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Competition for Specific Sequences Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11095-11099, November 1995 Evolution Restriction-modification systems as genomic parasites in competition for specific sequences (selfish gene/methylation/plasmid maintenance/programmed cell death/meiosis) KOHJI KUSANO, TAKU NAITO, NAOFUMI HANDA, AND ICHIZO KOBAYASHI* Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Shiroganedai, Tokyo 108, Japan Communicated by James F. Crow, University of Wisconsin, Madison, WI, July 31, 1995 ABSTRACT Restriction-modification (RM) systems are tight linkage of cognate restriction and modification genes. (iii) believed to have evolved to protect cells from foreign DNA. No sequence homology among restriction enzyme genes, However, this hypothesis may not be sufficient to explain the except for a few pairs, implying their independent evolution. diversity and specificity in sequence recognition, as well as (iv) Their ubiquitous occurrence throughout the prokaryotic other properties, of these systems. We report that the EcoRI world, and their virtual absence from the eukaryotic world. restriction endonuclease-modification methylase (rm) gene We previously reported that some type II restriction endo- pair stabilizes plasmids that carry it and that this stabiliza- nuclease gene (r)-modification methylase gene (m) (rm) gene tion is blocked by an RM of the same sequence specificity pairs increase stability of plasmids that carry them by causing (EcoRI or its isoschizomer, Rsr I) but not by an RM of a the death of cells that have lost them (7). Here, we demonstrate different specificity (PaeR7I) on another plasmid. The PaeR7I competitive exclusion between such selfish rm gene pairs: two rm likewise stabilizes plasmids, unless an rm gene pair with RM systems of the same sequence specificity cannot simulta- identical sequence specificity is present. Our analysis sup- neously force their maintenance on a host. We propose that ports the following model for stabilization and incompatibil- selfishness and competition underlie the evolution of diversity ity: the descendants of cells that have lost an rm gene pair and specificity in sequence recognition by RM systems. expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modifi- cation by another RM system of the same specificity protects MATERIALS AND METHODS these sites. Competition for specific sequences among these Bacteria. Escherichia coli strains AB1157 (rec+ racdel), selfish genes may have generated the great diversity and JC5519 (racdel recB21 recC22), JC8679 (recB21 recC22 sbcA23 specificity in sequence recognition among RM systems. Such rac+), and DH5 (recAl endA1 hsdR17) have been described altruistic suicide strategies, similar to those found in virus- (8). DH5 was used for propagation of plasmids. infected cells, may have allowed selfish RM systems to spread Plasmids. The plasmids driven by ColEl replication unit by effectively competing with other selfish genes. (their rm genotype; antibiotic resistance; source/comments/ references) are as follows: pBR322 [none; ampicillin (Amp), A type II restriction endonuclease makes a double-strand tetracycline (Tet); laboratory collection], pMB4 [EcoRI r+m+; break within or near a specific recognition sequence in duplex Amp; F. W. Stahl (9)]; pIK166 (EcoRI r+m+; Amp), pIK167 DNA. A cognate modification enzyme methylates the recog- (EcoRI r-m+; Amp), pPAORM3.8 (pIK137) [PaeR7I r+m+; nition sequence to protect it from the cleavage (1, 2). It is Amp; T. Gingeras (7, 10)], pTN4 [PaeR71 r-m+; Amp (7)], widely accepted that the evolution and maintenance of restric- pIK180 (pTZ18R) [none; Amp; K. Mizobuchi (11)], and tion-modification (RM) systems have been driven by the pIK181 (pTZ18U-Rsr I rm) [Rsr I r+m+; Amp; A. L. Bari and protection from foreign DNA that they afford to cells. The RM R. I. Gumport (12)]. The plasmids driven by pSClOlts repli- systems do protect cells from infection with some viruses by cation unit are as follows: pHSG415 [none; Amp, chloram- cleaving their DNA (for example, see ref. 3) and are likely to phenicol (Cam), kanamycin (Kan) (46)], pIK172 [EcoRI be responsible both for the evolution of antirestriction mech- r+m+; Amp, Cam (7)], pIK173 (EcoRI r-m+; Cam; Amps anisms and for the paucity of some restriction sites in certain version of pIK172), pIK179 (EcoRI r-m+; Cam; Amps version viruses and plasmids (4). of pIK173), pTN9 [PaeR7I r+m+; Amp, Cam, Kan (7)], pTN11 Recent experimental and theoretical analyses (5, 6), how- [PaeR7I r-m+; Amp, Cam, Kan (7)], and pTN18 (PaeR7I ever, seem to us to bring into question the efficacy of virus- r+m+; Cam, Kan; Amps version of pTN9). The restriction and mediated selection for RM systems. Defense by RM systems is modification phenotype was verified by plaque assays using A short-lived because invading viral DNA will occasionally es- cI- 71. cape restriction and will become modified, thus affording pIK163 was made by replacing a BamHI-Sal I fragment of protection from restriction to itself and its descendants (5, 6). pBR322 with a BamHI-Sal I fragment of pMB4. pIK166 was Bacteria will more likely develop other, longer-lasting means made from pIK163 by deleting a BamHI-HindIII fragment, of resistance to viruses, such as alterations in the receptor which stabilizes plasmids (data not shown). pIK164 was made required for infection (5, 6). Although RM systems can provide by cleavage ofpIK163 with Hindlll in the r gene, digestion with bacteria with advantage when they are invading new habitats T4 DNA polymerase, and self-ligation. This should delete 4 bp full of phages, it is not clear whether such colonization (bp 526-529) in the r coding region, change the reading frame selection is realistic under natural conditions (5, 6). after amino acid 68, and generate a stop codon (TAG) 25 It is also unclear whether the above "cellular defense" amino acids downstream. pIK167 was made by deleting a hypothesis can account for the following properties of type II BamHI-HindIII fragment of pIK164. pIK178 was made by RM systems (1, 2). (i) Their individual high specificity and collective wide diversity in the sequence recognition. (ii) The Abbreviations: R, restriction; M, modification; r, restriction endonu- clease gene; m, modification methylase gene; Amp, ampicillin; Cam, The publication costs of this article were defrayed in part by page charge chloramphenicol; Kan, kanamycin. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed (e-mail: ikobaya@ accordance with 18 U.S.C. §1734 solely to indicate this fact. hgc.ims.u-tokyo.ac.jp). 11095 Downloaded by guest on September 23, 2021 11096 Evolution: Kusano et aL Proc. Natl. Acad. Sci. USA 92 (1995) inserting a BamHI linker (5'-CCGGATCCGG) into a HinclI a second plasmid inhibited the stabilization, whereas an rm site in the bla (Ampr) gene, which confers resistance to Amp. pair of a different sequence specificity (EcoRI) on a second pIK179 was made similarly. pTN18 was made by cleaving the plasmid did not. The inhibition was observed when the second bla gene of pTN9 with Pst I, removal of 4-bp equivalents with plasmid carried a mutant version of PaeR7I rm, defective in T4 DNA polymerase, and self-ligation. restriction but proficient in modification. This result suggests that the methylation of the recognition sequence is responsible for inhibition of stabilization (Fig. 2C). The kinetics of plasmid RESULTS loss in these experiments are consistent with a simple partition "Incompatibility" Between Plasmids Carrying rm Gene model (15). Pairs of the Same Sequence Specificity. We found that a Thus, two RM systems of the same sequence specificity plasmid is stabilized in an E. coli rec+ strain when it carries the cannot simultaneously enjoy stabilization. We call this phe- EcoRI rm genes (Fig. 1). The fraction of host cells that retain nomenon incompatibility between RM systems since it is the plasmid was measured after prolonged growth in the reminiscent of incompatibility between plasmid replication absence of selection for the plasmid. Our vector, pHSG415, a units. We can say that the EcoRI, Rsr I, and, presumably, their plasmid driven by the pSC101 replication unit, was more stable isoschizomer RMs, which recognize the sequence 5'-GAATTC, when it carried the EcoRI rm gene pair than when it carried form one incompatibility group with respect to plasmid stabili- only the modification component (r-m+) or neither compo- zation. The PaeR7I and its isoschizomer RMs, which recognize nent (r-m-). This restriction-enzyme-dependent stabilization the sequence 5'-CTCGAG, may form another incompatibility was also observed in a recBC mutant (data not shown) and in group. a recBC sbcA mutant (7). The r-m+ version was consistently Host Killing Model for Stabilization and Incompatibility. less stable than the r-m- version (Fig. 1; unpublished data), These and other observations (7) have led us to hypothesize a presumably because of the toxic effect of the EcoRI methylase. Plasmid Second Stabilization of the plasmid carrying the EcoRI rm gene pair measured plasmid was abolished in the presence of a second plasmid carrying the pHSG41 5 pBR322 same EcoRI rm gene pair and driven by a different replication (Camr) (Ampr) unit (ColEl as opposed to pSC101) (Fig. 2A). However, the EcoRI r+m+ pBR322 stabilization was not affected by the presence of a second EcoRI r-m+ pBR322 plasmid carrying the PaeR7I rm gene pair (10), which recog- E 10-2 EcoRI r+m+ PaeR71 r+m+ nizes a different sequence (Fig. 2A). These and the following I> EcoRI r+m+ EcoRI r+m+ experiments used a recBC sbcA strain due to the general E 10-3 instability of plasmids in this strain (13). E The Rsr I rm and EcoRI rm cut and methylate at the same (a positions of the same target sequence, although the sequences 0 1 of the genes encoding them are only distantly related (12, 14).
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