A complementation analysis of the restriction and modification of DNA in Escherichia coli

J. Nol. Biol. (1969) 41, 459472

A Complementation Analysis of the Restriction and Modification of DNA in Escherichia coli

HERBERT W. BOYER AND DAISY ROVLLAND-DVSSOIX

Department of Microbiology, University of California

San Francisco .MedicaE Center, Ban Prancisco, CaliJ 94122, U.S.A.

(Received 28 October 1968, and in revised form 39 January 1969)

A complementation analysis of host-controlled modification and restriction of DNA by Escherichia coli has been carried out by examining the restriction and modification phenotypes of partial, permanent diploids containing various arrangements of wild type and mutant restriction and modification alleles. Interoistronic complementation was observed between three classes of restriction and modification mutants of E. coli B, indicating that at least three cistrons (the ram cistrons) are involved in the genetic control of the /restriction and modification of DNA. Mutations in one cistron (ramA) result in a loss of restriction activity but not in modification activity (r-m+). Mutations in a second cistron (ramC) result in a loss of restriction and modification activities (r-m-). Muta- tions in a third cistron result in a loss of modification activity and appear to be lethal unless accompanied by a mutation in the ramA or ramC cistrons. A fourth class of mutations, which are linked to the other ram cistrons and are expressed phenotypically as r-m- mutants, are tram dominant to the wild-type ram

alleles. It is not known if this latter class of mutants represents a fourth cistron of the rum locus. Complementation was observed between E. COG K12 and B ramA and ramC mutations and the host specificity of the restored restriction activity was dependent on an intact ramC cistron. However, complementation was not detected between the Pl and K12 or Pl and B ram alleles. A general model for the genetic control of the restriction and modification properties of E. coli strains and their episomes is presented.

1. Introduction Escherichia coli strains K12 and B (among others) have a mechanism for rejecting non-homologous DNA, i.e. DNA originating in a cell of a different strain (for a review on the subject see Arber, 1965a). Bacteriophage, bacterial and episomal DNA, when transferred from E. coli K12 to E. coli B, or vice versa, are subject to host-controlled restriction, or specific degradation by the recipient cell (Dussoix & Arber, 1962; Boyer, 1964). This destruction is brought about by an endonuclease which introduces a limited number of double-strand scissions at defined sites along the DNA molecule (Meselson & Yuan, 1968; Linn & Arber, 1968; Roulland-Dussoix & Boyer, manuscript in preparation). Intrastrain transfer of DNA is not subject to restriction because these sites are modified. This latter process is known as host-controlled modification of DNA. Several experiments (Arber, 19653, 1968) suggest that the chemical basis of modification is the secondary methylation of purine and/or pyrimidine bases. Recently, Kiihnlein $ Linn (personal communication) have demonstrated an in vitro modification of the replicative form of the phage fd which requires X-adenosyl-L-methionine in addition to an extract of a modifying strain.

459

460 H. W. BOYER AND D. ROULLAND-DUSSOIX

A thorough genetical analysis of the restriction and modification mechanism has been difficult because no efficient methods are available for selection of the various mutant and wild-type phenotypes. The methods that are available are sufficient for isolation of restriction mutants and estimation of this mutant frequency (Wood, 1966) but cannot be used in the selection of genetic recombinants. Despite these difficulties several important observations have been made about the genetic control of the restriction and modification mechanisms. Boyer (1964) and Stacey (1965) demonstrated that the wild-type restriction and modification properties of E. co.5 strains K12, B and 15 are controlled by alleles (the ram loci) that map near the thr and serB loci. Wood (1966) demonstrated that restriction mutants of E. coli K12 and B map in the same region and that the mutants unable to restrict DNA fell into two categories with roughly equal frequencies, r -m + and r -m -. The same distribution and frequency of restriction mutants were found for the prophage Pl-directed restriction and modification properties (Glover, Schell, Symonds & Stacey, 1963).

With the development of a new technique for the isolation of I?-merogenotes by Low (1968), we have been able to extend the genetic analysis of the restriction and modification of DNA. Thus, it was possible to construct partial permanent diploids of E. coli with various arrangements of mutant and wild-type restriction and modification alleles. Analysis of the restriction and modification properties of these diploids has led to the suggestion that there are at least three cistrons involved in the genetic control of the restriction and modification of DNA.

2. Materials and Methods

(a) Media

L-broth is (g/l.) : Bacto-tryptone (lo), yeast extract (5), and NaCl (10); 56 is (g/l.) : Na,HPO.7H,O (16.4), KH,PO, (5.4), (NH&SO4 (20), MgS0,.7H,O (0.2), Ca(NO& (O*Ol), and FeSO,7H,O (0.025); X- dil is (g/l.): Na2HP0.7H,0 (105), NaCl (4), and KH,PO, (3). Minimal agar medium contains equal volumes of 56 and 1.5% Bacto-agar with the following concentration of supplements when needed (mg/l.) : DL-threonine (82), I,-leucine (41), L-methionine (25), r,-arginine (146), L-histidine (42), L-proline (166), L-

tryptophan (18), thiamin (0.166), n-glucose (2000) andgstreptomycin sulfate (200). Tryptone broth is (g/l.): Bacto-tryptone (10) and NaCl (5). Tryptone agar is Tryptone broth with 15 g of Bacto-agar/l. Soft agar is Tryptone broth with 7.5 g of Bacto-agar/l. Nutrient agar contains 23 g of Bacto-nutrient agar/l. MacConky agar is 40 g of Bacto-MaoConky agar supplemented with 2 g of carbohydrate/l.

(b) Bacterial strains and nomenclature

The following nomenclature (as recommended by Demerec, Adelberg, Clark & Hartman, 1966) is used in this paper to describe the restriction and modification properties of E. COG strains and their episomes. We will use ram (restriction and modification) to designate the locus or gro~xp of genes linked to thr and serB, controlling the restriction and modification of DNA. Wood (1966) used the symbol hs, which does not conform to the Demerec et al. proposals. The host specificity of a ram locus will be designated by a prefix describing the host strain, e.g. K ram, B ram, Pl ram, etc. The following abbreviations, recommended by Arber (personal communication), are used to designate the various wild-type (WT) and mutant phenotypes of the ram loci: rgrni, rgrng and rP:rn& designate the WT restriction and modification phenotypes of E. coli B, K12 and the prophage Pl; rim:, rim,+ and r;,m& designate the phenotypes of restriction mutants which retain the WT modification property; rim;, r;rn, and r&rnpl designate the phenotypes of restriction and modification mutants. The designation of rOmO is reserved for strains such as E. co&i C which has no known restriction or modification property.

RESTRICTION AND MODIFICATION IN E. COLI

TABLET Pertinent bacterial strains

461

Restriotion and modification

Strain IlO.

Pheno- Geno- tme tYPe

Other characteristics Parental strain and derivation

HB81 HB16

HB76 HB77 HB78 HB82 HB91 HB153

HBlOO HBlOl HB103 HB104 HB105 HB94 HBlSO HB156 HB157 HB233 HB232 HB74

HB165

ramc1 ramA ramA ramA ramA ramC3 ram02 ramA ramC4 ramA ramA ramA ramB1

F- F- Pro- Gal- Stra Thi-

F- F’ HfrH Thi- Hfr (311) Ret- Thi- HfrH Gal- Thi- HfrH GaI- Thi-

F- Pro- Gal- StrR Rec- F- Pro- Gal- StrR Rec- F- Pro- Gal- Stra Rec- F- Pro- Gal- Stra Ret- F- Pro- Gal- St+ Rec- HfrH Gal- HfrH Gal- Hfr H Gal - Hfr H Gal - HfrH Gal- F- Gal - F- Pro- Gal- StrR

tion of rim: alleles with thr+ Derived from HBl6 and HB82 Derived from HBlOO by method IIt Derived from HBIOO by method IIt Derived from HBlOO by method IIt Derived from HBlOO by method II? Derived from HB91 by method 17 Derived from HB91 by method It Derived from HB153 by method I Derived from HB153 by method I Derived from HB1.53 by method I Derived from E. coli K12 by method I Derived from HB16 by method I

F- Pro- Gal- Stra Rec- Derived from HB104

E. coli C (from Bertani) rim: alleles co-transduced with thr+

to AB266 (E. co& K12) from Adelberg

E. coi!i K12 from Adelberg KLF4 from Low E. coli K12 from Adelberg AB3045 from Clark Mutant of HB78 Derived from HB150 by co-transduo-

HB174 r;rni ramA ramB2 F- Pro- Gal- Stra Derived from HB74

F- PI lysogen of HB76

H7397 rAm& rarA6 =- r; rnz Gal - Pl Iysogen of HB232

HB226 rh& Cm&

‘ramA F- Pro- Gal- Stra Pl lysogen of HB74

The parental strains of the diploids are listed in this Table but not the diploid strains. The parents of the diploid strains listed in subsequent Tables can be identified from the information given in the appropria,te Tables. Only the mutant phenotypes are listed. All of the restriction mutants described here yielded efficiency of plating values of I.0 for unmodified phage A.

t See Materials and Methods.

An E. coli K12 strain carrying the B ram locus was used to circumvent the poor adsorp- tion of h to E. coZi B (HB16 and derivatives). For the sake of brevity these strains will be referred to as E. coli B strains.

(c) Bacteriophage

Phage X + + was obtained from the laboratory of W. Arber and Xvir was obtained from the laboratory of E. A. Adelberg. Plvir was obtained from the laboratory of J. Tomizawa.


(d) Isolation of restriction mutants

Restriction mutants were selected by one of two methods. (I) The first method is that described by Wood (1966), which uses a stock of unmodified X dg gal” to transduce a restricting host. A large number of the Gal + transductants of a restricting host are restriction mutants. (II) We also found that the transfer and establishment of an unmodified F-merogenote in recipients of a Ret- restricting population results in the selection of restriction mutants. This is a variation of the procedure used by Glover et al. (1963). About 5 x lo8 cells of an F’ strain (HB77) and 5 x lo8 cells of a Ret- restricting strain (HBlOO) were mixed and allowed to incubate for 1 hr at 37°C. Pro+ or Leu+ Ret- StrR clones were usually (90 to 100%) males on the basis of sensitivity to male-specific phage. Spontaneous segregation (5 x 10V4) of the Leu- or Pro- phenotype was accompanied by a loss of sensitivity to male specific phage. Segregants of different Leu+ or Pro+ clones were usually restriction mutants.

(e) Isolation of modification mutants

Modification mutants of the r;rni (HB74) and rzrng (HB16) strains were isolated from nitrosoguanidine-treated cultures in the following way. Survivors of the mutagenized cultures were cloned and used to inoculate 1 ml. of Tryptone broth. These were incubated for 16 hr at 37°C. About lo4 hvir.B phage particles were added to each cultured clone and incubated for an additional 2.5 hr at 37°C. A drop of CHCI, was added to each infected culture. Each lysate was spotted with a loop on a plate containing an rimi strain and a plate containing an r;m; strain to test for B modification.

(f) Preparation of phage stocks

Stocks of Avir, Plvir and fd were made by standard plate methods. Phage Xf + stocks were prepared by the plate method or induction of lysogens with ultraviolet light. The notations of Arber & Dussoix (1962) are used to denote the host modification of the phage, e.g. h prepared on E. coli K12 is designated h.K.

(g) Eficiency of plating experiments

Experiments on the efficiency of plating were done with hvir stocks with C, B and K modifications. Indicator bacteria were prepared from Tryptone broth cultures in late log phase (5 to 8 x lOa cells/ml.) by centrifugation, resuspension in 0.01 M-MgSO, and incu- bation at 37°C with aeration for 1 hr. The phage were pre-adsorbed to indicator bacteria for 15 min at 37°C. E. coli C was used to titrate all phage stocks for calculations of efficiency of plating values.

(h) Mutagenesis

Bacteria were mutagenized with 2-aminopurine (by growing bacteria in L-broth supplemented with 1 mg of 2-aminopurinelml.) or N-methyl-N-nitrosoguanidine according to the procedure of Adelberg, Mandel & Chen (1965).

(i) Generation. of stable partial diploids

Diploids are generated from HfrH derivatives by a technique discovered by Low (1968). Crossing an HfrH population to a recA mutant strain and selection for early markers, such as Leu + or Pro + , selects for spontaneously occurring F-merogenotes. The episome isolated from HfrH usually carries a fragment of genome extending from the origin of transfer of HfrH to a point between proA and gal. A cross between HfrH and a recA mutant strain yields a low number of Leu+ or Pro + clones in comparison to a Ret + recipient (Low, 1968). The Leu+ or Pro+ clones are usually sensitive to male-specific phage (90 to 1OOo/o) and transfer Zeu+ or pro+ at high frequencies to appropriate recipients. The pro + or Zeu + markers spontaneously segregated from the Ret- strain at a low frequency (10L3) with a concomitant loss of sensitivity to male specific phage. Segregation of these F’ episomes from Ret+ strain is more frequent ( ~10~~). These F’ episomes contain the ram locus linked to thr and serB and the ram marker segregates with the leu + or pro + markers. We therefore used the Ret- strains in most cases to stabilize our diploids and to avoid ambiguous results with the efficiency of plating experiments, although the results with Ret + diploids were identical.

RESTRICTION AND MODIFICATION IN E. COLI 463

The E. coZi B rgrn$ allele was introduced into an E. coli K12 Thr- Gal- rKrn< HfrH by Plvir co-transduction with thr + , to yield strain HB153. This strain, crossed to the appropriate Ret- recipients, s&&s for the F’ rimi episome. Various r,rn+, and rim, mutants of HIS153 were isolated by method I and the F’ r<rng episomes selected by crossing to the appropriate Ret- recipient. In a similar manner, a Gal- derivative of HB78 was used to generate F’ r;rnG and F’ rim; episomes. The HB77 strain (KLF4 from Low) was the source of the F’ rzrn,+ episome. This episome was introduced into recipient strains by selection of Pro+ and counter selection with StrR.

3. Results (a) Complemenation between Tim: and r;m; alleles

Glover et al. (1963) and Wood (1966) found that restriction mutants of the prophage Pl and E. coli K12 and B were either r m+ or r m - and that these two mutant phenotypes occurred with approximately equal frequencies. Wood (1966) suggested that the restriction and modification processes are carried out by factors that have a common gene product as a possible explanation for the x-m - mutants. In order to explore this possibility and characterize the ram cistrons, we constructed a number of partial permanent diploids with different arrangements of mutant and wild-type B ram alleles and examined them for their ability to restrict and modify the phage A.

Four independent r;m,+ mutants (HB103, HB104 and HB105 and HB157) and three independent r;rn; mutants (HBlOl, HB156 and HB233) were isolated from the HBlOO and HB153 strains. Various diploid strains were constructed from these mutants as described in Materials and Methods. These r;m,’ mutant alleles were recessive to the wild-type rgrni allele (Table 2) ; however, not all of the r -m -mutant

TABLE 2 Complementation between rgm,’ and r;m; mutants

Strain no.

Haploid phenotype of mutant or WT allele on

resident F’ apisome genome

Efficiency of Phenotype plating of h*C of diploid on diploid

HB159 rim; (153) r:rnz (104) rim; 1 x IO-4 HBl60 rim; (153) r;rnB (101) r,+m,+ 1x10-4 HB163 rim; (157) rimi (104) rim; 1.0 HB212 rim,+ (157) rimi (103) rimi 1.0 HB213 rim,+ (157) rimi (105) rimi 0.8 HB167 r;rn; (156) r;rnB (101) rim; 1.0 HB237 rimi (233) rB,rni (101) rim; 1.0 HB162 rim< (156) rim:; (104) rimi 1 x 10-4 HB210 rimi (156) rim,+ (103) rimi 8X 10-4 HB211 GpB W-5) rim; (105) rim; 1 x 10-z HB238 rim, (233) rim,+ (103) rimi 4x 10-3 HB239 rimi (233) rim: (104) rfmi 2x10-3 HB240 rimi (233) rim: (105) rim; 3x 10-z HB214 rBrnB+ (157) rims (101) rzrni 2x IO-2

Stocks of hvir with C, B or K modification were assayed on the sdiploid strain as described in Materials and Methods. The numbers in parentheses following the phenotype designations identify the parental strains used to construct the diploids, e.g. the HB159 diploid which was derived from HB153 (rim;) and HB104 (r,,mi). The modification phenotype of the diploid strains was determined by efficiency of plating experiments with hvir stocks made on each of the diploids.


alleles we examined were recessive to the wild-type allele. We have classified the r-m - mutants with alleles that are recessive to the wild-type alleles as type I r-m - mutants and those r -m - mutants with alleles that are tralzs dominant to the wild- type alleles as type II r -m - mutants. The type II r -m - mutants are described in section (e) below.

Diploids (HB163, HB212, HB213) with independent mutations on the episome and resident genome leading to the r;]rni phenotype had no restriction activity (Table 2) but, of course, imparted B modification to phage X. Two diploid strains (HB167 and HB237) with independent type I r;m; mutations on the episome and resident genome failed to restrict or modify phage X (Table 2). On the basis of these results we conclude that the four mutants with the r;rn,+ phenotype have mutations in the same cistron, and the three mutants with the type I r&m; have mutations in the same cistron. However, the two mutant phenotypes must be the result of mutations in two different cistrons since seven diploids (HB162, HB210, HB211, HB214, HB238, HB239, IIB240) carrying these four independent rgmg+ and three independent r;rn; mutant alleles in heterozygous configurations restricted unmodified h (Table 2). All of these diploids modified X with B specificity. It should be pointed out that the levels of restriction in the different diploids were quite variable, ranging from efficiency of plating values for unmodified h of 1 x lo-* (wild-type levels) to 1 x 10 -a, We conclude from these experiments that the mutations leading to the rim,+ and r;rn; phenotypes complement one another at the intercistronic level, and that there are at least two cistrons cont8rolling the restriction and modification mechanism.

(b) Isolation of a mutant representing a third ram c&on

There is a third possible ram. phenotype, r + m -, which has not been isolated because it is likely that such a mutation would be lethal. To test this possibility, we mutagenized a culture of HBl6 (rzm,+) with nitrosoguanidine and tested the surviving clones for the ability to modify h. Five independent modification mutants were recovered, and all five of these were unable to restrict unmodified h. This is consistent with the idea that an r + m - mutation would be lethal. Therefore, we used two rim,+ mutant strains (HB104 and HB74) as a source of modification mutants. One modification mutant was recovered from the HB104 strain, and six independent modification mutants were isolated from the HB74 strain. Phage X prepared on these mutants had efficiency of plating values on a host carrying the B ram locus (HBlOO) ranging from 1 x 10-l (almost fully modified) to 5 x 10 -* (no modification).

We expected some of the modification mutants to have alterations in the cistron(s) leading to the type I and type II r-m- mutants. However, if some of the two-step r -m - mutants were the result of alterations in a third cistron, they could be identified on the basis of their complementation response. The two-step r-m - mutants were made diploid by the introduction of an F’ episome (HB77) carrying the r,‘mz alleles and the diploids examined for their restriction and modification properties. Diploids carrying the rimi alleles and type I r;m; alleles have the K restriction and modification specificity, but diploids carrying the rgrng and r;m,’ alleles have K and B restriction and modification specificities (see section (d) below). On the basis of an analysis of these diploids, we found two of the rim; mutants to be similar to the type I r;m; mutants and three to be similar to the type II r;rn; mutants. Two of the diploids, constructed from the HB165 and HB174 mutants, modified h with K and B host specificities and restricted h not having K and B modifications (Table 3).


TABLE 3

Complementation responses of modification mutants

Strain IIO.

Haploid phenotype of mutant or WT allele on Phenotype

resident of haploid Efficiency of plating of phage X F’ episome genome or diploid x.c X.B X.K

HB174 r;m; rim; 1.0 1.0 1.0

HB165 r;m; r;rnB 1.0 1.0 1.0

HB156 rim; rimi 1.0 1.0 1.0

HB178 I-,’ mKf r; mg

GmB (165) rimi 1x10-1 6x 1O-2 9x 10-Z

HB179 r,+mi (77) 8x 10-Z 2x10-I 2 x 10-l

HB215 rimi (156) 3x10-a 1.0 4x 10-a

Stocks of Xvir with C, B, or K modification were assayed on the diploid strains as described in Materials and Methods. The numbers in parentheses following the phenotype designations identify the parental strains of the diploids. Phage Xvir prepared on HI3174 has an efficiency of plating of 10-l on E. eoli B, and X prepared on HB165 has an efficiency of plating of 5 x 10m4 on E. coli B.

Phage Xvir prepared on the HB178 and HB179 diploid strains plate with an efficiency of 1.0 on E. coli B and K12. Phage h prepared on the HB215 diploid strain plate with an efficiency of 1.0 on E. coli B and 1 x low4 on E. coli K.

The results obtained with the HB174 mutant are not particularly striking because the mutation is so leaky (i.e. phage h prepared on this strain had an efficiency of plating value of 10 -l on a host carrying the B ram locus), but the HB165 mutant, which is unable to modify h phage (i.e. phage X prepared on this strain had an efficiency of plating value of 10m4 on a host carrying the B ram locus), shows normal B and K modification in the diploid state with the rgrng alleles. Since the HB165 strain is Ret -, it was possible to construct a diploid with this strain and an F’ episome (derived from HB157) carrying a mutation resulting in the type I rim; “phenotype. This diploid restricted unmodified X and imparted B specificity to A. Since the HB165 mutant and HB157 mutant do not restrict or modify X in their haploid states, we conclude that the second mutation in the HB165 mutant (and most likely in the HB174 mutant) is in a third cistron of the ram locus. Preliminary transduction experiments indicate that this mutation is closely linked to the serB locus.

(c) Dirploids with r + m + alleles

It was of interest to examine the restricting ability of wild-type homozygous and heterozygous diploids. The diploid (HB77) homozygous for the WT K ram alleles restricted X * C and h * B phage by a factor of 20 to 100 over the haploid strain (Table 4), but there was no change in the modification pattern. Similarly, the diploid (HB155) homozygous for the WT B ram alleles restricted h * C and X * K phage by a factor of 10 to 20 over the haploid strain (Table 4), but there was no change in the modification pattern. These results suggest that in the E. coli B and K haploid strains, the cellular levels of restriction endonuclease are limiting factors in the restriction of h phage. The diploid (HBllO) heterozygous for the WT K and B ram alleles restricted h - C, h . K and h * B, but the efliciency of plating values were about one order of magnitude lower than those obtained with the haploid strains (Table 4).


(a) Complementation between the B and K alleles

The restriction and modification properties of E. coli K12 and B are very similar (Arber & Dussoix, 1962; Bayer, 1964; Wood, 1966), and it was of interest to examine

TABLET

Restriction and modi$cation properties of di$oids with WT ram alleles

Strain no.


resident F’ episome genome

Efficiency of plating of phage X h.C X-B h.K

HB78 HBlOO HB155 HB77 HBllO

rim: (153) r,+m: (77) rim; (77)

r,’ mK+

ri,$ s,+rnz (100) r,‘mG (77) rgrni (100)

1x10-4 1 x 10-4 1.0 1 x 10-4 1.0 1x10-4 9x 10-s 1.0 6X IO-6 3x 10-e 3x10-s 0.9 1x10-s 2x 10-s 1x10-3

Stocks of hvir with C, B or K modification were assayed on the diploid and haploid strains as described in Materials and Methods. Numbers in parentheses following the phenotype designation identify the parental strains of the diploids. Stocks of Xvir prepared on the HB77 and HB155 diploids had normal host modification and Xvir prepared on HBllO had B and K host modifications. Pro- segregants of HBllO had rzrni phenotypes and transfer of the F’episome of HBllO to HBlOl resulted in a diploid with an r:rnz phenotype.

TABLE 5

Complementation between K ram and B ram alleles

Strain no.


resident Phenotype of Efficiency of plating of phage h F’ episome genome diploid X.C X.B h.K

HBlO9

HBlll

r:rn$ (77) rGrni (101) r$m? 1 x 10-d 1x10-4 1.0 “:.“y

r;rnI: (77) r,rnz (103) :2n$ 2x10-3 3x10-3 2x10-3 R B

rgrn$ (77) r;ml;: (104) rjrnj 1x10-3 1 x 10-s 1x10-3

HB113 rzm,’ (77)

HB116 r,rn: (94) r&m; (103) $21 1.0 B B

HB115 r;rnz (94) rim; (101) $2 1 x 10-s 1 x10-3 1.0 B B

HB149

HB225

r;rnG (150) rsrn; (101) z!!?&? 1.0 B - -

rim: (150) r.gm$ (103) 1 x 10-a 1.0

1.0

Stocks of Xvir ‘with C, B or K modification were assayed on the diploid strains as described in Materials and Methods. Numbers in parentheses following the phenotype designation identify the parental strains of the diploids. The modification phenotype of the diploid strains was determined by efficiency of plating experiments with Xvir stocks made on each of the diploids.


the complementation between the K and B ram alleles. The restriction and modification properties of these heterodiploids are summarized in Table 5.

A diploid (HBI16) heterozygous for the alleles leading to the rim,: and ‘r;rnL phenotypes modified h with K and B host specificity but did not restrict unmodified X. A diploid (HB149) heterozygous for the alleles leading to the r;m; and rim; phenotypes did not restrict or modify X. However, a diploid heterozygous for the rim, and r;rni alleles had the restriction and modification properties of B, while a diploid (HB115) heterozygous for the $rnKf and r;rn; had the restriction and modification properties of K. These results indicate that the cistronic products of the K and B ram loci are interchangeable, and that a type I r m - mutant has a defect in a cistron controlling the specificity of the restriction and modification processes. The r m + mutant must be defective in another cistron which controls some aspect of the restriction process.

A diploid (HBlll) heterozygous for the r,‘m& and rim,’ alleles restricted and modified h with B and K host specificities while a diploid (IIBlQQ) heterozygous for the rgrni and r;rn; alleles only exhibited K specificity. We have not examined any diploids with various arrangements of the mutant alleles of the K ram locus but, on the basis of the above results, expect them to respond in the same way as the B mutant alleles.

(e) Trans dominant r -m - mutations

We were surprised to hnd that in many cases when the one-step or two-step r -m - mutants were made diploid by conjugation with the HB77 strain, they were unable to restrict unmodified h, although some of these diploids modified X with K host specificity. A number of attempts were made to construct restricting diploids with these r-m - mutants and the 3” episome carrying the WT ram alleles but without success. We crossed two of these diploids, one being r;rng and the other r;rn; to an rim; recipient (HBlOl) known to be recessive to the rgrn; allele. Establishment of the F’ episome of these non-restricting diploids in the HBlOl recipient resulted in diploids with the normal K restriction and modification properties. We conclude then that there is a class of mutations leading to the r-m - phenotype which are tram dominant to the r + allele. We refer to mutants of this type as type II r -m - mutants; they carry type II r-m- mutations.

Three r;m; mutants derived from the HB16 rBrni strain through mutagenesis and screening for the modification mutant phenotype were found to be type II rim; mutants. We have examined a number of rim; and r;m;; mutants and found several more type II r -m - mutants. For example, three mutants derived from the HB74 r;rnz strain by mutagenesis and screening for the modification mutant phenotype turned out to be type II r m - mutants. We have found type II r -m - mutations in rim& and rim; mutants derived through mutagenesis and selection for the restriction mutant phenotype by h dg gal + transduction.

The nature of the trans dominant r-m - mutation is now under investigation. Preliminary experiments (Roulland-Dussoix & Boyer, unpublished observations) indicate that crude extracts of a type II rim; mutant or a type II mutant with WT K ram alleles on the 3” episome do not inhibit the in vitro activity of a crude extract containing the B restriction endonuclease or a purified preparation of the B restriction endonuclease. The rim; mutation trans dominant to the rgrn& allele is also dominant to the rgrni and r&m,+, alleles (Lyon & Boyer, unpublished observations). About

32


Q (7/16) of the r-m- mutants we have examined are type II r-m - mutants. We have determined that the type II I‘ -m- mutation is linked to the serB locus.

(f) Relationship of PI ram locus to B and K ram loci

It has been demonstrated (Meselson & Yuan, 1968; Linn & Arber, 1968; Roulland- Dussoix & Boyer, manuscript in preparation) that the restriction endonucleases of E. coli B, K12 and the prophage Pl are very similar. That is, they require ATP, X-adenosyl-L-methionine and Mg ’ + for enzymic activity, and they all have estimated molecular weights of 225,000 to 240,000 daltons. Glover et al. (1963) demonstrated another similarity, namely, r;,m,‘, and i&m& mutants were recovered with approximately equal frequencies.

Since the r;,rnG1 mutants were recovered in a PI lysogen of E. coli K12 (Glover et al., 1963), it appeared that the wild-type alleles of E. coli K12 could not complement the r;,m& mutation. However, the selective pressures may have selected for a non-complementing mutation. We therefore lysogenized r;rni and r;m,f mutants with Pl and examined the lysogens for their restriction and modification properties (Table 6). If the cistronic products of the Pl and K12 or Pl and B rawz loci are interchangeable like the K12 and B ram loci (see Results section (d)), we would expect

TABLE 6

Restriction and modification properties of Pl Eysogens with r;;rni and rimi alleles


Strain resident Phenotype of Efficiency of plating of phage h IlO. prophage genome lysogen x.c h . K (Pl) X . B (Pl)

HB74 rY$n;: 1.0 1.0 1.0

HB226 Gdl r,rni (74) $$’ 5x10-3 1.0 1.0 B B

HBl6 rimi 1x10-4 1x10-4 1.0 HBO6 &dl r:rng (76) 2x 10-V 1.0 2x 10-4

HB97 && rid (232) rhA rKm+ 1x10-4 1.0 1.0

K

Stocks of hvir with C, K(P1) or B(P1) modifications were assayed on the non-lysogenic and lysogenic strains as described in Materials and Methods. The numbers in parentheses following the phenotype designation identify the parental strains of the lysogens. The modification phenotypes were determined by efficiency experiments with Xvir stocks prepared on the lysogens.

restoration of the K12 and B restriction properties in these lysogens. Clearly, these lysogens have no B or K12 restriction, and it appears that the Pl ram allele does not complement the B or K12 ram alleles.

4. Discussion

Recent investigations in a number of laboratories have begun to elucidate the bio- chemical basis of host-controlled restriction and modification of DNA. The restriction enzymes under the genetic control of E. coli K12, B and the prophage Pl are endonucleases that* introduce a limited number of double-strand scissions in unmodified DNA (Meselson & Yuan, 1968 ; Dussoix & Boyer, manuscript in preparation). Several


experiments (Arber, 19653,1968; Kiihnlein & Linn, personal communication) suggest that methylation of DNA is involved in the modification m8echanism. The modification and restriction of DNA by these various strains are mutually exclusive. Thus modification of DNA by E. coli B renders the modified DNA insusceptible to the restriction endonuclease of E. coli B, but not to the restriction endonucleases of other strains, e.g., E. coli K12, and the prophage PI. This implies that a unique site, estimated to consist of a sequence of at least six or seven nucleotide base pairs (Arber, 1968) acts as a substrate for these enzymes.

The investigations reported here were undertaken to analyze the nature of the mutation leading to the r -m - phenotype. Wood (1966) suggested that the restriction and modification processes are carried out by factors that have a common gene product to explain the r-m - mutation. Because the restriction endonuclease and modification methylase appear to recognize the same substrate, a unique sequence of nucleotide base pairs, the common gene product might be a polypeptide subunit involved in the formation of enzyme-substrate complexes. A mutation in the cistron coding for the common subunit would result in the r -m .- phenotype.

The complementation data reveal two classes of rim; mutants. The type I rimi mutants have the properties predicted by Wood’s (1966) explanation, i.e. the rim; mutant allele is recessive to the WT B ram alleles and complements the r;m,’ mutant allele to restore the B restriction activity. Furthermore, the type I rimi mutant allele complements the r;m,f mutant allele to restore the K restriction activity but not the B restriction activity. This is in good agreement with the recent report of Linn & Arber (1968) that they detected in vitro restriction activity with a mixture of an rim,’ mutant extract and an rim; mutant extract. Since independent type I rim; mutants do not complement one another and independent r;rni mutants do not complement one another, the mutations leading to these two phenotypes must be in different cistrons.

A third ram cistron was implied by Wood’s (1966) suggestion which should result in an r +m - phenotype. A modest attempt to isolate this mutant phenotype failed, possibly because a mutation of this type would be lethal. However, at least one (and possibly two) modification mutant derived from an rimi mutant was complemented by the WT r,‘,mg alleles and the type I rimi mutant allele. Therefore, these mutants define at least three cistrons involved in the B ram locus. As yet we have not determined if the type II rimi mutants represent a fourth cistron.

We propose the following general model for the genetic control of host-controlled modification and restriction of DNA by E. coli B and extend the model to other related systems. Various aspects of this model have been discussed by others (Wood, 1966; Arber, 1968; Linn & Arber, 1968). At least three cistrons (collectively known as the B ram locus) linked to the thr and serB loci code for three polypeptides which are involved in. the restriction and modification of DNA. The number of each type of polypeptide per enzyme molecule is not known. The three cistrons are designated ramA, ramB and ramC and code for a restriction polypeptide, a modification polypeptide, and a recognition polypeptide. The relative order of these cistrons on the genome is not known. These three polypeptides can be considered to interact in one of two ways: (1) the three polypeptides form an oligomeric protein which has restriction and modification activities associated with it ; or (2) two oligomers are formed from the three polypeptides with one having restriction activity and one having modification activity. In the latter case, both enzymes would have one common


polypeptide (the recognition polypeptide) which is responsible for the formation of enzyme-substrate complexes. Although there is yet no strong evidence for one or the other of these possibilities, we prefer the latter on the basis of simplicity. It should be pointed out, however, that the requirement of X-adenosyl-methionine for restriction endonuclease activity is more compatible with the first possibility. We will consider the model in terms of the restriction and modification activities being associated with two distinct proteins, although either consideration is consistent- with the oomplementation data.

In the simplest terms of the model, we would predict that the different systems of restriction and modification, such as E. wli K12, B, and very likely the prophage Pl, are controlled by very similar ramA and ramB cistrons. Of course, the ramC oistrons must differ, since they primarily control the different substrate specificities of these organisms. New ram specificities could be generated by alterations of the ramC cistron. However, the results suggest that some changes also occur in the ramA oistron, and we will discuss this point later.

This model accounts for the mutant phenotypes (with perhaps the exception of type II r -m - mutant phenotypes) and the qualitative data of the oomplementation studies. Thus, a mutation in the ramA oistron leads to an r-m+ phenotype and a mutation in the ramC oistron leads to the type I r-m- phenotype. Mutations in the ramB oistron oan be recovered only if they are associated with a mutation in the ramA or ramC oistrons, since it appears that the r + m - phenotype is lethal. A mutation in the ramC oistron might also lead to the type II 1: -m - mutation if the product of this oistron also had some role as a controlling element in the expression of the restriotion and modification function. Alternately, the type II r m - mutation may define a fourth ram oistron.

We must consider some of the possible modes of interaction between the mutant and wild-type recognition, modification and restriction polypeptides in order to interpret the quantitative oomplementation data. For example, a mutant restriction polypeptide might interact with a wild-type recognition polypeptide in one of three ways :

(a) as completely as the wild-type restriction polypeptide to form an oligomerio protein similar to the wild-type enzyme in all respects (i.e. association and dissociation of subunits, etc.) with the exception of restriction activity; or

(b) it may be altered so that no oligomer can be made with the mutant restriction polypeptide and wild-type recognition polypeptide; or

(0) the mutant restriction polypeptide could partially interact with the wild-type recognition polypeptide to form an oligomer with properties intermediate between (a) and (b). In terms of the three-oistron model presented above and some reasonable assump- tions, we might expect then that diploids with ramC -/ramA -arrangements would have restriction activities similar to or less than the wild-type haploid. The other assump- tions are:

(a) in the diploid state there are about twice the number of restriction, modification and recognition polypeptides as in the haploid state;

(b) the restriction endonuolease is an oligomer composed of two or a multiple of two non-identical subunits; the estimated molecular weights of the K12 and B restriction endonuoleases (200,000 to 250,000) could accomodate four polypeptides (Meselson & Yuan, 1968; Roulland-Dussoix & Boyer, manuscript in preparation);


(c) the level of restriction endonuclease molecules in the haploid cell is limiting in the restriction of unmodified )r in the efficiency of plating experiments; this assumption was supported by the finding reported above (Results section (c)).

Thus the ramA -/ramC - diploids would have wild-type levels of restriction activity, if the mutant restriction and/or recognition polypeptides were not capable of interacting with the wild-type restriotion and/or recognition polypeptides. They would have less than wild-type restriction activity if the mutant restrictionand/or reoognition polypeptides were capable of interacting to some degree with the wild-type restriction and/or modification polypeptides. This is indeed what is observed with the F’ diploids of this type. This might also account for the efficiency of plating values of about 10 -3 for unmodified h on the restricting diploids with complementing mutant alleles of the K and B ram loci. It should be pointed out that in the latter situation the wild- type K and B restriction, modification, and recognition polypeptides might not be freely interchangeable, so that an additional factor has to be considered in these cases.

The restriction of h by the diploid carrying the WT K and B ram alleles was less than either haploid wild type and considerably less than either wild type homodiploid. This result is perhaps easier to explain within the consideration already developed if the restriction endonuclease is imagined to be composed of two recognition and two restriction polypeptides rather than one recognition and one restriction polypeptide. Thus, one-half of the endonuclease molecules would be hybrid with respect to the recognition polypeptide and presumably inactive. The remaining molecules (equivalent to the haploid number of molecules) would be subdivided into four classes with respect to the restriction polypeptide and perhaps not all of these molecular subclasses are equally effective as restriction endonuclease molecules. Other explanations are of course possible but are outside of the considerations we have developed here.

One of the inherent features of this model is that it explains the generation of new restriction and modification specificities through one-step mutational alterations of the ramC cistron. We might therefore expect that the various systems of restriction and modification, such as E. coli K12, B, 15, Pl, etc., differ only in the ramC cistron. This appears to be the case for E. coli K12 and B where the restriction polypeptides at least can substitute for one another rather effectively. In the Pl system of restriction and modification, both the ramA and ramC cistrons may have evolved to a point where their products are unable to interact effectively with the K12 or B ramA and ramC products. Despite this divergence, the genetic control of the Pl system must be similar to the B and K systems since it has been found that the type II r;rnG mutants trana dominant to the r,’ phenotype are also trans dominant to rG1 phenotype (Lyon & Boyer, unpublished observations). A similar genetic control probably exists for the restriction and modification of DNA by E. coli 15, although information on this system is rather limited (Stacey, 1965). It will be interesting to see if this model is applicable to related systems of restriction and modification such as the RTF-2 system (Watanabe, Nishida, Ogata, Arai & Sato, 1964).

Phage h prepared on diploids imparting B and K modification to the X phage demon- strates that both modifications can be imparted to one phage genome. This confirms an independent observation by Kellenberger, Symonds & Arber (1966). However, this phage is still restricted by a K12(Pl) strain, indicating that the sequence of nucleotide base pairs recognized by the restriction endonuclease of Pl is not affected by the K and B modifications in tandem.


In conclusion, we propose that at least three distinct cistrons are involved in the restriction and modification of DNA. The products of these cistrons might interact to form (1) one oligomeric protein molecule with restriction and modification activities or (2) two oligomeric protein molecules with a common subunit. In the latter case, one enzyme has restriction endonuclease activity and the other has modification activity, and the polypeptide shared by the two enzymes would be involved in the formation of enzyme-substrate complexes. The generation of new substrate specificities would arise through alterations of the ramC cistron so that both the restriction and modification specificities would change as the result of one mutational event. The generation of recognition polypeptides with different substrate specificities may alter the interaction of the recognition polypeptide with the restriction polypeptide and/or the modification polypeptide so that some evolution of the ramA and/or ramB cistron accompanies or follows the evolution of the ramC cistron. The evolution of the Pl ram locus vis ic via the E. coli K12 and B ram loci may be so extensive that the Pl polypeptides can no longer interact with the K or B polypeptides, although the gross characteristics (co-factor requirements, molecular weight, etc.) of the enzyme and other genetic features of those systems are similar.

This investigation was supported by U.S. Public Health Service Research Grant no. 5 ROl GM 14378; by U.S. Public Health Service Training Grant no. 5 TO1 AI 00299; and by a Jane Co&n Childs Memorial Fund for Medical Research award to one of us (D. R-D.).

We would like to thank the Misses L. Goeldner and J. Nakao for excellent technical assistance.

REFERENCES Adelberg, E. A., Mandel, M. & Chen, G. C. C. (1965). Biophys. Biochem. Res. Comm. 18,788. Arber, W. (1965a). Anm. Rev. Microbial. 19, 365. Arber, W. (19656). J. Mol. Biol. 11, 247. Arber, W. (1968). Sylrvp. Sot. Gen. Microbial. no. 18, p. 295. Arber, W. & Dussoix, D. (1962). J. Mol. Biol. 5, 18. Boyer, H. (1964). J. Bad. 88, 1652. Demerec, M., Adelberg, E. A., Clark, A. J. I% Hartman, P. E. (1966). Genetics, 54, 61. Dussoix, D. & Arber, W. (1962). J. MOE. Biol. 5, 36. Glover, S., Schell, J., Symonds, N. & Stacey, K. (1963). Genet. Res., Cambridge, 4, 480. Kellenberger, G., Symonds, N. & Arber, W. (1966). 2. Vererbungslehre, 98, 247. Linn, S. & Arber, W. (1968). Proc. Nat. Acad. Sci., Wa.sh. 59, 1300. Low, B. (1968). Proc. Nat. Acad. Sci., Wash. 60, 160. Meselson, M. & Yuan, R. (1968). Nature, 217, 1110. Stacey, K. (1965). B&t. Med. Bull. 21, 1211. Watanabe, T., Nishida, H., Ogata, C., Arai, T. & Sato, S. (1964). J. Bad. 88, 716. Wood, W. (1966). J. Mol. Biol. 16, 118.

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A complementation analysis of the restriction and modification of DNA in Escherichia coli

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