Plasmid-Controlled Variation in the Content of Methylated Bases in Bacteriophage Lambda Deoxyribonucleic Acid

The N(6)-methyladenine (MeAde) and 5-methylcytosine (MeC) contents in deoxyribonucleic acid (DNA) of bacteriophage lambda has been analyzed as a function of host specificity. The following facts have emerged: (i) lambda grown on strains harboring the P1 prophage contain ca. 70 more MeAde residues/DNA molecule than lambda grown either in the P1-sensitive parent, or in a P1 immune-defective lysogen which does not confer P1 modification; (ii) lambda grown on strains harboring the N-3 drug-resistance factor contain ca. 60 more MeC residues/DNA molecule than lambda grown on the parental strain lacking the factor; (iii) lambda grown in Escherichia coli B strains is devoid of MeC, whereas lambda grown in a B (N-3) host contains a high level of MeC; (iv) the MeAde content in lambda DNA is not affected by the N-3 factor. These results suggest that P1 controls an adenine-specific DNA methylase, and that the N-3 plasmid controls a cytosine-specific DNA methylase. The N-3 factor has been observed previously to direct cytosine-specific methylation of phage P22 DNA and E. coli B DNA in vivo; in vitro studies presented here demonstrate this activity.     

In vivo methylation of bacteriophage phi X174 DNA.

A mutant (designated mec(-)) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage lambda against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec(-) mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec(+) and C mec(-) cells infected with bacteriophage phiX174. phiX. mec(-) RFI is sensitive to in vitro cleavage by R.EcoRII and is cut twice to produce two fragments of almost equal size. In contrast, phiX.mec(+) RFI is relatively resistant to in vitro cleavage by R.EcoRII. R.BstI, which cleaves mec(+)/RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R. EcoRII. These results demonstrate that phiX.mec(+) RFI is methylated in vivo by the host mec(+) enzyme and that this methylation protects the DNA against cleavage by R.EcoRII. This is consistent with the known location of two mec(+)/ RII sequences (viz., [Formula: see text]) on the phiX174 map. Mature singlestranded virion DNA was isolated from phiX174 propagated in C mec(+) or C mec(-) in the presence of l-[methyl-(3)H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that phiX.mec(+) DNA had a 10-fold-higher ratio of [(3)H]5-methylcytosine to [(3)H]cytosine compared to phiX.mec(-). Since phiX.mec(+) contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of phiX174 is mediated by the host mec(+) enzyme only. These results are not consistent with the conclusions of previous reports that phiX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.     

Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes.

Phages lambda and fd were propagated in Escherichia coli strains that have either host K-12 or the N-3 R-factor deoxyribonucleic acid-cytosine methylase activity. Pyrimidine tracts containing 3H-labeled 5-methylcytosine (MeC) were analyzed; in all cases, the major methylated sequence was 5' ... C-MeC-T ... 3'.     

Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes.

Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K 12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro. Phage lambda and fd were propagated in the presence of L-[methyl-3H]methionine in various host bacteria. The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment. The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively. The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec minus (N-3) cells. For phage lambda the major 5-methylcytosine containing tract was the tripyrimidine, C2T; for both fd-mec minus (N-3) DNA and fd-mec+DNA, C2T was the sole 5-methylcytosine-containing tract. When various lambda DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec minus (N-3) cells, the extent of cytosine methylation was the same. This is in contrast to in vivo methylation where lambda-mec minus (N-3) DNA contains twice as many 5-methylcytosines per genome as lambda-mec+ DNA. Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.     

In vivo methylation by Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase protects against in vitro cleavage by the RII restriction endonuclease (R. Eco RII).

We have analyzed the susceptibility of the deoxyribonucleic acid (DNA) of phage fd replicative form (RF) and of Escherichia coli to in vitro cleavage by purified RII restriction endonuclease (R. Eco RII). The results are summarized as follows: (i) fd, mec- RFI, isolated from infected E. coli K-12 mec- bacteria (a mutant strain lacking DNA-cytosine methylase activity), is cleaved into at least two fragments, whereas fd. mec+ RFI, isolated from the parental mec+ strain, is not cleaved. (ii) E. coli mec- DNA is extensively degraded, whereas mec+ DNA-cytosine methylase acts as an RII modification enzyme.     

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.     

Mutants of the N-3 R-Factor Conditionally Defective in hspII Modification and Deoxyribonucleic Acid-Cytosine Methylase Activity

The N-3 drug resistance (R) factor specifies a deoxyribonucleic acid (DNA)-cytosine methylase and a DNA restriction-modification (hspII) system. We have isolated three independent mutants that are conditionally defective in their ability to modify bacteriophage lambda and to methylate DNA-cytosine residues. The ratio of 5-methylcytosine to N(6)-methyladenine in bacterial DNA and in the DNA of phages lambda and fd was determined after labeling with [methyl-(3)H]methionine at various growth temperatures. Although the ability of the wild-type N-3 factor to modify phage lambda and to methylate DNA-cytosine residues was unaffected with increasing temperature, two of the mutants exhibited a parallel loss in modification and cytosine methylation ability. The ability of the third mutant to carry out these functions was dependent on the presence or absence of an amber suppressor mutation in the host genome. These results offer further support for the notion that hspII modification is mediated by a DNA-cytosine methylase. Evidence is also presented that the modification methylase is responsible for the in vivo methylation of phage fd DNA (which is not subject to hspII restriction in vivo).     

Isolation and partial characterization of three Escherichia coli mutants with altered transfer ribonucleic acid methylases.

Seven transfer ribonucleic acid (tRNA) methylase mutants were isolated from Escherichia coli K-12 by examining the ability of RNA prepared from clones of unselected mutagenized cells to accept methyl groups from S-adenosylmethionine catalyzed by crude enzymes from wild-type cells. Five of the mutants had an altered uracil-tRNA methylase; consequently their tRNA's lacked ribothymidine. One mutant had tRNA deficient in 7-methylguanosine, and one mutant contained tRNA lacking 2-thio-5-methylaminomethyluridine. The genetic loci of the three tRNA methylase mutants were distributed over the E. coli genome. The mutant strain deficient in 7-methylguanosine biosynthesis showed a reduced efficiency in the suppression of amber mutations carried by T4 or lambda phages.     

Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties.

A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.     

Cloning and characterization of the dcm locus of Escherichia coli K-12.

The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K-12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones.     

EcoK restriction during in vitro packaging of coliphage lambda DNA

The K restriction system of Escherichia coli works in vitro [Meselson and Yuan, Nature 217 (1968) 1110-1114]. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-175]. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

Influence of DNA adenine methylase on the sensitivity of Escherichia coli to near-ultraviolet radiation and hydrogen peroxide

Near-ultraviolet (NUV) radiation and hydrogen peroxide (H2O2) inactivation studies were performed on Escherichia coli K-12 DNA adenine methylation (dam) mutants and on cells that carry plasmids which overexpress Dam methylase. Lack of methylation resulted in increased sensitivity to NUV and H2O2 (a photoproduct of NUV). In a dam mutant carrying a dam plasmid, the levels of Dam enzyme and resistance to NUV and H2O2 were restored. However, using a multicopy dam+ plasmid strain, increasing the methylase above wildtype levels resulted in an increase in sensitivity of the cells rather than resistance.     

Repression of lambda-Associated Enzyme Synthesis After lambda(vir) Superinfection of Lysogenic Hosts.

Lisio, Arnold L. (National Institutes of Health, Bethesda, Md.), and Arthur Weissbach. Repression of lambda-associated enzyme synthesis after lambda(vir) superinfection of lysogenic hosts. J. Bacteriol. 90:661-666. 1965.-Phage lambda(vir) is a multiple mutant of lambda which is capable of overcoming the immunity of a host lysogenic for lambda, and initiating normal vegetative replication of the superinfecting phage genome. Superinfection of Escherichia coli K-112 (lambda(22)) with lambda(vir) results in a normal phage yield, lysis time, and H(3)-thymine incorporation compared with infection of the sensitive host, K-112 (S). However, the production of the lambda phage-specific early protein, lambda-exonuclease, after superinfection of E. coli K-112 (lambda(22)) with lambda(vir) is only 25 to 50% of that obtained from corresponding infection of a nonlysogenic host, E. coli K-112 (S). This repression of lambda-exonuclease synthesis is dependent on the C(1) cistron of the prophage and is overcome if the lysogenic host cells are induced prior to superinfection. The data are interpreted as evidence for partial repression of lambda(vir) by the host immunity.     

Repression of damage-inducible (din) genes by the lexA3 mutation or by plasmid carrying the lexA gene; effect on pyrimidine dimer excision in UV-irradiated Escherichia coli

Dimer excision was followed in Escherichia coli K-12 AB1157 DM49 lexA3 mutant (whose repressor is not cleavable with RecA protease), and in E. coli K-12 AB2497[pGC3] carrying the cloned lexA gene. In either case din genes could not be efficiently derepressed. In such cells ultraviolet (UV) irradiation caused an extensive DNA degradation, which was not observed in cells with derepressed din genes. Even after a high UV dose (70 J/m2) dimers were being excised efficiently. However, progressive DNA degradation interfered with the precise detection of unexcised dimers. We conclude that induction of din genes is required for filling some of the gaps and for prevention of DNA degradation, but not for excision itself.     

Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda

Weissbach, Arthur (National Institutes of Health, Bethesda, Md.), Allan Lipton, and Arnold Lisio. Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda. J. Bacteriol. 91:1489-1493. 1966.-Infection of either the sensitive or lysogenic strain of Escherichia coli K-112S by lambda(+) leads to the formation of a new phage deoxyribonucleic acid (DNA) species having the properties of a twisted circular DNA duplex. This new phage DNA species is also seen in cells infected with clear or virulent mutants of lambda which cannot lysogenize, or do so at a low frequency. The sedimentation rate of circular lambda DNA duplex at various pH values and its lability were examined.     

Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli.

The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively. Even though the E. coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E. coli K-12, as well as in several members of the E. coli reference (ECOR) collection which are closely related to K-12. For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source. Most E. coli strains, including E. coli B, are naturally Phn+; a few E. coli strains are Phn- and are deleted for phn DNA sequences. The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E. coli K-12 delta phn mutant. The phn(EcoB) DNA hybridized to overlapping lambda clones in the E. coli K-12 gene library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location. The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence. However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus.