Host mutants resistant to phage lambda killing

Host mutants have been isolated which are resistant to killing by the kil protein of an induced λ prophage. In general, such host mutants are defective in adsorption and production of lambdoid phages, and show altered sensitivities to deoxycholate, colicins, and antibiotics. Most of the mutants are cold sensitive and form filaments at the restrictive temperature. These properties seem to implicate the cell envelope in the kil phenomenon. The cold-sensitive host mutants cannot be cured of their prophage; furthermore, growth of the mutants at different temperatures shows a marked negative correlation with the degree of repressor activity of the temperature-sensitive cI857 repressor. These observations suggest that some phage product, perhaps kil protein, is required for cell survival in these mutants.     

Isolation of ultravirulent mutants of phage lambda.

Ultravirulent mutants of phage λ able to grow in the presence of high cellular levels of repressor, have been isolated from λν2ν3. These phages can be divided into three groups of increasing virulence reflecting the number of mutational steps required for their isolation. Multiple mutations in the operator regions leading to a decrease in the affinity of the λ repressor for the operator sites seen to be responsible for the ultravirulent phenotype.     

Inactivating effect of o-methylhydroxylamine on phage lambda and its mutants]

The kinetics of inactivation of lambda+, lambda C160, lambda Nsus7, lambda Gsus9, lambda Tsus6 phages under the effect of O-methylhydroxylamine was studied. Inactivation curves of all the phages under study were found to be of a complex character and early in the reaction (up to 4 hours) to deviate from exponential dependence of the inactivation rate upon the time of incubation with the mutagen. All the phages under study showed differences in the inactivation rates early in the reaction. Constants of the inactivation rate in the linear part of the curve vary from 30 hours-1 for lambda+ to 0.14 hours-1 for lambda C160. The possible causes of the complicated pattern of inactivation courves of lambda phage and its mutants are discussed.     

A sedimentation analysis of DNA found in Escherichia coli infected with phage lambda mutants

A study was made of the phage DNA produced in Escherichia coli infected with mutants of λ unable to produce infectious particles because of defects in genes controlling head formation. Bacteria were irradiated with UV to eliminate host DNA synthesis and then infected with various λ sus mutants under conditions where subsequent radioactive labelling appeared exclusively in phage DNA. The intracellular DNA was extracted and examined by sedimentation in neutral and alkaline sucrose gradients. Mutants in genes A, B, C, D, and E cause blocks in DNA maturation which result in the accumulation of “rapidly sedimenting” species. Mutants in genes W and F accumulate much less of the rapidly sedimenting DNA and synthesize about 50–100% of the normal amounts of mature molecules as found in control infections. Intracellular phage DNA was purified from host material by density labelling and examined by electron microscopy. The “rapidly sedimenting” species appeared to be linear concatamers with lengths up to and exceeding four units. In an A mutant circular molecules were also found; these were both of monomer and multimer length (dimers and trimers). The proportion of immature DNA found by electron microscopy (long molecules) and by sedimentation (fast sedimenting DNA) agreed very well.     

A mutant of Escherichia coli that prevents growth of phage lambda and is bypassed by lambda mutants in a nonessential region of the genome

A mutant strain of E. coli (rap^?) has been isolated which blocks the growth of phages λ, 434, and ?80. Phage 424 and various non-lambdoid phages are not affected. λ mutants that can bypass the rap block are of two general types. First, a presumed point mutant mapping between h and att provides partial bypass of the block. Second, all deletions and substitutions that eliminate or substitute one or both recognition regions of the attachment site restore a high level of growth. However, a point mutation in the crossover region of the attachment site does not bypass the block, and the rap mutation does not affect Int-promoted λ recombination. While the mechanism by which rap blocks phage development is unknown, the block implies a new and potentially interesting form of phage-host interaction.     

DNA damage and mutagenesis of lambda phage induced by gamma-rays

Lambda phage DNA was gamma-irradiated in aqueous solution andthe amount of radiation-induced strand breakage [double- andsingle-strand breaks and alkali-labile sites (dsb, ssb, als)]was determined. Twice as much minor structural damage (ssb andals) per lethal hit was found in this DNA compared with DNAfrom irradiated phage suspensions. The in vitro irradiated DNAwas re-packaged into infectious particles (‘pack-phage’).The induction of mutations in the cI or cII cistron was scoredusing SOS-induced host cells. The in vitro prepared particleswere found to have second-order kinetics for mutagenesis inducedby gamma-rays indicating that two pre-mutational events werenecessary to produce a mutation. In contrast, bacteria-freephage suspensions (‘lys-phage’) showed single hitkinetics for mutagenesis after irradiation. The increase inthe mutation rate in the phage particles was mainly due to minorlesions, i.e. ssb, als and unidentified base damage. In lys-phage,mutagenesis might be enhanced by clustered DNA damage —a configuration which does not exist in pack-phage. The lossof infectivity was analysed in comparison with the extent ofstructural damage. All lesions investigated here contributedto biological inactivation. Minor lesions were tolerated bylambda phage to a limited extent. Major lesions, such as dsb,contributed most to the loss of infectivity and can be consideredas lethal events.     

N-ethylmaleimide and the induction of phage lambda

Uv induction of the normal uv-inducible prophage lambda was inhibited by treatment of cells with N-ethylmaleimide (NEM) prior to uv irradiation. Neither uv induction of the cIts mutant prophage nor heat induction of the cItsind^? mutant prophage was affected by NEM treatment.     

Phage lambda DNA injection into Escherichia coli pel- mutants is restored by mutations in phage genes V or H.

Mouse cells in culture contain two distinct forms of thymidine kinase enzyme activities. These two enzymes have been separated by polyacrylamide gel electrophoresis into a 0.2 R_f and a 0.5 R_f activity. The 0.2 R_f enzyme was found only in actively growing cells, while the 0.5 R_f form of thymidine kinase is the mitochondrial-associated enzyme and is most prominent in resting cells in culture. SV40 infection of these resting cells results in an increased specific activity of only the 0.2 R_f form of this enzyme.SV40 wild-type and SV40 tsBC and tsC mutants stimulated the levels of the 0.2 R_f thymidine kinase in resting cells after viral infection at either the permissive temperature (32°) or the nonpermissive temperature (41°). Five different SV40 tsA mutants (tsA7, 28, 30, 58, and 209) and two different SV40 tsD mutants (tsD202, 270) only stimulated thymidine kinase activity at the permissive temperature. Little or no 0.2 R_f thymidine kinase activity could be detected in tsA or tsD mutant-infected cells at the nonpermissive temperature. The SV40 tsA255 mutant appeared to be an exception to the A mutant class in that it stimulated the 0.2 R_f thymidine kinase activity at both permissive and nonpermissive temperatures.These results indicate that the SV40 A gene product may be required directly, and the D gene product indirectly, in the stimulation of cellular enzyme activities following viral infection.     

Regulatory circuit design and evolution using phage lambda

Bistable gene regulatory circuits can adopt more than one stable epigenetic state. To understand how natural circuits have this and other systems properties, several groups have designed regulatory circuits de novo. Here we describe an alternative approach. We have modified an existing bistable circuit, that of phage lambda. With this approach, we used powerful genetic selections to identify functional circuits and selected for variants with altered behavior. The lambda circuit involves two antagonistic repressors, CI and Cro. We replaced lambda Cro with a module that included Lac repressor and several lac operators. Using a combinatorial approach, we isolated variants with different types of regulatory behavior. Several resembled wild-type lambda--they could grow lytically, could form highly stable lysogens, and carried out prophage induction. Another variant could form stable lysogens in the presence of a ligand for Lac repressor but switched to the lytic state when the ligand was removed. Several isolates evolved toward a desired behavior under selective pressure. These results strongly support the idea that complex circuits can arise during the course of evolution by a combination of simpler regulatory modules. They also underscore the advantages of modifying a natural circuit as an approach to understanding circuit design, systems behavior, and circuit evolution.