Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

Spontaneous temperature-sensitive mutations in bacteriophage T7.

Attempts to recover temperature-sensitive mutations affecting genes 13 and 14 (virion proteins) in bacteriophage T7 by analysis of amber revertants were confounded by the frequent occurrence of spontaneous temperature-sensitive mutations in other genes. These incidental temperature-sensitive mutations are physically distinct from but may be functionally related to genes 13 and 14, as shown by complementation and recombination studies. The possibility that these incidental temperature-sensitive mutations represent secondary-site suppressors of the pseudonormal suppressed amber products is discussed.     

Bacteriophage tracer experiments in groundwater

Three tracer experiments employing three different bacteriophage were performed at one groundwater site near Beverley, Humberside. In two of the experiments the bacteriophage were injected into the aquifer by a borehole at a distance of 366 m from the pumping borehole. In the other experiment they were injected at a distance of 122 m. Regular samples were taken of water abstracted at the pumping boreholes as well as from the injection boreholes. The objectives were to: (1) investigate the pattern of bacteriophage recovery from the aquifer; (2) calculate the total number of bacteriophage recovered and the rate of their migration; and (3) detect any differences in bacteriophage behaviour which could be directly related to the morphology of the three bacteriophage. In all experiments the pattern of recovery was similar, exhibiting a peak of high numbers reaching the pumping borehole soon after injection. The highest percentage of original inoculum recovered was 1.9%. In the majority of cases, however, recovery was usually one log₁₀ lower than this. The fastest migration rates were very rapid, reaching 2.8 cm/s in one experiment. No variation in percentage recovery or transit time could be directly attributed to morphology of bacteriophage. The most important factor governing the pattern of migration was undoubtedly the hydrogeological conditions.     

Bacteriophage tracer experiments in groundwater

Three tracer experiments employing three different bacteriophage were performed at one groundwater site near Beverley, Humberside. In two of the experiments the bacteriophage were injected into the aquifer by a borehole at a distance of 366 m from the pumping borehole. In the other experiment they were injected at a distance of 122 m. Regular samples were taken of water abstracted at the pumping boreholes as well as from the injection boreholes. The objectives were to: (1) investigate the pattern of bacteriophage recovery from the aquifer; (2) calculate the total number of bacteriophage recovered and the rate of their migration; and (3) detect any differences in bacteriophage behaviour which could be directly related to the morphology of the three bacteriophage. In all experiments the pattern of recovery was similar, exhibiting a peak of high numbers reaching the pumping borehole soon after injection. The highest percentage of original inoculum recovered was 1.9%. In the majority of cases, however, recovery was usually one log10 lower than this. The fastest migration rates were very rapid, reaching 2.8 cm/s in one experiment. No variation in percentage recovery or transit time could be directly attributed to morphology of bacteriophage. The most important factor governing the pattern of migration was undoubtedly the hydrogeological conditions.     

Bacillus subtilis Bacteriophage SP01, SP82, and φe Require Host Lysyl- and Tryptophanyl-tRNA Synthetases for Phage Development

Infection of Bacillus subtilis mutants having temperature-sensitive lysyl- or tryptophanyl-tRNA synthetases with bacteriophage SP01, SP82, or phie indicates that both host enzymes are essential for phage development. No apparent modification of the temperature-sensitive phenotype of the mutant host enzymes occurs during phage infection.     

Intragenic Complementation Between Temperature-Sensitive Mutants of Gene 42 (dCMP Hydroxymethylase) of Bacteriophage T4

Interallelic complementation between certain temperature-sensitive mutants of gene 42 of bacteriophage T4 was demonstrated by measuring the incorporation of labeled thymine into DNA.     

Conditional Temperature-sensitive Restriction of Pseudomonas Bacteriophage CB3

Restriction of Pseudomonas bacteriophage CB3 growth on some Pseudomonas aeruginosa hosts was studied. On restricting hosts, growth of this phage was severely inhibited below 32 C and hence was temperature-sensitive. Investigation of this phenomenon revealed that restricting hosts were not killed as a consequence of their infection under nonpermissive conditions. The ability of some hosts to restrict showed segregation in sexual crosses between restricting and nonrestricting hosts. However, the pattern of restriction among various hosts differed with the phage in question when other phages were compared with CB3. Temperature-shift experiments indicated that blockage of an early event in the phage lytic cycle occurred when restricting conditions were imposed on cells infected with CB3. This blockage could be eliminated by holding at permissive conditions until the cold-sensitive step was bypassed or by pulsing restricting cells for 5 min at 37 C.     

Phenotypic mixing of pyocin R2 and bacteriophage PS17 in Pseudomonas aeruginosa PAO.

Previous results indicate that a group of bacteriocins in Pseudomonas aeruginosa, named R-type pyocins, have a structure resembling bacteriophage tails and share some serological homology with certain bacteriophages. This paper presents genetic evidence which strongly suggests that components of pyocin R2, an R-type pyocin of P. aeruginosa PAO, and tail components of bacteriophage PS17 are interchangeable. Complementation tests with pyocin R2-deficient mutants of PAO and ts mutants of PS17 revealed that various phenotypic interactions occur between the pyocin and bacteriophage in PAO cells lysogenized or infected with PS17. (i) Certain pyocin R2-deficient mutations were phenotypically suppressed in cells carrying PS17 prophage. (ii) A temperature-sensitive mutant of PS17, tsQ31, was phenotypically suppressed in PAO cells treated with mitomycin C. (iii) Phenotypically mixed phages with receptor and serological specificities of pyocin R2 were formed in PS17 lysogens of certain pyocin R2-deficient mutants.     

Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids.

Conjugative temperature-sensitive plasmids were derived from pSC101. These plasmids are useful in genetic analysis for two reasons: (i) they render possible the construction of new Hfr lines by plasmid integration at predetermined chromosomal loci via Tn10 inverse transposition, and (ii) the Hfr characters are transducible via bacteriophage P1. We also showed that replication from pSC101 origin is deleterious for the plasmid-chromosome fusion.     

Distribution of pyrimidine sequences of different length and base composition in bacteriophage T5 DNA]

Distribution of pyrimidine tracts of different length (isopliths) with general formula PynPn+1 in bacteriophage T5 DNA was studied. The first seven isoplith fractions were subfractionated by the chain length and the quantity of the resulting non-isomeric oligonucleotides was determined. The pattern of distribution of pyrimidine tracts of various length and base composition in bacteriophage T5 DNA is different from that previously observed in the DNAs of bacteriophages T3 and T7. The observed differences in distribution of pyrimidine nucleotides are in accordance with the other peculiarities of bacteriophage T5 genome.     

Structural studies of mutants of the lysozyme of bacteriophage T4. The temperature-sensitive mutant protein Thr157----Ile

To understand the roles of individual amino acids in the folding and stability of globular proteins, a systematic structural analysis of mutants of the lysozyme of bacteriophage T4 has been undertaken. The isolation, characterization, crystallographic refinement and structural analysis of a temperature-sensitive lysozyme in which threonine 157 is replaced by isoleucine is reported here. This mutation reduces the temperature of the midpoint of the reversible thermal denaturation transition by 11 deg.C at pH 2.0. Electron density maps showing differences between the wild-type and mutant X-ray crystal structures have obvious features corresponding to the substitution of threonine 157 by isoleucine. There is little difference electron density in the remainder of the molecule, indicating that the structural changes are localized to the site of the mutation. High-resolution crystallographic refinement of the mutant lysozyme structure confirms that it is very similar to wild-type lysozyme. The largest conformational differences are in the gamma-carbon of residue 157 and in the side-chain of Asp159, which shift 1.0 A and 1.1 A, respectively. In the wild-type enzyme, the gamma-hydroxyl group of Thr157 participates in a network of hydrogen bonds. Substitution of Thr157 with an isoleucine disrupts this set of hydrogen bonds. A water molecule bound in the vicinity of Thr155 partially restores the hydrogen bond network in the mutant structure, but the buried main-chain amide of Asp159 is not near a hydrogen bond acceptor. This unsatisfied hydrogen-bonding potential is the most obvious reason for the reduction in stability of the temperature-sensitive mutant protein.     

Gene 19 of bacteriophage T7. Overexpression, purification, and characterization of its product

Gene 18 and 19 proteins of bacteriophage T7 are essential for DNA maturation and packaging. The phage capsid is the site of both maturation and packaging of T7 DNA. Both gene 18 and 19 proteins bind to capsid intermediates during DNA packaging but are not found in mature virions, suggesting that they play a direct role in the enzymatic mechanisms of DNA maturation and packaging. As part of an effort to reconstitute T7 DNA maturation and packaging with purified components, we have cloned and overexpressed T7 gene 19 in Escherichia coli. Gene 19 has been inserted downstream from the bacteriophage PL promoter controlled by the temperature-sensitive lambda repressor encoded by c1857. Upon thermal induction, most of the overproduced gene 19 protein is insoluble and inactive. However, by attenuation of the expression of gene 19 from the PL promoter, significant levels of soluble and active gene 19 protein are produced. Soluble gene 19 protein can be monitored by its ability to complement extracts of T7-infected cells for packaging of exogenous DNA. We have used this assay to monitor the activity of gene 19 protein during purification. The native protein is a monomer of molecular weight 66,000. We have also tested for the formation of a stable complex between gene 18 and 19 proteins. Coproduction of gene 18 and 19 proteins has no effect on either the solubility or activity of gene 19 protein, despite the fact that gene 18 protein is produced at at least 10-fold greater rates. Furthermore, we find no evidence for any interaction between soluble gene 18 and 19 proteins in extracts or between the purified proteins.     

The asparaginyl-tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316.

Escherichia coli strain N4316 is a mutant that exhibits temperature-sensitive growth at 43 degrees C and temperature-sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called 'rescue', isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild-type parental strain; the purified protein is a homodimer of 54 kDa. The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature-sensitive growth phenotype of the mutant at the non-permissive temperatures. The cloned gene was sequenced, mapped to the 20.9-min region of the E. coli chromosome and shown to code for a 466-amino-acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl-tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read-through of nonsense codons and protein copy number is discussed.     

Involvement of DNA gyrase in bacteriophage T7 growth.

We have found that the burst size of bacteriophage T7 was decreased in two Escherichia coli temperature-sensitive gyrase mutants incubated at the restrictive temperature. This reduction in burst size indicates that gyrase may be required for T7 growth.     

Thymidine Triphosphate Nucleotidohydrolase and Deoxyuridylate Hydroxymethylase Induced by Mutants of Bacillus subtilis Bacteriophage SP82G

Assays on extracts of Bacillus subtilis infected by temperature-sensitive mutants of bacteriophage SP82G indicate that thymidine triphosphate nucleotidohydrolase may not be an essential SP82G enzyme and that deoxyuridylate hydroxymethylase is an essential enzyme coded for by SP82G gene 2.     

Structural changes during the transformation of bacteriophage T4 polyheads: II. Evidence that a conformational change in the major capsid protein precedes the expansion

The shell of the bacteriophage T4 prehead is transformed after the maturation cleavages from a fragile to a highly chemically resistant structure. A “cleaved but anchored” shell, in which the capsid protein has been cleaved but expansion to the mature structure has not yet occurred, is thought to be an intermediate in the transformation. We have compared native, trypsinized, temperature-sensitive mutant, and cleaved but anchored polyheads for differences and similarities in their capsomeres. Our results show that the altered capsomeres of the cleaved but anchored state must be attributed to a conformational change in the subunits, and not simply to the loss of the amino-terminal peptide by proteolysis.     

Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein

Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.     

A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.     

Evidence against the reversion of mutation in the Haemophilus influenzae phage HP1c1 by preinfection treatment of host cells or phage with MNNG.

N-Methyl-N'-nitrosoguanidine (MNNG) causes reversion of a temperature-sensitive mutation in a bacteriophage of Haemophilus influenzae if exposure to the mutagen takes place after infection but before lysis. However, neither pre-infection treatment of the phage DNA, host cells, or both will cause reversion. The reasons for this are discussed in relation to the somewhat different results in the Escherichia coli lambda phage system and in relation to error-prone repair and replication processes.