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.     

In vivo restriction of Escherichia coli-transforming DNA by endonuclease R.M.EcoRI

Transformation of Escherichia coli K-12 for various chromosomal markers was accomplished by using AB1157 recBC+ strain as a recipient. The yield of transformants was reduced 10-fold, as compared with that obtained in JC7623 recBC sbcB recipient. Elimination of transformation has been obtained for arg, pro, his markers in AB1157 (pSA14) harbouring the R.M.EcoRI coding plasmid. Production of restriction endonuclease in this strain did not affect the efficiency of transformation for thr, leu markers. The presence of pSA25 which is isogenic to pSA14 but devoid of R.M.EcoRI genes has been irrelevant to transformation for leu, arg, pro, his, thr markers. Correlation between the restriction of transformed markers in vivo and in vitro is discussed.     

In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12.

Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene product by an enzyme or enzymes that reduce the protein to an active form.     

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.     

nfi, the gene for endonuclease V in Escherichia coli K-12.

Endonuclease V is specific for single-stranded DNA or for duplex DNA that contains uracil or that is damaged by a variety of agents (B. Demple and S. Linn, J. Biol. Chem. 257:2848-2855, 1982). Thus, it may be a versatile DNA repair enzyme. The protein was purified to apparent homogeneity, and from its N-terminal sequence, its gene, nfi, was identified. nfi is immediately downstream of hemE, at kb 4208 (90.4 min) on the current chromosomal map of Escherichia coli K-12. This region was cloned, and plasmid insertion and deletion mutants were used to study its molecular organization. Although nfi is the third of four closely spaced, codirectional genes, it is expressed independently.     

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.     

Characterization of endonuclease III (nth) and endonuclease VIII (nei) mutants of Escherichia coli K-12.

The nth and nei genes of Escherichia coli affect the production of endonuclease III and endonuclease VIII, respectively, glycosylases/apurinic lyases that attack DNA damaged by oxidizing agents. Here, we provide evidence that oxidative lethal lesions are repaired by both endonuclease III and endonuclease VIII and that spontaneous mutagenic lesions are repaired mainly by endonuclease III.     

Localization of a genetic region involved in McrB restriction by Escherichia coli K-12.

A 5,500-base-pair BglII-EcoRI fragment proximal to the hsd genes of Escherichia coli K-12 has been cloned in the plasmid vector pUC9. The resultant hybrid plasmid was shown to complement the mcrB mutation of E. coli K802. The presence of the hybrid plasmid in strain K802 caused an 18.3-fold drop in transformation efficiency with AluI-methylated pACYC184 relative to unmethylated pACYC184. These results indicate that the cloned DNA is involved in the McrB system restriction of 5-methylcytosine DNA.     

Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12.

The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.     

In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.     

Analysis of the genetic requirements for viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants.

RecBCD protein, necessary for Escherichia coli dam mutant viability, is directly required for DNA repair. Recombination genes recF+, recN+, recO+, and recQ+ are not essential for dam mutant viability; they are required for recBC sbcBC dam mutant survival. mutH, mutL, or mutS mutations do not suppress subinduction of SOS genes in dam mutants.     

In vitro proteolytic cleavage of the Escherichia coli Ada protein by the ompT gene product.

Down regulation of the adaptive response to alkylation damage in Escherichia coli has been proposed to occur by proteolytic cleavage of the regulatory Ada protein. In this paper, it is shown that proteolysis of the Ada protein as observed in cell extracts is caused by the ompT gene product. This protease, however, was not involved in switching off the adaptive response in vivo.     

The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis.

The nucleotide sequence recognized and cleaved by the restriction endonuclease MboI is 5' GATC and is identical to the central tetranucleotide of the restriction sites of BamHI and BglII. Experiments on the restriction of DNA from Escherichia coli dam and dam+ confirm the notion that GATC sequences are adenosyl-methylated by the dam function of E. coli and thereby are made refractory to cleavage by MboI. On the basis of this observation the degree of dam methylation of various DNAs was examined by cleavage with MboI and other restriction endonucleases. In plasmid DNA essentially all of the GATC sequences are methylated by the dam function. The DNA of phage lambda is only partially methylated, extended methylation is observed in the DNA of a substitution mutant of lambda, lambda gal8bio256, and in the lambda derived plasmid, lambdadv93, which is completely methylated. In contrast, phage T7 DNA is not methylated by dam. A suppression of dam methylation of T7 DNA appears to act only in cis dam. A suppression of dam methylation of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely methylated. The results are discussed with respect to the participation of the dam methylase in different replication systems.     

Analogs of the dnaB gene of Escherichia coli K-12 associated with conjugative R plasmids.

The dnaB266(Am) mutation in Escherichia coli K-12 is an amber mutation such that strains carrying this mutation are not viable in a sup+ strain. With five different R plasmids, it has been possible to construct viable R+ derivatives of this amber mutant and show that the plasmids themselves do not carry amber suppressors. This is interpreted as evidence for the presence of dnaB analog genes associated with these plasmids. Plasmid-positive strains carrying these genes often showed some degree of cryosensitivity of DNA synthesis and colony-forming ability. These observations indicate that the presence of dnaB analog genes in association with R plasmids must be relevant to the plasmid state or to some aspect of conjugative ability.