Various characteristics of the anti-restriction mechanism in bacteriophage T5

Bacteriophage T5 is not confined by the restriction systems of the second type EcoRII and EcoRV. Bacteriophage T5 DNA is not modified by EcoRII and EcoRV methylases in vivo. The sites of recognition for restriction endonuclease EcoRV are mapped at 24.4; 57.6; 68.5; 70.2% of T5 DNA, while the sites at 5.1; 7.6% are recognized by EcoRII, the sites at 5.75; 6.0 and 6.5% are recognized by HpaI in FST. A high activity of restriction endonucleases EcoRI and EcoRV is demonstrated in crude extracts of E. coli B834 (RI) and E. coli B834 (RV) cells infected by bacteriophage T5. The simultaneous infection of E. coli B834 (RI) or E. coli B834 (RV) cells by the amber mutants of bacteriophage T5 and the suppressing phage lambda NM761 does not result in the protection of lambda DNA by the T5 anti-restriction mechanism. The presented data support the hypothesis that the anti-restriction mechanism of bacteriophage T5 is based on prevention of T5 DNA contacts with restriction enzymes by a specific phage protein.     

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.     

Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.     

Partial purification of the Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase: in vitro methylation completely protects bacteriophage lambda deoxyribonucleic acid against cleavage by R-EcoRII.

A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their Km for S-adenosylmethionine and their pH (in tris (hydroxymethyl)aminomethane buffer) and NaCl concentration optima. In vitro methylation of various phage lambda DNA substrates by the mec'r RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R-EcoRII). These results are consistent with our earlier proposal that the mec8ethylase recognizes RII host specificity sites.     

Overproduction of the EcoRII endonuclease and methylase by Escherichia coli strains carrying recombinant plasmids constructed in vitro

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the Bam HI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the Bam HI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from ApRSuR-resistant clones, which restricted and modified phage lambda imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su-, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37 degrees C is increased by 10--50-fold.     

Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.     

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.     

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases of Escherichia coli C and Klebsiella pneumoniae M5a1 show very high N-terminal sequence homology

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) dehydrogenase from Escherichia coli C and Klebsiella pneumoniae M5a1 have been purified and some of their properties studied. The apparent Km values for NAD and CHMS were 11.7 +/- 1.5 microM and 5.2 +/- 1.9 microM, respectively, for the K. pneumoniae enzyme, and 19.5 +/- 2.7 microM and 9.2 +/- 1.4 microM, respectively, for the E. coli enzyme. Both enzymes were optimally active at pH 7.5 in sodium phosphate buffer. They had subunit molecular weights of 52,000 (+/- 1000) and the native enzymes appeared to be dimers of identical subunits. The first 20 residues of their N-terminal amino acid sequences were 90% homologous. A degenerate oligonucleotide probe constructed to a six amino acid sequence common to both enzymes gave strong hybridization with DNA from E. coli strains B and W as well as with E. coli C and K. pneumoniae but little or no hybridization to DNA from E. coli K12 or Pseudomonas putida.     

Purification and properties of T4 phage thymidylate synthetase produced by the cloned gene in an amplification vector

We have introduced the T4 thymidylate synthetase gene, resident in a 2.7-kilobase EcoRI restriction fragment, into an amplification plasmid, pKC30. By regulating expression of this gene from the phage lambda pL promoter within pKC30 in a thyA host containing a temperature-sensitive lambda repressor, the T4 synthetase could be amplified about 200-fold over that after T4 infection. At this stage, a 20-fold purification was required to obtain homogeneous enzyme, mainly by an affinity column procedure. The purified plasmid-amplified T4 synthetase appeared to be identical with the T2 phage synthetase purified from phage-infected Escherichia coli in molecular weight, amino end group analysis, and immunochemical reactivity. The individual nature of the phage and host proteins was revealed by the fact that neither the T2 nor the T4 enzyme reacted with antibody to the E. coli synthetase, nor did antibody to the phage enzymes react with the E. coli synthetase. These differences were corroborated by DNA hybridization experiments, which revealed the absence of apparent homology between the T4 and E. coli synthetase genes. The techniques and genetic constructions described support the feasibility of employing similar amplification methods to prepare highly purified thymidylate synthetases from other sources.     

The biological activity of bacteriophage DNA, prepared by the cationic detergent dilution technique

The preparation of phage lambda DNA infecting E. coli K 12 with cationic detergent is described. This DNA infects E. coli spheroblasts with the same efficiency as DNA prepared by phenol methods.     

The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases

DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.     

Cloning of fragments of lambda phage DNA, containing red and gam genes

On the base of plasmid pCV20 (Apr, Tcr mol. weight 5.2 x 10(6) a recombinant plasmid pEH60 (Apr, mol. weight 17.0 x 10(6) with BamHI fragment of phage DNA, containing red+ and gam+ genes was constructed. Selection was found on the ability of phage red- and gam- to propagate in strain E. coli K12 recA-, which was transformed by recombinant plasmid with active red and gam genes. Influence of recombinant plasmid pEH60 on processes of repair and recombination of phage lambda DNA and bacterial DNA was studied. It was shown that red gene in plasmid pEH60 compensates deficiency of redA gene in these processes with phage lambda DNA; in the case of E. coli K12 AB2480 uvr- recA- (pEH60) the processes of multiple reactivation and decombination of phage red- were presented. In the case of bacterial cells, plasmid pEH60 did not compensate deficiency of recA function of bacteria, although it partly compensates deficiency of recBC function. Increase of survival after introduction of plasmid pEH60 in the cell was obtained only for recBC- strain, but not for wild type and recA- strains.     

Pleiotropic effects of a DNA adenine methylation mutation (dam-3) in Escherichia coli K12.

The dam-3 mutation results in a five-fold reduction in the number of 6-methyl-adenine (6-meA) residues in the DNA of E. coli K12 or phage lambda. The DNA of phage fd appears to be devoid of 6-meA when propagated on dam-3 bacteria. The phenotypic differences between dam-3 and dam+ bacteria include: (i) increased free phage in lysogenic dam-3 cultures, (2) increased sensitivity to methyl methanesulfonate (MMS), (3) inviability of dam-3 lex-I strains, (4) lower molecular weight of DNA in dam-3 bacteria in the absence of DNA ligase and (5) increased rate of DNA degradation in dam-3 recA strains.     

Cloning of Thermomonospora fusca genes coding for beta 1-4 endoglucanases E1, E2 and E5

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI and fragments in the size range from 4 to 15 kb were isolated, ligated into lambda gtWES.lambda B arms, packaged, and the recombinant phages plated on Escherichia coli. The plaques were screened for carboxymethyl cellulase (CMCase) activity by a gel overlay procedure, and 25 plaques were positive among the 15,000 plaques that were screened. Positive phages were amplified and used to prepare infected E. coli extracts which were assayed for CMCase activity before and after treatment with antisera prepared against five purified T. fusca beta 1-4 endoglucanases (E1-E5). One phage produced an enzyme that was inhibited by E1 antiserum, nine of the phages produced enzymes that were inhibited by E2 antiserum, 14 produced enzymes that were inhibited by E5 antiserum and the enzyme produced by the other phages was not inhibited by any of the five antisera. The DNA insert present in the phage coding for E1 was cut into a number of different fragments which were subcloned into E. coli first using lambda gtWES.lambda B and then plasmid pBR322. The smallest active subclone, pTE12, contained a 3.1-kb insert. The insert present in one of the phages coding for E2 was also subcloned and the smallest active subclone pTE23 contained a 2-kb insert. E. coli HB101 containing plasmid pTE12 or pTE23 produced enzymes that were identical to E1 and E2, respectively, in all the properties tested.     

Location and molecular cloning of the structural gene for the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli

The structural gene for deoxyguanosine triphosphate triphosphohydrolase (dGTPase) (EC 3.1.5.1) and its regulator, optA, have been located on a lambda phage carrying a 17.5kb Escherichia coli DNA insert. The DNA fragment has been excised and ligated into pBR325 and also transferred to another lambda vector. From the results of transduction and transformation experiments, we find that the structural gene for dGTPase is very closely linked to optA and dapD, which locates it at approximately 3.6 minutes on the genetic map of E. coli K12. We propose the mnemonic dgt as the designation for the structural gene for this enzyme.     

Genetic and physiological studies of an Escherichia coli locus that restricts polynucleotide kinase- and RNA ligase-deficient mutants of bacteriophage T4.

The RNA ligase and polynucleotide kinase of bacteriophage T4 are nonessential enzymes in most laboratory Escherichia coli strains. However, T4 mutants which do not induce the enzymes are severely restricted in E. coli CTr5X, a strain derived from a clinical E. coli isolate. We have mapped the restricting locus in E. coli CTr5X and have transduced it into other E. coli strains. The restrictive locus seems to be a gene, or genes, unique to CTr5X or to be an altered form of a nonessential gene, since deleting the locus seems to cause loss of the phenotypes. In addition to restricting RNA ligase- and polynucleotide kinase-deficient T4, the locus also restricts bacteriophages lambda and T4 with cytosine DNA. When lambda or T4 with cytosine DNA infect strains with the prr locus, the phage DNA is injected, but phage genes are not expressed and the host cells survive. These phenotypes are unlike anything yet described for a phage-host interaction.     

Replacement of potassium chloride by potassium glutamate dramatically enhances protein-DNA interactions in vitro

Although protein-nucleic acid interactions exhibit dramatic dependences on both ion concentration and type in vitro, large variations in intracellular ion concentrations can occur in Escherichia coli and other organisms without apparent effects on gene expression in vivo. E. coli accumulates K+ and glutamate as cytoplasmic osmolytes. The cytoplasmic K+ concentration in E. coli varies from less than 0.2 to greater than 0.9 m as a function of external osmolarity; corresponding cytoplasmic glutamate concentrations range from less than 0.03 to greater than 0.25 m. Only low levels of chloride occur in the cytoplasm of E. coli at all osmotic conditions. Since most in vitro studies have been performed in chloride salts, whereas glutamate is the more relevant physiological anion, we have measured the effects of the substitution of potassium glutamate (KGlu) for KCl on the kinetics and equilibria of a variety of site-specific protein-DNA interactions in vitro. Both the interaction of E. coli RNA polymerase with two phage lambda promoters and the interactions of various restriction enzymes with their DNA cleavage sites are enhanced by this substitution. Using the abortive initiation assay, we find a greater than 30-fold increase in the second-order rate constant for open complex formation at the lambda PR promoter and a 10-fold increase at the lambda PR' promoter, when KGlu is substituted for KCl. Replacement of KCl by KGlu does not affect the strong salt dependences of these interactions; increasing either KCl or KGlu concentrations decreases both reaction rates and extents. Substitution of glutamate for chloride does, however, shift the range of salt concentrations over which these interactions are observable to higher K+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two glutamyl-tRNA reductase activities in Escherichia coli

delta-Aminolevulinic acid (ALA) is the first committed precursor for tetrapyrrole biosynthesis. ALA formation in Escherichia coli occurs in a tRNA-dependent three-step conversion from glutamate. Glu-tRNA reductase is the key enzyme in this pathway. E. coli K12 contains two Glu-tRNA reductase activities which differ in their molecular weights. Here we describe the purification of one of these enzymes. Four different chromatographic separations yielded a nearly homogeneous protein. Its apparent molecular mass under denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation and gel filtration) is 85,000 +/- 5,000 Da. This indicates a monomeric structure for the active enzyme. Gel filtration and glycerol gradient centrifugation indicate that the other activity has a molecular mass of 45,000 +/- 5,000 Da. In the presence of NADPH both enzyme activities converted E. coli Glu-tRNA(2Glu) to glutamate 1-semialdehyde. Addition of GTP or hemin did not affect the reductase activity. Both enzymes display sequence-specific recognition of tRNA; E. coli Glu-tRNA(2Glu) is a good substrate while the Chlamydomonas reinhardtii, Bacillus subtilis, and Synechocystis Glu-tRNA(Glu) species are poorly recognized.     

Lambda phagemids and their transducing properties

Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1. This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with lambda gt::pMB9 was obtained when E. coli cells lysogenic for lambda were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.