Margaret Lieb

Studies of heat-inducible lambda bacteriophage: I. Order of genetic sites and properties of mutant prophages

When a lysogen containing a heat-inducible λ mutant is subjected to an appropriate heat treatment, phage is usually produced and the bacterium dies. Under certain conditions, the heated bacterium dies although phage production does not take place. Non-lysogenic cells, and lysogens containing wild-type λ, do not die when heated. Therefore, death after heating must be due to an activity of the mutant prophage. In this study, killing of the lysogen is called induction, whether or not phage is produced. Mutations resulting in heat-inducible prophage have been mapped at 12 different sites in the λ c 1 cistron. Some of the heat-inducible prophages with mutations in the left side of the cistron are extremely sensitive to ultraviolet induction. A heat-inducible mutant that is simultaneously resistant to ultraviolet induction maps on the left side of c 1 as does λ ind − . Prophages with mutations in the right side of c 1 are not induced to kill the lysogen when heated in media containing chloramphenicol or puromycin, or in the absence of a carbon or nitrogen source. Protein synthesis during the heating period appears to be required for induction of these mutants. However, when the mutation resulting in heat-inducibility is in the left part of c 1 induction occurs under conditions that block protein synthesis. In a bacterium containing phage genomes of both types, heat induction requires protein synthesis. It is proposed that the c 1 product is a protein molecule with two functions. The part of the molecule corresponding to the right side of c 1 represses transcription of λ DNA. The part of the c 1 product corresponding to the left side of the c1 is assumed to inhibit a phage-specified enzyme(s) present in the lysogenic bacterium.

Specific mismatch correction in bacteriophage lambda crosses by very short patch repair

SummaryIn crosses under rec+, red+, gam+ conditions, mutation am6 in the cI (repressor) gene of bacteriophage λ recombines with other cI mutations much more frequently than predicted by the physical distances involved. In four-factor crosses of am6 with mutations located 22–60 base pairs to the left, cI+ recombinants that are expected to require three crossovers (triple recombinants) are more frequent than recombinants that require only one crossover. However, when am6 is crossed with large insertions in cI, which may be expected to interfere with the formation of heteroduplexes by branch migration, the frequency of cI+ triple recombinants is very low. In addition, cI+ recombinants in crosses between am6 and adjacent mutations have a high probability of retaining the flanking markers of the am6 parent. These findings suggest that am6 is particularly susceptible to mismatch repair in heteroduplexes are presumed to be the result of branch migration from crossovers occurring at some distance from am6. The absence of co-repair when am6 is crossed with adjacent cI mutations indicates that most repair tracts extend no farther than about 20 bp to either side of the mismatch.The am6 mutation arose in the glutamine codon in a CCAGG sequence, in which the central cytosines are methylated in K12 strains. Their location in methylated sequences may make certain amber mutations susceptible to a specific very short patch (VSP) repair.

Very short patch repair: reducing the cost of cytosine methylation

In Escherichia coli and related bacteria, the product of gene dcm methylates the second cytosine of 5′‐CCWGG sequences (where W is A or T). Deamination of 5‐methylcytosine (5meC) results in C to T mutations. The mutagenic potential of 5meC is reduced by a system called very short patch (VSP) repair, which replaces T with C. T:G and U:G mispairs in the methylatable sequence and in related sequences are recognized by the product of vsr, a gene adjacent to dcm. Vsr creates a nick just 5′ of the mispaired pyrimidine to initiate the repair. Additional products known to be required for VSP repair are DNA polymerase I and DNA ligase. MutS and MutL have a stimulatory role but are not required. The ability of Vsr to recognize T:G mispairs in sequences related to CCWGG is probably responsible for over‐ and under‐representation of certain tetranucleotides in the E. coli genome. Although VSP repair reduces spontaneous mutations at 5meCs in replicating bacteria, mutation hot‐spots persist at these sites. Under conditions that more accurately mimic the natural environment of E. coli, VSP repair appears to be effective in preventing mutation at 5meC.

Enhancement of ultraviolet-induced mutation in bacteria by caffeine

SummaryThe addition of caffeine or theophylline to the growth medium of irradiatedE. coli B/rtry− resulted in a 10-fold or greater increase in the frequency oftry+ mutants. These observations extend those ofWitkin (1958). Caffeine produced a slight reduction in the rate of RNA and protein synthesis, and a somewhat greater but temporary reduction in the rate of DNA synthesis. The analogue must be added immediately after UV-irradiation to produce its optimal effect, and the ability of an irradiated culture to respond to caffeine was lost completely after 20 min incubation in broth. Normal purine ribosides did not compete with caffeine. The optimal exposure time to caffeine was correlated with the time of DNA doubling, but marked increases of mutation frequency resulted when caffeine was present for 30 min in the absence of DNA synthesis. Incubation in caffeine before irradiation had no effect. Caffeine also reduced mutation frequency decline caused by incubation of irradiated bacteria in chloramphenicol. It is suggested that caffeine interfers with a “dark repair” enzyme system which removes a UV photoproduct (s) whose presence during DNA synthesis leads to mutation.

Cooperation and competition in mismatch repair: Very short-patch repair and methyl-directed mismatch repair in Escherichia coli

In Escherichia coli and related enteric bacteria, repair of base‐base mismatches is performed by two overlapping biochemical processes, methyl‐directed mismatch repair (MMR) and very short‐patch (VSP) repair. While MMR repairs replication errors, VSP repair corrects to C•G mispairs created by 5‐methylcytosine deamination to T. The efficiency of the two pathways changes during the bacterial life cycle; MMR is more efficient during exponential growth and VSP repair is more efficient during the stationary phase. VSP repair and MMR share two proteins, MutS and MutL, and although the two repair pathways are not equally dependent on these proteins, their dual use creates a competition within the cells between the repair processes. The structural and biochemical data on the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays a role similar to MutH (also an endonuclease) in MMR. Biochemical and genetic studies of the two repair pathways have helped eliminate certain models for MMR and put restrictions on models that can be developed regarding either repair process. We review here recent information about the biochemistry of both repair processes and describe the balancing act performed by cells to optimize the competing processes during different phases of the bacterial life cycle.

Ultraviolet Sensitivity of Escherichia coli Containing Heat-Inducible λ Prophages

The c1-t mutants of bacteriophage λ can form prophage at 36�C but cause lysis of sensitive bacteria at temperatures above 42�C. Growth of cultures at 42� to 46�C induces prophage replication and lysis in Escherichia coli K12 (λ c1-t); lysogenic strains containing wild-type prophage are not induced to lyse at these temperatures. Heat induction is prevented by chloramphenicol. Strains containing heatinducible prophage are much more sensitive to killing by ultraviolet light than is K12 (λ+).

A fine structure map of spontaneous and induced mutations in the lambda repressor gene, including insertions of IS elements

SummaryMutations at over 70 sites in the cI gene have been mapped by 4-factor crosses and assigned precise or approximate positions in the DNA sequence. 16 of 25 spontaneous mutations were insertions of IS1, IS3 or IS5 into AT-rich regions of cI. The 5-methylcytosine in the sequence Cm5CAGG is a hot spot for spontaneous cI amber mutations. Recombination frequencies between mutations were proportional to distance with the exception of amber mutations at 4 sites, including the host spot for spontaneous mutations. Mutations with a given phenotype are clustered on the genetic map. No missense mutations affecting repressor activity were found in the central one-third of cI, but 5 of 6 ind- mutations were located in this region. The aminoterminal third of the gene contains the sites of most trans-dominant cI- mutations, and of all ts mutations that result in repressors that are reversibly inactivated at high temperatures.

Dark repair of UV induction in K12 (λ)

Abstract UV induction of K12(λ) is increased when proflavine (5 μg/ml or caffeine (1 mg/ml) is added to the medium in which bacteria are grown after irradiation. These substances prevent intracellular repair of UV damage that occurs in the dark (DR). They do not prevent photoreactivation of induction, but they interfere with the increased dark repair that is made possible by PhR.

Studies of heat-inducible λ mutants: II. Production of C1 Product by superinfecting λ+ in heat-inducible lysogens

Abstract When a λCI+ bacteriophage infects a lysogenic bacterium containing a heat-in -ducible λ prophage, the lysogen becomes heat resistant. Heat resistance is taken to indicate the presence of a heat-stable product of the CI+ gene. Some bacteria of a heat-sensitive culture are heat resistant 3 minutes after the addition of a CI+ phage. Twelve minutes after superinfection, most of the superinfected bacteria are heat resistant. Heat-inducible lysogens superinfected with λCI+ segregate progeny that are not induced at 43 ° although they no longer contain the CI+ gene. The persistence of heat resistance in lysogens several generations removed from the superinfected cell suggests that the λCI+ product is a stable substance. A single superinfecting λ+ genome can prevent heat induction of a lysogen that contains several heat-inducible prophages. It is proposed that the substance that prevents induction of λ is an oligomer of several molecules of CI product. One or more heat-stable subunits are assumed to prevent heat inactivation of an oligomer containing one or more subunits of heat-labile CI product.

λcI mutants: Intragenic complementation and complementation with acI promoter mutant

SummaryComplementation for the maintenance of lysogeny was studied by superinfecting λcIts lysogens at 34° C, and then heating to 43° C. With certain exceptions,ts mutants with defects in the left half of the repressor complementedts mutants with defects in the right half to produce a less heat-labile repressor (Fig. 3). AllcIamber mutants failed to complementcIts mutants. ThecI mutantc50 complements allts mutants. Mutations in Pre (cy) or genescII andcIII do not significantly affect the expression ofcI by a superinfecting λ genome in an immune lysogen. Mutants with very heat-labile repressors failed to complement λcy42 for the establishment of lysogeny at elevated temperatures, while those with less heatsensitive repressors apparently did complementcy.According to a suggested model, the left side of thecI product is concerned primarily with subunit aggregation, while operator binding is the function of the right side of the oligomer.