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(Rgla) and Mcrb (Rglb) Loci of Escherichia Coli K-12

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(Rgla) and Mcrb (Rglb) Loci of Escherichia Coli K-12 Copyright 0 1989 by the Genetics Society of America Genetic and Physical Mapping of the mcrA (rglA)and mcrB (rglB) Loci of Escherichia coli K-12 E. A. Raleigh,* R. Trimarchi" and H. Revelt *New England BioLabs, Beverly, Massachusetts 01915-9990, and tDepartment $Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637 Manuscript received June 13, 1988 Accepted for publication March 10, 1989 ABSTRACT We have genetically analyzed, cloned and physically mapped the modified cytosine-specific restric- tion determinants mcrA (rglA)and mcrB (rglB)of Escherichia coli K-12. The independently discovered Rgl and Mcr restriction systems are shown to be identical by three criteria: 1) mutants with the RglA- or RglB- phenotypes display the corresponding McrA- or McrB- phenotypes, and vice versa; 2) the gene(s) for RglA and McrA reside together at one locus, while gene@) for RglB and McrB are coincident at a different locus; and 3) RglA+ and RglB' recombinant clones complement for the corresponding Mcr-deficient lesions. The mcrA (rglA) gene(s) is on the excisable element e14, just clockwise of purB at 25 min. The mcrB (rglB)gene(s), at 99 min, is in a cluster of restriction functions that includes hsd and mrr, determinants of host-specific restriction (EcoK) and methyladenine-specific restriction respectively. Gene order is mcrB-hsdS-hsdM-hsdR-mrr-serB.Possible models for the acqusi- tion of these restriction determinantsby enteric bacteria are discussed. HE wide variety of restriction systems elaborated 1960). Wild-type T-even phage with the glucose mod- T in different bacterial species allow bacteria to ification are insensitive to Rgl restriction, as is a T4 assess the origin of incoming DNA and determine its triplemutant that synthesizes DNA with ordinary fate (forreviews see ARBER1974; BICKLE1982; MOD- cytosine (TAKAHASHI,SAITO and IKEDA1978). In RICH and ROBERTS1982). In the most familiar type I contrast, T-even phage with HMC-DNA, but lacking and type I1 systems, exemplified by EcoK and EcoRI, the normal glucose modification, are restricted. These endonucleases recognize specific unmodified nucleo- phage arise when the glucosyltransferase enzymes are tide sequences and cleave the DNA into fragments, defective, as in phage gt mutants (REVEL, HATTMAN thusdestroying its biological activity. DNA is pro- and LURIA 1965; GEORGOPOULOS1967), or when tected from digestion when modified at the endonu- wild-type phage are grown on a mutant galU host, clease recognition site by an associated modifying which cannotprovide the glucosyl donor, UDPG function, usually by methylation of a specific adenine (SHEDLOVSKYand BRENNER 1963; HATTMANand Fu- (at N6) or cytosine [at C5 (SMITHand KELLEY1984) KASAWA 1963). Evidence for limited cleavage of non- or N4 (BUTKUSet al. 1985)lresidue. In this way, glucosylated HMC-DNA by RglB in vivo (DHARMAL- endogenous DNA is immune to endonucleolyticcleav- INGAM and GOLDBERG1976) and in vitro (FLEISCH- age, while foreign DNA is recognized and destroyed, MAN, CAMPBELLand RICHARDSON1976) suggests a providing protection from invading phages grown on role for recognition of sequence as well as of specific heterologous hosts, as well as from foreign plasmids base modification. Nevertheless, it has been ques- (ARBER 1974). tioned whether the Rgl functions represent true re- A less familiar type of restriction, aimed specifically striction enzymes, because sequence specificity has not at modified DNA in T-even phages, was historically been clearly demonstrated, and because there is no the first restriction system to be described (LURIAand cognate methylase activity (KRUGER and BICKLE HUMAN1952). Two functions specifying this restric- 1983). tion activity are present in Escherichia coli K-12 (REVEL More recently, sequence-specific, modification-de- 1967). Originally called Rs and RP,~,they were sub- pendent restriction systems that attack DNA with the sequently renamed RglA and RglB, for restriction of common base, 5-methylcytosine, were found in E. coli glucoseless phages (see review, REVEL 1983). During K-12 (RALEIGHand WILSON1986; NOYER-WEIDENER, T-even phage replication the unusual base 5-hydrox- DIAZand REINERS1986). N4-Methylcytosine also can ymethylcytosine (HMC) completely substitutes for cy- confer sensitivity to one of these systems (BLUMEN- tosine in phage DNA, and the hydroxymethyl group THAL, GREGORY andCWPERIDER 1985; BUTKUSet al. is subsequently glucosylated in phage-specific patterns 1987). These Mcr (for modified cytosine restriction) atthe polynucleotide level (LEHMANand PRATT systems are able to res& phage h modified by some, Genetics 122: 279-296 (June, 1989) 280 A. E. Raleigh, R.H. Trimarchi and Revel butnot all, sequence-specific cytosine modification Phage Plvir was used for transductions (MILLER1972). methylases foreign to E. coli K-12. Plasmid clones Xvir or XcIb2 were used for assays for K and B restriction. carrying the corresponding methylase genes are also T2H, T4D, andT6 (hereafter called T2, T4, and T6) and their derivatives T2gt2, T4agt57Dgt14 and T6gt41 [(REVEL, restricted. HATTMANand LURIA 1965;GEORGOPLOULOS 1968; REVEL In the present communication we show, by compar- 1983), hereafter called T2gt, T4gt, and TGgt] were used to ative genetic and physical mapping and cloning, that assay for Rgl phenotype. Nonglucosylated wild type T-even the newly identified McrA and McrB restriction sys- phages (T* phage) were prepared by growth on the galU host W4597 (HATTMANand FUKASAWA1963). tems are genetically identical to the previously de- Mutant isolation: Isolation of RglA- and B- mutants was scribed RglA and RglB functions active against non- as described previously (REVEL 1967).Briefly, cells unable glucosylated HMC-DNA in T-even phages. The map- to restrict T6gt phage (RglA- cells) yield nibbled, irregular ping reveals the interesting facts that the mcrA (rglA) colonies when nitrosoguanidine-mutagenized wild-ty e cul- locus is found on an accessory determinant, e14, and turesare spread onto plates seeded with about10 T6gt r phage. RglB- cells were selected similarly from mutagenized that themcrB (rglB)locus is part of a clusterof restric- RglA- cultures using T2gt as the selective agent. Cells with tion functions that may have been acquired from (an) the RglA' RglB- phenotype, notpreviously described, were accessory element(s). selected from mutagenized RglA- RglB- cultures by screen- ing for smooth, nonnibbled, insensitive colonies in the pres- MATERIALSAND METHODS ence of T6gt.Alternatively, this phenotype could be se- lected, though less efficiently, from wild-type RglA+ RglB+ Nomenclature: The restriction systems described here cells as nibbled colonies in the presence of phenotypically were originally characterized independently by two differ- Glu- T4* phage that are obtainedas above. HsdR- mutants ent kinds of assays, using two different mnemonic designa- were isolated in similar fashion, using unmodified XcIb2 tions, as described in the Introduction. A major point of (X.0) as the screening agent. this paper is that the two kinds of assays test the same McrB- mutants, spontaneous or induced by TnlO with underlying function,restriction of DNA modified at partic- XNK561 as described (KLECKNERet al. 1978), were isolated ular cytosine residues. Accordingly, we must initially retain among thesurvivors of transformation of a restricting strain a separate nomenclature for these two assays, but finally with pHaeII4-I1 or pER82, plasmids that carry haellM, the choose a single preferred designation for the loci. We will gene for theHue11 methylase, M .HaeII.' Survival followin retain the original designations, RglA, RglB and rglA, rglB transformation for spontaneous mutants was 1 to 5 x IO- k. in discussing those experiments in which only T-even phages Of these, 20-35% had lost plasmid methylase activity, and are at issue, and will use the terms McrA and McrB specifi- 65-80% were host mutants permissive for the methylase cally to refer to restriction of methylated (not hydroxy- plasmid. TnlO insertion with Tet' selection yielded survivors methylated) plasmids and phages. In discussing transduction at afrequency of 2 X per infected cellwith about 2% experiments, in some of which both assays were used, we auxotrophs as reported in the literature (KLECKNER,ROTH will use both genotypic designations, placing first the mne- and BOTSTEIN1977; KLECKNERet al. 1978). Twenty inde- monic for the phenotypeprincipally used in the experiment, pendent pools of insertions were transformed with pER82, and in parentheses that for the phenotypechecked second- yielding survivors at frequencya of about 6 X All arily. In discussing physical mapping experiments, and in three independent survivors had insertions in mcrB. future work, we will use exclusively the mnemonic mcr for To obtain plasmid-free mutantsfor further analysis, genotypic designations, since we believe that this more ac- either the mcrB mutation was backcrossed to the original curately reflects the function of these loci. The phenotypic host by transduction, or the permissive mutant strains were designations,Rgl, will be employed when it is useful to cured of the haeIlM plasmid by growth in the presence of distinguish restriction of T-even phage from restriction of the incompatible Cm' plasmid, pNK259, without Ap' selec- methylated phages or plasmids. tion (SHEN, RALEIGHand KLECKNER1987). pNK259 was Bacterial strains, plasmids and media: Strains used are then cured from theCm' Ap" derivatives by penicillin selec- given in Table
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