Chelating Agent Shock of Bacteriophage T5

When two strains of phage T5 (heat-susceptible form T5st(+) and its heat-resistant mutant T5st) were placed in solutions containing various high concentrations of chelating agents (sodium citrate and ethylenediaminetetraacetic acid) at room temperature, they could be effectively inactivated by rapid dilution in distilled water of relatively low temperatures (2 to 37 C). This phenomenon has been termed "chelating agent shock" (CAS). The susceptibility of phage T5 to CAS increased with an increase in the concentration of chelating agents and with an increase in temperature of the water used for rapid dilution. Under any given condition, T5st(+) was much more sensitive to CAS than was T5st. Phage T5 was protected against inactivation by the addition of monovalent or divalent metal salts, but not by the addition of nonionic solutes, to the shocking water prior to CAS treatment. This finding is compatible with the view that cations combined with the phage protein are removed by the chelating agent, although no metal ion has been identified in the phage protein. Alternatively, since the chelating agents used are polyanions, they may bind relatively tightly to the protein subunits in the head of T5, thereby distorting the structure of the phage head. Rapid dilution of these distorted particles could lead to loss of phage DNA. No evidence for recovery of phage activity could be obtained by the addition of metal salts to the inactivated phage after CAS. The morphological properties of phage inactivated by CAS are similar to those of heat-inactivated T5 phage. Electron micrographs showed that most of the phage particles consisted of empty head membranes; some of the particles had lost their tails. Both heritable and nonheritable resistance to heat was accompanied by resistance to CAS in phage T5. The sensitive element detected by each test seemed to be the same.     

Bacteriophage T5 DNA ejection under pressure

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for λ and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and λ, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.     

Polarized Injection of the Bacteriophage T5 Chromosome

Examination of the first-step-transfer DNA of T5ris mutants which carry new EcoRI sites showed that the left end of the chromosome is injected first.     

High Negative Interference and Recombination in Bacteriophage T5

The process of close recombinant formation in bacteriophage T5 crosses has been studied by examining the structure of internal heterozygotes (HETs), the immediate products of recombination events. The T5 system was chosen because it permits the study of internal heterozygotes exclusively, thus avoiding the ambiguities inherent in previous studies with T4. The heterozygotes were obtained by the nonselective screening of progeny phage in a prematurely lysed sample from an eight-factor cross. The molecular structure of each HET was inferred from the strand genotypes displayed among its progeny. This investigation presents unequivocal evidence that both overlap and insertion HETs are intermediates in recombinant formation and that insertion HETs are a significant source of close double recombinants. There is evidence suggesting that mismatch repair of overlap HETs could be the source of close triple exchanges. Thus, a significant part, and perhaps all, of the high negative interference for close-marker recombination observed in this system is a direct consequence of the fine structure of the recombinational intermediates. These findings are compatible with recombination models proposed by others, in which a single branched intermediate can give rise to HETs of both the overlap and insertion types.     

The Evolution of Epistasis and the Advantage of Recombination in Populations of Bacteriophage T4

Experiments reported here test two hypotheses about the evolution of recombination: first, the Fisher-Muller concept that sexual organisms respond to selection more rapidly than do asexual ones, and second, that epistasis is more likely to evolve in the absence of recombination. Populations of bacteriophage T4 were selected by the drug proflavine in discrete generations and the change in mean population fitness was monitored. Three separate selection series yielded results supporting the Fisher-Muller hypothesis. The amount of epistasis evolved was measured by partitioning the T4 map into regions and comparing the sum of the proflavine resistances of each region with the resistance of the whole. Significantly more interactions were found in phage isolated from the populations with lower total recombination than in those from populations with higher recombination. The degree to which these experiments fit preconceived notions about natural selection suggests that microorganisms may be advantageously used in other population genetics experiments.     

Regulation of Bacteriophage T5 Development by ColI Factors

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb(+) cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb(+) cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis.     

Bacteriophage T5 Mutants Carrying Deletions in tRNA Gene Region

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2–35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.     

The Arrangements of Nucleotide Sequences in T2 and T5 Bacteriophage DNA Molecules

The genetic map of T4 (and T2) bacteriophage is circular but the DNA molecule that is liberated by phenol extraction is a linear duplex of polynucleotide chains. If the genetic map is related to the physical structure of the DNA molecule, the problem arises as to how a linear molecule can give rise to a circular map. An explanation can be made on the basis that the bacteriophage liberate molecules which have nucleotide sequences which are circular permutations of each other. Thus, markers which are most distant on one molecules are closest together on another. To test this hypothesis, the middles of T2 and T5 DNA molecules were mechanically deleted and the absence of certain nucleotide sequences was tested by “renaturation” or “reannealing” experiments using columns containing denatured DNA immobilized in agar beads. The results indicate that when the middles are deleted from the T5 DNA molecule, some special sequences are removed; whereas, when the middles are deleted from the T2 DNA molecule, no special group of sequences is removed. This would indicate that T2 molecules begin at different points in their nucleotide sequence, while T5 molecules all begin at the same point. It is likely that this permutation of sequences of T2(T4) molecules is related to the circularity of their genetic map.     

Genetic Heterogeneity in Algerian Human Populations

The demographic history of human populations in North Africa has been characterized by complex processes of admixture and isolation that have modeled its current gene pool. Diverse genetic ancestral components with different origins (autochthonous, European, Middle Eastern, and sub-Saharan) and genetic heterogeneity in the region have been described. In this complex genetic landscape, Algeria, the largest country in Africa, has been poorly covered, with most of the studies using a single Algerian sample. In order to evaluate the genetic heterogeneity of Algeria, Y-chromosome, mtDNA and autosomal genome-wide makers have been analyzed in several Berber- and Arab-speaking groups. Our results show that the genetic heterogeneity found in Algeria is not correlated with geography or linguistics, challenging the idea of Berber groups being genetically isolated and Arab groups open to gene flow. In addition, we have found that external sources of gene flow into North Africa have been carried more often by females than males, while the North African autochthonous component is more frequent in paternally transmitted genome regions. Our results highlight the different demographic history revealed by different markers and urge to be cautious when deriving general conclusions from partial genomic information or from single samples as representatives of the total population of a region.     

Evidence Against Phenotypic Mixing Between Bacteriophage T4 Wild Type and T4v?

In a recent publication Shames et al. (1973) concluded that the UV-specific T4 endonuclease (a repair enzyme coded for by the gene v of wild-type T4) is a component of extracellular phage, which is injected into the host cell and can perform an early repair step without requiring gene expression. This notion is, however, not supported by results presented in this paper. Lysates obtained from mixed multiple infection of Escherichia coli cells with T4v(1) (-) and T4v(+) (or T4v(2) (-) and T4v(+)) failed to show the expected phenotypic mixing, i.e., incorporation of UV endonuclease into capsids of v(-) phages resulting in recognizable repair. The fraction of v(+) and v(-) particles in such lysates was determined by single-plaque analysis before and after irradiation with a UV dose at which virtually all survivors are particles having undergone repair. Even though our mixed infection conditions were most favorable for the possible occurrence of phenotypic mixing, none out of several hundred individual plaques from survivors were found to be genotypically v(-), whereas 30 were expected in the case that phenotypically mixed v(-) particles were repaired like T4v(+). Our failure to observe phenotypic mixing suggests that the data by Shames et al. reflect intracellular synthesis of endonuclease after phage infection.     

Cadaverine in Bacteriophage T4

Cadaverine was found in bacteriophage T4 when the host cells of Escherichia coli K-12 were grown in complex media and aerated by agitation. Only traces of cadaverine were found if the host was grown and agitated in synthetic medium or was aerated by vigorous bubbling in a complex medium. When the host cells were grown anaerobically in a complex medium, cadaverine became the major polyamine in the progeny phage. The polyamine content comprised 80% cadaverine, 14% spermidine (or its recently discovered homologue, N-3-aminopropyl-1, 5-diaminopentane), and the remainder putrescine. The conditions that favored appearance of cadaverine are known to be required for induction of lysine decarboxylase. It was shown that lysine was the sole source of bacterial cadaverine.     

Genetic Evidence for Two Protein Domains and a Potential New Activity in Bacteriophage T4 DNA Polymerase

Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.     

Stable Heterogeneity for the Production of Diffusible Factors in Cell Populations

The production of diffusible molecules that promote survival and growth is common in bacterial and eukaryotic cell populations, and can be considered a form of cooperation between cells. While evolutionary game theory shows that producers and non-producers can coexist in well-mixed populations, there is no consensus on the possibility of a stable polymorphism in spatially structured populations where the effect of the diffusible molecule extends beyond one-step neighbours. I study the dynamics of biological public goods using an evolutionary game on a lattice, taking into account two assumptions that have not been considered simultaneously in existing models: that the benefit of the diffusible molecule is a non-linear function of its concentration, and that the molecule diffuses according to a decreasing gradient. Stable coexistence of producers and non-producers is observed when the benefit of the molecule is a sigmoid function of its concentration, while strictly diminishing returns lead to coexistence only for very specific parameters and linear benefits never lead to coexistence. The shape of the diffusion gradient is largely irrelevant and can be approximated by a step function. Since the effect of a biological molecule is generally a sigmoid function of its concentration (as described by the Hill equation), linear benefits or strictly diminishing returns are not an appropriate approximations for the study of biological public goods. A stable polymorphism of producers and non-producers is in line with the predictions of evolutionary game theory and likely to be common in cell populations.