Pseudomonas aeruginosa bacteriophages with DNA structure similar to the DNA structure of Mu1 phage. II. Evidence for similarity between D3112, B3, and B39 bacteriophages: analysis of DNA splits by restriction endonucleases, isolation of D3112 and B3 recombinant phages

It is found that bacteriophages B3 and B39 specific for Pseudomonas aeruginosa have the same genome structure as previously described phage D3112. On the right (S) end of their genomes a variable non-phage DNA is located (approximately 0.9-2.5 kilobases for different phages). It is probable that this variable DNa has its origin from different regions of bacterial chromosome. In genome of one of the phages, B3 phage, such variable DNA (not more than 150 base pairs) was found on the left end of DNA molecule. Isolation of a viable B3XD3112 recombinant phage and analysis of its genome with restriction technique and with studies of homo- and heteroduplex molecules had confirmed genetical relationship of B3 and D3112. Some essential non-homology of B3 and D3112 DNAs have been found on the right ends of genomes of the phages.     

Singlet oxygen mutagenicity induced in the lac operon

We have studied the specificity of singlet oxygen (1O2) mutagenesis in single-stranded DNA phage by analysing 1O2-induced mutations in the lac insert of the M13 mp 19 hybrid phage. 107 lac mutants were analysed showing mainly single-base substitutions with a total of 93% and 7% of 40-50 base deletion mutations. Most of the substitutions are G----T and C----A transversions with respectively 27 and 54% of the mutations. The replicative form of the M13 mp 19 DNA (RFDNA) was used as substrate for the 1O2 reactions, there are then two types of progeny phages DNA's. As guanine residues are the targets of the oxidation, it appears that both types of transversions are provided by one type of lesion: the guanine oxidised by 1O2 is read like a thymine by E. coli DNA polymerase-I.     

Use of a rapid DNA sequencing system to demonstrate the induction of frameshift mutations by bleomycin

The rapid DNA sequencing system based on the single-stranded bacteriophage M13 and the chain-terminator method has been used to look directly for mutational alterations. A small DNA fragment that primes DNA synthesis through the N-terminal 200 base pairs of the beta-galactosidase gene was prepared, and used to detect changes in base sequence among phages that give white plaques after treatment of the host cells with bleomycin. Bleomycin treatment of E. coli in which M13 mp2 was growing gave an increase in white plaque frequency. DNA sequence analysis of phage from 7 independent mutant plaques showed them all to have a frameshift mutation.     

Influence of base sequence on the stability of the double helix of DNA.

On the basis of published measurements of the melting transitions of synthetic polydeoxyribonucleotides with known sequences we have determined the parameters of the interplane (stacking) interactions of base pairs in DNA over the range of ionic strengths from 0.01 to 0.1 M Na+. We found that deviations of the stacking-interaction energy from the mean value of 7-8 kcal/mole were extremely small and did not exceed 0.2 kcal/mole. We report an analysis of the influence of the heterogeneity of the stacking interactions on the melting parameters of polynucleotides with random sequences (models of natural DNA's). Inclusion of this effect does not significantly distort the linear dependence of the melting temperature on the relative content of G-C pairs and insignificantly affects the width of the helix-coil transition in DNA under normal conditions. However it is the heterogeneity of the stacking interactions that plays the crucial role in the melting of DNA under conditions where the difference between the relative stabilities of the A-T and G-C pairs tends to zero, as in concentrated solutions of tetraethylammonium and tetramethylammonium salts.     

Genetic characteristics and genome structure of Streptomyces coelicolor actinophages]

Actinophage phi C31 of Streptomyces coelicolor A3 (2) and two novel temperate actinophages phi C43 and phi C62 isolated from strains of blue actinomycetes group are homoimmune, serologically and functionally related. DNA molecules of phages phi C31, phi C43 and phi C62 have cohesive ends; sizes of DNAs of these phages and some mutants have been determined. The extent of homology between the DNAs of three phages is 93-96% as shown by heteroduplex analysis. The regions of non-homology are of a deletion-insertion type and of approximately 1500 base pairs in the length. Location of deletions in DNAs of mutant phages phi C31 vd and phi C31 c5 has been shown. Structural modifications in phage dnas have been found only to occur in the right part of molecules. Heteroduplex maps have been constructed for all phages studied.     

Study of DNA melting in the region of the inversion of relative stability of AT and GC pairs]

Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.     

Thermodynamics of DNA containing very stable chemically modified base pairs

DNA chemical modifications caused by the binding of some antitumor drugs give rise to a very strong local stabilization of the double helix. These sites melt at a temperature that is well above the melting temperatures of ordinary AT and GC base pairs. In this work we have examined the melting behavior of DNA containing very stable sites. Analytical expressions were derived and used to evaluate the thermodynamic properties of homopolymer DNA with several different distributions of stable sites. The results were extended to DNA with a heterogeneous sequence of AT and GC base pairs. The results were compared to the melting properties of DNA with ordinary covalent interstrand cross-links. It was found that, as with an ordinary interstrand cross-link, a single strongly stabilized site makes a DNA's melting temperature (T(m)) independent of strand concentration. However in contrast to a DNA with an interstrand cross-link, a strongly stabilized site makes the DNA's T(m) independent of DNA length and equal to T(infinity), the melting temperature of an infinite length DNA with the same GC-content and without a stabilized site. Moreover, at a temperature where more than 80% of base pairs are melted, the number of ordinary (non-modified) helical base pairs (n) is independent of both the DNA length and the location of the stabilized sites. For this condition, n(T) = (2 omega-a)S/(1-S) and S = exp[DeltaS(T(infinity)-T)/(RT)] where omega is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and DeltaS, T, and R are the base pair entropy change, the temperature, and the universal gas constant per mole. The above expression is valid for a temperature interval that corresponds to n<0.2N for omega=1, and n<0.1N for omega>1, where N is the number of ordinary base pairs in the DNA chain.     

Characterization and comparison of virulent bacteriophages of Streptococcus thermophilus isolated from yogurt

Seven virulent bacteriophages of Streptococcus thermophilus were characterized at the molecular level and classified into 2 subgroups (A and B) by DNA/DNA hybridization experiments and analysis of their structural proteins. Two representatives of subgroups A and B were compared to 3 representatives of Neve's subgroups I, II and III (Neve et al, 1989) by Southern blot experiments. These isometric-headed phages possess a double-stranded DNA genome varying between 30-44 kilobase (kb) pairs. Subgroup A is composed of 3 phages (phi 57 as representative) with similar structural proteins as determined by sodium dodecyl sulfate-poly-acrylamide gel (SDS-PAGE) electrophoresis (estimated molecular weights of 31,000 and 27,500 for phage phi 57 and 32,000 and 27,000 for the 2 others). A common structural protein of 43,000 was found for phages of subgroup B. Phages phi 57 (subgroup A) and a10/J9 or PO (Neve's subgroups I or II, respectively) belonged to the same subgroup as determined by DNA/DNA hybridization experiments. Partial DNA homology was detected among all the phages tested except for phage phi ST27 of AW Jarvis. Phage-host interactions were also investigated by cross-propagation of the 7 studied phages on different indicator strains. A complete lack of correlation existed between the DNA homology grouping of the phages and their host range. Various restriction-modification systems were detected in some of the Streptococcus thermophilus strains.     

Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.     

Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D

Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases. The nucleic acids isolated were not digested by RNase A, but were digested by DNase I and exonuclease III, indicating that they were double-stranded DNA. On the basis of the restriction endonuclease digestion patterns on 0.8% agarose gel electrophoresis, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 kilobase pairs and that of c-203 and d-1873 was about 150 kilobase pairs. The digestion patterns of c-st, c-468, and c-n71 phage DNAs by PstI and HindIII were very similar. High homology was observed in the dot hybridization test. For other phages and nucleases, a good similarity was not observed. Only a little similarity was observed between c-203 and c-d6f phages. The existence of the structural genes for the toxin in both c-st and c-n71 phages was confirmed by the hybridization test with these phage DNAs and the oligonucleotide probe which represented the DNA sequence predicted for the N-terminal amino acids (2 to 17) of C. botulinum type C toxin. The loss of the converting ability of c-n71 phage may be caused not by the deletion of the tox+ gene but rather by the base mutation in c-st phage DNA.     

Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D.

Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases. The nucleic acids isolated were not digested by RNase A, but were digested by DNase I and exonuclease III, indicating that they were double-stranded DNA. On the basis of the restriction endonuclease digestion patterns on 0.8% agarose gel electrophoresis, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 kilobase pairs and that of c-203 and d-1873 was about 150 kilobase pairs. The digestion patterns of c-st, c-468, and c-n71 phage DNAs by PstI and HindIII were very similar. High homology was observed in the dot hybridization test. For other phages and nucleases, a good similarity was not observed. Only a little similarity was observed between c-203 and c-d6f phages. The existence of the structural genes for the toxin in both c-st and c-n71 phages was confirmed by the hybridization test with these phage DNAs and the oligonucleotide probe which represented the DNA sequence predicted for the N-terminal amino acids (2 to 17) of C. botulinum type C toxin. The loss of the converting ability of c-n71 phage may be caused not by the deletion of the tox+ gene but rather by the base mutation in c-st phage DNA.     

Electron microscopic study of the interaction between phages and Bacillus thuringiensis cells

The interaction of phages belonging to different morphological groups with the cells of Bacillus thuringiensis var. galleriae R and S variants was studied. No adsorption of phages Tg11 and Tg18 on the cells of R variant was found upon infection in a liquid medium. What is characteristic of phage Tg11 is that it is predominantly adsorbed at the poles of S variant cells. Phage Tg18 particles are uniformly distributed along the perimeter of S variant cells. Phage Tg13 is adsorbed on the both variant cells. Phage aggregates with the elements of cell walls having a tetrahonal assembly of the subunits can be revealed in phage Tg13 lysates. The size of the subunits is 7 nm and the distance between their centers is 11 nm. A structured element, apparently the T-layer, is involved in the adsorption of phage Tg13 on the cells.     

Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion

A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.     

Classification of virulent bacteriophages of Streptococcus salivarius subsp. thermophilus isolated from yoghurt and Swiss-type cheese

Summary Twelve isometric-headed bacteriophages virulent against Streptococcus salivarius subsp. thermophilus were differentiated into three subgroups by analysis of the phage genomes and the structural proteins. Subgroup I is composed of two phages (P6 and P8) with a genome size of 41.2 and 44.2 kb pairs, respectively, complete DNA homology, and identical protein composition (main proteins of sizes 39.8, 24.0, 14.8 kilodaltons in sodium dodecyl sulphate-polyacrylamide gel electrophoresis). One phage (a10/J9) with low DNA homology to the other phages was classified into subgroup II. Subgroup III consists of nine phages with a genome size of 33.8 to 36.7 kb pairs and two major structural proteins (30.9 and 24.0 kilodaltons, or 30.9 and 26.3 kilodaltons). In general, phages with different host spectra revealed different restriction enzyme patterns, and DNA homologies of various degrees were detected among all phages tested.     

Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry.

To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated. Phage numbers were estimated to be between 3 x 10(9) and 1.6 x 10(10) particles ml of ruminal fluid-1. The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components. A broad region of DNA between 30 and 200 kb was always present on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage. It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages. The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of filamentous phage M13 in protein engineering

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.     

Some Properties of Five New Salmonella Bacteriophages

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C(1). Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.     

DNA relatedness among bacteriophages of the morphological group C3

Six bacteriophages with an elongated head and a short, noncontractile tail were compared by DNA-DNA hybridization, seroneutralization kinetics, mol% G+C and molecular weight of DNA, and host range. Three phage species could be identified. Phage species 1 containedEnterobacter sakazakii phage C2,Erwinia herbicola phages E3 and E16P, andSalmonella newport phage 7–11. These phages had a rather wide host range (4 to 13 bacterial species). DNA relatedness among species 1 phages was above 75% relative binding ratio (S1 nuclease method, 60°C) when labeled DNA from phage C2 was used, and above 41% when labeled DNA from phage E3 was used. Molecular weight of DNA was about 58×10⁶ (C2) to 67 ×10⁶ (E3). The mol% G+C of DNA was 43–45. Anti-C2 serum that neutralizes all phages of species 1 does not neutralize phages of the other two species. Species 2 contains only coliphage Esc-7-11, whose host range was only oneEscherichia coli strain out of 188 strains of Enterobacteriaceae studied; it was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage Esc-7-11 had a base composition of 43 mol% G+C and a molecular weight of about 45×10⁶. Species 3 contains onlyProteus mirabilis phage 13/3a. Its host range was limited to swarmingProteus species. Species 3 was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage 13/3a had a base composition of 35 mol% G+C and molecular weight of about 53×10⁶. It is proposed that phage species be defined as phage nucleic acid hybridization groups.     

Melting of crosslinked DNA: VI. Comparison of influence of interstrand crosslinks and other chemical modifications formed by antitumor compounds on DNA stability

A computer modeling of thermodynamic properties of a long DNA of N base pairs that includes omega interstrand crosslinks (ICLs), or omega chemical modifications involving one strand (monofunctional adducts, intrastrand crosslinks) has been carried out. It is supposed in our calculation that both types of chemical modifications change the free energy of the helix-coil transition at sites of their location by deltaF. The value deltaF>0 corresponds to stabilization, i.e., to the increase in melting temperature. It is also taken into account that ICLs form additional loops in melted regions and prohibit strand dissociation after full DNA melting. It is shown that the main effect of interstrand crosslinks on the stability of long DNA's is caused by the formation of additional loops in melted regions. This formation increases DNA melting temperature (T(m)) much stronger than replacing omega base pairs of AT type with GC. A prohibition of strand dissociation after crosslinking, which strongly elevates the melting temperature of oligonucleotide duplexes, does not influence melting behavior of long DNA's (N>/=1000 bp). As was demonstrated earlier for the modifications involving one or the other strand, the dependence of the shift of melting temperature deltaT(m) on the relative number of modifications r = omega/(2N) is a linear function for any deltaF, and deltaT(m)(r) identical with 0 for the ideal modifications (deltaF=0). We have shown that deltaT(m)(r) is the same for periodical and random distribution if the absolute value of deltaF is less 2 kcal. The absolute value of deltaT(m)(r) at deltaF>2 kcal and deltaF<-2 kcal is higher for periodical distribution. For interstrand crosslinks, the character of the dependence deltaT(m)(r) is quite different. It is nonlinear, and the shape of the corresponding curve is strongly dependent on deltaF. For "ideal" interstrand crosslinks (deltaF=0), the function deltaT(m)(r) is not zero. It is monotone positive nonlinear, and its slope decreases with r. If r<0.004, then the entropy stabilizing effect of interstrand crosslinking itself exceeds the influence of a distortion of the double helix at sites of their location. The resulting deltaT(m)(r) is positive even in the case of the infinite destabilization at sites of the ICLs (deltaF--> -infinity). In general, stabilizing influence of interstrand crosslinks is almost fully compensated for by local structural distortions caused by them if 0相似文献   

DNA heteroduplex analysis of the relation between bacteriophages phiX174 and S.13

When observed in the electron microscope using the isodenaturing methods of Davis &; Hyman (1971), only one small segment (4.7 ± 1.9%) of the DNA of phage φX174 is highly homologous with phage S.13 DNA; the rest is partially homologous with an over-all average 36% base mismatch. The two phage DNA molecules appear to be identical in length and have no regions of complete base non-homology. The phage-coded proteins were compared by electrophoresis on slab polyacrylamide gels and only one of the S.13 coded proteins migrated identically with its φX174 counterpart. The other eight S.13 coded proteins varied in size from their φX174 counterparts by +4.6% to ?6.0% (± ten amino acid residues). The relevance of these data to the complementation and recombination between these two phages is discussed.