Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 DNAthe cellular defense hypothesis. The specificity and diversity were understood in terms of frequency-dependent selection in the cellular defense (Levin 1988
). However, we have advocated an alternative hypothesisthe selfish gene hypothesisfrom 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 1995
). This resistance turned out to be due to the death of cells that have lost the RM gene complex (Handa and Kobayashi 1999
). This host killing was demonstrated for several type II RM gene complexes examinedPaeR7I, EcoRI, EcoRV (Kusano et al. 1995
; Naito, Kusano, and Kobayashi 1995
; Nakayama and Kobayashi 1998
). 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. 2000
). 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 1998
) and from bacterial genome analysis (Kobayashi et al. 1999
).
One type of competition between RM systems is mutual exclusion (Nakayama and Kobayashi 1998
). 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 systemscompetition for a recognition sequencewas demonstrated in the absence of any invading DNA (Kusano et al. 1995
). 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, 1999b
). Some pairs of R genes show weak amino acid sequence homology (Jeltsch, Kroger, and Pingoud 1995
). 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.
|
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 cI857Sam7 with an EaeI-XbaI fragment from pRMS1.
|
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. 1989
; Kusano, Nakayama, and Nakayama 1989
; Handa and Kobayashi 1999
). 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 1990
).
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 inhibitiondecrease of cell viability, to be more precisewas stronger than that observed with any of the type II RM systems we analyzedPaeR7I (Naito, Kusano, and Kobayashi 1995
), EcoRI (Naito, Kusano, and Kobayashi 1995
), and EcoRV (Nakayama and Kobayashi 1998
). The growth inhibition was accompanied by change in cell shape, as in the case of EcoRI RM (Handa and Kobayashi 1999
; Handa et al. 2000
). 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).
|
|
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
).
|
|
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 46-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. 1997
). 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.
|
|
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. 1995
; Naito, Kusano, and Kobayashi 1995
; Nakayama and Kobayashi 1998
; Handa and Kobayashi 1999
; Handa et al. 2000
; 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 enzymesactivity, 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 factorincreased virulence by decrease in sequence specificitymay 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, 1996
; Owen et al. 1996
), 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 componentsR and Mwhich 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. 1995
). 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 generalto be a generalist or a specialist.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
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.
2 Keywords: restriction and modification
molecular evolution
sequence recognition
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
![]() |
literature cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Borck, K., J. D. Beggs, W. J. Brammar, A. S. Hopkins, and N. E. Murray. 1976. The construction in vitro of transducing derivatives of phage lambda. Mol. Gen. Genet. 146:199207.[ISI][Medline]
Bujnicki, J. M., and M. Radlinska. 1999a. Molecular evolution of DNA-(cytosine-N4) methyltransferases: evidence for their polyphyletic origin. Nucleic Acids Res. 27:45014509.
. 1999b. Molecular phylogenetics of DNA 5mC-methyltransferases. Acta Microbiol. Pol. 48:1930.
Game, J. C., K. C. Sitney, V. E. Cook, and R. K. Mortimer. 1989. Use of a ring chromosome and pulsed-field gels to study interhomolog recombination, double-strand DNA breaks and sister-chromatid exchange in yeast. Genetics 123:695713.
Gillen, J. R., D. K. Willis, and A. J. Clark. 1981. Genetic analysis of the RecE pathway of genetic recombination in Escherichia coli K12. J. Bacteriol. 145:521532.[ISI][Medline]
Grant, S. G., J. Jessee, F. R. Bloom, and D. Hanahan. 1990. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc. Natl. Acad. Sci. USA 87:46454649.
Handa, N., A. Ichige, K. Kusano, and I. Kobayashi. 2000. Cellular responses to post-segregational killing by restriction-modification genes. J. Bacteriol. 182:22182229.
Handa, N., and I. Kobayashi. 1999. Post-segregational killing by restriction modification gene complexes: observations of individual cell deaths. Biochimie 81:931938.
Hashimoto-Gotoh, T., F. C. H. Franklin, A. Nordheim, and K. N. Timmis. 1981. Specific-purpose plasmid cloning vectors: I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors. Gene 16:227235.
Jeltsch, A., M. Kroger, and A. Pingoud. 1995. Evidence for an evolutionary relationship among type-II restriction endonucleases. Gene 160:716.
Karyagina, A. S., V. G. Lunin, and I. I. Nikolskaya. 1990. Characterization of the genetic determinants of SsoII-restriction endonuclease and modification methyltransferase. Gene 87:113118.
Karyagina, A., I. Shilov, V. Tashlitskii, M. Khodoun, S. Vasil'ev, P. C. K. Lau, and I. Nikolskaya. 1997. Specific binding of SsoII DNA methyltransferase to its promoter region provides the regulation of SsoII restriction-modification gene expression. Nucleic Acids Res. 25:21142120.
Kobayashi, I. 1998. Selfishness and death: raison d'etre of restriction, recombination and mitochondria. Trends Genet. 14:368374.[ISI][Medline]
Kobayashi, I., A. Nobusato, N. Kobayashi-Takahashi, and I. Uchiyama. 1999. Shaping the genomerestriction-modification systems as mobile genetic elements. Curr. Opin. Genet. Dev. 9:649656.[ISI][Medline]
Kusano, K., T. Naito, N. Handa, and I. Kobayashi. 1995. Restriction-modification systems as genomic parasites in competition for specific sequences. Proc. Natl. Acad. Sci. USA 92:1109511099.
Kusano, K., K. Nakayama, and H. Nakayama. 1989. Plasmid-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sbcBC mutant. J. Mol. Biol. 209:623634.[ISI][Medline]
Levin, B. R. 1988. Frequency-dependent selection in bacterial populations. Philos. Trans. R. Soc. Lond. B Biol. Sci. 319:459472.[ISI][Medline]
Marinus, M. G. 1973. Location of DNA methylation genes on the Escherichia coli K-12 genetic map. Mol. Gen. Genet. 127:4755.[ISI][Medline]
Murray, N. E., W. J. Brammar, and K. Murray. 1977. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150:5361.[ISI][Medline]
Naito, T., K. Kusano, and I. Kobayashi. 1995. Selfish behavior of restriction-modification systems. Science 267:897899.
Nakayama, Y., and I. Kobayashi. 1998. Restriction-modification gene complexes as selfish gene entities: roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion. Proc. Natl. Acad. Sci. USA 95:64426447.
Owen, P., M. Meehan, H. De Loughry-Doherty, and I. Henderson. 1996. Phase-variable outer membrane proteins in Escherichia coli. FEMS Immunol. Med. Microbiol. 16:6376.[ISI][Medline]
Roberts, R. J., and S. E. Halford. 1993. Type II restriction endonucleases. Pp. 3588 in S. M. Linn, R. S. Lloyd, and R. J. Roberts, eds. Nucleases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Takahashi, N., and I. Kobayashi. 1990. Evidence for the double-strand break repair model of bacteriophage l recombination. Proc. Natl. Acad. Sci. USA 87:27902794.
van der Woude, M. W., B. A. Braaten, and D. A. Low. 1992. Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap. Mol. Microbiol. 6:24292435.[ISI][Medline]
. 1996. Epigenetic phase variation of the pap operon in Escherichia coli. Trends Microbiol. 4:59.[ISI][Medline]
Wilson, G. G., and N. E. Murray. 1991. Restriction and modification systems. Annu. Rev. Genet. 25:585627.[ISI][Medline]