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 tothe 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 thatthe 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 torefer 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 1. Plasmids used and their sources are shown tion (MILLER1972) in the presence of chloramphenicol. in Table 2. All are based onthe vector pBR322 or its Ampicillin kills the growing Cm' Aps cells, but does not kill relative pBR313, except forpGP325, pCP326 (+) and the nongrowing Cms Ap"cells that arise by spontaneous pGP326 (-), which are based on the vector pACYC184. plasmid loss. These plasmidless cells are not killed by the Media were routinely Luria-Bertani medium (LB; SILHAVY, bacteriostatic agent chloramphenicol either, and will subse- BERMAN, andENQUIST 1984) or M9 minimal salts (MILLER quently formcolonies when washed cells are plated on drug- 1972) supplementedwith drugs (ampicillin, 100 /.tg/ml; tet- free plates. racycline, 20 /.tg/mI; streptomycin, 100 /.tg/ml) or nutrients Mcr assays: Three tests, a X growth assay and two plasmid (L-amino acids, 40 /.tg/ml; bases, 20 /.tg/ml; or vitamins, 1 transformation assays, were used to measure Mcr activity. /.tg/ml) as required. Glucose was used in minimal media at I: The X assay tested the ability of strains to restrict X 0.2%. In some cases, TG minimal medium (ECHOLSet al. bearing a foreign modification pattern. X was modified by 1961) was used instead of M9. Phage titrations and plate growth on a host carrying a cloned methylase gene for M. stocks were doneon X medium (KLECKNERet al. 1978) HaeII, M.MspI, or M.HpaII. Modified X titers were deter- except for P1, for which LB with 5 mMCaC12 was used. mined either by spotting 25 and 50p1 of 10-fold serial phage Phages were diluted in Xdil (10 mM Tris-HCI, pH 7.5, 10 dilutions on a bacterial lawn in soft agar on X medium agar mM MgSO4). andrecording the endpoint forplaque formation(for Chemicals were from Sigma or Nutritional Biochemical X.HaeII and X.MspI) or by full plate counts (X.HpaII). Corporation, Cleveland, Ohio, except for streptomycin sul- Plating efficency (EOP) is defined as the lysate titer on the fate (E. L. Squibb and Sons, New York) and nitrosoguani- dine (N-methyl-N'-nitro-N-nitrosoguanidine;Aldrich Chem- M.Haell, Haell-specific modification methylase. This form of designa- ical Co., Inc., Milwaukee, Wisconsin). tion (SMITHand NATHANS 1973)will be used for all methylases cited here. Mapping of mcr(rgl) A and B Loci 28 1

TABLE 1 Bacterial strains

rgl/mcr genotype" Other relevant Strain Background A B genotypeb Source' 1100 + + F-X-supE44 thi-1 rfbDf?spoTI? I. R. LEHMAN (1) endAl C600 1 + N. KLECKNER (1) CH731 + NT e14+ H. BRODY(2) C H13 32 CH73 CH1332 1 - NT el4' H. BRODY(2) C H 1372 CH731 1372 CH 1272::TnlO NT el4-1272::TnlO H. BRODY(2) E R1370 NK7254 ER1370 + + F-X-leu+ tonA2 This work A(1acZ)rl supE44 trp31 his-1 argG6 rpsLIO4 xyl-7 mtl-2 metBl hsd+ serB28 ER1372 ER1370 + 1 zjj202::TnlO This work ERI 378 ER 1370 + 1 hsdR2 serB+ This work ER 1380 ER1370 + + hsdR2 zjj202::TnIO serB+ This work ERl38 1 ER1370 + + hsdR2 serB+ This work ER1394 "294 + 1 hsd+ zjj202::TnlO serB28 This work ER 1398 ER 1394 + 1 hsdR2 This work EK1467 ERI 370 + 4::TnlO hsdR2 serB+ This work; TnlO induced EK 1468 ER-I 370 + 5::TnIO hsd+ serB+ This work; TnlO induced ER 1469 ER 1370 + 6::TnIO hsdR2 serB+ This work: TnlO induced ERl486 ER 1370 + A2 A(mcrB-hsd-mrr)2::TnlO This work; spontaneous, from ER1380, containing ZJ,i202::TnIO ER1489 ER 1370 + 3::IS hsdR2 serB+ This work; spontaneous ERI 509 H680 - NT zcg2043::TnIOpurB51 This work; H680 from P. G. DE HAANvia B. BACHMANN ER151 6 ER1370 ER1516 - 1 zcg2043::TnIO purB51 hsdR2 This work serB+ EK 1564 ER 1370 1272::TnlO + hsdRP serB+ This work EK 1565 ERl370 1272::TnIO 1 hsdR 2 serB+ This work ER1576 JK268 - NT zcf2033::TnlO purB58 This work; UK268, see 3) ER1648 ER1370 1272::TnlO A2 A(mrr-hsd-mcrB)2::TnlO serB+ This work; argf ER 1656 ER 1370 + 1 hsdR 2 This work HBlOl K/B + B hsdS20s J. BROOKS(4) Hfr Cavalli K12 + 1 hsd+ B. BACHMANN(I) HRI 10 K12 + + K12 F' (X) hsd' K-12 wild type, Stanford; (1) HRl11 HR110 2 + P1' REVEL (5,6); was Kr6- or Kr6-r~.r+ HRl12 HKI 11 2 8 REVEL(5, 6, 7); was = K,gI HRll6 1100 ts4 + This work, NGd, T6gt selection HR 140 HK112 + 8 SUP REVEL (6, 7);was Kr6+r2.4-~~+ HR184 "294 3 + hsdR 17 This work, NGd, T6gt selection

a +: restricts modified DNA; -: does not restrict modified DNA; NT: not tested. Where assigned allele numbers have been assigned these are given in lieu of -: all alleles are defective for restriction. All mcrB alleles have been mapped. Not all mcrA mutations have been mapped, but we assume that they are allelic (see text). Not every strain has been tested for both Rgl and Mcr phenotypes; see text and RALEIGHet al. (1988) for those that have. Complete genotypes and details of strain construction are available from the authors upon request. Genotypes shown are complete for recipients used in strain construction. For strains prefixed ER and HR, the genotype is given only where it differs from the original strain (given under "Background"); mcrA and mcrB genotypes are given in the two columns so labelled. For strains not constructed here, only the genotype relevant to this paper is given. ' Numbers in parentheses refer to the following reports: (1) BACHMANN (1987b):(2) BRODY, GREENERand HILL (1985); (3) HUGHES, SIMONSand NUNN(1988); (4) BOYER and ROULLAND-DUSSOIX(1969): (5) REVEL(1 967); (6) REVELand GEORGOPOLILOS(1969): (7) GEORGOPOULOSand REVEL (197 1); (8) TAYLORand TROTTER(1972); (9) HEITMANand MODEL(1987): (10) BLUMENTHAL, GREGORYand COOPERIDER(1985): (1 I) ROBERTSet al. (I 985); (12) BOLIVARet al. (1977); (13) HATTMANand FUKASAWA (1963); (14)WOOD (1966); (15) SIGNER,BECKWITH and BRENNER (1 965);6) (1BOYER (1 966). NG, nitrosoguanidine-inducedrgl mutation. teststrain divided by lysatetiter on a permissivestrain. ylation pattern. Transformation was as described (MANDEL McrBrestriction reduced EOP to 0.02-0.001 for h.HaeI1 and HICA 1970). Briefly, 50 pI of transformation mix was and X.MspI; McrA restriction reduced EOP to 0.3-0..5 for added to 1.5 ml LB broth and grown for 20 min at 37 "C X.HpaII. to allowexpression phenotypic aliquotsspreading prior to IIA: Transformation assays tested the ability of strains to on ampicillin selective medium to determine the number of restrict transformation by plasmids bearing a foreign meth- transformed cells. In method IIA, pBR322 was methylated 282 E. A. Raleigh, R. Trimarchiand H. Revel

TABLE 1-Continued

rgllmcr genotype" Other relevant Strain Background A B genotypeb Source' genotypeb B A Background Strain H R186 AT2459 HR186 - 9 hsd+ serB22 This work, (8) NGd, T2gt selection JH83 GW 1040 ' NT + hsd+ mrr2::Tn5 J. HEITMAN(9) zjj202::TniO JM 107 - + hsdR7 1 R. M. BLUMENTHAL(1 0) JM 107MA2 JM107 - 7 R.hsdR 1 7 M. BLUMENTHAL(1 0) LCK8 1 WA802 zjj202::TnIO1 hsdR2 L. COMAIvia B. BACHMANN "294 1100 + 7 + hsdR 1 J. BROOKS(1) NK7254 JC1552 + + F- leu-6 tonA2 A(1acKLECKNER Z)rlN. (1 1) supE44 trp-3 1his-1 argG6 str-104xyl-7 mtl-2 metBl X- RRl HBlOl + B asbut HBlOl recAf G. WILSON(1 2) W3110 + + N. KLECKNER(1) w4597 + + galU S. E. LURIA(1 3) W6 + + B. BACHMANN(1) WA802 1 1 hsdR2 N. MURRAY(1 4) WA803 1 1 hsdS3 N. MURRAY (14) X149 - + F- lac-2 purB pyrF trp A E. SIGNER (15) has tyr mal thi h' E. coli B derivatives B + + S. E. LURIA AC2522 + + HfrBl(unstable Hfr) H. BOYER(1 6) AC25 19 + + F- progallactrp leu BOYERH.thr 6)(1 met his str' HR 45 AC2519 HR45 5 + This work, T6gt selection Shigella sonnei SH - - S. E. LURIA (12)

TABLE 2 Plasmids used

P lasmid Functions carried Vector carried Functions Plasmid Source or construction pB R322 Ap'M.HaeIIpBR322 R.Hae1I BOLIVARet al. (1 977) pHaeIIAp'M.HaeII 4-1 R.HaeIIpBR322 1 R. CROFT and G. WILSON pH ha11 2-13pHha11 Ap'M.HhaIIpBR322R.HhalI D. NWANKWO andG. WILSON pHpal1 pBR322 Ap' M.HpaII C. CARD andG. WILSON pER82 pBR322Ap' M .HaeII in vitro deletion of pHaelI4-1 1 pM sp 1-30pMsp Ap'pBR322R.MspI M.MspI NWANKWO andWILSON (1988) pPstI Reg1 pPstI pBR322Ap'R.PstI M.Pstl G. WILSON pBg3 Ap' HsdRMS' pBR322 SAIN and MURRAY (1 980) PB g6 Ap' Hsd'S Ap' PBg6 McrB/RglB pBR322 SAIN and MURRAY(1 980) pER 105 Ap' McrB/Rgl B pBR322 This work; mcrB BamHI clone, Figure 3 pER106 Ap' McrA/RglA Pin pBR322 This work; mcrA BamHI clone, Figure 5 pER137 Ap' McrA/Rgl A Pin pBR322 This work; subcloneof pER106, Figure 5 PER 146 Ap'Pin pBR322 This work; subclone of pERI06, Figure 5 pNK290 Ap' IS 10 pBR333 FOSTERet al. (198 1) pNK259 Cm'Ap' pBR322 SHEN, RALEIGHand KLECKNER(1 987) pAG2 Ap' 'e 14 pBR3 13 GREENER and HILL(1 980) pHBlO6 Ap' 'e 14 pBR3 13 BRODY, GREENER and HILL (1 985) pHB107 Ap' 'e14 Pur B pBR3 13 BRODY, GREENER and HILL (1985) Tc'Pin+ pACYC 184 PCP325 pACYC Tc'Pin+ PLASTERK and VAN DE PUTTE (1985) pGP326 (-) Tc'Pin- (- orientation)pACYC184 PLASTERKand VAN DE PUTTE(1 985) pCP326 (+) Tc'Pin- (+ orientation)pACYC184 PLASTERKand VAN DE PUTTE(1 985) in vitro (designated, e.g.,pBR322 .MspI) to render sensitiveit for a methylase (the plasmid is designated, e.g., pHaeZZM or to restriction. Restriction was inferred when the fully meth- pER82) that confers sensitivity to restriction. In this case, ylated plasmid gave less than one-tenth as many transform- relative transformation efficiency (relative EOT) was used ants as the unmethylated plasmid (efficiency of transforma- as the index of methylase-specific restriction. This was cal- tion (EOT) 50.1). McrA' restricted pBR322.HpaII with an culated as the ratio of the transformation efficiency (trans- EOT of 0.1; McrBf restricted pBR322 .MspI and pBR322. formants per ml in test strain/transformants per mi in per- AM, with an EOT of 0.003-0.02. missive strain) of the test plasmid to thetransformation ZZB: In method IIB, a pBR322 derivative carries a gene efficiency of the vector alone. This procedure controls for Mappingmcr(rgl) of A and B Loci 283 effects on intrinsic transformability of strain background, another probe, since the autoradiograms can be superim- plasmid size, and DNA concentration. posed. Hybridization and the first wash were carried out at Mrr assays: Measurement of Mrr restriction activity was 65’ (high stringency). Size standards were T7 digested with as for Mcr activity method I, except that the targetX phages DraIII or with BglII. Probes were nick-translated (RIGBYet were modified by M.HhaIIor M.PstI, as described by al. 1977) pBg6 or pNK290(to detect chromosomal se- HEITMAN andMODEL (1987). Mrr+ restricted X.HhaII and quences) each mixed with nick-translated T7 (to detect the X.PstI with an EOP of 0.1 to 0.3. size standard). Rgl assays: Rgl restriction activity was measured by the Digested chromosomal DNA from twelve strains was growth properties of wild type and gt mutant T-even phages probed with pBg6. Three different enzymes (HindIII, Sal1 determined by spot titer, as described above (see Table 3) and ApaI) were used to digest chromosomal DNA of or by crpss-streak tests. For the RglA cross-streak test, a ER1380, ER1381, ER1489, ER1468, and W3110. DNAs toothpicked colony was streaked at right angles across a fromER1370, ER1372, ER1378, ER1486, ER1467, dried loop-streak of T6gt phages. RglB activity, in a strain ER1469 and LCK8 were digested with these and also with known to be RglA-, was assayed similarly by cross-streaking NcoI. These digests were probed with pBg6. The HindIII, against T2gt or T4gt. In an RglA+ host, RglB activity was NcoI, and ApaI digests were also probed with pNK290, assayed by full-plate titration assays of wild-type T2 EOP. which carries one of the inverted repeats of TnlO and will RglA and RglB activity can also be measured by spot test therefore detect fragments that contain TnlO ends. analysis with phenotypically Glu- wild-type T-even phages Cloning of McrA and McrB Donor DNA (about 25 pg; (T*) that have grown on galU hosts (see RESULTS). The from ER1370) was prepared by the MARMUR(1961) pro- restriction patterns are the same but efficiencies of plating cedure, digested with BamHI (16 units for 2 hr in a total aregreater (about as compared to for gt phages), volume of 30 pl) and then precipitated with isopropanol in since progeny phage, now modified with glucose, are no the presence of 2 M NH4-acetateand resuspended in 10 mM longer restricted. Tris, 1 mM EDTA, 5 mM NaCI, pH 7.5. The DNA frag- Hsd assays: EcoK and EcoB restriction was assayed by ments were ligated to BamHI-digested dephosphorylated cross-streak against several dilutions of unmodified X or by pBR322 (about 5 pg; NEB) at a final DNA concentration of spot titer, as described inMcr assays method I. M.EcoK 25pg/ml, with 10,000 units of T4 DNAligase in the activity was determined by picking X plaques grown on the recommendedbuffer and incubatedovernight at16”. strain of interest into 1 ml of diluent, and then spot titering ERI 5 16 was transformed (MANDELand HICA 1970) with the suspension on Hsd+ and hsdR strains. Plaques from hsdM the ligation mixture (5.25 ml total transformation volume) or hsdS strains, which lack M.EcoK, were restricted. and the transformation mixture was subcultured 1:7 into Pin assays: Pin function (see RESULTS)was tested using a LB with 40 pg/ml adenine (required to supplement thepurB PstI digest. There is a PstI site asymmetrically disposed mutation in ER1516) and grown with vigorous aeration at within the invertible DNA of e14, yielding one pair of PstI 37” for 1.75 hr. T4gt was then adsorbed for 20 min at a fragments when the invertible segment is in one orientation multiplicity of about 1 phage per cell, and portions were and another pair when it is in the other orientation. Pin is plated on LB ampicillin plates. Survival was about 2 X requiredto invert the segment.Preparations of a Pin+ after overnight incubation, and 7 colonies, all clones of mcrA plasmid carrying the invertible segment will contain both or mcrB, were obtained. Reconstruction experiments orientations, yielding the sumof the two possible digest showed that RglA+B- or RglA-B+ strains carried through a patterns. pGP326(+) and pGP326(-) are Pin- plasmids in “mock” procedure in parallel (no DNA added during the which the invertible segment is frozen in different orienta- transformation protocol) survived at a frequency of about tions unless complemented by a Pin+ host. pGP325 is the 30%. Pin+ parent of these. pGP325 (positive control), pGP326 (+)and (-)(negative controls), pAG2 (e14 plasmid), pERIO6 RESULTS (mcrA clone),pER137 (McrA+ subclone) and pER146 (McrA- subclone), all prepared in Pin- CH1332, were ana- Phenotypes conferred by the Rgl and Mcr sys- lyzed with PstI. Only one pair of diagnostic PstI fragments or the other was present in the digests of pGP326 (+) or tems: RglA and RglB phenotypes are defined by the (-), verifying that CHI332 is Pin-. pGP325, pAG2, plating properties of T-even wild-type phages, andof pERlO6, pERl37 and pER146 all displayed two pairs of their glucosyl transferase deficient mutant derivatives bands, demonstrating that they all carry functional pin genes (gt mutants) that fail to add glucose to phage HMC- and invertible DNA segments (not shown). DNA (Table 3). In K-12 strains all four combinations Methylation reactions and restriction digests were done as described (RALEIGHand WILSON1986). All enzymes were of Rgl phenotypes can be distinguished with just T6gt obtained from NEB and were used as recommended. and wild type T2. T6gt is restrictedonly by RglA Mapping studies by transduction or mating were done (comparelines 1 and 2), while T2, with 25% of its essentiallyas described in MILLER (1972) except in Hfr HMC residues not modified by glucose (LEHMANand crosses with E. coli B (BOYER1966). In these crosses, the PRATT1960) is restricted only by RglB (lines 1 and rich broth was washed out before plating the mating mixture for recombinants on minimal medium (since the efficiency 3). SinceRglA strongly restricts T6gt growth, the of mating is lower). All recombinants were purified before RglA phenotype of a strain can be determined une- scoring unselected markers. quivocally. Incontrast, the weak but reproducible Southern blotting was done as described (MANIATIS, RglB restriction of T2 is less definitive. However, in FRITSCHand SAMBROOK 1982)but using a mirror blotting the absence of RglA function, both T2gt and T4gt procedure. In this method, DNA present in the gel is provide a stringent assay for RglB function (compare transferred to nitrocellulose in both directions normal to the plane of the gel, producing two blots. In this way, it is lines 2 and 4). In K-12, T2gt and T4gt plate with an easier to be confident that a fragment hybridizing with one efficiency of 10-5-10” when the wild-type allele of probe is identical in size with a fragment hybridizing with either rglA or rglB is present. Restriction by the two 284 E. A. Raleigh, R. Trimarchiand H. Revel

TABLE 3 TABLE 4 Growth of T-even phages and their gt mutants on strains Efficiency of transformation of strains carrying mcrA and mcrB carrying rglA or rglB mutations mutations by plasmids bearing methylase genes

Plating efficiency of T-even Phenotype/genotype" ofRelative transformation efficiency' Phenotype/genotype" phagesb: K-12 strainsb of plasmids

RglAlrglA RglBlrglB T2 T6gtT4gtT2gt PM. Of K12 strain' McrAlmcrAMcrBlmcrB HpaIl pHaeII 4-1 1 pMspI 1-30 + + 0.1 10-~ 4 X 10-6 + + 0.002 0.0005 0.001 - + 0.1 10" 1.0 - + 0.8 0.0007 0.0009 + - 1.0 4 X 10"10-5 4 X 10-6 + - 0.001 0.3 0.4 - - - - 1.0 1.0 1 .o 1.o (1) (1) (1) Of B straind ' We use the genotype designation because the mutations have + + 1.0 lo+ 10-6 10-5 - been mapped to distinct loci (seebelow). However, each Mcr + 1.0 0.5 1 0.05 .o function could be encoded by multiple closely linked genes. Mcr assay method IIB. a We use the genotype designation because the mutations causing * the RglA and RglB phenotypes have been mapped to distinct loci ' The prototype recipient strains used were: ER138 1 mcrA+ (see below). However, two or more closely linked genes could be mcrB+; ER1378 mcrA+mcrB1; ER1564 mcrA1272::TnlO mcrB+; re uired for each of these functions. ER1565 mcrA1272::TnlO mcrB1. Values represent the titer on agiven strainltiter on the permis- sive host Shigella sonneiSH. The plating efficiency of wild-type T4 McrB- cells (Table 4, lines 1 and 2). The McrA and and T6 was 1.O in all cases. McrB functionsact completely independently. The ' Prototype E. coli K-12 strains were: HRl 10 rglA+ rglB+,HRl11 rglA2 rglB+; HR140 rglA+ rglB8; HR112 rglA2 rglB8. presence or absence of McrB has no effect on trans- Prototype E. coli B strains were: B rglA+rglB+; HR45 rglA- formation with hpaZZM, and McrA has no effect on rglB+. transformation with haeIIM. Assay of Mcr functions with in vivo methylated phage (method I) or in vitro functions is not additive, because there always are gt+ methylated pBR322 (method IIA)yielded similar pat- revertants in the phage stocks that determine a lower terns, but restriction was reduced (not shown). limit of plating efficiency. The RglA and RglB phe- We have not characterized Mcr functions that may notypes can also be assayed using the growth proper- be present in E. coli B, but we have tested two K-B ties of phenotypically Glu- T-even phages (T*),wild hybrid strains that carry the mcrB locus from E. coli type phage that have been grown on galU hosts (data B, HBl 0 1 and its RecA+ derivative, RR 1. We find no not shown). The restriction pattern is the same as for detectable McrB-dependent restrictionin these strains gt phages, but the EOPs are increased by a factor of (not shown). This observation is consistent with the 103-1 04, because the T* phages become modified attenuated RglB restriction phenotype of E. coliB. with glucose during replication and are notrestricted in subsequent growth cycles. However, most of the methylase clones used to char- E. coli B also has two Rgl restriction functions. RglA acterize McrB were isolated in RR1(RALEIGH and restricts strongly like its K-12 counterpart (lines 5 and WILSON1986) and it is conceivable that the E. coli B 3 in Table 3), but the RglB activity is very weak (line McrB has a different specificity, not observable with 6). Asin K-12, T6gt is restricted only by RglA. In our tests. striking contrast to K-12, however, T2gt and T4gt are Characterization of mutantstrains and coinci- only slightly restricted by RglB (EOP of 0.5 and 0.05, dence of phenotype: The rglA2 mutant, the rglB8 respectively, compared with EOPfor the K-12 mutant and the mutantalleles mcrBI-mcrB7, as well as RglB). This weak effect has prevented isolation of A(hsdS HindIII 1.8) (SAIN and MURRAY1980), were rglB mutants in E. coli B by procedures successful in shown to display correlated Mcr and Rgl phenotypes K-12. (datanot shown). The mcrB alleles testedincluded McrA and B restriction phenotypes are defined by five isolated spontaneously or following TnlO muta- the acceptance or rejection of DNA modified by se- genesis (MATERIALS AND METHODS; tested in ER1467, quence-specific methylases (RALEIGHand WILSON ER1468, ER1469, ER1486 and ER1489), the mcrBl 1986). While the two phenotypes are readily separable allele found in WAS02 (see below) andthe mcrB7 and can be assayed by several procedures (see MATE- allele of JM107MA2, isolated by BLUMENTHAL,GRE- RIALS AND METHODS), the largest effect occurs when GORY and COOPERIDER(1985; selection was for ac- the restrictiontarget is a cloned methylase gene ceptance of PvuZZM). The allele A(hsdS HindIII 1.8) (Table 4). A plasmid carrying the HpaII methylase present in strain NM496 (SAINand MURRAY1980; N. gene, hpaZZM, transforms McrA+ cells at about MURRAY,personal communication) was constructed relative to transformation of McrA- cells (Table 4, in vitro and transferredinto the chromosome of C600 lines 1 ane 3). Similarly, plasmids carrying haeZZM or by recombination. The rgl and mcr mutations were mspZM transform McrB+ cellsless efficiently than isolated using three different selective methods (for Mapping of mcr(rgl)A and B Loci 285 sensitivity to T-even gt phages, for MaHaeII accept- notypes. The rglA gene was linked to trp, near 27 ance, and for M .PvuII acceptance) in a variety of min; the rglB gene was linked to thr, around 0 min strainbackgrounds (HR110 (K-12 wild type), (datanot shown). In E. coli B, rglB is in a similar ER1370, W3 110, and JM107). In addition, a large location. However, the E. coli B rglA gene maps in a number of laboratory strains have been tested forRgl different position than in K-12, but we are unable to or Mcr phenotypes (RALEIGHet al. 1988), and those locate the position exactly. Except for ts mutations, tested by both methods (25 morestrains) also yielded the rglA mutants obtained in this background were correlatedphenotypes. Many laboratorystrains are simultaneously Thi- as previously noted(REVEL McrA- and a few are McrB-. 1967). One of these apparent deletions was mapped We believe that McrA- laboratory variants are prev- by F+ crosses to the vicinityof his. The F+ donor, alent because agene essential for McrA activity is AC2322, was crossed with the rglA thi multiple aux- carried by a UV-inducible prophage-like element, e14 otroph HR45 as described by BOYER(1966). RglA+ (GREENER and HILL 1980; BRODY, GREENER andwas HILL transferred at high frequency (63%) when His+ 1985; and see below) and that this has been cured on was selected and was 100% linked to Thi+ (400 sco- one or moreoccasions during construction of labora- red), but was essentially unlinked to Trp+ (0.5%) or tory strains by mutagenic treatments. Three lines of Pro+ (1 %). In K-12, his is at 44 min, some 20 min evidence support this view. First, the loss or alteration away from trp, where rglA is found in K-I 2. A roughly of e14 was correlated with the loss of McrA restriction. mapped thi locus at 46 minin K-12 (BACHMANN, e14+ CH731 is McrA+, while isogenic strains cured of 1987a) might correspond to the site of rglA in E. coli e14 (CH1332) or carrying one of three transposon B. The relative locations of his, trp and thi in E. coli B insertions in it (CH 1372) areMcrA- (RALEIGHet al. are not well known. 1988).Second, RglA- strains can bederived from Transductional mappingof rgZA (mcrA):Pl trans- "294 or ER1378 easily by a UV curing protocol duction of rglA revealed that it is linked only to PurB, (data not shown), consistent with the inducible nature of four tested auxotrophic markers in the region of of the e14 element. Third, cloning experiments sup- the E. coli chromosomenear trp (23-28 min). In port this assignment (see below). crosses a-c (Figure lA), 67-75% of selected PurB+ As far as we have determined by pedigree analysis, transductants received thedonor RglA phenotype, allMcrB- laboratory K-12 strains (except the K/B while trpA and pyrF were unlinked. In a fourth cross hybrids RRl and HBIO 1) ultimately derive the 99- (not shown) a pyrC rglA recipient was transduced with min mcrB region (see below) from the Cavalli Hfr or Pl(HR110). No donor RglA+ activity was detected from its hsd descendants,WA802 (also known as among 200 PyrCC transductants. Since purB shows K802) or WA803(K803). W6, theparent of the 4% linkage with PyrC (SIGNER,BECKWITH and BREN- Cavalli Hfr, is McrA+McrB+. The mcrBI mutation NER 1965) the data suggested, butdid not prove, that seems to have appeared in the Cavalli Hfr, which is the rglA locus is clockwise of purB. McrA restriction McrA+McrB-. This strain was crossed with C600 was unknown when these experiments were per- (McrA-McrB+) to yield WA704 (WOOD1966). The formed. recombinationevent selected is consistent with an With the discovery of the McrA and McrB restric- McrA-RglA-, McrB-RglB- phenotypefor WA704 tion systems (RALEIGHand WILSON1986) and dem- (not tested), and thus for its spontaneous HsdR- de- onstration of coincidence of McrA/RglA and McrB/ rivatives WA802 and WA803, as is observed. RglB phenotypes in more than 25 laboratory strains A spontaneous mcrB mutation selected from (RALEIGHet al. 1988) it was of interest definitively to ER1380 (hsdR2 zjj202::TnZO) and now designated localize the rglA/mcrA genes with respect to purB and A(mcr3)2, deserves comment because it differs from to document apossible coincidence of the geneticloci. its parent in four ways. Complementation (see Table The analysis used strains with TnlO insertions at two 5) and transduction analysis (not shown) reveal that in lociin the purB-trp region(HUGHES, SIMONSand addition to theselected loss ofMcrB activity, A(mcrB)2 NUNN1988). Tet' Tn10-2 transductantsshowed weak lacks EcoK methylase and mrr gene activity, and the linkage with rglA and pur3 (Figure IA, crosses d-f). linkage between the nearby TnlO tetracycline-resist- Though the number of recombinants exhibiting do- ance marker and these phenotypeshas increased from nor activity at these loci was low (18 total), the results 30%to >98%. Simultaneous elimination of three consistently placed rglA on the trp side of purB, irre- functions and reduced frequency of recombination spective of strain background. RglA and McrA phe- between two markers are properties of a deletion,and notypes, assayedas described in MATERIALS AND this has been confirmed by Southern analysis (see METHODS and illustrated in Tables3 and4, were below). coincident. In addition, the similarity of the results in Conjugational mappingof rgZA and B: These two the presence or absence of RglB+ (McrB+) confirms loci were initially mapped in K-12 using the Rgl phe- the genetic independence of the A and B restriction 286 E. A. Raleigh, R. Trimarchi and H. Revel A. Summary of P1 transduction crolucs. A. Map position relative to hsdR and scrB Damr Rriplcnt #, &Wed Damr mrktr OIhrr phcw(ypu rglB hsdR scrB # of h.la drain pbaolypc mcd, % wleclcd or rortd transductants eU purB Donor + - + Recipient - + rPl(1100) X149 1MW 61 lo0 """11 309 """"""""".""""""""24 b. Pl(IlRll0) X149 108W 75 100 ...... 112 -1"" """"1 3 s.Pl(HIl116) X149 15OW 75 100 Total 450 &Pl@Rl509)Wl378 94T& 4 3 e.Pl@RlS09) WIN1 ZWTa' 2 1 tPI(ERlS09) ER1398 lo0Ta' 9 4 B. Map position relative to a202::TnZO and scrB 8.PlW1576) W1378 94Td 70 M mcrB llji202::TnIO scrB Donor + + Recipient - + """"1 67 (43) B. Genetic nap of the rnerlrgfA region. .------.------._.------.--71 (54) """""""""."""""""""""24 (23) """""11 """"1 s 164 (122)

8.7- : +! Map position relative to mrr2::TnS and I 0.02 ' C. hsdR mcrB hsdR mrr O.O* i Donor + + - 4-'e 0.7 Recipient - -+ "" 20 FIGURE1 .-P 1 crosses mapping the mcrA/rglA region of E. coli """""- 4 K-12. (A) Summary of P1 crosses. Strains used with relevant """""1."""" """""1."""" 51 genotype wereas follows. Crosses (a-c), donors-1100: purBf .I""" "" 0 rglA', HR110: purB' rglA+,HRI 16: purBf rglA"; recipient-X149: 75 pyrF purB trprglA. Crosses (d-g), donors"ER1509:purB51 zcg2043::TnIO (Tet') rglA trp45, ER1576: purB58 zcf2033::TnIO FIGURE2.-PI crosses mapping the mcrB/rglB region of E. coli (Tet') rglA trpA trpE; recipients"ER1378: rglBl purB+ rglA' trp3 1 K-12. Representative crosses determining the positionsof mcrB, Tet', ER 138 1: mcrB+ purB' rglA+ trp3 1 Tet", ER 1398: rglBl purB+ hsdR, mrr, zj202::Tn10, and serBareshown. Dashed lines represent rglA+ trp' Tet'. Thirty transductants from the three recombinant donor DNA transferred to the transductant, and each row repre- classes in cross g (10 each of phenotypes Pur+ RglA-, Pur- RglA-, sents one class of recombinant, with the number of recombinants and Pur+RglA-) were tested for McrA phenotype; it coincided with in that class given on theright. In cross (B), thenumbers in the RglA phenotype. (B) Genetic map of the mcrA/rglA region of parentheses are the numberof recombinants in each class that were E. coli K-12. Arrows begin at the marker selected and point to the tested for RglB phenotype as well as McrB phenotype. In all cases marker scored. Numbers are the average fraction of selected re- the two coincided. Cross (A), Pl(HR184) X HR186: selection was combinantsinheriting the donor allele atthe unselected locus. for Ser+, the recipient was RglA-; tests of restriction phenotype TnfO-1and Tn10-2 are zcg2043::Tn10, from ER1509, and were: RglB, T2gt; HsdR, X.0. Cross (B), Pl(ER1381) X ER1394: zcf2033::TnI0, from ER1576, respectively. These insertions are selection was for Ser+; the recipient was RglA+; tests of restriction described in HUGHES,SIMONS and NUNN(1988); theoriginal strains were: McrB, h.MspI; RglB, T2 wild type. Cross (C), PlUH83) X containing them were RS3245 and RS3232, respectively. ER1378: selection was for Kan'; the recipient was Kan'; tests of restrictionwere: McrB, X.Mspl; HsdR, X.O; Mrr, X.PstI. 5 Mrr+ activities. Using the TnlO-1 insertion, which is more transductants were excludedfrom the analysis (BIEKand ROTH closely linked to rglA (mcrA) (Figure 1 A, cross g), we 1980). obtained greater numbersof Tet' transductants bear- ing the donor alleles of rglA(mcrA) and purB. The shown), and all 10 transductants tested were also ratio of transductants with the donor rglA allele to McrA-. CH 1372 and its parent CH73 1 are T6' and those with the donor PurB allele confirmed the pre- thus could not be tested directly for RglA phenotype. vious placement of rglA clockwise of purB. The RglA Transductional mapping of rgZB (rncrB): Repre- and McrA phenotypes of 30 recombinant transduc- sentative transductional crosseswhich establish that tants (10 of each type) were shown to be coincident mcrB (rglB) is tightly linked to two other K-12 restric- (not shown). tion systems, in the gene order mcrB (rg1B)-hsd-mrr- We also showed that the e14-1272::TnIO insertion zjj202::TnlO-serB proceeding clockwise on the host isolated by BRODY,GREENER and HILL (1985) is the chromosome, are summarized in Figure 2. direct cause of the McrA- phenotype of CH1372: The gene order rglB (mcrB)-hsdR-serB comes from Tet' was >98% cotransducible withRglA- (not the type of cross shown in Figure 2A. Here, Serf Mapping of mcr(rgl)A and B Loci 287 transductants were selected and RglB and HsdR were mutant of a clone complementing eitherrgl mutation scored using T2gt andX. 0 (MATERIALS AND METHODS). should confer resistance. Accordingly, a population rglB and hsdR are tightly linked (as previously noted of transformed ER15 16cells carrying randomBumHI by REVEL1983), in agreement with thereport of fragments of ERl370 DNA in pBR322 was grown for RAVI, SOZHAMANNANDHARMALINCAM and (1 Of985). a generation, infected with T4gt, and plated on am- Ser+ recombinants that received one of the two re- picillin plates (MATERIALS AND METHODS). Under these striction loci, 79% received both (1 12/141). The re- conditions, survival of isogenic singly mutant rgl combinant class with the donor allele at hsdR in con- strains transformed with vector alone was 30-50%. junction with the recipient allele at rglB was larger Of 23 survivors, seven retained a T4gf phenotype than its converse (24:5), suggesting that hsdR is in the upon purification. One of these, pER106, contained middle. In five other crosses with the rglB (mcrB) and the mcrA gene, since italso conferred resistance to hsd markers in various configurations, Ser+ recombi- T6gt and displayed the McrA+ phenotype. The other nants were scored for HsdRand either RglB or McrB six, which retained sensitivity to T6gt and restored phenotypes (notshown). The results agreed with those restriction of X.MspI and X.HueII, contained the mcrB shown, yielding a linkage of rglB (mcrB)to serB of 15- gene. All six mcrB clones contained a common 8-kb 26% and to hsdR of 80-95%. All possible combina- fragment. The fragment in PER 105 was mapped to a tions of hsdR and rglB(mcrB) genotypes were ob- region that includes hsdS and aboutseven kb of down- tained, confirming the independence of the two re- stream sequences (Figure 3). This fragment, aswell striction systems. as those in the other five mcrB clones, were in the The order mcrB-zjj202::TnlO-serB was from the same orientation with respect to thevector as in pBg6, type of cross represented in Figure 2B. The location with hsdS distal to the Tet promoterof pBR322. One of the transposoninsertion agrees with a previous of the six also contained a second BamHI fragment placement of zjj202::TnIO clockwise of hsd (FREITAG from elsewhere in the genome. and EISENSTEIN1983). The observed 58% linkage of Complementation of mutations in the mcrB-hsd-mrr Tet' to serB when the TnlO was in the recipient was region by pER105 and by the two hsd clones pBg3 reduced slightly to 40% when the insertion was in the and pBg6 (SAINand MURRAY1980) is shown in Table donor. All transductants were initially screened for 5 and correlated with the physical map in Figure 3B. McrB phenotype by method I. Whena randomly In addition to supplying the missing mcrB gene func- selected large fractionof the recombinants were tested tion in the deletion strain ER 1648 (mcrBA2) and the also for RglB phenotype by T2 plating efficiency (see mcrBl point mutation in ER1656 (restriction of X. Table 3), the two phenotypes coincided. MspI, line l), pER105 complemented all the remain- Crosses of the type shown in Figure 2C confirmed ing mcrB alleles in our collection (not shown). pBg3, the gene ordermcrB-hsdR-mrr, (HEITMAN andMODEL which was known to include all of hsdR and hsdM and 1987) and showed the same tight linkage between the proximal part of hsdS (Figure 3), complements the hsdR and mcrB seen in Figure 2A. Of Kan' Mrr- hsdR2 mutation in ER1656 (Table 5, line 3). Although transductants that received either unselected restric- the overall effect of Mrr restriction is small (three- tion locus, 90% received both; four received the do- fold), it is clear that pBg3 also complements the Mrr nor hsdR allele only, butnone received the donor deficiency in ER1648 mcrBA2 (Table 5, comparelines mcrB allele only, placing hsdR in the middle. 4 and 10). In contrast, pBg6,which includes the distal Genetic organization and restriction map of the part of hsdS and at least 8 kb of added downstream mcrB-hsd-mrr region: To verify this transductional sequences, complemented only the mcrB mutations. order and to establish the close physical proximity of These data are consistent with the placement of hsdR the threegenes, we first cloned the mcrB gene de novo. in the middle of the mcrB-hsd-mrr genecluster by We then carried out a comparative restriction map- transduction analysis (Figure 2C). ping and complementation analysis ofone mcrB clone, Combining our observed gene order mcrB-hsd-mrr- pERlO5,and of two previously characterized hsd serB with the known orientation andphysical positions clones that carry extra genomic sequences flanking of the hsd genes on pBg3 and pBg6 permits us to the host-specific restriction loci, pBg3 and pBg6 (SAIN orient the hsdRMS genes relative to the chromosome and MURRAY1980; see Figure 3). as shown in Figure 3A. It is important to note that Clones of both mcr loci were selected using resist- this orientation is reversed from all published versions ance to T4gt (Rgl+ phenotype). DonorDNA was from of the hsd region. A consequence is that the hsdRMS ERI 370 (McrA+McrB+), while the recipient host was genes (SAINand MURRAY1980; LOENENet al. 1987) the double mutant strainERl516 (mcrA mcrBl), which as well as the downstream genes putatively identified is completely sensitive to T4gt (Table 3). Since both with mcrB (ROSS,ACHBERCER and BRAYMER 1987), single mutants are resistant to T4gt due torestriction are transcribed and translated in a counterclockwise by the remaining rgl+ locus, acquisition by the double direction on the E. coli map. Our independent deri- 288

FIGURE3.-Physical and genetic organization of the mrr-hsd-mcrB region. (A) Summary of the genetic organi7ation of the region, aligned with the restriction map in part C. serB lies to the right of the figure, uxu to the left. Arrows: positions of transposon insertions (the Tn10 insertions can be positioned within +IO0 bp, Figure 4; the mrr::Tn5 insertion isolated by HEITMAN andMODEL (1 987) was mapped by them to a 2-kb EcoRI fragment upstream of hsdR, but the position of the mrr::Tn5 insertion has not been mapped within the EcoRI fragment); open boxes: DNA shown to be sufficient but not necessarily wholly required for the indicated activities; filled box: HindIIl fragment that is altered in the McrB mutants analyzed in Figure 4; hatched boxes: sequenced genes within the sequenced region; dashed box: sequenced region (COUCHand MURRAY1983; LOENENet al. 1987). (B) Summary of genetic data. Stippled boxes indicate the DNA carried by the constructs named. Box at left summarizes the phenotypes shown to be conferred by each construct. Data in parentheses are from SAIN and MURRAY(1980) (hsd phenotypes) or Ross and BRAYMER(1987) (mcrB phenotype). Other data are in Table 5 or not shown. The McrB' phenotypes of X1048 and pBg6 allow us to limit the extent of DNA required for McrB activity (vertical dashed lines). (C) Restriction map. Scale is numbered in kilobasepairs. On the scale are placed sites for enzymes (B, H3,R, S, Hp, Bg) mapped by SAINand MURRAY(1980) and confirmed by us, but reversed to conform with the orientation shown in Figure 2. Boxes below the scale show sites not mapped by SAINand MURRAY(1980). B = BamHI; H3 = HindIII; R = EcoRI; P = Pstl; Pv = PuuII: S = SalI; N = NcoI; A = Apal; Hp = Hpal; Bg = Eglll. The restriction map was generated by sizing 30 (pBgS), 26 (pBg6) or 18 (pER105) fragments generated by single and double restriction digests of plasmid DNA, or from Southern blots of singly digested DNA from wild type cells, using pBg6as probe. The scale is numbered to coincide with the numbering of the hsdS sequence (COUGHand MURRAY1983). with 0 at one end of the BamHI fragment carried by pER105, and increasing in the direction of mcrB. (*) This EamHl site mapped by us does not appear in SAINand MURRAY(1980) but appears in LOENEN et al. (1987). (t)We did not attempt to confirm the EcoRl and Sal1 doublets identified by SAINand MURRAY(1980). (4) We did not order the Hpal, EcoRl and HindIIl sites between 6 kb and 7 kb. (tt)We did not attempt to confirm the EcoRI site at 11.7 kb. vation of this orientation of the hsd genes produces a CR63 (data not shown). Thus we conclude that the restriction map that is in accord with that of a region mcrB region is conserved in four different K-12 back- between 98 and 99 min on the E. coli genome map grounds. In addition, it appears that the mcrBl mu- obtained by KOHARA, AKIYAMAISONO and (1987). tation in LCK8 is either a point mutation or a very

Southern blot analysis of mcrB mutants: To ex- small deletion. or rearrangement,- since thefragment aminethe stability of the mcrB region, tofurther patterns were identical with those from strains carry- characterize our mcrB mutations and to localize the ing the wild type mcrB+ allele. gene(s), we used pBg6 and pNK290 (which carries When the pBg6 probe was hybridized to blots of TnlO homology) as hybridization probes to parental our fivenewly-isolated spontaneousand TnlO-in- and mutant chromosomal DNAs. We first confirmed duced mcrB mutants (mcrBZ-mcrBb),the fragment pat- the restriction map of the insert DNA from K-12 tern was changed. Specifically, all mutations altered strain CR63 in pBg3 and pBg6 (SAINand MURRAY the 3.9-kb HindIII fragment just downstream from 1980) and located sites for four additional enzymes the 3' end of hsdS (Figures 3 and 4, black box). This (Figure 3C). Labeled pBg6 was then hybridized to band was replaced by a new 5.1-kb fragment in mcrB3, DNA of K-12 strainsW3110, ER1370, LCK8 and by a 5.6-kb fragment in mcrB5 and mcrB6, by two ER1380 digested with SaZI, NcoI, HindIII, or ApaI. fragments of 5.4 and 2.6 kb in mcrB4, and was missing The pBg6 probe gave fragment patternsin each digest altogether in mcrB2 (Figure 4). In addition, mcrBZ consistent with the restriction map developed from showed no homology to the flanking 5.6- and 1.8-kb Mapping of mcr(rg1) A and B Loci 289

TABLE 5

Complementation of mcrB, hsdR and mrr

Efficiency X plating on host strain with of Inferred plasmid genotype indicated plasmid6

Tester ER 1648 A(mcrB- ER1656 HsdS+ Plasmid phage" hsdSMR-mrr) HsdM+ hsdR2 mcrBl mcrB hsdR' mrr 1. ~ER105~ X. MspI 0.03 0.05 NT + NT - 2. pBg3 X. Mspl 0.8 0.6 3. X.0 0.6 0.03 + 4. X. PstI' 0.27 2 0.07 NT + X. MspI 0.04 0.02 + 5. pBg6 - 6. X.0 0.9 1.4 7. X. PstY 0.85 & 0.3 NT - 8. pBR322 X. MspI (1.O) 0.9 - 9. 1.0 (1.0) 0.6 - 10. X. PstI' (1.0) & 0.1 NT

a X.Mspl tests the McrB phenotype of the host: X.0 tests HsdR: X.Pstl tests Mrr. Numbers (Eop) are the ratioof the titer of the phage on a given strain to the titer of the same phage on ER1648/pBR322. Numbers in boldface indicate positive restriction activity. NT = not tested. ' Note that EcoK restriction activity requires the presence of all three gene products: HsdR, HsdM, andHsdS. pBg3 carries R and M, but ER1648 does not provideHsdS. pER105 was tested in separate experiments; the hosts tested were ER1486 and ERI 516 (Table 1). Numbers are normalized to the same strains with pBR322, as in other experiments. Mrr tests were a separate experiment. Four independent transformants foreach plasmid were tested for Mrr; values are the averageand standard deviation of EOP normalized to average titer on ER1648/pBR322.

HindIII fragments detectedby pBg6 in the remaining probe. None of the HindIII, SaZI, or NcoI fragments mutants, consistent with its genetic behavior as a dele- homologous to pBg6 was disrupted.Further, two tion. pNK290 hybridized to the same new bands as IS10-containing HindIII bands (4.9 and 4.1 kb) de- did pBg6 in the three TnlO-induced mutants mcrB4, tected by pNK290 in the parental strain ER1380 did mcrB5 and mcrB6, as expected if the insertions caused not hybridize with the mcrB-specific probe pBg6. The the McrB- phenotype. The single exception, a 2.4-kb disappearance of the 4.9-kb band in the mcrB2 dele- band in mcrB5 and mcrB6 detected by pNK290 but tion strain ER1486 suggests that one end of the dele- not by pBg6, probably has about 100 bp of sequence tion begins at the TnlOelement. homologous to pBg6 that went undetected in the The mcrA gene is on the e14 excisable element: exposures used. Our attention was drawn tothe e14 element by genetic Combining the blot data from HindIII, ApaI and observations. Transductional anomalies in some NcoI digests with the well-characterized TnlO restric- strains, suggesting the presence of a large deletion tion map (KLECKNER1988) allowed us to locate the near mcrA and PurB, led tothe E. coli K-12 map three TnlO insertions as shown in Figure 3A. The (BACHMANN 1987a) awareness and of att e14. We then mcrB4::TnlO insertion is about 300 bp to the left of observed that strains known to lack e14 (e.g., C600) the first Hind111 site located downstream of hsdS; both were McrA-, while those known to carry it were mcrB5::TnlO and mcrB6::TnlO insertions map about McrA+. As noted above (transduction), we showed 200 bp closer to the same HindIII site. In view of the that one of three strains with TnlO insertions in the well-known site specificity of TnlO insertion (KLECK- element (BRODY, GREENERand HILL 1985) had also NER et al. 1979), the occurrence of two independent become McrA-RglA-. We designatedthe insertion insertions at the same site is not surprising. The IS mcrA1272::TnIO (Table 1). This mutation is comple- insert in mcrB3::IS is unknown, and its location within mented for both RglA and McrA phenotypes by our the 3.9-kb HindIII fragment in Figure 4 is arbitrary. mcrA clone, pERIO6. The observation that TnlO promotes loss of adja- Physical and genetic analysis of pERlO6 positions cent DNA (Ross, SWAN andKLECKNER 1979), com- the mcrA gene at the right end of the el4 excisable bined with an analysis of our Southern blot data (not element, immediately adjacent to the pin gene and its shown) suggests that the deletion in A(mcrB-hsd-mrr)2 associated invertible DNA, as summarized in Figure was probably mediated by the zjj202::TnIO insertion 5. We compared restriction digests of pERlO6 with in the parental strain, ER1380. Specifically, we can digests of plasmids known to carry portions of e14. conclude that zjj202::TnlO is some distance from pHBlO6, pHBIO7 and pAG2 together carry most of mcrB, since its presence in LCK8 or ERl380 did not e14 and some flankingchromosomal sequences alterthe fragments detected by the mcrB-specific (BRODY,GREENER and HILL 1985), while pGP325 290 E. A. Raleigh, R. Trimarchi and H. Revel

mcrB:: Tn 10 genehrdR end lnsertlon locrtlons p896* H3 tragmentr ' 5.6 kb

Relevantfragments detected Allele blottedStralns H3 A N H3 l"-l wild type, ER1370, mcrBl W3110, LCK8 L3.9 kb I

H3A N

mcrB3::IS. 111,:" ER1489 5.1 kd ,- ,- 73 A, 73 ;r"" mcrB5::n T 10, ER1468, mcrB6:: Tn 10 . ER1469 15.6kb 1

H3 A N N H3A H3 N H3

mcrB4:: T n 10. ER1467

~ I5.4kb GZJ

""""""" A (mcrB-hsd-mrr)2 ~~1486

(no homology) FIGURE4.-Example of Southern blot analysis of parental and mcrB mutant DNA. HindIII digests of chromosomal DNA of the indicated strains were electrophoresed, blotted, and probed with labeled pBg6. At top is the organization of the mcrB-hsd-mrr region, redrawn from Figure 3. Ticks below the genetic map indicate the four Hind111 (H3) sites that define fragments that hybridize to pBg6. The black box highlights the single HindIII fragment altered by all the mutations except mcrB1. The flanking 1.8- and 5.6-kbp Hind111 fragments are conserved in all strains exceptin deletion strain, A(mcrB-hsd-mrr)P,which had lost all pBg6-homologous sequences (represented by the dotted line), as well as McrB, Hsd, and Mrr activity. Structures below the black box indicate the wild-type 3.9-kbp fragment and alterations in this fragment in insertion and deletion strains. Here thin lines represent chromosomal sequence; thin open boxes represent inserted DNA; thick open boxes represent the IS10 inverted repeats of TnlO, which also hybridize with pNK290 as discussed in the text. Positions of restriction sites, abbreviated as in Figure 3, are shown. Shaded box below each schema are fragments detectedby the pBg6 probe. This blot and similar blots to ApaI and NcoI digests (data not shown) allowed placement and orientation of the TnlO insertions. Placement of the unknown insertion in ER1489 (mcrB3::IS) is arbitrary, since no new sites for these restriction enzymes appeared. carriesa segment of e14 encoding the site-specific only PER 106 complemented mcrA defects. recombination function, Pin, together with the inver- The mcrA gene must lie downstream of pin, strad- tible DNA on which it acts (PLASTERKand VAN DE dling the HindIIIsite at 3.9 kb onthe mapof pERlO6 PUTTE1985). InBamHI-Hind111 digests, pERlO6 dis- (Figure 5C), since pGP325 and pAG2 are McrA- due played a 4.1-kb fragment common to pHBlO7 and to truncation at this position, yet retain the site of the pHBlO6, and a 3.9-kb fragmentin common with the mcrA 1272::TnIO insertion (triangle b) thatinactivates right ends of the pAG2 and pGP325 inserts (Figure the gene. We verified this hypothesis by analysis of 5B). In the same digests, pAG2 and pGP325 shared a subclones of pERlO6 that eliminated the DNA to the 1.7-kb fragment not present in pERlO6. pAG2 car- right of the HindIII site (pER146) or to the right of ried an additional 5.5-kb BamHI-Hind111 fragment, the EcoRI site in the flanking region(pER137); pERl37 retained McrA activity but pERl46 did not. while pGP325 carried instead an additional, 2.1-kb Both plasmids retained Pin function and theinvertible EcoRI-Hind111 fragment..Thus, pERlO6 DNA in- DNA. cludes the HindIII site at 13.9 kb (Figure 5A). The inference that pERlO6 carriespart of the e14 element was confirmed by functional complementation analy- DISCUSSION sis (Figure 5B) and by further restriction digests (Fig- rgl and mcr are genetically identical: To provide ure 5C). These data show that pER106, like pAG2 agrounding for biochemical characterization, we and pCP325, carries a functional pin gene along with went to considerable lengths to establish the identity its associated invertible DNA segment.pHB107, of the Rgl and Mcr systems. In particular, we wished which carries att e14 and flanking chromosomal se- to know whether RglB is specific for hydroxymethyl- quences, complemented only purB as expected, while cytosine-containing DNA, because there are available pHBlO6, which retains theright end of eI4, was biochemical data on RglB suggesting that at least two inactive in all functional tests. Of the five plasmids, components are involved in restriction (FLEISHMAN, Mapping of mcr(rgl) A and B Loci 29 1

-10 -5 0 5 >O !I5 i 20 kb purB pin mcrA I I I I I I I 1 I plasmid B. I I I """""""" pHB107 + nt - I I I pHBIO6 - -I I I I! I I I I I pAG2 -+- I I I 1': ,I I - + - I I I pGP325 I I pER146 - + - - I -I I I I pER137 - + + pERlO6 - + + "-- """ --" """""" b C,,' -." C. "" v B P P(+) P(-) 89 P :H31*Y R1 <"B I I D. '8 kb e14 chromosomal

FIGURE5.--Summary of the organization of the mcrA region. (A) Restriction map in kilobasepairs, redrawn from BRODY,GREENER and HILL (1985). The map shows the e14 excisable element (wide filled box), the approximate position of purl3 (dashed line above the scale), positions of restriction enzyme sites (abbreviated as in Figure 3 and Av = AuaI) and positions of three el4::TnlO insertions (triangles: a = insertion 1279; b = 1272; c = 1290) as determined by BRODY,GREENER and HILL (1985). Note that these are not the same insertions indicated in Figure 1. (B) Parts of the region carried by various plasmids (Table 2) and the genetic loci associated with each, assayed as described in MATERIALS AND METHODS. pHB107 does not carry the DNA corresponding to the dashed line. Vertical dashed lines, and ticks on pAG2 and pGP325, indicate the restriction fragments used to correlate the various plasmid maps (see text). (C) Expanded map of the pERlO6 insert with PstI (P) and BglII (Bg) sites added. P(+) and P(-) refer to PstI sites in the two orientations of the invertible DNA. AuaZ was not mapped. (D) Inferred positions of the various genetic elements on the 8.0-kb pERlO6insert. The mcrA1272::TnIO insertion (triangle b) maps to the left of the HindIII site at 3.9 kb on the lower map (BRODY,GREENER andHILL 1985). The e14::1290 insertion (triangle c) maps to the right of the HindIII site, but is in el#, because it can be excised and reinserted with the element (BRODY,GREENER and HILL 1985); its precise position within these limits is not known. However, it does not inactivate mcrA (data not shown).

CAMPBELLand RICHARDSON1976). We find that the phenotypes are mediated by nonidentical proteins. RglA- and McrA- phenotypes always coincide, as do For example,the mcrB locus could encode asubunit(s) the RglB- and McrB- phenotypes, whether upon mu- common to both an RglB enzyme and an .McrB en- tant isolation, upon genetic mapping (Figures 1 and zyme; or it could encode a positive regulator of two 2), or by complementation analysis of the single mu- such enzymes; or all mcrB alleles isolated so far could tants isolated so far. We will henceforth refer to these be polar mutations eliminating expression of two en- loci as mcrA and mcrB, since the mnemonic (modified zymes simultaneously. Further genetic and biochemi- cytosine restriction) more accuratelydescribes the ob- cal analysis will be required to resolve this question. servedrestriction target than does the alternative The wandering restriction locus, mcrA: The mcrA (restricts glucoseless phage). gene is located near 25 min onthe E. coli K-12 These loci encode factors requiredfor both Rgl chromosome, tightly linked to purB (Figure l), on the and Mcr restriction activity to be expressed, not re- excisable prophage-like element e14 (Figure 5). We pressors or inhibitors of restriction activity that are suspect that the many mcrA mutations in laboratory activated by mutation, because presumed null alleles strains originate as excisions of the e14 genetic ele- (insertions and deletions) at each locus abolish the ment, which is lost after DNA damage (BRODY, GREE- restriction characteristic of that locus (Figures 3 and NER and HILL 1985; GREENERand HILL 1980), and 5, and not shown). These required functions might these mutations may thus all be the same allele. The be positive regulatory factors or the restriction func- position of mcrA in E. coli B is consistent with an tions themselves. Isolation of a thermosensitive allele accessory element-mediated acquisition. It is not lo- at the mcrA locus (ts4 in HRI 16, Figure 1, cross c) cated near trp, as it is in K-12, but is linked with his suggests that the gene productis most likelya protein. in Ff X F- crosses (conjugational mapping). There is It is still possible that the Rgl and Mcr restriction no report of a test of E. coli B itself for eI4; the B 292 E. A. Raleigh, R. Trimarchi and H. Revel derivative B/5 does not carry e14 homology, but it region is mcrB-hsdS-hsdM-hsdR-mrr-rjj202::TnlOserB. does carry homology to the unoccupied (e14’) K-12 The genes in thecluster are fairly tightly packed: attachment site (GREENERand HILL 1980; BRODY, mcrB straddles the Hind111 site that lies 1 kb down- GREENERand HILL1985). Attachment of an e14- stream of hsdS (Figure 3), since insertions to the left related element at a different position in E. coli B and inactivate mcrB, and a deletion of the righthand frag- imprecise excision from it could explain why mcrA ment (SAINand MURRAY1980) also does so; and mrr mutants also acquire a non-revertible thiamine auxo- must be containedin the -2 kb between the beginning trophy (REVEL 1967). It remains to be seen whether of the hsdR gene and the upstream end of the pBg3 E. coli B carries e14 at a different location (unlike B/ insert (Table 5). 5), or carries a related elementat a different location, Our results are broadly consistent the data of oth- or carries mcrA in virtue of some otherroute of ers, except with respect toorientation relative to acquistion. outside markers. Similar data have been reported in It is curious to note that mcrA of e14 and mom of parts by other laboratories: RAVI,SOZHAMMANAN and bacteriophage Mu occupy similar positions. Both DHARMALINGAM (1 985) mappedrglB relative to hsd; genes lie between pin or gin, respectively, and the end ROSS,ACHBERGER and BRAYMER(1987) located mcrB of the prophage. Mom is a DNA-modifying function downstream of hsdS; HEITMANand MODEL (1987) active only late in lytic .infection, and it acts to protect derived the order mcrB-hsd-mrr; and KOHARA, AKI- the infective stage from host restriction (KAHMANN YAMA and ISONO(1987) reported a restriction map and HATTMAN 1987); it represents the converse of including this region. ROSS,ACHBERGER and BRAY- McrA, which seems to act during lysogeny to protect MER (1987) infer the reverse orientation for reasons the host from infective phage. notstated. HEITMAN and MODEL (1987)infer the The identity of mcrA: There are fourknown func- reverse orientation by referenceto an unpublished tions encoded by e14. One, the site-specific inversion restriction map of the E. coli chromosome. The ori- recombinase, pin, we have shown to be distinct from entation we show is consistent with both our own mcrA by subcloning experiments (Figure5B). Another transducational data and those of RAVI, SOZHAMAN- e14 function, the product of gene lit, also interferes NAN and DHARMALINGAM (1985), andalso with the with growth of phage T4; but lit is not the same as restrictionmap of KOHARA, AKIYAMAand ISONO mcrA, both because it maps at the other end of the (1987). On the other hand,we have made one contra- element and because it must be activated by mutation dictory observation. Hybridization of pBg3 to South- before it will inhibit phage (KAO and SNYDER 1988). ern blots of the mcrB2 deletion suggested that this The e14 integrase (BRODY, GREENERand HILL 1985; deletion spared some DNA homologous to pBg3 (E. BRODYand HILL 1988)is carried by the element, has A. RALEIGHand R. TRIMARCHI,unpublished data). If not been localized on it, and is presumably a DNA the deletion were a simple onepromoted by the cutting-joining enzyme, as are restriction enzymes; but zjj202::Tn10, this fact would require the reverse ori- no requirement for cytosine modification of the at- entation of theregion. However, since TnlO may tachment sites has been suggested and we think this promote coupled multiple rearrangements (such as an unlikely to be mcrA. The gene sjC has been mapped inversion followed by deletion:,a RALEIGH and to the element but notlocalized (MAGUINet al. 1986). KLECKNER1984), andbecause the autoradiogram was It is involved in the coupling ofcell division with of poor quality, we consider these dataunreliable. replication and is thoughtto act by inhibiting the The agreement of two independent kinds of evidence division protein FtsZ (MAGUIN, LUTKENHAUSand (the restriction map of KOHARA,AKIYAMA andISONO, D’ARI 1986). Since nothing is known about mode the and the transductional data of ours and of others) action of mcrA, we cannot rule out identity with sjC. seems to us compelling. The region specifying mcrA is sufficient to encode one Addition of mrr and mcrB to hsd (EcoK) increases average-sized protein or two small ones. It must lie the precision with which this locus can monitor the between the end of pin and the position of the e14- modification pattern of incoming DNA. One system 1290::TnlO (triangle c in Figure 5C), which did not recognizes an unmodified sequence, one recognizes a inactivate mcrA. This leaves a maximum of about 1 methyladenine-containing sequence, and one recog- kb of e14 sequence in which mcrA might reside. We nizes a methylcytosine-containing sequence.Recent have not ruled out that some other part of our clone, data suggest even more complexity: the mcrB specific- including the pin-associated invertibleDNA, might ity of K-12 may be the sum of two different specifici- also be required but not be sufficient for McrA activ- ties encoded by adjacent genes (D. DILAand E. A. ity. RALEIGH, unpublishedobservations). With this many “Immigration control” in E. coli: The mcrB (rglB) restriction systems of different types in one region, an gene(s) are foundin a clusterof restriction genes near “immigration control” genecomplex emerges. Strains 99 min (Figures 2 and 3). The order of genes in this might differentiate genetically in consequence of shuf- Mapping of mcr(rgl)A and B Loci 293 fling the specificities at these loci, which could be a of invading phage,a population of sensitive cells useful strategy (see below). would do better if it were differentiated for restriction Is there evidence for geneshuffling in this complex? specificities, so that each phage burst faces a variety In the descent of K-12 strains, the structure of the of restriction spectra and is not protected from most region is quite stable, in contrast to the situation with of them. Addition of restriction specificities recogniz- the mcrA locus. Southern blot experiments (Figure 4 ing methylated bases, as K-12 hasdone, both increases and text) tested four strain backgrounds representing possibilities for differentiation and vitiates the phage threeindependent derivations from K-12 wild-type strategy of incorporating modified bases in place of (BACHMANN, 1987b);all gave the same structure. The the normal ones, as does XP12 (HUANGet al. 1982). mcrBI allele, which is probably present in all common One can also argue that restriction plays a role in nonhybrid McrB- K-12 strains (see text), is a point the regulation of gene flow among bacteria. Contin- mutation or astructural alteration too small to be uingdifferentiation of restriction specificity within detected by our method. the population might promotegenetic exchange Whendifferent wild isolates are examined, how- wheregenetic differences exist butare small, and ever,there is considerable variability in structure inhibit it where differences are large. If two strains (DANIELet al. 1988). The three loci seem to vary differ in some restriction systems but not others, al- independently, and there may be additional variation lowing some relatively low level of restriction upon in theshort region separating hsd and mcrB. For DNA transfer, restriction would act to increase the example, the organization of the E. coli B, C, and 15 levels ofboth the substratesand theenzymes required T- regions(among others) has beenexamined by for recombination. Double strand breaks can be re- DANIELet al. (1988). The center of the cluster, hsd, is paired by recombination (KRASINand HUTCHINSON at the same genetic location in E. coli B and 15T- as 1981 ; PICKSLEY,ATTFIELD and LLOYD 1984; SYMING- in K-12,but hsdR B hybridizes with hsdR K, while TON, MORRISONand KOLODNER 1985; STAHL 1986; hsdR A (from 15T-) does not; E. coli C has no such THALER,STAHL and STAHL1987); restriction must restriction system, and nohomology. On theleft flank promote synthesis of some recombination functions, (as in Figure 3), genetically there is a weak mcrB in since the action of mrr and mcrB (HEITMANand the B strain (Table3), andDNA in this region hybrid- MODEL 1987; DHARMALINCAMand GOLDBERG1980) izes with the K-12 mcrB-specific probe, pRH2. C and as well as hsd (DAY 1977;THOMS and WACKERNACLE 15T- have not been tested genetically for mcrB, but 1982) induces the SOS response and some recombi-. no hybridization with this probe is observed; yet in nation functions (recF, ruu) are SOS-inducible (LOV- between mcrB and hsd, 15T- hybridizes to K-12 ETT and CLARK 1983);so restriction could promote (pRH3), as though hsd wereinserted at the same its own recombination-dependent repair. However, if position independently twice, once for K and B and donor and recipient differed very much in restriction once for A. C has no homology at all. On the right spectrum, incoming DNA would bedegraded too flank, E. coli B lacks mrr activity (HEITMAN andMODEL rapidly or to pieces too small for recombination to 1987); 15T-has homology with K-12 by hybridization occur,and restriction would become abarrier to with pBg3; and C lacks all homology. If more E. coli genetic exchange. At some point the sequence diver- (GOUCHand MURRAY1983; FULLER-PACE,COWAN gence would become so great that lack of homology and MURRAY1985) and Salmonella (BULLAS,COLSON would limit geneticexchange, and restriction spec- and NEUFELD 1980;BULLAS and COLSON1975) data trum would no longer be particularly relevant. Thus, are added, even more genetic variation is seen. All of paradoxically, differentiation of restriction systems this is difficult to put into a simple tree, even if we within a population would promote genetic homoge- assume that the cluster was assembled from accessory nization in closely related strains, but it would rein- elements like transposons and phages. There could be forcegenetic isolation of more distantly related an active shuffling process. strains. Why such a complex restriction cluster?Two types There aredifficulties with both of these models. If of pressures might maintain the high level of poly- protection from phages is the primary role of restric- morphism described above: pressurefor protection tion systems, it is hard to see why many of them have from invading phages, and regulation of gene flow such small effects. On the other hand, the general between strains. In protecting against phage it would consensus seems to be that genetic exchange via ho- be advantageous for members of a sensitive popula- mologous recombination of chromosomalgenes tion to carry multiple, and variable, restriction func- rarely occursin nature (LEVIN198 1 ; CAMPBELL198 1 ; tions. Because phage survivors of restriction occur at SELANDER, CAUCANTWHITTAM and 1988). Diversity biologically plausible levels (1 0-‘- 1O-‘), cells would of restriction spectra might contribute to the rarityof do better with multiple restriction systems; and be- recombination, but then some other explanation for cause restriction systems act only on the “first wave” the diversity itself must be sought. Perhaps thesetwo 294 E. A. Raleigh, R. Trimarchiand H. Revel potential roles-protection from phage infection and type of specificity towardmodification of substrate. Nucleic regulation of genetic exchange-work togetherto Acids Res. 13: 5727-5746. maintain the diversity of restriction systems in enteric BUTKUS,V., S. KLIMASAUSKAS,L. PETRAUSKIENE,Z. MANELIENE, A.LEBIONKA and A. JANULAITIS,1987 Interaction of AluI, bacteria. Further examination of the structure of the Cf.61, and PuuII restriction-modificationenzymes with sub- mcrB-hsd-mrr region should helpus to understandhow strates containing either N4-methylcytosine or 5-methylcyto- this cluster evolved. sine. Biochim. Biophys. Acta 909: 201-207. CAMPBELL, A.,1981 Evolutionary significance of accessory DNA We thank NOREENMURRAY for generousassistance with strains, elements in bacteria. Annu. Rev. Microbiol. 39: 55-83. plasmids, and discussion; GEOFFWILSON, CHARLES HILL andPIETER DANIEL,A. S., F. V. FULLER-PACE,D. M. LEGGEand N. E. MURRAY, 1988 Distribution and diversity of hsd genes in some enteric VAN DE PUTTEfor sharing plasmids; JOE HEITMAN for sharing data and strains;ELISABETH LATIMER andCATHY COMEAU forassistance bacteria, particularly Escherichia coli. J. Bacteriol. 170: 1775- 1782. with transductions; JOAN BROOKS,WILLIAM JACK, NANCYKLECK- DAY,R. S., 1977 UV-induced alleviation of K-specific restriction NER, PAULRIGGS and IRA SCHILDKRAUT forcritical review of the manuscript, discussions and moral support. of bacteriophage lambda. J. Virol. 21: 1249-1 25 1. DHARMALINGAM,K., and E. B. GOLDBERG,1976 Phage-coded protein prevents restriction of unmodified progeny T4 DNA. LITERATURECITED Nature 260 454-456. ECHOLS,H., A. GAREN,S. GAREN andA. TORRIANI, 1961 Genetic ARBER,W., 1974 DNAmodification andrestriction. Prog. Nu- control of repression of alkaline phosphatase in E. coli. J. Mol. cleic Acids Res. Mol. Biol. 14: 1-37. Biol. 3: 425-438. BACHMANN,B., 1987a Linkage map of Esherichia coli K-12, Ed. 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