C.R. Fuerst

Genetics and physiology of defective lysogeny in K12 (λ): Studies of early mutants

Abstract The properties of a number of strains of K12 (λ) which are defective in their ability to form lambda DNA after induction have been examined by genetic, physiological, and biochemical tests. The strains fall into four classes, and include mutants in cistrons N, O, and P, previously identified by Campbell (1961), and a fourth new class, called x, in a region between C I and C II . Mutants in each of the classes can be distinguished genetically, by their relative ability to form the λ-exonuclease, and by their tendency to show curing of their prophage after induction. Mutants in the P and O cistrons form normal levels of λ-exonuclease, whereas mutants in the N cistron form very low levels, and mutants in the x region, relatively high levels of this enzyme. Curing is very efficient for mutants in the x region, less efficient for mutants in the O and P cistrons, and is seldom observed with mutants in the N cistron. These patterns of curing are reflected in the curves for UV survival of the strains carrying defective mutations in the various cistrons. The significance of these findings in respect to the early events in phage development are discussed.

Mutations in bacteriophage lambda affecting host cell lysis.

Nine temperature-sensitive and defective mutants of phage lambda deficient in lysis of the host cell have been analyzed in respect to location of the site of mutation and phenotypic properties. Seven of the isolates are mutant in cistron R and produce either a thermolabile endolysin or no detectable enzyme. The remaining two mutants, one temperature-sensitive and one defective, produce high levels of apparently normal enzyme. These mutants denote the involvement of at least one phage cistron other than R in the lytic process.

Mutations in bacteriophage lambda affecting particle morphogenesis.

Abstract A number of temperature-sensitive and defective mutants of λ bacteriophage, located to the left of the homology region, have been isolated and characterized. Genetic mapping and complementation tests have demonstrated the existence of two new cistrons, in addition to those previously described by Campbell. Analyses of mutant phenotype show that the cistrons to the left of the homology region are involved in particle morphogenesis. There is distinct clustering of genes governing formation of phage heads and tails.

PURIFICATION OF MURINE ENCEPHALOMYOCARDITIS VIRUS.

Abstract Methods are described for the concentration and purification of EMC virus grown in L cells in tissue culture. Virus concentrates obtained by treatment of lysates with protamine sulfate and methanol were purified with little or no loss of infectivity by a sequence of steps including digestion with trypsin, treatment with sodium pyrophosphate and ribonuclease, and column chromatography on hydroxylapatite.

Genetic and physiological characterization of the J gene of bacteriophage lambda.

Abstract More than 20 temperature-sensitive and defective J mutants of λ have been analyzed in terms of the location of the mutations and their effect on the gene product. All of the defective sites and most of the temperature-sensitive sites are confined to a comparatively small region at the right-hand end of the J map. This is the region in which genetic determinants of the distinctive host-range and serological characteristics of phages λ and 434 are located. Although most of the λ defective mutants do not produce an active J protein, two mutants with closely linked mutations produce material that is serologically active and can be incorporated into intact particles. Leakiness of the temperature-sensitive J mutants is highly variable, in a way that suggests that the location of the codon change has a bearing on how readily polypeptides produced at high temperature undergo transition to a temperature-insensitive state.

Involvement of the htpR gene product of Escherichia coli in phage λ development

Growth of phage lambda at high temperature requires a functional htpR host gene. The stages of the phage growth cycle shown to be dependent on htpR gene function include prophage excision and particle morphogenesis. Two types of morphogenetic abnormalities have been detected. One is a defect in phage tail assembly that results from a deficiency in tail fibers even though gpJ is produced. The severity of this defect is phage-strain specific. The second morphogenetic defect is less clearly defined, but results in formation of aberrant phage head structures. These abnormalities in lambda reproduction are presumed to be caused by the absence in htpR mutant host cells at high temperature of one or more of the heat-shock proteins of Escherichia coli whose synthesis is known to be regulated by the htpR gene.

Temperature sensitivity in Esherichia coli K12: Mutants unable to support normal growth of γ phage at high temperatures

Abstract Physiological analyses of seven temperature-sensitive mutants of Escherichia coli K12 suggest that the inability of two of the mutants to support normal λ reproduction at high temperature is associated with a defect in λ DNA synthesis. Failure of one of the mutants to support the growth of λ and two other phages is attributed to a temperature-sensitive block in protein synthesis. Abnormalities in λ development in the remaining mutants appear to reflect participation of different temperature-sensitive host components in processes occurring comparatively late in the phage growth cycle. The approximate map positions of mutations responsible for temperature sensitivity of four of the mutants have been determined.

Indications of an involvement of heat-shock proteins in restoration of repression of temperative-inducible λ prophage

Characteristics of lambda c/ts857 prophages that can be attributed to the ability of the temperature-sensitive phage repressor to renature at low temperature are not apparent in host cells that contain a mutation in the htpR gene. Host killing by prophages that are N- or are blocked in DNA synthesis is not prevented by the return of mutant cells to low temperature, and recovery of cells in which the phage remains derepressed is not delayed if the prophage is a mutant that cannot kill. These and other findings suggest that the phage repressor protein is unusually susceptible to inactivation in cells that are unable to respond to heat shock.

Mutations in bacteriophage λ that alter phage dependence on the htpR gene product of Escherichia coli

Abstract Six mutants of λ having reduced dependence on the htpR function of Escherichia coli were isolated from λcIts857. Burst sizes in htpRts cells at 40.5° were in the range of 10 to 20 particles per cell. Mapping and complementation analysis of one of the mutants suggested that the mutation in this isolate is in gene J. Additional evidence that the mutations in most of the isolates are in J was provided by the finding that all but one of the mutants differ from the parental phage in properties pertaining to extended host range.

Plating efficiencies of modified λbio particles on temperature-sensitive hsd mutants of Escherichia coli K12

Two mutants of Escherichia coli K12 that are temperature sensitive in cell growth and lambda phage production are shown to contain at least two mutations. One of the mutations in each of the isolates is in the hsd locus, and modification and restriction of lambda exhibits temperature sensitivity. One of the hsd mutations causes plaque formation by modified lambda bio particles that do not contain an intact ral gene to be temperature dependent.