Mechanism of Replication of φX174 Single-Stranded DNA IX. Requirement for the Escherichia coli dnaG Protein

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage phiX174 by P1 transduction. phiX174 reproduces in this strain at 30 C but not at 40 C. All three stages of phiX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of phiX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.     

Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides

The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.     

Requirement of Protein Synthesis for Bacteriophage φX174 Superinfection Exclusion

Within 5 to 10 min at 37 C, bacteria infected by bacteriophage phiX174 acquire the ability to exclude superinfecting phiX particles from reproducing themselves. The superinfecting phage are blocked at a stage prior to synthesis of the parental replicative form molecule; the superinfecting deoxyribonucleic acid remains as intact (infective) single strands. Establishment of superinfection exclusion and its maintenance require protein synthesis.     

Recognition of receptor lipopolysaccharides by spike G protein of bacteriophage phiX174

The spike G protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the phiX174-sensitive strains, and did not bind to LPSs of the phiX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.     

Location of the 5-Methylcytosine Group on the Bacteriophage φX174 Genome

Bacteriophage phiX174 DNA was labeled in vivo with [methyl-(3)H]methionine. The methyl-labeled progeny DNA was extracted from purified bacteriophage phiX174 particles and was used as template for in vitro synthesis of the complementary strand in the presence of the nucleoside triphosphates and Escherichia coli polymerase I. The resultant replicative form DNA was then cleaved, in separate experiments, with restriction endonucleases from Haemophilus influenzae and H. aegyptius. The DNA fragments were analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methylcytosine in the viral DNA is located in a specific region of the phiX174 genome, very likely in gene H.     

Growth and reduction of microorganisms in sediments collected from a greywater treatment system

AIMS: To study the effects of competitive microbiota, temperature and nutrient availability on Salmonella, Enterococcus, Campylobacter spores of sulphite reducing anaerobes and bacteriophages MS2 and phiX174 in sediments from a greywater treatment system. METHODS AND RESULTS: Standard culture methods were used. Bacteria died off rapidly under normal conditions (20 degrees C, competitive microbiota) but remained stable or grew in the other conditions studied. When the sediments became nutrient depleted after 2 weeks, a log-linear die-off was observed for Salmonella, which was higher at 20 degrees C than at 4 degrees C. Bacteriophage decay was shown to be log-linear from day 0, with T90 values ranging from 9 (phiX174, 20 degrees C) to 55 days (phiX174, 4 degrees C). The MS2 phage had a significantly higher decay rate in tyndallized sediments (T90 = 17 days) than in original sediments (T90 = 47 days) (P < 0.001), with temperature not shown to affect the decay rate. Spores of sulphite-reducing anaerobes were not significantly reduced during the study period (35 days). Campylobacter died-off rapidly or entered a viable but non-culturable state and subsequently results were not provided. CONCLUSIONS: Competition was the most important factor to suppress pathogenic bacterial growth in an eutrophic environment. When nutrient depleted conditions prevailed, temperature was more important and log-linear decay of microorganisms could be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the normally occurring microbiota will suppress pathogenic bacterial growth in nutrient rich sediments. With lower nutrient status, temperature is the more important factor in reducing pathogens.     

Rifampin inhibition of bacteriophage phiX174 parental replicative-form DNA synthesis in an Escherichia coli dnaC mutant.

The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage phiX174 parental replicative-form synthesis (Kranias and Dumas, 1974). However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, phiX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in phiX174 parental replicative-form DNA synthesis, presumably in its initiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of phiX174 DNA are discussed.     

In vivo methylation of bacteriophage phi X174 DNA.

A mutant (designated mec(-)) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage lambda against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec(-) mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec(+) and C mec(-) cells infected with bacteriophage phiX174. phiX. mec(-) RFI is sensitive to in vitro cleavage by R.EcoRII and is cut twice to produce two fragments of almost equal size. In contrast, phiX.mec(+) RFI is relatively resistant to in vitro cleavage by R.EcoRII. R.BstI, which cleaves mec(+)/RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R. EcoRII. These results demonstrate that phiX.mec(+) RFI is methylated in vivo by the host mec(+) enzyme and that this methylation protects the DNA against cleavage by R.EcoRII. This is consistent with the known location of two mec(+)/ RII sequences (viz., [Formula: see text]) on the phiX174 map. Mature singlestranded virion DNA was isolated from phiX174 propagated in C mec(+) or C mec(-) in the presence of l-[methyl-(3)H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that phiX.mec(+) DNA had a 10-fold-higher ratio of [(3)H]5-methylcytosine to [(3)H]cytosine compared to phiX.mec(-). Since phiX.mec(+) contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of phiX174 is mediated by the host mec(+) enzyme only. These results are not consistent with the conclusions of previous reports that phiX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.     

Ultraviolet-Induced Cross-Links in the Deoxyribonucleic Acid of Single-Stranded Deoxyribonucleic Acid Viruses as a Probe of Deoxyribonucleic Acid Packaging

Deoxyribonucleic acid (DNA) from ultraviolet (UV)-irradiated phiX174 sediments in alkali at rates up to 1.7 times that of unirradiated phiX174 DNA and is observed as a condensed, cross-linked structure when examined in the electron microscope by the formamide spreading technique. This structure appears to result from multiple cross-links induced in the tightly coiled DNA contained within the spherical phiX174 capsid. In contrast, the DNA extracted after UV irradiation of the filamentous bacteriophage M13 is not strikingly altered in its sedimentation properties and appears by electron microscopy to be rod-shaped as a result of side-to-side association of the circular DNA. The differences in these UV-induced structures reflect the differences in the packaging of the single-stranded DNA in the two virions.     

Specific Fragments of φX174 Deoxyribonucleic Acid Produced by a Restriction Enzyme from Haemophilus aegyptius, Endonuclease Z

A restriction-like enzyme has been purified from Haemophilus aegyptius. This nuclease, endonuclease Z, produces a rapid decrease in the viscosity of native calf thymus and H. influenzae deoxyribonucleic acids (DNA), but does not degrade homologous DNA. The specificity of endonuclease Z is different from that of the similar endonuclease isolated from H. influenzae (endonuclease R). The purified enzyme cleaves the double-stranded replicative form DNA of bacteriophage phiX174 (phiX174 RF DNA) into at least 11 specific limit fragments whose molecular sizes have been estimated by gel electrophoresis. The position of these fragments with respect to the genetic map of phiX174 can be determined by using the genetic assay for small fragments of phiX174 DNA.     

Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.     

Microviridae, a family divided: isolation, characterization, and genome sequence of phiMH2K, a bacteriophage of the obligate intracellular parasitic bacterium Bdellovibrio bacteriovorus.

A novel single-stranded DNA phage, phiMH2K, of Bdellovibrio bacteriovorus was isolated, characterized, and sequenced. This phage is a member of the Microviridae, a family typified by bacteriophage phiX174. Although B. bacteriovorus and Escherichia coli are both classified as proteobacteria, phiMH2K is only distantly related to phiX174. Instead, phiMH2K exhibits an extremely close relationship to the Microviridae of Chlamydia in both genome organization and encoded proteins. Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies. These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages.     

Heterologous transfection with bacteriophage phiX174 DNA: and improved system.

A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage phiX174 DNA was established. By mild washing of the speroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of phiX174 progeny phage production and not on those of phiX174 dougle-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage phiX174 DNA can be detected and the number of molecules can be determined with accuracy.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.     

Evolution of bacteriophage phi C174. V. Alignment of the phi X174, G4, and St-1 restriction enzyme cleavage maps

The restriciton enzyme cleavage maps of bacteriophage phiS174, G4, and St-1 were aligned by two-dimensional filter hybridization. These studies show that the basic genome structure of phiX174 is conserved in the other two bacteriophage. However, the data also suggest the existence of regions of nonhomology.     

Effect of freezing modes on the survival of Escherichia coli bacteriophages

The object of this work was to study the effect of freezing down to--196 degrees C at different cooling and warming rates on the survival of T3, T4 and phiX174 phages. Phage particles survived when T3 phage was frozen at a rate of 20-400 degrees/min and phiX174 phage at a rate of 20-45 degrees/min. The survival rate of T4 phage was highest when it was frozen at a rate of 45 degrees/min. The survival of the phages depended also on the regime of warming. The susceptibility of the phages to freezing correlated with their sensitivity to osmotic shock in NaCl and sucrose solutions.     

Cold-Sensitive Mutants of Bacteriophage φX174. II. Comparison of Two Cold-Sensitive Mutants

Cold-sensitive bacteriophage phiX174 mutants, another class of conditional lethals, were examined with regard to growth parameters, DNA synthesis, and particle properties. Two mutants, cs70 and cs82, were examined. Mutant cs70 was eclipse defective, showing altered eclipse kinetics at permissive temperature (40 C) and failing entirely to eclipse at restrictive temperature (25 C). Mutant cs70 replicated well at 25 C if allowed prior eclipse at 40 C. Mutant cs82 had wild-type eclipse at both temperatures but was defective in single-strand synthesis at 25 C, which led to delayed progeny phage appearance, decreased progeny phage synthesis rate, and greatly reduced burst size. The cs82 block could not be bypassed by temperature shift. Since complementation analysis of cs70 and cs82 was not feasible due to the unique properties of these mutants, those phiX174 properties affected by the virus coat were examined as an index of a mutation in a coat protein gene. Mutant cs70 had aberrant attachment kinetics at both 25 C and 40 C, evidence of a coat protein alteration. Mutant cs70 also exhibited significantly decreased thermal stability, further evidence of an altered virus structure. Mutant cs82 had increased thermal stability, but the difference was not sufficient to allow unequivocal assignment of this mutant to a coat protein gene. Both mutants had wild-type antiserum inactivation and host range, although cs70 was subject to less of (low-level) plating restriction by endogenous F(+) factors.     

Synthesis of complex forms of bacteriophage phiX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli C.

Fast-sedimenting forms of bacteriophage phiX174 double-stranded replicative-form DNA observed in normal infections continued to accumulate at the nonpermissive temperature in a temperature-sensitive dnaC mutant of Escherichia coli. These complex molecules accounted for up to half of the DNA synthesized during short pulses at the nonpermissive temperature. They were the dead-end products of DNA synthesis, not intermediates in normal replicative-form replication. The data suggest that these higher-than-normal-molecular-weight DNA molecules result from abnormal initiation of phiX174 replicative-form DNA replication.     

Heterologous transfection with bacteriophage phiX174 DNA. Unusual heterogeneous products.

Hydroxylamine-resistant infectious materials (HARIM) synthesized in natural non-host and progeny phage low productive bacterial spheroplasts upon transfection with bacteriophage phiX174 DNA were found to be unusually heterogeneous in their forms. Using Pseudomonas aeruginosa as a source of HARIM, it was shown that they have the following unusual features. (1) Almost all of the HARIM are denser than normal single-stranded (SS)- and double-stranded replicative form (RF)-DNAs of phiX174 found usually in the phage-infected host cells. (2) A great part of these heavy HARIM (approximately 84%) contain a variable length of single-stranded RNA associated with their infectious elements. (3) For most of the HARIM (approximately 80% of total molecules as the infectious elements of the heavy HARIM), the infectious elements are phiX-RFI-DNA. The wide-spread system for phiX-HARIM synthesis was shown to be present in many gram-negative bacterial cells.