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PubMed2003

Mechanisms of replication and telomere resolution of the linear plasmid prophage N15.

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularizes via cohensive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). Purified protelomerase alone processes circular and linear plasmid DNA containing the target site telRL to produce linear double-stranded DNA with covalently closed ends in vitro. N15 protelomerase is necessary for replication of the linear prophage through its action as a telomere-resolving enzyme. Replication of circular N15-based miniplasmids requires the only gene repA that encodes multidomain protein homologous to replication proteins of bacterial plasmids replicated by theta-mechanism, particularly, phage P4 alpha-replication protein. Replication of the N15 prophage is initiated at an internal ori site located within repA. Bidirectional replication results in formation of the circular head-to-head, tail-to-tail dimer molecule. Then the N15 protelomerase cuts both duplicated telomeres generating two linear plasmid molecules with covalently closed ends. The N15 prophage replication thus appears to follow the mechanism distinct from that employed by poxviruses and could serve as a model for other prokaryotic replicons with hairpin ends, and particularly, for linear plasmids and chromosomes of Borrelia burgdorferi.

Coliphages↗
PubMed1971

Viricidal activity of open air.

Airborne Semliki Forest virus and T coliphages were inactivated at a considerably enhanced rate in open air compared with enclosed air. Open air exerts its maximum sterilizing activity on viruses contained in the smallest sized particles.

Aerosols↗
PubMed1972

Host specificities of RNA phages.

Host ranges of members of four groups of male-specific RNA coliphages were determined by plating on hosts carrying various derepressed plasmids. An RNA phage originally isolated on Pseudomonas aeruginosa failed to form plaques on any of the strains of Escherichia coli.

Coliphages↗
PubMed1980

Inactivation of viruses in municipal effluent by chlorine.

The influence of pH and temperature on the efficiency of chlorine inactivation of two unrelated picornaviruses in a typical urban wastewater effluent was examined. Temperature, unlike pH, had relatively little effect on the rate of inactivation. The pH effect was complex and the two viruses differed. The f2 coliphage was more sensitive to chlorine at low pH, but at all values there was a threshold above which additional chlorine resulted in very rapid inactivation. The amount of chlorine required for this was less at low than at high pH, although at pH values above 7 the extent of inactivation was about the same. There was no apparent correlation between pH and rate of inactivation of poliovirus but there was a suggestion that at a pH close to the isoelectric point of the virus it was less sensitive to chlorination.

Chlorine↗
PubMed1984

Inactivation of human rotavirus, SA11 and other enteric viruses in effluent by disinfectants.

A preparation of infectious human rotavirus, isolated from faeces and resuspended in wastewater effluent, was shown to be inactivated by chlorine, chlorine dioxide, ozone and peracetic acid. Infectivity was assayed in MA 104 cells by the detection of cell-associated viral antigen by immunofluorescence. The inactivation curves were similar to those reported for other enteric viruses. Human rotavirus was at least as resistant as poliovirus, coxsackievirus, echovirus and f2 coliphage and was strikingly less sensitive to inactivation than the simian rotavirus, SA11. The latter was generally the most sensitive of the six tested viruses yet is often taken as being representative of the human rotaviruses.

Chlorine↗
PubMed1991

Mechanisms of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient RecA-independent mutagenic noninstructional lesion.

It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed1992

Assignment of amide 1H and 15N NMR resonances in detergent-solubilized M13 coat protein: a model for the coat protein dimer.

The major coat protein of the filamentous coliphage M13 is a 50-residue integral membrane protein. Detergent-solubilized M13 coat protein is a promising candidate for structure determination by nuclear magnetic resonance methods as the protein can be prepared in large quantities and the protein-containing micelle is reasonably small. Under the conditions of our experiments, SDS-bound coat protein exists as a dimer with an apparent molecular weight of 27,000. Broad lines and poor resolution in the 1H spectrum have led us to adopt an 15N-directed approach, in which the coat protein was labeled both uniformly with 15N and selectively with [alpha-15N]alanine, -glycine, -valine, -leucine, -isoleucine, phenylalanine, -lysine, -tyrosine, and -methionine. Nitrogen resonances were assigned as far as possible using carboxypeptidase digestion, double-labeling, and an independent knowledge of the amide proton exchange rates determined from neighboring assigned 13C-labeled carbonyl carbons. 1H/15N heteronuclear multiple quantum coherence (HMQC) spectroscopy of both uniform and site-selectively-labeled proteins subsequently correlated amide nitrogen with amide proton chemical shifts, and the assignments were completed sequentially from homonuclear NOESY and HMQC-NOESY spectra. The most slowly exchanging amide protons were shown to occur in a continuous stretch extending from methionine-28 to phenylalanine-42. This sequence includes most of the resonances of the hydrophobic core, although it is shifted toward the C-terminal end of the protein. Strong NH to NH (i,i+1) nuclear Overhauser enhancements are a feature of the coat protein, which appears to be largely helical. Between 20 and 25 residues give rise to 2 juxtaposed resonances which can be seen clearly in the HMQC spectrum of uniform 15N-labeled coat protein. These residues are concentrated in a region extending from the beginning of the membrane-spanning sequence through to the disordered region near the C-terminus. We propose that dodecyl sulfate-bound M13 coat protein consists of two independent domains, an N-terminal helix which is in a state of moderately fast dynamic flux and a long, stable, C-terminal membrane-spanning helix, which undergoes extensive interactions with a second monomer. Amide 1H chemical shifts are consistent with this picture; in addition, a marked periodicity is observed at the C-terminal end of the molecule.

Amides↗
PubMed1986

Backbone dynamics of a model membrane protein: 13C NMR spectroscopy of alanine methyl groups in detergent-solubilized M13 coat protein.

The filamentous coliphage M13 possesses multiple copies of a 50-residue coat protein which is inserted into the inner membrane of Escherichia coli during infection. 13C nuclear magnetic resonance (NMR) spectroscopy has been used to probe the structure and dynamics of M13 coat protein solubilized in detergent micelles. A comparison of backbone dynamics within the hydrophobic core region and the hydrophilic terminal domains was obtained by biosynthetic incorporation of [3-13C]alanine. Alanine is distributed throughout the protein and accounts for 10 residues (i.e., 20% of the total). Similar 13C NMR spectra of the protein have been obtained in two anionic detergents, sodium deoxycholate and sodium dodecyl sulfate, although the structures and physical properties of these solubilizing agents are quite different. The N-terminal alanine residues, assigned by pH titration, and the penultimate residue, assigned by carboxypeptidase A digestion, give rise to analogous peaks in both detergent systems. The pKa of Ala-1 (approximately 8.8) and the relaxation parameters of individual carbon atoms (T1, T2, and the nuclear Overhauser enhancement) are also generally similar, suggesting a similarity in the overall protein structure. Relaxation data have been analyzed according to the model-free approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall correlation times were obtained by fitting the three experimental relaxation values for a given well-resolved single carbon atom to obtain a unique value for the generalized order parameter, S2, and the effective correlation time, tau e. The former parameter reflects the spatial restriction of motion, and the latter, the rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1990

Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: use of the INEPT experiment to follow individual amides in detergent-solubilized M13 coat protein.

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1H nuclear magnetic resonance (NMR) study [O'Neil, J. D. J., & Sykes, B. D. (1988) Biochemistry 27, 2753-2762], multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow "kinetic sets" containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10(5)-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein we use 15N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiment [Morris, G. A., & Freeman, R. (1979) J. Am. Chem. Soc. 101, 760-762] can be used to transfer magnetization to the 15N nucleus from a coupled proton; when 15N-labeled protonated protein is dissolved in 2H2O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H+ and OH- ions. Base catalysis is significantly more effective, resulting in a characteristic minimum rate in model peptides at pH approximately equal to 3. Rate versus pH profiles have been obtained by using the INEPT experiment for the amides of leucine-14, leucine-41, tyrosine-21, tyrosine-24, and valines-29, -30, -31, and -33 in M13 coat protein. The valine residues exchange most slowly and at very similar rates, showing an apparent 10(6)-fold retardation over poly(DL-alanine). A substantial basic shift in the pH of the minimum rate (up to 1.5 pH units) was also observed for some residues. Possible reasons for the shift include accumulation of catalytic H+ ions at the negatively charged micelle surface or destabilization of the negatively charged transition state of the base-catalyzed reaction by either charge or hydrophobic effects within the micelle. The time-dependent exchange-out experiment is suitable for slow exchange rates (kex), i.e., less than (1-2) x 10(-4) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1976

Physicochomecial studies on interactions between DNA and RNA polymerase. Isolation and mapping of a T7 DNA fragment containing the early promoters for Escherichia coli RNA polymerase.

The cleavage sites in the early promoter region of coliphage T7 have been mapped for four restriction enzymes. They are, from the left end in base pairs, 1100 and 740 for Hinf; 680, 320, 530, 240, 77, and 67 for Hind II; 620 and 530 for Hpa II; 790 for Alu I. The nucleotide sequence between the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end has been determined, from which the start point of the promoter A3 is located at 720 base pairs from the left end. The start points of the other two major promoters A1 and A2 are deduced to be at 460 and 580 base pairs from the left end, respectively, from the chain lengths of the in vitro transcripts off the 1100 base-pairs long Hinf fragment. Similar to the sequences of a pL and pR promotors of phage lambda and a sequence in Simian Virus 40 used by Escherichia coli RNA polymerase as a promotor, the sequence of the A3 promotor of T7 also has a Hind II restriction site approximately 30 base pairs upstream to the start point of RNA synthesis. No such Hind II sites exist, however, for the A1 and A2 promoters. Experiments on the protection of some of the restriction sites on the 1100 base-pairs-long Hinf fragment by RNA polymerase binding support the electron microscopic observations of others that, in addition to the three sites A1, A2 and A3, there is at least a fourth site at which E. coli RNA polymerase can bind strongly. In addition to the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end, which are presumably protected by the binding of a single RNA polymerase at the A3 site, the Hind II site at 240 base pairs from the left end is also protected at a level of 5 polymerase molecules/DNA fragment. The possible existence of several minor promotor sites in the early promotor region, in addition to the three major promotor sites, is discussed.

Base Sequence↗
PubMed2004

Domain structures and roles in bacteriophage HK97 capsid assembly and maturation.

Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.

Asparagine↗
PubMed1980

Recovery frequency of phages lambda and M13 from human and animal faeces.

Derivatives of bacteriophages lambda and M13 are in common use as vectors in recombinant DNA RESEARCH. These laboratory-derived phages have been designed to allow cloning of DNA fragments, but to be unable to survive outside a defined laboratory and/or host-cell environment. To assess the availability of wild-type lambda or M13 phages in the environment which might potentially rescue debilitated derivative phages, we have now examined the frequency of these and other bacteriophages in human and animal faeces. We detected coliphage in over two-thirds of the faecal samples. Of these, 1.2% of the samples contained lambda-like phage and 3.5% had phage indistinguishable from M13.

Animals↗
PubMed1972

The amino acid sequence of the B-protein of bacteriophage ZJ-2.

1. Bacteriophage ZJ-2 is a filamentous male-specific coliphage. Methods are given for its preparation and separation into nucleic acid and the major coat-protein component (B-protein). 2. The determination of the amino acid sequence of the B-protein of bacteriophage ZJ-2 is described. The principal features of this aspect of the work were the special procedures dictated by the insolubility of the protein and the extreme insolubility of its fragments. The sequence is compared with that obtained by other workers for the coat protein of the related bacteriophage fd.

Acylation↗
PubMed1996

Recognition mechanisms of the minus-strand origin of phage f1 by Escherichia coli RNA polymerase.

BACKGROUND: The primer RNA for the synthesis of the minus strand of filamentous coliphages is produced by host RNA polymerase at a specific site on the plus strand template. The mechanism used by the enzyme in recognizing the origin is unknown, but minus strand replication requires the holoenzyme form of RNA polymerase. The origin contains two inverted repeats which can form hairpins. RESULTS: When the origin sequence is drawn arranging the two hairpins horizontally, it resembles a stretch of a mostly double-stranded molecule. The nucleotides protected from nucleases by the holoenzyme were found to be located in two regions on this drawing: one around the 35th nucleotide upstream of the RNA start site, and the other ranging from the 10th nucleotide upstream to the 10th downstream, of the start site. The core enzyme did not show any protection. Mutational analyses of the origin indicated that the base-paired structure in the former region was important for origin activity. In the region around the 10th nucleotide upstream of the start site, specific base(s) in the non-template strand were required for origin activity, while the base sequence of the template strand was irrelevant. CONCLUSIONS: It is likely that the recognition mechanism of the origin by RNA polymerase shares common features with that of transcriptional promoters.

Base Sequence↗
PubMed1999

Occurrence and numbers of bacteriophages and bacterial indicators in faeces of yellow-legged seagull (Larus cachinnans).

Faeces from feral populations of yellow-legged seagulls from the northern coastal area of Catalonia (North-eastern Spain) contained variable amounts of faecal coliforms, faecal streptococci, somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. Occurrence and numbers of bacterial indicators and bacteriophages in the faeces of yellow-legged seagulls are in the ranges described in the faeces of different animals. The ratios between numbers of bacterial indicators and numbers of bacteriophages are much higher in faeces of seagulls than in treated or raw sewage contributed by out-falls of the same area.

Animals↗
PubMed2000

Bacteriophages as indicators of enteric viruses and public health risk in groundwaters.

Low concentrations of all types of bacteriophages in groundwater limit their power to predict the presence of enteric viruses. There is little concordance in the literature regarding phage detection methods, thus making comparisons extremely difficult. Different authors have used different hosts, phage concentration methods, and end-point determinations. Also, markedly different volumes of sample have been employed, varying from 1 litre to 400 l. Bacteriophage concentration methods are not reproducible. There has been marked variability among groups in the natural substrates used (for example, beef extract), the type of adsorbing filter used, centrifugation instruments and conditions, and the delivery of the concentrate to the host cells. There is no consensus on the best bacterial host strain. Currently, several are employed with each showing differential sensitivities and specificities. In particular, host stability must be considered. Host stability has two components: the ability of the host to continue to be receptive to the bacteriophage after continued sub-culture, and the lack of lysogenic or temperate bacteriophage in the host cell line which may be randomly and unpredictably activated. There is a lack of consistent recovery of bacteriophages from individual faecal specimens. In particular, only approximately 3% of individual humans carry the FRNA phages. While there is some evidence to indicate that the phages multiply in sewage, it is not clear how they do so since the host pili should not be produced at lower temperatures. These ecological factors need to be understood. Of all the phages thus far studied, Bacteroides fragilis HSP40 has the highest recovery rate from individual people. However, Bacteroides, being an anaerobe, is a difficult host for routine laboratory analysis. Methods for the enumeration of F(+)-specific phages and Bacteroides phages are complex, time-consuming, costly and not reproducible. Conversely, somatic coliphage methods are simpler and results can be available in 4-6 h. The occurrence of phages and viruses in groundwater depends on physicochemical characteristics that control their fate and transport in the groundwater/aquifer environment. There are very little actual data taken from the field that allow an understanding of the ecology and life span of phages in their natural environment. Moreover, the ability of phages to serve as a source of food for other microbes needs to be understood. There has been a lack of association of bacteriophage recovery with gastroenteritis outbreaks due to enteric viruses. There is only a small epidemiological database concerning the occurrence of enteric viruses in groundwater.

Animals↗
PubMed2003

Usefulness of different groups of bacteriophages as model micro-organisms for evaluating chlorination.

AIMS: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfection processes based on chlorination. METHODS AND RESULTS: Naturally occurring bacterial indicators, bacteriophages and enteroviruses were determined to evaluate the effect of chlorination in groundwater and secondary sewage effluents. Additionally, the effect of chlorinating on selected bacteriophages, enteroviruses and Escherichia coli was also tested in spiked samples of bottled water and sewage effluents. Results indicate that chlorination inactivates more efficiently bacteria than phages and enteroviruses. Among the human viruses, phages infecting Bacteroides fragilis and selected somatic coliphages belonging to the Siphoviridae family were the most persistent to chlorination. CONCLUSIONS: The three groups of bacteriophages studied were all more resistant to chlorination than bacteria and some of the phages were more resistant than enteroviruses. Results presented here indicate that it is very risky to generalize from information obtained with inactivation experiments done with single isolates of any phage or virus. If possible, inactivation studies should be done with naturally occurring populations. Phages offer a good opportunity for studying naturally occurring populations. Thus, the bacteriophages offer a range of resistance to chlorination that may represent most of the viruses that can be found in water. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported in this study support the inclusion of bacteriophages as additional indicators of the efficiency of water chlorination processes and water quality.

Bacteriophages↗
PubMed1998

Viral escape from antisense RNA.

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.

Antiviral Agents↗