peptidoglycan was covalently attached to it. In the present study we demonstrate the adjuvant effect of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) in this system. Immunization with a mixture of MDP and P2-A -- L brought about only slight enhancement in the titer of neutralizing antibodies, as compared to the immunization with P2-A -- L in saline. The best results were achieved when the MDP was chemically conjugated to P2-A -- L. This completely synthetic material, when administered in aqueous solution, yielded highly inactivating antiserum with a titer similar to that obtained with complete Freund's adjuvant in the absence of MDP. MDP-P2-A -- L elicited also a humoral immune response to MDP, but with much lower titer than that induced by complete Freund's adjuvant containing P2-A -- L only. It was also observed that the capacity of MDP-P2-A -- L to increase resistance against infection was more than a 100-fold greater than that of unconjugated MDP.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1980

Procoat, the precursor of M13 coat protein, requires an electrochemical potential for membrane insertion.

The coat protein of coliphage M13 spans the host cell cytoplasmic membrane prior to its assembly into extruding virus. It is made as a soluble cytoplasmic precursor, termed "procoat," with 23 extra amino acid residues at the NH2 terminus. Procoat binds to the cell membrane and is converted proteolytically to coat protein. When the electrochemical gradient of an infected cell is rapidly dissipated by uncouplers, procoat still binds to the plasma membrane but is not converted to coat. We report here that membrane-bound procoat is only detected at the inner face of the cytoplasmic membrane and that uncouplers prevent it from integrating into a transmembrane conformation.

Coliphages↗
PubMed1987

Site-specific insertion of DNA into a pseudorabies virus vector.

A simple, efficient method for introducing recombinant DNA into a herpesvirus vector and retrieving it at a later time has been developed. By using the Cre-lox site-specific recombination system of coliphage P1, DNA can be readily inserted in vitro into a pseudorabies virus (PRV) vector containing the lox recombination site. The vector PRV42 contains a lox site within the nonessential gIII gene, which encodes a virion envelope glycoprotein. Incubation in vitro of PRV42 DNA with Cre protein and a circular plasmid containing a lox site generates approximately 5% recombinant molecules having the plasmid integrated into the PRV genome at the lox site. Transfection of the reaction mixture into cultured cells allows recovery of the infectious recombinant virus, which is readily identified by a nondestructive "black-plaque assay" using a gIII-specific monoclonal antibody. PRV42 plaques stain black when treated with the gIII monoclonal antibody and a peroxidase-linked second anti-antibody because the lox site placed within the gIII gene of PRV42 does not destroy the gIII epitope. However, Cre-mediated integration of heterologous DNA at the lox site disrupts the gIII epitope so that the resulting recombinant virus produces white plaques. The recombinant virus is infectious, stable, and grows as well as the parental PRV42 vector. The inserted plasmid can be efficiently excised (greater than 50%) from viral DNA by Cre and recovered by transformation of Escherichia coli.

Coliphages↗
PubMed1995

Identification, cloning, and characterization of the bacteriophage N4 gene encoding the single-stranded DNA-binding protein. A protein required for phage replication, recombination, and late transcription.

The coliphage N4-coded single-stranded DNA-binding protein (N4SSB) is essential for phage replication and for expression of the phage late genes, which are transcribed by the Escherichia coli sigma 70 RNA polymerase. As a first step in investigating the role of N4SSB in replication and transcriptional activation, we have identified and sequenced the N4SSB gene. The gene encodes a 265-amino acid protein with no apparent sequence homology to other single-stranded DNA-binding proteins. We present data indicating that N4SSB is also essential for phage recombination. Mutational analysis of the carboxyl terminus of the protein indicates that this region is required for protein-protein interactions with the N4 replication, N4 recombination, and E. coli transcriptional machineries, while the rest of the protein contains the determinants for single-stranded DNA binding.

Amino Acid Sequence↗
PubMed2000

The lysis protein E of phi X174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis.

Coliphage phi X174 encodes a single lysis protein, E, a 91-amino acid membrane protein. Dominant mutations have been isolated in the host gene mraY that confer E resistance. mraY encodes translocase I, which catalyzes the formation of the first lipid intermediate in bacterial cell wall synthesis, suggesting a model in which E inhibits MraY and promotes cell lysis in a manner analogous to cell wall synthesis inhibitors like penicillin. To test this model biochemically, we monitored the effect of E on cell wall synthesis in vivo and in vitro. We find that expression of Emyc, encoding an epitope-tagged E protein, from a multicopy plasmid inhibits the incorporation of [(3)H]diaminopimelic acid into cell wall and leads to a profile of labeled precursors consistent with MraY inhibition. Moreover, we find that membranes isolated after Emyc expression are drastically reduced in MraY activity, whereas the activity of Rfe, an enzyme in the same superfamily, was unaffected. We therefore conclude that E is indeed a cell wall synthesis inhibitor and that this inhibition results from a specific block at the MraY-catalyzed step in the pathway.

Bacterial Proteins↗
PubMed2003

Qbeta-phage resistance by deletion of the coiled-coil motif in elongation factor Ts.

Elongation factor Ts (EF-Ts) is the guanine-nucleotide exchange factor of elongation factor Tu (EF-Tu), which promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome in prokaryotes. The EF-Tu.EF-Ts complex, one of the EF-Tu complexes during protein synthesis, is also a component of RNA-dependent RNA polymerases like the polymerase from coliphage Qbeta. The present study shows that the Escherichia coli mutant GRd.tsf lacking the coiled-coil motif of EF-Ts is completely resistant to phage Qbeta and that Qbeta-polymerase complex formation is not observed. GRd.tsf is the first E. coli mutant ever described that is unable to form a Qbeta-polymerase complex while still maintaining an almost normal growth behavior. The phage resistance correlates with an observed instability of the mutant EF-Tu.EF-Ts complex in the presence of guanine nucleotides. Thus, the mutant EF-Tu.EF-Ts is the first EF-Tu.EF-Ts complex ever described that is completely inactive in the Qbeta-polymerase complex despite its almost full activity in protein synthesis. We propose that the role of EF-Ts in the Qbeta-polymerase complex is to control and trap EF-Tu in a stable conformation with affinity for RNA templates while unable to bind aminoacyl-tRNA.

Coliphages↗
PubMed2006

Characterization of a novel intramolecular chaperone domain conserved in endosialidases and other bacteriophage tail spike and fiber proteins.

Folding and assembly of endosialidases, the trimeric tail spike proteins of Escherichia coli K1-specific bacteriophages, crucially depend on their C-terminal domain (CTD). Homologous CTDs were identified in phage proteins belonging to three different protein families: neck appendage proteins of several Bacillus phages, L-shaped tail fibers of coliphage T5, and K5 lyases, the tail spike proteins of phages infecting E. coli K5. By analyzing a representative of each family, we show that in all cases, the CTD is cleaved off after a strictly conserved serine residue and alanine substitution prevented cleavage. Further structural and functional analyses revealed that (i) CTDs are autonomous domains with a high alpha-helical content; (ii) proteolytically released CTDs assemble into hexamers, which are most likely dimers of trimers; (iii) highly conserved amino acids within the CTD are indispensable for CTD-mediated folding and complex formation; (iv) CTDs can be exchanged between proteins of different families; and (v) proteolytic cleavage is essential to stabilize the native protein complex. Data obtained for full-length and proteolytically processed endosialidase variants suggest that release of the CTD increases the unfolding barrier, trapping the mature trimer in a kinetically stable conformation. In summary, we characterize the CTD as a novel C-terminal chaperone domain, which assists folding and assembly of unrelated phage proteins.

Bacillus Phages↗
PubMed1975

Further studies of DNA damage and lethality from the decay of iodine-125 in bacteriophages.

The DNA of coliphages T4 and T1 was labelled with 125I-iododeoxyuridine. 125I decay is known to cause severe molecular damage via vacancy cascades (the Auger effect). We have compared the induction of both single- and double-strand breaks (SSBs and DSBs) in 125I-labelled T4 DNA stored at - 196 degrees C during decay, either as intact phage or as free DNA. These comparative experiments indicate that, in addition to one DSB which apparently results directly from the Auger effect, each decay in an intact phage also give rise to an additional 0-05 DSBs, as well as 1-6 SSBs, as a result of ionizing radiation absorbed in the same phage particle where the decay occurs. An examination of T4-killing by 125I decay reveals a two-phase survival curve, whose initial slope corresponds to a lethal efficency per 125I decay of 0-95 +/- 0-05, which is considerably higher than values previously determined. The results for phage T4, and of a more limited comparison of 125I suicide and DNA damage in phage T1, support the hypothesis that the vacancy cascades which accompany each 125I decay in DNA result in a double-strand break at the decay site and that each such break is a lethal event.

Coliphages↗
PubMed1978

Quantitative determination of cross-linkage of bacteriophage DNA and protein by ionizing radiation.

Coliphage T7 was dissolved in tryptone broth and exposted to 60C gamma-radiation. Cross-linkage of DNA and protein of the virion was assayed using phenol-water countercurrent distribution. The results are interpreted in terms of a statistical model of cross-linkage and double-strand breaks. It was found that protein--DNA cross-links accumulate linearly with dose at a rate of o.74 X 10(-11) cross-links per rad per nucleotide pair, which is of the order of 5 per cent of the formation rate of double-strand breaks.

Cobalt Radioisotopes↗
PubMed1979

Double strand-breaks and DNA-to-protein cross-links induced by fast neutrons in bacteriophage DNA.

Coliphage T7 was suspended in tryptone broth and exposed to a mixture of fast neutrons and gamma radiation. Plaque survival, double strand-breaks and DNA-to-protein cross-linkage were examined and the results compared with those found in phage exposed to gamma radiation alone. Neutral sucrose density sedimentation patterns indicate that neutron-induced double strand-breaks sometimes occur in clusters of more than 100 in the same phage and that the effeciency with which double strand-breaks form is about 50 times that of gamma-induced double strand-breaks. Neutron-induced protein-to-DNA cross-links probably also occur in clusters with enhanced efficiency relative to low LET radiation.

Coliphages↗
PubMed2006

The use of bacteriophages for monitoring the microbiological quality of sewage sludge.

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated sludge is presented in this study. Raw and anaerobically digested sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analyzed for total coliforms, E. coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. E. coli concentrations of > or =10(3) cfus g(-1) and <10(3) cfus g(-1) comprise a threshold for the presence of FRNAPH and BFRPH, respectively. Likewise, intestinal enterococci concentrations of > or =10(4) cfus g(-1) and <10(3) cfus g(-1) comprise a threshold for the presence of FRNAPH and BFRPH, respectively. In the case of SOMCPH, it was not possible to define a threshold, since they were detected with the lowest observed indicator concentrations in all samples.

Bacteriophages↗
PubMed1975

Studies of mutations in T4 control genes 33 and 55.

Available mutations in transcriptional control genes 33 and 55 of coliphage T4 have been examined. By complementation analysis and map position, 15 mutants (13 in T4D, 2 in T4B) have been shown to lie in gene 33 and 6 (5 in T4D, 1 in T4B) in gene 55. According to patterns of suppression and recombination, these mutants define three distinct amber sites in gene 33 and also three distinct amber sites in gene 55. All of these mutations are true amber mutations, in apparent contrast to some traditional T4 "amber" mutants which grow in su+ E. coli CR63 but not in su minus E. coli B because of a strain difference other than the su+ determinant. Evidence is presented that, contrary to previous suggestions (BOLLE et al. 1968; pulitzer and geiduschek 1970), the gene 33 product is absolutely essential for T4 development.

Coliphages↗
PubMed1984

Nucleotide sequence of the essential region of bacteriophage P4.

Nucleotide sequence of one-third of the genome of coliphage P4 has been obtained and mutations virl, epsilon am104, cI405, sidl, and delta 35 identified. The epsilon gene likely encodes a 10 kd protein with epsilon am104 being located at the beginning of the gene. cI405, a proposed repressor gene mutation, is located in a sequence capable of coding for a 15 kd protein. A new class of P4 mutations, ash, is located in the neighborhood of cI405. Two TATA-like sequences are mapped 5' to this cI (ash) sequence. Virl is possibly a promoter-up mutation and is located near or within the replication origin, which is about 400 bp long and AT rich. A sidl mutation is amber that shortens the sid protein by 9 amino acids. The delta gene may encode a 17 kd protein and appears to be coupled with the sid gene translationally. In the 5' side of the sid gene a sequence of CACAAT is the best TATA-like sequence. Sequences of two possible genes that are previously unrecognized and part of the alpha and psu genes are also identified.

Amino Acid Sequence↗