Department of Microbiology, University of Gdask, K
adki 24, 80-822 Gda
sk, Poland
Correspondence
Tadeusz Kaczorowski
kaczorow{at}biotech.univ.gda.pl
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ABSTRACT |
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Nomenclature. The nomenclature for restriction endonucleases and methyltransferases in this paper follows the recommendations of Roberts et al. (2003b).
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INTRODUCTION |
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Our research is focused on the nature of the isospecificity phenomenon among type II RM systems, where we are especially interested in finding out what these enzymes have in common. As a model in our study we decided to use a group of systems isospecific to HindIII, an RM system from Haemophilus influenzae Rd (Roy & Smith, 1973; Old et al., 1975
). This group consists of over 30 RM systems isolated from different bacteria, all of them recognizing the same specific palindromic sequence 5'-AAGCTT-3' (Roberts et al., 2003a
). To date, except for HindIII (Nwankwo et al., 1994
), only two other systems, EcoVIII from Escherichia coli E1585-68 and LlaCI from Lactococcus lactis subsp. cremoris W15, have been cloned and sequenced (Kaczorowski & Szybalski, 1998
; Mruk et al., 2001
; Madsen & Josephsen, 1998
).
In the present study, we investigated the properties of the M.LlaCI. The presence and distribution of nine highly conserved amino acid sequence motifs and a putative target recognition domain in the enzyme structure suggests that this enzyme belongs to the N6-adenine -class MTases. These motifs can be grouped into three clusters which are responsible for three principal functions: (i) sequence-specific DNA recognition (TRD, target recognition domain); (ii) binding of the methyl group donor S-adenosyl-L-methionine (AdoMet) (motifs X, I and II); and (iii) catalysis of the methyl group transfer (motifs III, IV, V, VI, VII and VIII). The same motif organization was also observed in the case of M.HindIII and M.EcoVIII, both being isospecific to the M.LlaCI enzyme (Nwankwo et al., 1994
; Mruk & Kaczorowski, 2003
). Biochemical characterization of M.LlaCI led us to discover its unusual sensitivity to Mg2+. This particular property is shared with a few other MTases (e.g. M.EcoRI, Hanish & McClelland, 1988
; M.FokI, Kaczorowski et al., 1999
). We argue that this finding has far-reaching biological consequences. Most probably, the intracellular concentration of Mg2+ inhibits M.LlaCI activity without affecting the potency of the cognate restriction ENase. These circumstances clearly promote DNA restriction over DNA methylation, a scenario which seems to be profitable for bacteria fighting with invading DNA. However, it must be stressed that the observed inhibition of the modifying enzyme apparently does not affect the cell's viability. In this article, we also argue that observed sensitivity of some MTases to Mg2+ can encourage the process of genetic recombination. Involvement of RM enzymes in this process was raised in several reports (Chang & Cohen, 1977
; McKane & Milkman, 1995
; Kobayashi et al., 1999
, 2001
).
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METHODS |
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DNA isolation and manipulation.
Molecular cloning experiments were performed by standard techniques (Sambrook et al., 1989). After cloning, hybrid plasmids were transformed into an appropriate E. coli strain. Recombinant plasmids were examined by restriction analysis and automated DNA sequencing by the dideoxy chain-termination method using Applied Biosystems ABI Prism BigDye Terminators and an ABI Prism 310 Genetic Analyser. Restriction ENases and DNA-modifying enzymes were purchased from either New England Biolabs or MBI Fermentas. Enzyme reactions were carried out under conditions suggested by the suppliers. PCR reactions were performed with DyNAzyme II DNA polymerase from Thermus brockianus (Finnzymes).
The LlaCI MTase assay.
The M.LlaCI protection assay was performed in a 20 µl reaction mixture containing 0·5 µg DNA, 80 µM AdoMet, 10 mM Tris/HCl (pH 7·0), and 2 µl of column fractions (1 h, 37 °C). The reaction was stopped by heating the mixture (60 °C, 10 min). Following cooling, 2 µl NEB2 10x reaction buffer (New England Biolabs) and 5 units R.HindIII were added (1 h, 37 °C), and the DNA was analysed by 0·8 % agarose gel electrophoresis. One unit of the modification activity was defined as the minimum amount of MTase that conferred complete resistance to cleavage by the cognate restriction ENase, to 1 µg
DNA in 1 h at 37 °C, under the assay conditions described above. The second assay for M.LlaCI activity was based on monitoring of the enzyme-catalysed transfer of 3H-labelled methyl groups from [methyl-3H]AdoMet to DNA. Methylation was performed in a 30 µl reaction mixture containing 5 µg
DNA (or synthetic oligonucleotide), 10 mM Tris/HCl (pH 7·0), 0·5 µM [methyl-3H]AdoMet (3x1012 Bq mmol-1, Amersham), and an aliquot of the enzyme (1 h, 37 °C). The reaction was stopped by adding 30 µl 50 % trichloroacetic acid. The sample was centrifuged (10 000 g, 10 min). The pellet was washed with 1 ml 70 % ethanol, centrifuged, and dried. Scintillation counting was used to estimate the incorporated radioactivity.
Protein purification.
The DNA MTases M.EcoVIII and M.HindIII used in this study were purified to apparent electrophoretic homogeneity using procedures described previously (Mruk & Kaczorowski, 2003).
Purification of M.LlaCI from recombinant E. coli.
The enzyme was prepared from E. coli BL21(DE3)[pLysS] transformed with pLlaMet3. This plasmid was constructed by cloning into a pT7-6 vector, previously linearized with BamHI and SmaI, a 1·15 kb DNA fragment carrying the M.LlaCI gene that was obtained in a PCR reaction followed by BamHI digestion and T4 polynucleotide kinase phosphorylation. The forward and reverse primers were 5'-GAGGATCCACGACATTTA-3' and 5'-ATGAATTCCAGTT TTGAT-3', respectively (the BamHI site is underlined). Plasmid pSX1 (LlaCI R-/M+; Madsen & Josephsen, 1998) was used as a template in the PCR reaction. In recombinant plasmid pLlaMet3 the start codon of the llaCIM gene is located 164 nt downstream from the
10 promoter of phage T7. Bacteria carrying the overproducing plasmid were cultivated at 37 °C in TY-broth (1 litre) supplemented with Ap and Cm to an OD600 of 0·3. At this time overproduction of the M.LlaCI was induced by adding IPTG to the culture to a final concentration of 1 mM. Induction proceeded for 2·5 h. Cells were harvested by centrifugation and stored at -70 °C. For enzyme purification, frozen cells (2·5 g) were resuspended in 10 ml buffer P (10 mM potassium phosphate pH 7·0, 50 mM KCl, 1 mM EDTA, 10 mM 2-mercaptoethanol and 5 %, v/v, glycerol) supplemented with PMSF (0·15 mM), and disrupted by sonication (60 bursts of 10 s at an amplitude of 12 µm). All procedures were carried out at 4 °C. Cellular debris was removed by centrifugation (14 000 g, 40 min), and clarified lysate was applied to a 2·5x5 cm P-11 column (Whatman) equilibrated with buffer P. Proteins bound to the column were eluted with a 150 ml linear gradient of KCl (0·051·0 M) in buffer P. Fractions enriched in M.LlaCI were collected and applied directly to a 1x5 cm blue-agarose column (Pharmacia) equilibrated with buffer B (10 mM potassium phosphate pH 7·0, 1 mM EDTA, 10 mM mercaptoethanol and 5 %, v/v, glycerol). Protein was eluted with a 100 ml linear gradient of KCl (01·0 M) in buffer B. The final step was chromatography on Superose 12HR (FPLC, Pharmacia) equilibrated with buffer S (10 mM potassium phosphate pH 7·6, 150 mM KCl, 1 mM EDTA, 10 mM mercaptoethanol and 2·5 %, v/v, glycerol). Fractions with the highest M.LlaCI activity were dialysed overnight against buffer E (10 mM potassium phosphate pH 7·5, 50 mM KCl, 10 mM mercaptoethanol, 0·5 mM EDTA and 50 %, v/v, glycerol) and stored at -20 °C.
Mr determination.
A purified preparation of the M.LlaCI was analysed by SDS-PAGE (Laemmli, 1970) in order to estimate purity and Mr under denaturing conditions. After electrophoresis, protein position was visualized by Coomassie brilliant blue R 250 staining. The Mr of the M.LlaCI was calculated using a calibration curve obtained with the following standard proteins (Pharmacia): phosphorylase b (Mr, 94 000), bovine serum albumin (67 000), ovalbumin (43 000), carbonic anhydrase (30 000), trypsin inhibitor (20 100) and
-lactalbumin (14 400).
N-terminal amino acid sequencing.
A 300 pmol sample of homogeneous preparation of M.LlaCI was separated by SDS-PAGE, electroblotted onto PVDF membrane and then analysed using an Applied Biosystems 491 gas-phase protein sequencer. The phenylthiohydantoin (PTH) derivatives of the amino acids were identified with an Applied Biosystems 140C PTH-analyser connected to the sequencer. The first 10 PTH-amino acids were unambiguously identified.
Protein cross-linking with glutaraldehyde.
In order to determine the state of aggregation in solution, M.LlaCI enzyme was subjected to chemical cross-linking, which was performed by using a previously described protocol (Schlossman et al., 1984). An aliquot of M.LlaCI enzyme (2·5 µg) was incubated at 30 °C for 10 min in 30 µl reaction mixtures in standard buffer. Glutaraldehyde (1·9 µl of a 2·5 %, w/v, solution) was added to the reaction mixture (2 min, 30 °C), followed by sodium borohydride treatment (2·6 µl of a 1 M solution, 20 min, 4 °C). The reaction was terminated by the addition of Tris/HCl pH 7·5 (9 µl of a 1 M solution) and incubation of the sample for 5 min at 4 °C. Proteins were analysed using SDS-PAGE (5 %) (Weber & Osborn, 1969
), and visualized by silver staining (Heukeshoven & Dernick, 1985
).
Determination of the methylated base.
To determine the base methylated by M.LlaCI the method employing type IIS restriction ENases was used (Pósfai & Szybalski, 1988). In order to obtain appropriate templates two synthetic DNA fragments carrying the M.LlaCI recognition site overlapped by the MboII cut site (5'-AATTCGAAGAATCGATCA-3'/3'-GCTTCTTA GCTAGTTCGA and 5'-AATTCGAAGATCGATCA-3'/3'-GCTTCTAGCTAGTTCGA-5') were cloned into plasmid pGEM3Zf(+), previously double-digested with HindIII and EcoRI, resulting in plasmids pIM1 and pIM2, respectively. These plasmids differ by one base-pair in the region between the LlaCI and MboII sites. Plasmid pIM3 without the LlaCI site was used as a control. This plasmid was constructed by digestion of pIM1 with R.HindIII followed by the filling-in of the 5' protruding ends with Klenow fragment in the presence of dNTPs. After ligation and transformation into E. coli MM294 cells, recombinants were checked by DNA sequencing analysis. To determine M.LlaCI methylation pattern, 366 bp (pIM1), 365 bp (pIM2) or 370 bp (pIM3) DNA fragments were amplified using a pair of primers complementary to pGEM3Zf(+) vector (ADE1, 5'-TTACGCCAGCTGGCGAAAG-3' and ADE2, 5'-CATTAATGCAGCTGGCAC-3'). DNA fragments carrying synthetic oligomer isolated from PCR reactions where either pIM1 (366 bp) or pIM2 (365 bp) served as template, were methylated by M.LlaCI. In the control experiment a 370 bp DNA fragment without an LlaCI site, derived from plasmid pIM3, was used. Methylation was performed in a 30 µl reaction mixture containing 0·5 µg of a particular DNA fragment (365, 366 or 370 bp), 10 mM Tris/HCl pH 7·0, 3·7x105 Bq [3H]AdoMet (3x1012 Bq mmol-1, Amersham) and 50 ng M.LlaCI (1 h, 37 °C). In the experiment investigating the effect of cleavage by the MboII enzyme, the 3H-labelled DNA fragment was cut either between adenine and adenine (366 bp DNA fragment) or between adenine and guanine (365 bp DNA fragment) within the M.LlaCI recognition site. After electrophoresis, particular DNA fragments were extracted from 1·5 % agarose gel using a method employing DEAE-cellulose membrane (Kaczorowski et al., 1993
). Scintillation counting was used to estimate the radioactivities of the DNA fragments.
Western blot analysis.
Homogeneous preparations of the enzymes (M.LlaCI, M.EcoVIII and M.HindIII) were separated by SDS-PAGE (10 %) and then electroblotted onto nitrocellulose membrane. After blocking with 3 % skimmed milk, the membrane was incubated with a 1 : 25 dilution of anti-M.EcoVIII rabbit polyclonal antibodies, which were prepared according to standard procedures (Harlow & Lane, 1988). The primary antibodies were tagged successively with a 1 : 10 000 dilution of goat anti-rabbit polyclonal antibodies conjugated with alkaline phosphatase (Sigma). All antibodies were diluted in phosphate-buffered saline (Sambrook et al., 1989
). Reactive bands were visualized by using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the alkaline phosphatase substrate and nitro blue tetrazolium (NBT) as the colour development reagent.
Effect of divalent cations on activity of DNA MTases.
The activity of M.LlaCI and its isomethylomers was measured in a reaction buffer (10 mM Tris/HCl pH 7·0) containing divalent metal ions (Mg2+, Mn2+, Ca2+, Zn2+) at different concentrations. Enzyme activity of M.LlaCI and its isospecific homologues (M.EcoVIII and M.HindIII) was assayed by measuring incorporation of 3H-labelled methyl groups from [methyl-3H]AdoMet into 2·5 pmol of 29 bp double-stranded oligonucleotide (5'-TGCAGTCGCGAAGCTTGGTCACCTTGAGG-3'/3'-GTCAGCGCTTCGAACCAGTGAACTCCGT-5'; the LlaCI site is underlined) using 35 ng of the enzyme (30 min, 37 °C). Each experiment was repeated at least three times. For each cation the value of a 50 % inhibitory concentration (IC50) was determined.
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RESULTS |
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Chemical cross-linking of M.LlaCI
The state of aggregation of the M.LlaCI enzyme was tested using a glutaraldehyde cross-linking reaction under optimal conditions (10 mM potassium phosphate pH 7·0, 80 µM AdoMet and 10 mM EDTA) with M.LlaCI at 2·7 µM (2·5 µg). Chemical intermolecular cross-linking, catalysed by glutaraldehyde, is strongly dependent on the presence of lysine residues in the protein structure. A molecule of M.LlaCI contains 21 lysine residues as predicted from the nucleotide sequence of the llaCIM gene. As a result of a cross-linking experiment, we have found that M.LlaCI exists in solution predominantly as a dimer (see Fig. 2, lane 2). A well-known complex-forming protein, E. coli DnaK, was used as a control. Upon cross-linking, DnaK, which is known to produce multimeric forms (Liberek et al., 1990
), formed three distinct bands corresponding to DnaK monomers, dimers and trimers (Fig. 2
, lane 1). Most of the type II restriction ENases exist in solution as dimers. On the other hand, type II DNA MTases usually function as monomers. M.LlaCI makes an exception to this rule, existing in solution predominantly as a dimer. We have shown previously that an isomethylomer of M.LlaCI, M.EcoVIII, exists in solution as a monomer (Mruk & Kaczorowski, 2003
). A few other MTases possess dimeric structure: e.g. M.RsrI (Kaszubska et al., 1989
), M.DpnA, M.DpnM (de la Campa et al., 1987
), and M.KpnI (Bheemanaik et al., 2003
). Structural elements required for dimer formation remain unclear. Only in the case of the M.RsrI enzyme, crystallographic analysis resulted in the proposition that the unusual fold of the putative DNA binding domain is most probably involved in the formation of M.RsrI dimers (Scavetta et al., 2000
). However, it is unclear how such a state of oligomerization can affect enzyme function. Detailed biochemical analysis of the M.KpnI enzyme did not provide any clues for understanding why a dimeric form could be superior when such factors as DNA binding preferences (unmethylated, hemimethylated DNA) or kinetic parameters were taken into consideration (Bheemanaik et al., 2003
).
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DISCUSSION |
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It is a common belief that RM systems evolved to protect bacteria against lethal infections by phages. This notion is supported by the fact that some phages have developed multiple mechanisms to avoid antiviral activity exerted by RM systems (Krüger & Bickle, 1983). As pointed out by some authors (Wilkins, 2002
) this defensive character of RM systems may facilitate bacteria which carry them to colonize a new habitat containing previously unencountered phages (Korona & Levin, 1993
; Korona et al., 1993
). However, it must be stressed that the barrier raised by RM systems is not absolute and the phages which evade it usually acquire protective methylation. However, this barrier can be raised higher when several RM systems are present within the bacterial cell. Therefore, it is reasonable to assume that restriction enzymes exert selective pressure directed towards decreasing the number of restriction sites present in phage DNA. Computational analysis revealed significant avoidance of short palindromic sequences not only in DNA of bacteriophages but also in bacterial chromosomes (Karlin et al., 1992
; Gelfand & Koonin, 1997
; Rocha et al., 2001
). In addition, it was shown that the sequences recognized by type II RM enzymes present in the bacterium under scrutiny were among the most avoided palindromes in its genome (Gelfand & Koonin, 1997
). In our opinion, the observed restriction site avoidance is a consequence of the aggressiveness of the ENases residing within the bacterial cell, whose restriction potential might be strengthened by particular properties of cognate MTases, a notion which we discuss below. Surprisingly, these critical conditions do not affect the viability of bacterial cells possessing a particular RM system. This might be a result of the functioning of the precise repair mechanisms whose existence was proven recently (Heitman et al., 1999
; Handa et al., 2000
).
An important question still remains open: what molecular mechanisms are responsible for maintenance and evolution of RM systems? It was proposed that these processes might be driven by phage-mediated selection (Sharp, 1986; Korona et al., 1993
; Korona & Levin, 1993
). A second mechanism might be based on involvement of RM systems in genetic recombination. The role of restriction ENases in this process can be defined as the processing of large DNA molecules entering cells into smaller recombinogenic fragments. In the case of an E. coli genome, it was observed that its mosaic character might be associated with incorporation of short DNA fragments most probably processed by restriction enzymes (McKane & Milkman, 1995
; Milkman, 1999
). Considerable genome plasticity was noted not only in E. coli but in the structure of other bacterial chromosomes sequenced to date (Casjens, 1998
). On the other hand, it was shown that the presence of a multitude of RM systems in some bacteria (e.g. Helicobacter pylori) can prevent genome rearrangements by chromosomal DNA from competing strains which are non-isogenic (Aras et al., 2002
). Lastly, RM systems may have evolved as molecular parasites, a notion which has only recently received much attention (Naito et al., 1995
).
Data obtained in our laboratory indicate that the restriction activity can be regulated by divalent metal ions. Among cations investigated by us, Mg2+ is the most abundant ion in bacterial cells, playing an important role in their metabolism. Cellular concentrations of the other divalent metal ions that we investigated are extremely low: Ca2+, 10-7 M (Gangola & Rosen, 1987); Zn2+, 10-510-12 M; and Mn2+, 10-8 M (Outten & O'Halloran, 2001
). Biochemical analysis carried out by us revealed that Mg2+ is a strong inhibitor of M.LlaCI. We found that this property is shared with two isospecific MTases: M.EcoVIII and M.HindIII. This is also true for a few other bacterial MTases, e.g. M.RsrI, M.BamHI, M.EcoRI, M.AluI, M.FokI, M.TaqI and M.EcoKDam (Kaszubska et al., 1989
; Hanish & McClelland, 1988
; Kaczorowski et al., 1999
). It is difficult to estimate whether the observed sensitivity to Mg2+ is general among type II MTases. The survey performed by Hanish & McClelland (1988)
was limited to only a few enzymes. The intracellular concentration of free Mg2+ in E. coli cells was estimated to be in the range of 14 mM (Lusk et al., 1968
; Alatossava et al., 1985
) and it is very likely that this value is similar for other bacterial species, as common mechanisms are responsible for the accumulation of Mg2+ inside bacterial cells (Moncrief & Maguire, 1999
). These mechanisms appear to be omnipresent in prokaryotes (Kehres et al., 1998
). Homologues of well-known Mg2+ transporters such as CorA or MgtA were found in L. lactis (Seegers et al., 2000
; Bolotin et al., 2001
). This means that in L. lactis cells, the activity of M.LlaCI is strongly inhibited [IC50 0·35 mM Mg2+] while the activity of the cognate restriction ENase is intact. This finding stresses the defensive character of type II RM systems. For the bacterial cell, high activity of the restriction ENase seems to be profitable. Clearly, it promotes DNA restriction over DNA methylation. This is facilitated by the fact that type II DNA MTases, unlike solitary enzymes such as M.EcoKDam, modify DNA in a non-processive manner (Surby & Reich, 1996
; Gowher & Jeltsch, 2000
; Urig et al., 2002
). Our results also explain experiments that have demonstrated the recombinogenic role of restriction ENase EcoRI in vivo (Chang & Cohen, 1977
; Stahl et al., 1983
; Silberstein et al., 1993
). It was shown previously that the activity of EcoRI MTase is strongly inhibited by Mg2+ (Hanish & McClelland, 1988
). Also, sensitivity of EcoKDam MTase to Mg2+ could explain why only 50 % of DNA molecules of phage
isolated from E. coli are methylated at dam sites (Dreiseikelmann et al., 1979
). Our interpretation is different from concepts offered by other authors. Some of them have argued that undermethylation of dam sites is a result of a low level of Dam MTase in the E. coli cells (Szyf et al., 1984
).
We also hypothesize that the fact that some type II RM systems [e.g. ScrFI (Davis et al., 1993), BcnI (Vilkaitis et al., 2002
), DpnII (de la Campa et al., 1987
), LlaDCHI (Moineau et al., 1995
), MboI (Ueno et al., 1993
)] possess two MTases with the same specificity can be linked to a possible sensitivity of one or both enzymes to the Mg2+. In such circumstances, to ensure proper methylation of genomic DNA, it would make sense to employ in this process two enzymes with the same methylating activity. Additional evidence supporting this notion is an apparent lack of substantial identity between pairs of isospecific MTases at the amino acid sequence level (M.ScrFIA/M.ScrFIB, 24 %; M.BcnIA/M.BcnIB, 11 %; M.DpnA/M.DpnM, 13 %; M.LlaDCHIA/M.LlaDCHIB, 13 %; M.MboIA/M.MboIB, 12 %). This indicates that each MTase of a particular RM system is not a product of gene duplication but was acquired/evolved independently. Recent studies on the diversity of RM systems in Helicobacter pylori provide facts endorsing such a notion (Nobusato et al., 2000a
, b
; Lin et al., 2001
). In vivo experiments with the BcnI RM system show that M.BcnIB alone is sufficient to support the growth of E. coli cells carrying the BcnI ENase gene; this could not be demonstrated for M.BcnIA (Vilkaitis et al., 2002
). In the light of our experiments, it is conceivable that this may be an effect of extreme sensitivity of M.BcnIA to Mg2+. Verification of this hypothesis in the case of the MTases listed above will need further validation by biochemical analysis using purified proteins. Another argument endorsing our hypothesis might be derived from biochemical analysis of the FokI MTase, an enzyme which possesses two catalytic centres, each responsible for methylation of an adenine residue located on a different strand of the asymmetric recognition site 5'-GGATG-3'/3'CCTAC-5' (Landry et al., 1989
). This suggests that the gene encoding M.FokI has most probably arisen by fusion of two non-homologous open reading frames. Physical separation of these two catalytic centres results in truncated derivatives which may function as independent MTases, each able to methylate only one strand within the recognition site. In our laboratory, we have shown that the two truncated derivatives possess different tolerance to Mg2+ (Kaczorowski et al., 1999
): M.FokIC (C-terminal derivative) is very sensitive to Mg2+ (IC50 0·4 mM), while the IC50 for M.FokIN (N-terminal derivative) is tenfold higher (4·3 mM).
The exceptional sensitivity of some type II MTases to Mg2+ provides evidence that this particular property of the modifying enzymes can effectively modulate the flow of genes among bacteria by strengthening the restriction activity of cognate ENases. Analysis of the sequenced genomes supports the notion that the process of acquisition of genes from the environment is fundamental for the evolution of bacteria (Arber, 2000; Ochman et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 13 June 2003;
revised 15 August 2003;
accepted 18 August 2003.
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