Evolution of Sequence Recognition by Restriction-Modification Enzymes: Selective Pressure for Specificity Decrease

Akito Chinen, Yasuhiro Naito, Naofumi Handa and Ichizo Kobayashi

Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Several type II restriction-modification (RM) gene complexes kill host bacterial cells that have lost them, through attack on the chromosomal recognition sites of these cells. Two RM gene complexes recognizing the same sequence cannot simultaneously enjoy such stabilization through postsegregational host killing, because one will defend chromosomal sites from attack by the other. In the present work, we analyzed intrahost competition between two RM gene complexes when the recognition sequence of one was included in that of the other. When the EcoRII gene complex, recognizing 5'-CCWGG (W = A, T), is lost from the host, the SsoII gene complex, which recognizes 5'-CCNGG (N = A, T, G, C), will prevent host death by protecting CCWGG sites on the chromosome. However, when the SsoII (CCNGG) gene complex is lost, the EcoRII (CCWGG) gene complex will be unable to prevent host death through attack by SsoII on 5'-CCSGG (S = C, G) sites. These predictions were verified in our experiments, in which we analyzed plasmid maintenance, cell growth, cell shape, and chromosomal DNA. Our results demonstrate the presence of selective pressure for decrease in the specificity of recognition sequence of RM systems in the absence of invading DNA.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
A type II restriction endonuclease makes a double-strand break within or near a specific recognition sequence in duplex DNA. A cognate modification enzyme methylates the same recognition sequence to protect it from cleavage (Wilson and Murray 1991Citation ; Roberts and Halford 1993Citation ). Why do restriction-modification (RM) systems have very specific recognition sequences? Why are these recognition sequences so diverse?

It has been widely believed that the evolution and maintenance of RM systems have been driven by the cell's need to protect itself from infection by foreign DNA—the cellular defense hypothesis. The specificity and diversity were understood in terms of frequency-dependent selection in the cellular defense (Levin 1988Citation ). However, we have advocated an alternative hypothesis—the selfish gene hypothesis—from the following observation.

We found that several type II RM gene complexes behave selfishly, in the sense that they kill host bacterial cells that have lost them. A plasmid carrying an RM gene complex could not be readily displaced by an incompatible plasmid (Naito, Kusano, and Kobayashi 1995Citation ). This resistance turned out to be due to the death of cells that have lost the RM gene complex (Handa and Kobayashi 1999Citation ). This host killing was demonstrated for several type II RM gene complexes examined—PaeR7I, EcoRI, EcoRV (Kusano et al. 1995Citation ; Naito, Kusano, and Kobayashi 1995Citation ; Nakayama and Kobayashi 1998Citation ). This "postsegregational killing" takes place because the restriction enzyme cuts the host chromosome at unmodified recognition sites that the modification methylase fails to protect (Handa et al. 2000Citation ). This phenomenon is similar to suicidal defense against phage infection, or phage exclusion, programmed by prophages and plasmids, and may well play a similar role. These findings, in addition to previous work, led us to hypothesize that these RM systems represent a form of life, as do transposons or viruses. They increase and maintain their own frequency through two strategies: by destroying unmodified "nonself" DNA, whether it is invading DNA or its host's chromosome, and by moving between genomes. There is increasing evidence for this "selfish gene" hypothesis from experimental analysis (Kobayashi 1998Citation ) and from bacterial genome analysis (Kobayashi et al. 1999Citation ).

One type of competition between RM systems is mutual exclusion (Nakayama and Kobayashi 1998Citation ). When an RM system establishes itself in a new host cell, it is necessary for it to have a regulatory mechanism that delays expression of the restriction enzyme so as to prevent chromosome cleavage. A resident RM system may abort establishment of the incoming RM system by forcing the incoming RM system to prematurely express restriction enzyme and kill the host (see the last section of Results and Discussion and the figures there).

Another type of competition between RM systems—competition for a recognition sequence—was demonstrated in the absence of any invading DNA (Kusano et al. 1995Citation ). Host killing by an RM gene complex did not operate when the second RM gene complex within the same cell shared the same sequence specificity. Two RM systems of the same specificity are unable to enjoy stabilization simultaneously. This type of incompatibility implies competition for specific sequences by RM systems. This would result in the specialization of each of these selfish-gene units in only one of many diverse sequences. This explains why their recognition sequences are so specific and diverse.

Modification methylase genes show homology with each other, as represented in the form of a phylogenetic tree. There is a relationship between their gene sequences and their recognition sequences (Bujnicki and Radlinska 1999a, 1999bCitation ). Some pairs of R genes show weak amino acid sequence homology (Jeltsch, Kroger, and Pingoud 1995Citation ). These findings support the notion that modification methylases and restriction enzymes have gradually changed their recognition sequences.

How, then, have RM systems evolved their sequence recognition? We need to examine elementary steps of evolution of their sequence recognition. Let us choose two RM gene complexes, one recognizing 5'-CCWGG (W = A, T) (such as EcoRII) and the other recognizing 5'-CCNGG (N = A, T, G, C) (such as SsoII), with the former's recognition sequences being included in the latter's less specific ones. A host that has lost either the former or the latter RM gene complex could die because of their attack on the chromosome (fig. 1A and B ). What will happen when these two RM systems are both present in the same host? When the CCWGG-recognizing RM gene complex is lost from the host, the CCNGG-recognizing RM would prevent host death by protecting these sites (fig. 1C-1 ). On the other hand, when the CCNGG-recognizing RM gene complex is lost, the CCWGG-recognizing RM would be unable to prevent host death (fig. 1C-2 ). These predictions were verified in the present work. Our results demonstrate the presence of selective pressure for decrease in the specificity of recognition sequences of RM systems in the absence of invading DNA.



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Fig. 1.—Experimental design. A, The SsoII RM gene complex, which recognizes 5'-CCNGG (N = A, T, G, C) on one plasmid. The cell loses this plasmid and, therefore, the SsoII RM gene complex. R and M decrease in concentration through cell growth and/or proteolysis. When M has become unable to effectively modify their recognition sites along the chromosome, R cuts the chromosome at the unprotected sites, and the cell dies. B, The EcoRII RM gene complex, which recognizes 5'-CCWGG (W = A, T) on one plasmid. Postsegregational cell killing occurs as in A. C, Two RM gene complexes, EcoRII, which recognizes CCWGG, and SsoII, which recognize CCNGG, on two separate plasmids. Loss of the plasmid carrying the EcoRII RM gene complex does not lead to cell killing, because the SsoII RM gene complex protects the CCWGG sites on the host chromosome (C-1). Loss of the plasmid carrying the SsoII RM gene complex leads to cell killing as in A, even in the presence of the plasmid carrying the EcoRII RM gene complex (C-2). SsoII R cuts 5'-CCSGG (S= G, C) sites which are not protected by EcoRII

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Plasmids
Plasmids used in this work are listed in table 1 . The plasmids that have an EcoRII RM gene pair (plasmid's RM genotype; replication unit; antibiotic resistance; comments/ references) are as follows. Plasmid pNY30 (EcoRII R+M+; ColE1; Ap) was constructed by inserting a fragment including the EcoRII RM gene pair that had been amplified by polymerase chain reaction (PCR) into the SmaI site of pUC19. A 3,026-bp fragment including the EcoRII RM gene pair was amplified with PCR primers RII-1 (5'-GGC CCG GGC ATA GTC GAG ATT GGT GCA GA-3') and RII-2 (5'-GGC CCG GGT CAT CCA TAC CAC GAC CTC AA-3') from plasmid N3 (EcoRII R+M+; unknown; tetracycline; S. Hattman [Univ. of Rochester]). Each PCR primer had a SmaI site at the 5' end (underlined). Two plasmids, pNY31 (EcoRII R+M+; p15A; Cm) and pNY33 (EcoRII R+M+; ColE1; Ap), were constructed by replacing an EcoRV-NruI fragment of pACYC184 or pBR322 with a SmaI fragment of pNY30. Plasmid pNY35 (EcoRII R+M+; pSC101ts; Cm) was constructed by replacing an FspI-SmaI fragment of pHSG415 (none; pSC101ts; Ap, Cm, Km; Hashimoto-Gotoh et al. 1981Citation ]) with a SmaI fragment of pNY30. The R- versions of these three EcoRII plasmids, pNY41 (EcoRII R-M+; p15A; Cm), pNY43 (EcoRII R-M+; ColE1; Ap), and pNY45 (EcoRII R-M+; pSC101ts; Cm), were constructed by inserting a KpnI linker (TAKARA) into the BamHI site in the R gene.


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Table 1 Plasmids Used in this Work

 
The plasmids carrying SsoII RM gene pair were as follows. Two plasmids, pNY32 (SsoII R+M+; p15A; Cm) and pNY34 (SsoII R+M+; ColE1; Ap), were constructed by replacing a ClaI-NruI fragment of pACYC184 or pBR322 with an NspV-HincII fragment of pRMS1 (SsoII R+M+; ColE1; Ap; Karyagina, Lunin, and Nikolskaya [1990Citation ]). The R- versions of these two SsoII plasmids, pNY42 (SsoII R-M+; p15A; Cm) and pNY44 (SsoII R-M+; ColE1; Ap), were constructed by inserting a KpnI linker into the BglII site in the R gene. Two plasmids, pNY49 (SsoII R+M+; pSC101ts; Cm) and pNY50 (SsoII R-M+; pSC101ts; Cm), were constructed by replacing a ClaI-NcoI fragment of pNY45 with an XbaI-NcoI fragment of pNY32 or pNY42. Cohesive ends made by ClaI or XbaI were converted to blunt ends by T4 DNA polymerase.

Bacteria and Bacteriophage
Escherichia coli K-12 strains and the bacteriophage used in this work are listed in table 2 . LIK1114 was made by replacing an Eco52I (19944)–XbaI (24505) fragment of {lambda} cI857Sam7 with an EaeI-XbaI fragment from pRMS1.


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Table 2 Bacteria and Bacteriophage Used in this Work

 
Postsegregational Killing
The procedure for postsegregational killing was as follows (Handa and Kobayashi 1999Citation ). Cells were aerated at 30°C in L broth containing antibiotics with aeration and grown to an OD660 of 0.3. The antibiotics selective for the plasmid of interest were then removed, and the cells were transferred to a temperature of 42°C. The culture was diluted every time its OD660 reached about 0.3. The total cell number was counted under a microscope. The number of viable cells was estimated by counting the number of colonies on L agar with appropriate antibiotics selective for the second plasmid at 30°C. Plasmid-carrying cells were counted by colony formation on L agar with antibiotics selective for the plasmid in question and for the second plasmid as appropriate.

Morphological Observation
Samples were viewed under a Nikon E600 microscope. Photomicrographs were taken through ARGUS-20 and FISH Imaging Software.

Pulsed-Field Gel Electrophoresis
The procedure for analysis of the chromosome by pulsed-field gel electrophoresis is as follows (Game et al. 1989Citation ; Kusano, Nakayama, and Nakayama 1989Citation ; Handa and Kobayashi 1999Citation ). Escherichia coli cultures were mixed with 2,4-dinitrophenol to block energy metabolism and treated as described. The DNA was electrophoresed through a 1.0% agarose gel at 14°C in 45 mM Tris-borate/1.25 mM EDTA with a pulse time of 50 s for 24 h by using hexagonal electrodes in a CHEF-DR III apparatus (BioRad).

Transformation
Plasmids were purified using a QIAGEN kit. They were used to transform E. coli strains by electroporation with a Gene-Pulser apparatus (BioRad) as described (Takahashi and Kobayashi 1990Citation ).


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Stable Maintenance of the EcoRII RM Gene Complex (Recognizing CCWGG) Is Inhibited by SsoII Methylase (Recognizing CCNGG)
An EcoRII RM gene complex, or its R- mutant version, was inserted into a plasmid vector (pACYC184) carrying a chloramphenicol-resistance gene. This plasmid was introduced into a bacterial strain (E. coli K-12, strain JC8679). When these cells were grown in the presence of chloramphenicol, most of the cells carried this plasmid, because the antibiotics killed cells that had lost the plasmid and, therefore, the chloramphenicol resistance gene. In the absence of such selection for the maintenance of the plasmid, the R- plasmid was gradually lost (fig. 2A ). However, the EcoRII RM plasmid was maintained at a high frequency in the absence of such drug selection (Fig. 2A ), as found earlier with several other type II RM systems (Kusano et al. 1995Citation ; Naito, Kusano, and Kobayashi 1995Citation ; Nakayama and Kobayashi 1998Citation ). This is probably because the cells that have lost the EcoRII RM gene complex are killed by EcoRII restriction enzyme through attack on their chromosomal EcoRII sites. This is a case of the phenomenon known as postsegregational killing.



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Fig. 2.—A, Stabilization of a plasmid carrying the EcoRII RM gene complex and its suppression by SsoII M. The bacterial cells (BIK11048, EcoRII R+M+ []; BIK11049, EcoRII R-M+ [{circ}]; and BIK11103, EcoRII R+M+, SsoII R-M+ [{blacktriangleup}]) were incubated with aeration at 37°C in LB broth with Ap and Cm until their OD660 reached 0.3. The cells were diluted 1:106 in LB with Ap lacking antibiotics and incubated at 37°C with aeration to saturation. The culture was continued with dilutions of 1:106. The cells were spread on an LB agar plate with Ap to determine viable cell number and on an LB agar plate with Ap and Cm to determine the number of cells carrying the EcoRII plasmid. The number of viable cells was used to calculate the generation number. B, Postsegregational killing by the EcoRII RM gene complex and its suppression by SsoII M. The bacterial cells (BIK11075, EcoRII R+M+ []; BIK11078, EcoRII R-M+ [{circ}]; and BIK11077, EcoRII R+M+, SsoII R-M+ [{blacktriangleup}]) were incubated at 30°C in LB broth with Ap and Cm until their OD660 reached 0.3. Cm was then removed, and the temperature was shifted to 42°C. B1, The total number of cells was counted under a microscope. B2, The number of viable cells was determined by counting colonies on LB agar plate with Ap. B3,s The number of plasmid-carrying cells was determined by counting colonies on LB agar plates with Ap and Cm

 
EcoRII RM recognizes 5'-CCWGG (W = A, T), while SsoII RM recognizes 5'-CCNGG (N = A, T, G, C). Because the EcoRII sites on the chromosome are expected to be modified by SsoII methylase, postsegregational killing by EcoRII would be inhibited by the presence of SsoII methylase in the same cell. This prediction was verified (fig. 2A ). The EcoRII-carrying plasmid is lost at the rate of the R- control plasmid in the presence of an SsoII methylase gene on a compatible plasmid.

Postsegregational Cell Killing by the EcoRII RM Gene Complex (Recognizing CCWGG) Is Inhibited by SsoII Methylase (Recognizing CCNGG)
In order to bring about simultaneous loss of the EcoRII RM gene complex for analysis of the process of postsegregational killing, we inserted the EcoRII RM gene pair (or its R- version as a negative control) into a temperature-sensitive replicon (pHSG415). This plasmid was established together with a plasmid driven by a compatible replicon (pBR322) in the same bacterial strain. After the temperature shift, bacteria carrying this plasmid showed a reduction in the number of viable cells (colony-forming units) and slowed down the increase in the total number of cells (microscopic observation) (fig. 2B1 and B2 ). The R- control did not show such inhibition of cell growth (fig. 2B1 and B2 ) although most cells lost the plasmid (fig. 2B3 ). This growth inhibition—decrease of cell viability, to be more precise—was stronger than that observed with any of the type II RM systems we analyzed—PaeR7I (Naito, Kusano, and Kobayashi 1995Citation ), EcoRI (Naito, Kusano, and Kobayashi 1995Citation ), and EcoRV (Nakayama and Kobayashi 1998Citation ). The growth inhibition was accompanied by change in cell shape, as in the case of EcoRI RM (Handa and Kobayashi 1999Citation ; Handa et al. 2000Citation ). Many cells became filamentous (fig. 31a and 1b ) in an R+-dependent manner (fig. 32a and 2b ). These results are consistent with strong postsegregational cell killing in a plate assay and in a liquid assay shown by this RM gene complex in other genetic backgrounds (unpublished data).



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Fig. 3.—Morphology of the cells losing the EcoRII RM gene complex. 1a and 1b, BIK11075, EcoRII R+M+. 2a and 2b, BIK11078, EcoRII R-M+. 3a and 3b, BIK11077, EcoRII R+M+, SsoII R-M+. 1a, 2a, and 3a, 0 h after temperature shift in the experiments of figure 2B. 1b, 2b, and 3b, 4 h after temperature shift in the experiments of figure 2B. Each plate is 271 x 204 µm.

 
Chromosomal DNA was analyzed by pulsed-field gel electrophoresis after loss of the EcoRII RM gene complex. Huge linear forms of the chromosome, which band just below the well, accumulated by 2 h after the temperature shift and then increased. Smaller DNA forms, which are detected as a smear in the lower part of the gel, increased. In the R- control, neither the huge linear forms nor the smaller forms accumulated (fig. 4 ).



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Fig. 4.—Cleavage and degradation of chromosomal DNA following loss of the EcoRII RM gene complex. DNA was prepared and subjected to pulsed-field gel electrophoresis. Marker lane contains Saccharomyces cerevisiae chromosomes. BIK11075, EcoRII R+M+; BIK11078, EcoRII R-M+; and BIK11077, EcoRII R+M+, SsoII R-M+

 
We then examined whether SsoII methylase (recognizing CCNGG) can block the postsegregational cell killing by EcoRII (recognizing CCWGG), presumably by protecting CCWGG sites on the chromosome. As expected, the SsoII methylase gene present on the pBR322 plasmid vector was able to suppress all of the examined death phenotypes caused by loss of the EcoRII RM gene complex. Cell growth inhibition as detected by viable cell count and total cell count was suppressed (fig. 2B1 and B2 ). Cell filamentation and chromosome degradation were also completely suppressed (figs. 33a, 33b, and 4 ).

Postsegregational Cell Killing by the SsoII RM Gene Complex (Recognizing CCNGG) Is Not Inhibited by EcoRII Methylase (Recognizing CCWGG)
We then tried a series of experiments in which the roles of SsoII and EcoRII were reversed: EcoRII, recognizing only CCW(= A, T)GG would be unable to methylate all of the CCN(= A, T, G, C)GG sites recognized by SsoII. SsoII showed the plasmid stabilization effect (fig. 5A ).



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Fig. 5.—A, Stabilization of a plasmid carrying the SsoII RM gene complex. The bacterial cells (BIK11050, SsoII R+M+ [], and BIK11051, SsoII R-M+ [{circ}]) were incubated without selection as in figure 2A. B, Postsegregational cell killing by the SsoII RM gene complex and failure of its suppression by EcoRII. The bacterial cells (BIK11137, SsoII R+M+ []; BIK11140, SsoII R-M+ [{circ}]; and BIK11139, SsoII R+M+, EcoRII R-M+ [{blacktriangleup}]) were incubated at 30°C in LB broth with Ap and Cm until the OD660 reached 0.3. Cm was then removed, and the temperature was shifted to 42°C as in figure 2B. B1, The total number of cells was counted under a microscope. B2, The number of viable cells was determined by counting colonies on an LB agar plate with Ap. B3, The number of plasmid-carrying cells was determined by counting colonies on LB agar plates with Ap and Cm, respectively

 
The increase in the number of viable cells carrying the SsoII RM gene complex on a ts plasmid was inhibited at about the same time as the number of plasmid-carrying cells stopped increasing (fig. 5B2 and B3 ). This growth inhibition was significant compared with the R- control and reproducible, although it was weaker than the inhibition observed with any type II system we have analyzed—EcoRII (unpublished data; see above), EcoRI (Kusano et al. 1995Citation ; Naito, Kusano, and Kobayashi 1995Citation ), PaeR7I (Naito, Kusano, and Kobayashi 1995Citation ), and EcoRV (Nakayama and Kobayashi 1998Citation ). Some of the cells became filamentous (fig. 61a and 1b ) in an R+-dependent manner (fig. 62a and 2b ), although their frequency was not as large as that with EcoRII (above) or EcoRI (Handa and Kobayashi 1999Citation ; Handa et al. 2000Citation ). Chromosome degradation was not detected by pulsed-field gel electrophoresis.



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Fig. 6.—Morphology of the cells losing the SsoII RM gene complex. 1a and 1b, BIK11137, SsoII R+M+. 2a and 2b, BIK11140, SsoII R-M+. 3a and 3b, BIK11139, SsoII R+M+, EcoRII R-M+. 1a, 2a, and 3a, 0 h after temperature shift in the experiments of figure 5B. 1b, 2b, and 3b, 4 h after temperature shift in the experiments of figure 5B. Each plate is 271 x 204 µm

 
The presence of EcoRII methylase did not suppress two postsegregational killing symptoms—growth inhibition (fig. 5B1B3 ) and cell filamentation (fig. 63a and 3b )—as expected from their recognition sequences. We concluded that EcoRII methylase (recognizing CCWGG) did not inhibit the postsegregational killing by the SsoII RM gene complex (recognizing CCNGG).

Absence of Exclusion Between EcoRII (Recognizing CCWGG) and SsoII (Recognizing CCNGG)
In the above situation, two RM gene complexes were present in the same host, and one of them was lost. In this case, the RM system with a less specific recognition sequence had a competitive advantage. This demonstrates selective pressure for decrease in the specificity of recognition sequence and, therefore, increase in the number of recognition sites on the chromosome. However, there are many RM systems with a 4–6-bp-long recognition sequence, and the number of the sites for a particular RM system remain finite. Therefore, we have to postulate some counterbalancing forces that prevent the recognition sequence from becoming less specific.

One candidate for such counterbalancing forces is mutual exclusion (see Introduction and fig. 7 ). It is known that RM gene complexes have some regulatory mechanism to delay R gene expression when they enter a new host (fig. 7A ). Some RM gene complexes have a regulatory gene, the C gene, in addition to the R and M genes. Because R gene expression requires the C gene product, the R gene is expressed after M and C gene expression. The other RM gene complexes do not have a C gene, but they have to have some mechanism for delaying R gene expression (Karyagina et al. 1997Citation ). If a resident RM system shares specificity of this expression delay mechanism with the incoming RM gene complex, an incoming RM system may express R prematurely (fig. 7B ). If this R recognized a different sequence, it would cut the chromosome and kill the cell. Establishment of the RM would be aborted. When the recognition sequence of an RM system has become less specific (recognizing more sites than its parent) by a mutation, it may well retain the same specificity in the regulatory mechanism that delays premature restriction, because it is unlikely that recognition sequence and regulatory mechanism change at the same time. Therefore, a new RM system that recognizes a less specific sequence would not spread in the bacterial population that already has its parental RM system. Hence, the change of recognition sequence to less specific would be a disadvantage for the RM system in some population.



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Fig. 7.—Mutual exclusion between RM gene complexes. A, An RM gene complex on a plasmid enters a cell. M is expressed first. After the modification enzyme has modified almost all of the chromosomal recognition sites, R is expressed. B, Entry of an RM gene complex on a plasmid into a cell harboring another RM gene complex. The two RM systems share the same specificity in the regulatory system to delay R expression but differ in the specificity of their sequence recognition. The resident RM system forces the incoming RM gene complex to express its R, which then cleaves its yet-unmodified chromosomal recognition sites and kills the cell

 
In table 3 , our early data showing strong exclusion of PvuII by BamHI was cited. The transformation efficiency of PvuII RM was reduced 104-fold by resident BamHI RM. We tried the same sort of experiments for EcoRII and SsoII, but we could not detect any difference in transformation efficiency (table 3 ). We concluded that EcoRII and SsoII do not have the ability to exclude each other. They may carry a different specificity in the mechanisms delaying R expression.


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Table 3 Absence of Mutual Exclusion Between EcoRII and SsoII

 
The above arguments regarding mutual exclusion as a counterbalancing force apply only when the two RM systems share the same specificity in the regulatory mechanism. This should be the case when an RM system with an altered sequence specificity arises as a result of mutation within a parental RM population and stays close to the parental RM population. However, this may not be the case when an RM system with a less specific sequence specificity appears in a host through horizontal transfer. It may well be of distant phylogenetic relationship with the resident RM and may carry an unrelated specificity in the R delay mechanism. If horizontal transfer of RM gene complexes is extensive, exclusion cannot play the role of a force counterbalancing decrease in the sequence specificity.

Further Discussion About Counterbalancing Forces
Here, we discuss two more possible forces counterbalancing the force of decrease in the specificity of sequence recognition inferred from the present work.

The second candidate is the strength of the postsegregational killing. Suppose that a mutation making a recognition sequence less specific arises in a population of an RM system. If other things were not altered by the mutation, this mutant RM would introduce many more breaks into the host chromosome than its parent. It would become more difficult for the host cell to repair these many double-strand breaks in the chromosome. One might expect that the mutant RM with the less specific recognition sequence would result in stronger postsegregational killing. This RM might cause cell killing at even a slight disturbance of gene expression. If the virulence of this RM system is too strong, it would be lost from the population of RM/host pairs, just as virulent pathogens will be lost.

The strength of postsegregational killing may be modified by factors other than recognition sequence, as one might expect. RM systems vary in strength of postsegregational killing even in comparable situations (Kusano et al. 1995Citation ; Naito, Kusano, and Kobayashi 1995Citation ; Nakayama and Kobayashi 1998Citation ; Handa and Kobayashi 1999Citation ; Handa et al. 2000Citation ; this work; unpublished data). Their strength of postsegregational killing did not parallel the number of recognition sites along the chromosome. An EcoRII plasmid construct showed very strong postsegregational killing (this work; unpublished data). An SsoII plasmid construct showed the weakest postsegregational killing, although SsoII has the shortest recognition sequence among these. Some properties of restriction or methylation enzymes—activity, half-life, etc.—might determine the particular features of postsegregational killing development in various RM systems. When there is extensive horizontal transfer of RM systems, this factor—increased virulence by decrease in sequence specificity—may not contribute as a counterbalancing force.

The third candidate for forces counterbalancing the force decreasing specificity in the sequence recognition is the cost of DNA methylation. There may be cost on the side of the RM system and that on the side of the host. The host genome would be methylated at more sites with a less specific and shorter recognition sequence. DNA methylation is known to affect gene expression in bacteria (van der Woude, Braaten, and Low 1992, 1996Citation ; Owen et al. 1996Citation ), as in many other organisms. This force will be effective even with horizontal transfer of RM systems. Further understanding of the natural history of RM systems and their horizontal transfer is necessary to evaluate the relative importance of these three forces.

There is one point of simplification in the above arguments. A type II RM system is composed of two components—R and M—which recognize the same specific sequence independently. Change of recognition sequence becomes possible only after a change in each of the two components. This may be achieved by sequential mutation in M and R or by recombination of R and M of different origin. Extensive horizontal transfer would favor the latter route.

From previous work demonstrating inhibition of one RM's postsegregational killing by another RM of the same sequence specificity, we have argued that there is competition for recognition sequences among different RM systems (Kusano et al. 1995Citation ). The present work on competition between two RMs with related recognition sequences describes an elementary force in the competition for sequences among RMs. The issue may be analogous to the issue of host specificity of parasites in particular, and the issue of adaptation to ecological niches in general—to be a generalist or a specialist.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
We thank S. Hattman, Anna Karyagina, M. G. Marinus, A. J. Clark, and Y. Kitamura for generous gifts of biological materials, and Kurt Nordstrom, Alfred Pingoud, Mike Yarmolinsky, and Anna Karyagina for comments on manuscripts. This work was supported by the Department of ESSC of the Japanese government (DNA repair, genome) and by NEDO.


    Footnotes
 
Naruya Saitou, Reviewing Editor

1 Abbreviations: Ap, ampicillin; Cm, chloramphenicol; E. coli, Escherichia coli; Km, kanamycin; M, modification methylase gene or enzyme; PCR, polymerase chain reaction; R, restriction endonuclease gene or enzyme. Back

2 Keywords: restriction and modification molecular evolution sequence recognition Back

3 Address for correspondence and reprints: Ichizo Kobayashi, Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Shiroganedai, Tokyo 108-8639, Japan. E-mail: ikobaya{at}ims.u-tokyo.ac.jp Back


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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
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Accepted for publication July 11, 2000.