Transmission of amber mutants of bacteriophage T4. III. Thermostability of the replication of amber mutants in cells of a non-permissive host is typical for the majority of phage tail genes

The article deals with determination of the spreading of the earlier discovered phenomenon of the temperature sensitivity of multiplication of T4 phage amber mutants. On the basis of the study of the dependence of multiplication of 50 amber mutants in 22 genes of T4 phage tail in the cells of non-permissive host on the incubation temperature in the range of 15-41 degrees C, the following conclusion is drawn: temperature sensitivity of multiplication of amber mutants appears to be gene-specific and is widely spread among T4 phage genes, i.e. in the case of amber mutants the burst size decreases, even for 14 tail genes, by several orders with the increase in incubation temperature. Temperature sensitivity of multiplication is typical of amber mutants in the genes whose proteins are either of small number in a phage particle (several molecules) or play the role of catalytic factors. Moreover, genes, amber mutants of which possess temperature sensitivity of multiplication, map in defined clusters.     

Transmission of bacteriophage T4 amber mutants. I. Temperature sensitivity of gene 26 T4 phage mutant amN131 multiplication in Escherichia coli B cells]

When studying the single cycle of the multiplication of gene 26 mutant amN131 of phage T4, like in temperature shift experiments, the yield of this mutant in non-permissive host depends greatly on the temperature. The burts size of phage in Escherichia coli B is found to be 3.3 phage particles at 25 degrees C, 1.6 at 30 degrees C, 0.051 at 37 degrees C and 0.0007 at 41 degrees C. In the case of permissive host (E. coli CR-63) the burst size per cell decreases from 158 to 49 phage particles at the same temperature interval. The results of the single-burst experiments indicate, that when the incubation temperature increases, the number of E. coli B cells, in which the phage particles maturate, also decreases. It results in the dependence of the transmission coefficient value on the temperature. The transmission coefficient in the conditions favourable for the maturation of the phage is found to be 0.80. It is shown by several methods that the temperature sensitivity of the multiplication of the mutant amN131 in bacterial cells is entirely due to amber mutation in genome of the phage. Therefore the amber mutants having high temperature sensitivity when maturating in non-permissive host cells exist among ordinary amber mutants of phage T4.     

Transmission of amber mutants in phage T4. V. Positive effect of heat shock proteins on the replication of amber mutants in gene 31

The effect of growth of Escherichia coli BE, prior to infection, on multiplication of double amber mutant amN54-amNG71 in gene 31, mutant amN131-amNG114 in gene 26 and T4D wild-type at different temperatures has been studied. In the case of gene 31 mutant the increase in phage burst size, along with increase in growth temperature, was only observed. And this dependence seems to have the same character as the known dependence of growth temperature on cellular levels of heat shock proteins. Possibly, the product of gene 31 might be substituted to some extent by some heat shock protein. An antiserum against gene 31 protein immunoprecipitates heat shock protein, the molecular weight of which is close to the molecular weight of gene 31 protein. So, it seems likely that, in addition to supposed ability of this heat shock protein for functional substitution of gene 31 protein, these proteins might have some structural homology as well.     

Recombination of amber mutants of bacteriophage T4B. II. Localization of amber mutants on maps of genes 30, 34, 35, 36 and 38]

Localization of 275 amber mutants of five genes of phage T4B (30, 34, 35, 36 and 38) on genetic maps allowed us to determine the recombination length of these genes. Gene 34 substantially differs from the rest studied genes by numbers of amber mutants isolated in each gene and by recombination frequency. In particular, according to the results of crossing the flank markers, the recombination length of gene 34 is 10 times greater than in gene 38; using the summary value of recombination frequencies between elementary intervals a 20-fold excess of the length of gene 34 compared with the length of gene 38 was receieved. Molecular weight of the product of gene 34 is only 6 times as great as in gene 38. An elevated recombination frequency was also detected in gene 35. The data obtained indicate a local recombination anomaly at the region of genes 34--35 of bacteriophage T4 genome.     

Characteristics of the intragenic recombination of T4B bacteriophage amber mutants under nonpermissive (su-) conditions

Intragenic recombination of bacteriophage T4B amber mutants in early genes 30, 32, 42, 43, 44, 56 and in late gene 7 in su- cells of Escherichia coli B was studied. The frequency of recombination under such conditions was increased in genes 30, 43 and 7, but it was lowered in genes 46 and 44, and was completely inhibited in genes 32, 42 and 56. The level of stimulation or inhibition of recombination frequencies in early genes was gene-specific and did not depend either on the distances between amber mutations, or on progeny phage maturation delay. On the other hand, the level of recombination stimulation in the late gene 7 was greatly influenced by the distances between amber mutations tested. Wild type alleles arising in su- cells during recombination proved to be functionally active, and their activity caused the increase in progeny phage yield and in a partial removal of phage maturation delay.     

Isolation and characterization of Escherichia coli dnaA amber mutants.

Specialized transducing phage lambda (formula, see text) dnaA-2 was mutagenized, and two derivatives designated lambda (formula) dnaA17(Am) and lambda (formula) dnaA452(Am) were obtained. They did not transduce such mutations as dnaA46, dnaA167, and dnaA5 when an amber suppressor was absent, but they did so in the presence of an amber suppressor. By contrast, they transduced the dna-806 and tna-2 mutations in the absence of an active amber suppressor. The dna-806 and tna-2 mutations are known to be located very close to the dnaA gene, but in separate cistrons. When ultraviolet light-irradiated uvrB cells were infected with the derivative phages and proteins specified by them were analyzed by gel electrophoresis, a 50,000-dalton protein was found to be specifically missing if an amber suppressor was absent. This protein was synthesized when an amber suppressor was present. The dnaA17(Am) mutation on the transducing phage genome was then transferred by genetic recombination onto the chromosome of an Escherichia coli strain carrying a temperature-sensitive amber suppressor supF6(Ts), yielding a strain which was temperature sensitive for growth and deoxyribonucleic acid replication. The temperature-sensitive trait was suppressed by supD, supE, or supF. We conclude that, most likely, the derivative phages acquired amber mutations in the dnaA gene whose product is a 50,000-dalton protein as identified by gel electrophoretic analysis.     

Genetic studies of coliphage 186. I. Genes associated with phage morphogenesis.

In coliphage 186, 22 essential genes were defined by complementation studies with amber mutants. Eighteen genes were associated with phage morphogenesis: 11 with phage tail formation, and 7 with phage head formation. The remaining four genes are discussed in the accompanying paper (S. M. Hocking and J. B. Egan, J. Virol. 44:1068-1071, 1982).     

Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli.

We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.     

Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin

Orias, E. (University of California, Santa Barbara), and T. K. Gartner. Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin. J. Bacteriol. 91:2210-2215. 1966.-Streptomycin-induced suppression of amber and ochre rII mutants of phage T4 was studied in a streptomycin-sensitive strain of Escherichia coli and four nearly isogenic streptomycin-resistant derivatives of this strain, in the presence and in the absence of an ochre suppressor. Most of the 12 rII mutants tested were suppressed by streptomycin in the streptomycin-sensitive su(-) strain. This streptomycin-induced suppression in the su(-) strain was eliminated by the independent action of at least two of the four nonidentical mutations to streptomycin resistance. In two of the su(+)str-r strains, streptomycin markedly augmented the suppression caused by the ochre suppressor. In those su(-)str-r hosts in which significant streptomycin-induced suppression could be measured, the amber mutants were more suppressible than the ochre mutants.     

Nonsense-suppressor mutants of Erwinia carotovora subsp. carotovora

Abstract A promiscuous plasmid (pLM2) carrying amber mutations in two antibiotic-resistance genes was transferred to a derivative of Erwinia carotovora subsp. carotovora strain SCRI193. Following mutagenesis, two putative amber-suppressing mutants of this strain were isolated. The genotype of these mutants was confirmed by use of rep _am plasmid-specific phage. This constitutes the first isolation of amber-suppressing mutants in Erwinia spp.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.     

Coliphage P1 morphogenesis: analysis of mutants by electron microscopy.

We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage.     

Recombination in amber-mutants of bacteriophage T4B. I. Recombination and complementation in phage T4 amber-mutants]

In studying intergenic and intragenic complementation in amber mutants in genes of phage T4 controlling the synthesis of phage tail fibres the data have been obtained indicating the dependency of the results of complementation tests on those of crosses of respective markers. The results obtained show that in complementation of amber mutants of phage T4 the phage yield varies widely and depends on the location of markers on the phage genetic map.     

Phenotypic suppression by streptomycin of amber mutants in the ribonucleic Acid bacteriophage coat protein cistron

After mutagenesis with nitrosoguanidine or ultraviolet light, 298 streptomycin high-resistant and 98 streptomycin high-dependent mutants were isolated from HfrC Su⁻. They were tested for their ability to phenotypically suppress five different amber ribonucleic acid (RNA) bacteriophage mutants in the presence of streptomycin. The phage mutants are all in the coat protein, which is 129 amino acids long; the uracil-adenine-guanine codons were at the following positions: sus3 and amB2, 6; amB11, 50; amB21, 54; sus11, 70. Only sus3 and amB2 could be phenotypically suppressed by streptomycin; this was clearly demonstrated in nine mutant strains, seven str-HR and two str-HD. The suppression was always dependent upon added streptomycin and was dose-dependent in all cases. None of the mutants showed measurable suppression in absence of the drug. Among revertants to streptomycin independence from streptomycin-dependent strains that could show phenotypic suppression, most of those that were still resistant to streptomycin (10 μg or more) retained the capacity to show phenotypic suppression; whereas among those revertants sensitive to 10 μg of streptomycin or less, none retained the capacity. Eight different amber polar mutants (strong and weak) in gene 34 of phage T4 were also tested for pleiotypic suppression by streptomycin in all the streptomycin-resistant and -dependent strains isolated. No suppression was found in any of the 396 strains tested.     

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.     

TRNA2Gln Su+2 mutants that increase amber suppression.

We selected mutants of lambda pSu+2 which had an increased ability to suppress on Escherichia coli trp B9601 amber mutation on translationally stringent rpsL594 streptomycin-resistant ribosomes. tRNA2Gin Su+2 molecules produced from eight independent mutants were purified, and their ribonucleic acid sequences were determined. Two types of mutations were mapped to the tRNA2Gin Su+2(glnV) gene by this method. Both altered the pseudouridine at position 37 of the tRNA anticodon loop. Seven of the isolates were transitions (pseudouridine to cytosine), and one was a transversion (pseudouridine to adenine). These mutations resulted in Su+ transfer ribonucleic acid molecules that exhibited higher transmission coefficients than their parent Su+2 transfer ribonucleic acids. As judged by their suppressor spectra on T4 amber mutants, which were almost identical to that of Su+2, the two mutant Su+ transfer ribonucleic acids inserted glutamine at amber sites.     

The mechanism of the mutagenic action of hydroxylamine XII. Phenotypic suppression of amber mutants of phage T7 by hydroxylamine and O-methylhydroxylamine.

The reproduction of phage T7 in the presence of hydroxylamine (HA) (mutagenesis in vivo) results in the phenotypic suppression of some amber mutants. The presence of O-methylhydroxylamine (OMHA) results in a similar effect, indicating a similar mechanism for the action of the two compounds. Since the rate of reaction of mutagen with nucleoside residues under these conditions in negligibly low, one of the most plausible explanations of this effect is the enzymic formation of modified precursors and their incorporation into bacterial tRNAs or phage-induced RNA.     

In vivo and in vitro "phenotypic mixing" with amber mutants of phages T3 and T7

Summary Gene 17 of phage T7, and a homologous gene of T3, were shown to code for the serum blocking power protein (SBP). Noninfective T7 particles lacking gene 17 product (SBP-less particles) could be rendered infective by incubation with extracts of nonpermissive host bacteria infected with amber mutants of T7, as well as of T3, defective in other genes. SBP-less T7 particles activated by extracts of T3-infected cells were characterized as coat chimeras by their specificity towards anti-T3 and anti-T7 sera.     

Temperature-sensitive mutants of bacteriophage SH-133 specific for the hydrogen bacterium Pseudomonas facilis: isolation, complementation, and partial characterization.

Seventeen temperature-sensitive mutants of bacteriophage SH-133 have been isolated following mutagenesis with UV-light, nitrosoguanidine, and ethyl methanesulfonate. The mutants were classified into 15 complementation groups according to their ability to complement each other at 32 degrees C, the nonpermissive temperature. Each mutant was studied with regard to the relationship between its ability to multiply in heterotrophically (H-) and autotrophically (A-) grown Pseudomonas facilis cells. At 27 degrees C, the permissive temperature, the plaque-forming ability of the 17 mutants and wild-type phage was reduced 10-fold in A-grown cells. At 32 degrees C, mutants belonging to 10 groups exhibited identical levels of multiplicity-dependent leak under both modes of growth. However, the infection of A-grown cells by mutants belonging to the remaining five groups resulted in as much as 500-fold inhibition of multiplicity-dependent leak when contrasted with the infection of cells grown heterotrophically. These observations indicate that the expression of five SH-133 phage cistrons is defective when multiplication proceeds under autotrophic metabolism. Seven mutants were found to differ from the wild-type phage with regard to thermal stability at 56 degrees C which suggests that they possess altered structural proteins. Four of the seven thermosensitive mutants exhibited reduced levels of multiplicity-dependent leak in A-grown cells. The data suggest that the reduction in plaque-forming ability of SH-133 in A-grown cells is caused by a defect in the expression of specific phage structural components.