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Journal of Bacteriology, March 2008, p. 2039-2049, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01319-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Maintenance Forced by a Restriction-Modification System Can Be Modulated by a Region in Its Modification Enzyme Not Essential for Methyltransferase Activity

Satona Ohno,^, Naofumi Handa, Miki Watanabe-Matsui, Noriko Takahashi, and Ichizo Kobayashi^*

Department of Medical Genome Sciences, Graduate School of Frontier Science and Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Received 14 August 2007/ Accepted 2 January 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Several type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called postsegregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of postsegregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by an L80P amino acid substitution (T239C nucleotide substitution) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme, although a deletion mutant lacking the N-terminal 83 amino acids did not lose activity at either of the temperatures tested. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical postsegregational killing systems.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Postsegregational killing by addiction modules coding for a toxin and an antitoxin was first identified as a maintenance mechanism for plasmids (15, 24, 30). The proteic toxin-antitoxin systems, forming one of the three currently recognized groups of postsegregational killing (or genetic addiction) systems, are typically composed of two adjacently located genes, one for a toxin protein and the other for an antitoxin protein (13). In the absence of the antitoxin or through ectopic overexpression, the toxin can inhibit cell growth or, in some cases, kill cells by inhibiting important cellular processes such as translation. The cognate antitoxin counteracts toxin action through direct interaction. The toxin is metabolically stable whereas the cognate antitoxin is unstable because of the degradation by a specific protease. Such differential stability was shown to be critically important for their roles in postsegregational killing process. When cells fail to retain the addiction module genes, degradation of the unstable antitoxin results in release of the toxin to attack its cellular target (6, 13, 37, 68, 69).

When an addiction module establishes itself in a new host cell, it should attempt to avoid cell killing by expressing its antitoxin prior to its toxin. In its maintenance phase, the antitoxin and the toxin should have tight autoregulation of the module so that together they can suppress the toxin activity until they receive some signal to attack the host. Many classical proteic systems are autoregulated at the level of transcription by the binding of the antitoxin to the operator-promoter region of the operon (1, 10, 57, 42). In many cases, the toxin serves as a corepressor with the antitoxin repressor.

The instability of the antitoxin is also the central feature of a second type of postsegregational killing systems (14). In these systems, the antitoxins are antisense RNAs that inhibit translation of toxin-encoding mRNAs. The antisense RNAs are unstable, whereas the toxin-encoding mRNAs are very stable. The selective killing of plasmid-free cells is based on the differential decay rates of the antisense RNAs and the mRNAs (for details of the molecular mechanisms used by the antisense antitoxin to counteract expression of the toxin as well the triggering of the system, see references 14 and 16).

An addiction module found on plasmids, prophages, integrons, and other mobile elements would help their stable maintenance, and, in general, an addiction module would stabilize maintenance of any linked genomic region (5, 8, 21, 32, 58, 64). Indeed, a theoretical work demonstrated that an addiction module can defeat its competitor and spread in a population in the presence of space structure (47). The role or biological significance of the addiction modules outside mobile elements remains controversial (30, 43). They have been proposed to play a role in prokaryotic stress responses that include cell death (33), and several works have tried to test this hypothesis (for example, see reference 67).

A third type of postsegregational killing systems consists of several type II restriction-modification (RM) systems (29, 31, 48). A type II RM system is typically composed of a gene encoding a DNA endonuclease (restriction enzyme), which cleaves double-stranded DNA at a specific recognition sequence, and a gene encoding a DNA methyltransferase (modification enzyme), which methylates the same recognition sequence to protect DNA from the cleavage. According to a hypothesis known as the cellular defense hypothesis, RM systems can provide a defense against invading foreign DNAs such as phage genomes and plasmids and may have been selected and maintained during evolution due to this benefit. Postsegregational host killing allows a plasmid carrying a type II RM gene complex to resist its elimination from host cells by a competitor genetic element (48). Such RM systems with demonstrated capacity for postsegregational killing include PaeR7I, EcoRI, EcoRV, and SsoII (7, 36, 48, 49). Chromosomally placed RM gene complexes (PaeR7I and BamHI) also resist replacement by an allelic DNA (21, 58). However, the level of such killing varies among RM systems, presumably reflecting the complexity of the parasite-host interaction (and we will analyze one example in the present work). Postsegregational killing has not been proven for type I RM systems examined so far (34, 53). Host attack by EcoKI, one of these type I restriction enzymes tested, is prevented by its proteolytic cleavage (11).

Analyses of various prokaryotic genomes provide evidence that RM genes move between prokaryotic genomes (29, 31). In fact, RM genes are often found on mobile genetic elements such as plasmids, viruses, transposons, and integrons (56). Comparison of closely related bacterial genomes also suggests that RM genes themselves behave as mobile elements and cause genome rearrangements (2, 7, 50). Therefore, we proposed a hypothesis that several type II RM gene systems represent selfish genetic elements, the so-called selfish-gene hypothesis (29, 31, 48). Their attack on invading DNA is not exclusive to their selfish nature; the host genome and the RM systems have a common interest when they encounter such an invasion.

The selfish-gene concept is strongly supported by the finding that the multiplication of a chromosomal RM gene complex is dependent on the functional restriction gene (58). Furthermore, type II RM systems show some similarity to viruses in their requirements for regulation of gene expression (30, 49, 60, 62, 66) in that, when they enter a new host, they have to establish themselves without excessive killing of the host cells. After establishment, type II RM systems, like the other types of postsegregational systems, are expected to tightly regulate their gene expression. Organization of their genes is quite variable and might also reflect each of their lifestyles (30). The regulation of RM gene complexes has been studied at the transcriptional level. One regulatory mechanism employs a small protein, the so-called C protein (49, 66). The C proteins specifically bind a DNA operator sequence to regulate expression of the restriction enzyme, modification enzyme, or both (70). This group of proteins is likely to delay the expression of the restriction enzyme to prevent cell death during establishment in a new host (49). The coordinated expression of RM genes in SsoII and EcoRII systems relies on the ability of the modification enzymes to repress the transcription of their own genes (4, 28, 63). In CfrBI and LIaJI RM systems, transcription regulation depends on the methylation status of cognate recognition site(s) within the promoter region (52). Expression of R.EcoRI and M.EcoRI is negatively regulated by intragenic reverse promoters (38, 39). It has not been examined how these regulatory mechanisms contribute to their postsegregational killing.

In a selfish type II RM system, the restriction enzyme plays the role of a toxin while the modification enzyme plays the role of an antitoxin. Rather than interacting directly with the toxin or inhibiting its expression, the antitoxin protects the target of the toxin, consisting of numerous copies of the recognition sequence along the chromosome. After gene loss, dilution of the antitoxin leads to the exposure of target sites along the newly replicated chromosomes to lethal attack by the remaining restriction enzyme (19, 48). This dilution is hypothesized to be the main mechanism of postsegregational killing by type II RM systems. This mechanism is in apparent contrast to mechanisms of the two other types of postsegregational killing systems, in which metabolic instability is the essential feature of the antitoxin. However, if the antitoxin methyltransferase is metabolically unstable, the host killing by the restriction enzyme would be accelerated. A recent test of this idea with the EcoRI RM system, however, revealed that the modification enzyme is as metabolically stable as the restriction enzyme in vivo (26). The other RM systems have not yet been examined with regard to this point.

We observed a related phenomenon during study of competition between RM systems. When two RM systems within one host recognize the same sequence, only one of them can force its maintenance through postsegregational killing. Loss of one RM system would not lead to chromosomal attack by its restriction enzyme because the modification enzyme of the other RM system would protect the recognition sequence. This prediction was verified by experiments in the absence of any invading elements (36). Such competition between RM systems for a recognition sequence may have accelerated the evolution of specificity and diversity in their sequence recognition. In a similar way, postsegregational killing by an RM system would be prevented by a solitary methyltransferase that recognizes the same sequence. This was demonstrated for the EcoRII RM system, a type II system with a frequently occurring recognition sequence of 5' CCWGG (where W is A or T), and Dcm, a solitary methyltransferase found in the chromosome of Escherichia coli and related bacteria (65). Dcm was hypothesized to have evolved to serve as a molecular vaccine against parasitism by EcoRII isoschizomers. In this and a related work (7), we noticed a unique feature of host killing by the EcoRII RM gene complex, which had been cloned from plasmid N3 (23). When a bacterial strain carrying a plasmid with thermosensitive replication machinery and an addiction module, such as a type II RM gene complex, is shifted to a nonpermissive temperature, there is a halt in the increase of viable and plasmid-carrying cell counts because a cell remains viable as long as it carries the plasmid. In such experiments, however, the EcoRII gene complex conferred an immediate and severe decrease in cell viability instead of growth arrest (65), a phenomenon called postdisturbance cell killing.

In the present study, we found that this phenomenon involved a mutation in the EcoRII modification enzyme which affects its stability. Our analysis demonstrated that the selfish maintenance of the EcoRII system can be modulated by a sequence in a dispensable region of its modification enzyme. This finding revealed a point of similarity between RM systems and the two other types of postsegregational killing systems.

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Bacterial strains, plasmids, phage, and media. The E. coli K-12 strains and plasmids used are listed in Table 1. BNH2586, a lysogen of lambda DE3 (Novagen), was made by a procedure provided by the supplier. Bacteria were grown in Luria-Bertani (LB) medium (46). When required, antibiotics were added at the following concentrations: ampicillin (Amp), 50 µg/ml; tetracycline (Tet), 15 µg/ml; chloramphenicol (Cml), 25 µg/ml; oxacillin, 100 µg/ml.

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TABLE 1. Bacterial strains and plasmids

Plasmids were constructed as follows. The ecoRIIRM genes were amplified from plasmid N3 using the primers RII1 and RII2 (65), which contain an XmaI site. The amplified fragment was digested with XmaI and inserted into the corresponding site of pUC19 or pHSG415 and designated pOS28 or pOS30, respectively. The larger fragments of a HindIII digestion were self-ligated to make a restriction-defective version [R(–)] pOS34 and pOS39, respectively. pOS22 and pOS48 were constructed from pNY30 by site-directed mutagenesis using the following primers: IKOS31 (5'-GACGTTAGCCGAAGAGGAACTTCTACGTAAAATGCTTCCGGAAGCGC-3') and IKOS32 (5'-GCGCTTCCGGAAGCATTTTACGTAGAAGTTCCTCTTCGGCTAACGTC-3') for pOS22; IKOS50 (5'-CTGACGTTAGCCGAAGAGGAACTTCTACGAAAAATGCTTCCGGAAGCGC-3') and IKOS50-r (5'-GCGCTTCCGGAAGCATTTTTCGTAGAAGTTCCTCTTCGGCTAACGTCAG-3') for pOS48. The italic letters indicate SnaBI sites introduced as markers, while the underlined letters indicate the mutant base pairs.

The EcoRII methyltransferase gene was cloned into pFLAG2 (Sigma-Aldrich Japan, Tokyo, Japan) vector as follows. The wild-type ecoRIIM gene was amplified from pNY30 using the primers IKOS46 (5'-GGGAATTCATGTCTGAATTTGAATTAC-3'; the italic letters indicate an introduced EcoRI site) and IKOS47 (5'-GGAGATCTTCAGATTCGTTCAACCTT-3'; the italic letters indicate an introduced BglII site). The amplified fragment was digested with EcoRI and BglII and ligated with pFLAG2 (pOS41). pOS43 was constructed in the same way from pOS28. The N-terminal deletion version (pOS45) was constructed in the same way with IKOS47 and IKOS48 (5'-GGGAATTCCTTCCGGAAGCG-3'; italics indicate restriction site) primers.

pOS51 and pOS54 were constructed from pNY41 and pNY31, respectively, by site-directed mutagenesis using the primers IKOS50 and IKOS50-r (see above). pOS57 was constructed from pNY31 by site-directed mutagenesis using the following primers: IKOS49 (5'-GCTTGATGAGCAAGGGGGGGACTGTAAGGAAGTAAATATTTGGGTATG-3') and IKOS49-r (5'-CATACCCAAATATTTACTTCCTTACAGTCCCCCCCTTGCTCATCAAGC-3'). The underlined letters indicate the mutant base pair.

pNH876 was constructed by the following method. PCR products using the primers EcoRII N1 (5'-GGCCATGGGTATGTCTGAATTTGAATTACTGGCGCAG) and EcoRII C (5'-GCGAGCTCGATTCGTTCAACCTTGCACGAATCGGCA) with pOS28 DNA as the template were connected between the NcoI and SacI sites of an expression vector, pET52b (Merck Ltd., Japan). pNH881 was constructed in a similar way but with pOS48 DNA as a template. The entire M.EcoRII gene was confirmed by sequencing, and methylation activity of both constructs at 30°C was confirmed in vitro by using their crude extract.

All the transformation experiments to construct plasmids were performed by the Rubidium method (18). All the plasmids were prepared with a GenElute Plasmid Miniprep kit (Sigma).

During these plasmid constructions, EcoRII restriction and modification activities were confirmed using lambda phage infection. EcoRII restriction activity in vivo was evaluated from the efficiency of plaque formation of unmodified and dcm-modified lambda vir phage (laboratory collection) on the E. coli strain. EcoRII modification activity in vivo was assessed by growing lambda vir phage on an E. coli strain carrying a plasmid to be tested and then determining the efficiency of plaque formation of the resulting phage on an E. coli strain carrying pNY30.

Postsegregational killing by temperature shift. A bacterial strain carrying an ecoRIIRM plasmid with thermosensitive replication machinery was grown with aeration at 30°C in LB liquid medium with selective antibiotics until the optical density at 660 nm (OD₆₆₀) of the culture reached 0.5. Then, the cells were collected by centrifugation, suspended in liquid LB medium without antibiotics, and grown at 42°C with aeration. The cells were diluted whenever the OD₆₆₀ of the culture reached 0.6. The total cell number was counted under a microscope. The number of viable cells was defined as the number of CFU on LB agar plate grown without antibiotics at 30°C. The number of cells carrying the ecoRIIRM plasmid was defined as the number of CFU on LB agar plate with antibiotics grown at 30°C.

Measurement of plasmid maintenance. All the cultivation procedures were carried out at 30°C with aeration. A fresh single colony grown on LB agar with the selective antibiotics (Cml) was suspended in 5 ml of liquid LB medium with antibiotics and grown overnight. The saturated culture was diluted and spread onto LB agar with or without the antibiotics for overnight incubation and colony counting. The overnight liquid culture was diluted to 10^–5 in LB broth without the antibiotic for overnight growth. The cycle of sampling/colony counting and dilution/overnight growth was repeated every day. The generation number was calculated from the viable cell count. The procedure has been detailed elsewhere previously (65).

Measurement of thermosensitivity of bacterial growth. A bacterial strain carrying one of the ecoRIIRM plasmids was grown at 30°C with aeration in liquid LB medium with selective antibiotics until the OD₆₆₀ of the culture reached 0.5. The culture was diluted and spread on an LB agar plate with and without antibiotics. The plates were immediately placed at 30°C or at 42°C for overnight incubation and colony counting.

In vitro methylation. Methylation activity of EcoRII methyltransferase was monitored using crude cell lysate from the dcm cells (BNH670) harboring either pOS41 (M.EcoRII wild type), pOS43 (M.EcoRII L80P), pOS45 (M. EcoRII with a mutation that deletes N-terminal 83 amino acid residues [M. EcoRII N83]), or pFLAG2 empty vector. The cell lysates were prepared from cells growing at logarithmic phase with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 2 h at 30°C. Cell culture (5 ml) was centrifuged and suspended in a buffer (50 mM Tris-HCl, pH 8.0, 10% sucrose) and sonicated on ice. The supernatant after centrifugation was incubated with pUC19 DNA substrate which was isolated from the dcm strain and with S-[methyl-¹⁴C]adenosylmethionine (56 mCi/mmol; Amersham Biosciences UK Limited, Buckinghamshire, United Kingdom) in a reaction buffer containing 40 mM Tris-HCl (pH 8.0), 1 mM EDTA, 5 mM dithiothreitol for 2 h at 30°C or 37°C. After the reaction, samples were subjected to 1% agarose gel electrophoresis. The ¹⁴C-methylated pUC19 DNA was visualized on a BAS-MS2025 imaging plate (Fuji Photo Film Co., Ltd., Tokyo, Japan) and analyzed using an FLA-5100 imaging system (Fuji Photo Film Co., Ltd.). As a positive control, M.EcoRI (New England BioLabs Inc., Ipswich, MA) was examined.

Detection of M.EcoRII-mediated plasmid methylation in vivo. BNH670 (dcm) carrying pOS41 (M.EcoRII wild type), pOS43 (M.EcoRII L80P), pOS45 (M.EcoRII N83), or pFLAG2 empty vector was grown at 30°C or 37°C with selective antibiotics (Amp and Tet) and 0.4% glucose until the OD₆₆₀ of the culture reached approximately 0.4. Two hours after the addition of 1 mM IPTG, the plasmid DNA was isolated. It was then treated with EcoRII restriction endonuclease (Nippon Gene) and subjected to agarose gel electrophoresis.

Measurement of M.EcoRII-mediated plasmid methylation under inhibited protein synthesis conditions. Strains carrying pOS41 or pOS43 were grown with aeration at 30°C in liquid LB medium with the selective antibiotic (Amp) and 1 mM IPTG until the OD₆₆₀ of the culture reached 0.5. Then Cml was added (final concentration, 25 µg/ml) to inhibit protein synthesis (9), and the incubation temperature was shifted to 42°C. The control cultures were grown at the same temperature (30°C or 42°C) before and after the addition of Cml. The plasmid DNA recovered from the culture was treated with EcoRII restriction enzyme. The products were analyzed by agarose gel electrophoresis.

Stability of M.EcoRII protein under inhibited protein synthesis conditions. Lambda DE3 lysogen of the dcm strain was constructed to express the M. EcoRII gene. The M.EcoRII expression plasmid, pNH876 (wild type), or pNH881 (L80P) carrying cells was grown with aeration at 30°C in liquid LB medium with the selective antibiotic (Amp) and glucose (0.4% final) until the OD₆₆₀ of the culture reached 0.3. Then, IPTG (0.5 mM final) was added to induce the gene under the T7 promoter. Thirty minutes after the addition of IPTG, Cml (340 µg/ml final concentration) was added to inhibit protein synthesis, and the culture was shifted to 42°C. Samples (0.1 ml) were collected every 15 min after the addition of Cml and boiled after centrifugation. Then a total protein sample, corresponding to the same OD units of the culture, was loaded onto each lane of a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. Total proteins were stained with Coomassie brilliant blue. The M.EcoRII bands were analyzed by using ImageGauge software (Fuji Photo Film Co., Ltd.), and the intensity of the M.EcoRII band was normalized to that of an internal constitutive band.

Sequence comparison with homologs. The N-terminal 120 amino acids of M.EcoRII were used as a query for BLASTP search in the nr database. The sequences were aligned with ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and visualized with Boxshade (http://www.ch.embnet.org/software/BOX_form.html).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
L80P mutation in the modification enzyme of the EcoRII gene complex confers thermosensitivity of cell growth. When we first characterized the EcoRII RM gene complex (65), it showed a drastic level of cell killing compared with the other RM systems examined (19, 20, 36, 48). The cell killing was observed by forcing plasmid loss by shifting to a nonpermissive temperature for the replicon with the RM gene complex. However, significant cell death still occurred at this higher temperature even when the EcoRII RM gene complex was connected to a thermoresistant plasmid such as pUC19 (Table 1, pNY30). These and the following experiments were carried out in E. coli strains mutated for the dcm locus (dcm-6 or dcm) (see Materials and methods) because Dcm protects the chromosome from EcoRII restriction (65) (see also Discussion).

When we sequenced this EcoRII RM gene complex for comparison with the published sequences (AJ224995 [55] for the restriction gene and X05050 [61] for the modification gene), we found that the formerly cloned modification gene carries a T239C mutation, which causes the L80P amino acid substitution (Fig. 1). The restriction gene was shown to carry a synonymous mutation, T402C, which does not lead to significant change in codon usage (http://www.kazusa.or.jp/codon/). These two mutations might have been introduced during cloning by PCR. On the other hand, the newly cloned EcoRII RM gene complex from plasmid N3 did not have any sequence alterations from the published ones. Therefore, hereafter we designate the newly cloned methyltransferase the wild type, and the former one is the L80P mutant.

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FIG. 1. EcoRII modification enzyme and the L80P (T239C) mutation. Conserved motifs of C5 DNA methyltransferases are indicated by Roman numerals (35, 54, 59). The nucleotide and amino acid substitutions in the mutant are shown below. a.a., amino acids.

Table 2 shows colony formation of E. coli strain BNH670 (dcm) or GM31 (dcm-6) harboring a plasmid with various versions of the EcoRII RM gene complex. The cells carrying pNY30, which has an EcoRII RM gene complex with the L80P mutation in the methylase gene (65), showed a drastic decrease in viability at 42°C, while the cells possessing its r^– version (pNY31) did not. We observed the same tendency in the absence of selective antibiotics with pUC derivatives (Table 2) and pACYC184 derivatives (data not shown). Thus, this thermosensitivity phenotype comes from the mutant form of the RM gene complex. To determine whether the cause of cell death at 42°C is the T239C (L80P) mutation in ecoRIIM or the T402C mutation in ecoRIIR, we replaced each by the wild-type sequence through site-directed mutagenesis (see Materials and Methods). As shown in Table 2, the E. coli strain GM31 carrying pOS22 [pUC19 with ecoRIIR(T402C) M⁺] is able to grow at 42°C. However, the E. coli strain GM31, carrying pOS48 [pUC19 with ecoRIIR⁺M(T239C)], failed to grow at this temperature (Table 2). E. coli strain GM31 (dcm-6) or BNH670 (dcm) carrying a plasmid (pOS28) containing the wild-type EcoRII RM complex is able to grow at 42°C. These results indicated that the T239C (L80P) mutation in ecoRIIM is responsible for the cell death at 42°C.

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TABLE 2. Thermosensitivity of bacterial growth conferred by T239C (L80P) mutant EcoRII gene complex

L80P mutation enhances the plasmid maintenance capacity of the EcoRII gene complex. Because our previous study (65) used the mutant form of the EcoRII RM gene complex as described above, we examined the effect of loss of the wild-type EcoRII RM gene complex during cell growth using a thermosensitive replication plasmid. The inhibition of plasmid replication following the shift of the wild-type EcoRII r⁺ and r^– cultures to the nonpermissive temperature arrested the increasing plasmid-carrying cell count (Fig. 2A). The count of the viable cells paralleled that of the plasmid-carrying cells in the r⁺ strain. The increase in the total cell number as estimated under a microscope eventually stopped in the r⁺ culture (Fig. 2A). We did not observe a drastic loss of cell viability as we did in the mutant form of the EcoRII RM gene complex (65). Following loss of the RM plasmid, cell morphology was observed under a microscope after DAPI (4',6'-diamidino-2-phenylindole) staining of the nuclei (Fig. 2B). Filamentous cells appeared 6 to 8 h after the temperature shift in an r⁺-dependent manner. A few of the r⁺ cells appeared anucleated. These features are common to postsegregational host killing after loss of other type II RM gene complexes (e.g., EcoRI and PaeR7I) (19, 20, 36, 48). These results suggested that the EcoRII RM gene complex mediates postsegregational killing similarly to the previously examined type II RM gene complexes.

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FIG. 2. Postsegregational killing by wild-type EcoRII RM gene complex. (A) Growth inhibition following loss of the EcoRII RM gene complex. E. coli strain BNH670 (dcm) carrying pOS30 (left; pHSG415 which has a thermosensitive replication unit with wild-type ecoRIIRM, Cmlr) or pOS39 (right; its r– version) was grown at 30°C in LB broth with antibiotic (Cml) selection until the OD660 reached 0.5. At time zero, the culture was shifted to 42°C and freed of the antibiotic selection. The total cells were counted under a microscope. The viable cells were counted on LB agar without the antibiotic at 30°C. The plasmid-carrying cells were counted on LB agar with the antibiotic at 30°C. Each point represents an average of two measurements. (B) Cell shape. The cells harvested at the indicated times after the temperature shift were fixed for staining with DAPI for visualization of nucleoids.

Insertion of a type II RM gene complex into a plasmid leads to its stable maintenance in the host cell because of the postsegregational killing by the RM system (7, 36, 48, 49). When we compared the plasmid stabilization capacity of two variants of the EcoRII RM gene complex, we found that a plasmid carrying the L80P mutant form of the ecoRIIRM gene was more stable than an isogenic plasmid carrying the wild-type ecoRIIRM (Fig. 3). Plasmid stabilization by ecoRIIR(T402C) ecoRIIM(L80P) was indistinguishable from that by ecoRIIR⁺ ecoRIIM(L80P) (data not shown). The stronger plasmid maintenance by the mutant EcoRII RM gene complex was also confirmed with another plasmid vector, pHSG415 (data not shown).

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FIG. 3. L80P mutant shows stronger capacity to maintain a plasmid. E. coli strain BNH670 (dcm) carrying pOS51 (filled triangle; pACYC184 carrying wild-type ecoRIIR(–)M, Cmlr), pOS54 (filled circle; pACYC184 carrying ecoRIIRM(L80P), Cmlr), or pOS57 (open circle; pACYC184 carrying wild-type ecoRIIRM, Cmlr) was grown at 30°C in LB broth without antibiotic selection with aeration. After overnight incubation, the culture was examined for plasmid-carrying cell counts (on LB agar with Cml at 30°C) and viable cell counts (on LB agar without Cml at 30°C). The culture was diluted 10–5-fold for overnight incubation. This cycle was repeated. The ratio of the plasmid-carrying cell count to the viable cell count was plotted against the generation number, which was calculated from the viable cell counts.

Thermosensitive methylation activity of the L80P mutant and thermoresistant methylation activity of the N-terminal 83-amino-acid deletion mutant. We then asked how the L80P mutation in the EcoRII RM gene pair makes its carrier cells thermosensitive for growth. One obvious possibility is that the mutant enzyme is thermosensitive, although the mutation lies outside of the domain with DNA methyltransferase motifs (Fig. 1).

Crude extracts from cells expressing the wild-type or the mutant version of the EcoRII methyltransferase were examined to evaluate their ability to transfer a methyl group to the target sequence on a plasmid (pUC19). As shown in Fig. 4, the extract from cells expressing the wild-type EcoRII showed the activity both at 30°C and 37°C. However, as expected, the extract from cells expressing the L80P mutant form showed activity at 30°C but not at 37°C. When we tried a mutant methyltransferase lacking the N-terminal 83 amino acids, it showed activity at both temperatures. This was unexpected because the deletion removes L80 (Fig. 1). This deletion was reported to decrease but not to abolish the methylation activity of M.EcoRII in vitro (12).

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FIG. 4. In vitro thermosensitivity of DNA methyltransferase activity. Crude cell lysate was prepared from a dcm strain (BNH670) harboring pOS41 (wild-type M.EcoRII), pOS43 (L80P mutant of M.EcoRII), pOS45 (N83 mutant of M.EcoRII), or pFLAG2 (empty vector) grown at 30°C. Transfer of the 14C-labeled methyl group from S-[methyl-14C]adenosylmethionine to pUC19 DNA (carrying five EcoRII sites) was assessed at the indicated temperatures. M.EcoRI was also examined as a positive control. There is one EcoRI site on pUC19.

In order to assess methyltransferase activity in vivo, we inserted the wild-type and the mutant versions of the modification gene into an IPTG-inducible vector and induced their expression at 30°C and 37°C. These plasmid DNA molecules were recovered and digested with EcoRII restriction endonuclease to evaluate the level of their methylation. The L80P mutant form of M.EcoRII protected the sites at 30°C but failed to do so at 37°C (Fig. 5), although the incompleteness of the digestion suggested some protection. The plasmid DNA from cells harboring the M.EcoRII N83 gene was methylated at both 30°C and 37°C (Fig. 5). The results so far indicate that L80P mutation confers thermosensitivity in methylation activity even though it is not essential to the methylation activity. There are two possible explanations for this, poor expression and ad hoc instability, as we will see in the next sections.

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FIG. 5. In vivo thermosensitivity of DNA methyltransferase activity in the L80P mutant. E. coli strain BNH670 (dcm) carrying a plasmid with ecoRIIM (wild-type; pOS41), ecoRIIM(L80P)(pOS43), or ecoRIIM (N83; pOS45) was grown at 30°C or 37°C. After the modification enzyme was induced with IPTG for 2 h, the plasmid DNA was prepared, treated with EcoRII restriction endonuclease in vitro, and electrophoresed through 0.7% agarose.

Decrease of the methylation activity of the L80P mutant after inhibition of protein synthesis and shift to a high temperature. Because the N terminus is implicated in the regulation of transcription of the EcoRII gene complex (62, 63), the above thermosensitivity could be explained by poor expression of the mutant enzyme. In order to examine this possibility, we followed the methylation activity after a temperature shift from 30°C to 42°C in the absence of de novo protein synthesis. The E. coli strains were grown with IPTG at 30°C, Cml was then added, and the culture was shifted to 42°C. Although Cml inhibits de novo protein synthesis and cell growth, it does not block replication of ColE1-origin plasmids (9). Figure 6 shows that the L80P mutant over time came to fail to methylate the plasmid DNA. The control cultures grown at the same temperature (30°C or 42°C) before and after the addition of Cml behaved as expected (data not shown). These results suggested that the thermosensitivity phenotype of the methyltransferase does not necessarily involve protein synthesis.

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FIG. 6. Loss of methyltransferase activity in the L80P mutant in vivo under the condition of protein synthesis inhibition. E. coli strain BNH670 (dcm) carrying a ColE1-based plasmid with ecoRIIM (wild-type; pOS41) or ecoRIIM(L80P)(pOS43) was grown in LB broth with selective antibiotic and IPTG at 30°C until the OD660 of the culture reached 0.5. Cml was added to inhibit protein synthesis and cell growth, and then the temperature of the culture was shifted to 42°C. Plasmid DNA prepared at the indicated time intervals was treated with EcoRII restriction endonuclease and electrophoresed through 0.7% agarose. U, not treated with EcoRII restriction enzyme in vitro; C, treated with EcoRII restriction enzyme in vitro; cc, closed circle; oc, open circle; M, DNA linear ladder marker (BRL-Invitrogen Japan, Tokyo, Japan).

Instability of the L80P mutant protein at a high temperature. In the classical proteic postsegregational killing systems, the instability of the anti-killer protein is central (see the introduction). Its specific region defines susceptibility to protease and determines its metabolic instability. It would be interesting if the L80 region outside of the methyltransferase domain plays an analogous role in the EcoRII system by modulating its stability. To address the question of whether the thermosensitivity of the L80P methyltransferase is due to enhanced thermoinstability, we monitored the M.EcoRII enzyme following inhibition of protein synthesis. Thirty minutes after induction of the methyltransferase from an expression vector by the addition of 0.5 mM IPTG, Cml was added to inhibit protein synthesis. At this time (time zero), the culture was shifted to 42°C. As shown in Fig. 7, both wild-type and L80P M.EcoRII proteins are well expressed after IPTG induction. After the addition of Cml, the amount of expressed M.EcoRII protein gradually decreased over time. Compared with the wild-type protein, the L80P mutant protein was significantly unstable (Fig. 7A and B). These results suggest that the thermosensitivity of the L80P activity may be due to instability of the protein. The rapid decrease in the amount of the mutant protein after the temperature shift coincides well with the rapid decrease of viability of cells carrying both EcoRII and the mutant M.EcoRII (65).

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FIG. 7. Stability of M.EcoRII protein in vivo under the condition of protein synthesis inhibition. (A) Total protein profiles after inhibition of protein synthesis. E. coli strain BNH2586 [dcm (DE3)] carrying a plasmid with ecoRIIM (wild-type)(pNH876) or ecoRIIM(L80P) (pNH881) was grown until early log phase at 30°C. IPTG was added to induce M.EcoRII, and the cultivation was continued for 30 min. Then, Cml was added to inhibit protein synthesis, and the temperature of the culture was shifted to 42°C. Whole protein samples were taken at the indicated time intervals and were run through a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. Total proteins were visualized by Coomassie brilliant blue staining. (B) Quantification of M.EcoRII protein. Intensity of the M.EcoRII band, as normalized by that of a constitutive band (indicated by an arrow in panel A) in the same lane, is plotted.

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We found that the previously characterized EcoRII RM gene complex (65) is a mutant carrying a T239C (L80P) substitution in the modification gene and a synonymous T402C substitution in the restriction gene (Fig. 1). The substitutions are likely to have been induced during PCR with ExTaq polymerase (Takara) for cloning. The L80P (T239C) mutation in the modification enzyme was found to be responsible for the thermosensitivity of the host growth (Table 2). The mutant methyltransferase was indeed found to be thermosensitive in vitro and in vivo (Fig. 4 and 5).

The phenotype observed after loss of the wild-type EcoRII RM gene complex on a thermosensitive vector by a temperature shift (Fig. 2) is similar to that observed with other type II RM gene complexes (19, 20, 36, 48). The drastic loss of viability observed earlier with the mutant EcoRII RM gene complex by the same protocol (7, 65) is likely to have been caused by the thermosensitivity conferred by the mutant modification enzyme.

The cell death after loss of the wild-type EcoRII RM gene complex was observed in a dcm background (Fig. 2) but was not significant in a dcm⁺ background (Table 3) or in a dcm-6 background (data not shown). This implies that postsegregational killing is not always obvious with a given type II RM system. Why was the cell killing following EcoRII RM gene loss not observed with the dcm-6 mutation (44), which abolishes the methyltransferase activity? Expecting that dcm-6 might produce a defective protein that is able to protect the CCWGG sites along the genome, we determined its sequence. It carries two mutations (underlined): TGG for the 45th tryptophan to TGA (W45stop) and GAA for the 407th glutamic acid to AAA (E407K). The E407 residue is likely to be located at the methyltransferase catalytic center if the structure resembles the hypothetical structure of homologous M.EcoRII (59). The mutation point is away from the DNA binding site so that the Dcm-6 protein might be able to bind the target sequence CCWGG if it is produced. We do not know, however, how efficient readthrough at UGA is in the GM31 background. We cannot exclude the possibility that the above difference between the dcm-6 background and dcm background derives from the state of vsr or other genes deleted together with dcm in the latter or other silent mutations.

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TABLE 3. Absence of postsegregational killing by wild-type EcoRII gene complex with wild-type E. colia

Why is the mutant modification enzyme thermosensitive for methylation? The L80P mutation lies outside of the motifs essential for the methylation reaction (Fig. 1). The N-terminal 83 amino acids are not essential for the catalytic function of M.EcoRII although the mutant shows a somewhat decreased activity in vitro (12, 63). Under our assay conditions, the mutant in which the N-terminal 83 amino acids (covering L80) are deleted showed methylation activity at the higher temperature tested while L80P failed to do so (Fig. 4 and 5). These observations defy several straightforward explanations, such as loss of some function of the N terminus necessary for methylation. Rather, the L80P mutation appears inhibitory to the methylase domains outside of the N terminus at the nonpermissive temperature. However, we failed to combine the N-terminal deletion form of the methyltransferase gene with a cognate restriction gene (N. Handa and I. Kobayashi, unpublished results) in an attempt to evaluate this possibility. The weak methylation activity (12, 63) may not be good enough to protect the target sequence along the E. coli genome against a cognate restriction endonuclease. The L80 residue is predicted to be in a loop region (59). The conservation of L80 among close homologs (Fig. 8) suggests some role. It would be interesting if this region is involved in modulation of the strength of the methylase activity and, therefore, selfishness of RM systems in these examples. The RM systems move horizontally and encounter various potential hosts. If the host attack by an RM system is too strong, it would be selected against. If it is too weak, it would be easily lost. The region could play the role of adaptation to a new host.

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FIG. 8. Comparison of N-terminal region in M.EcoRII homologs. L80 in M.EcoRII is indicated by an arrow. asterisk, identical in all the sequences; period, similar in all the sequences; black shading, identical; gray shading, similar. Homologs are identified as follows (name, annotation [strain name], and accession number): CKO_03470, hypothetical protein CKO_03470 [Citrobacter koseri ATCC BAA-895], YP_001454986; Yersinia, site-specific DNA methylase [Yersinia bercovieri ATCC 43970], ZP_00823657; Serratia, DNA-cytosine methyltransferase [Serratia proteamaculans 568], YP_001476807; Photorhabdus, DNA-cytosine methyltransferase [Photorhabdus luminescens subsp. laumondii TTO1], NP_927695; Pseudomonas, DNA-cytosine methyltransferase [Pseudomonas putida GB-1], ZP_01716858; DCM, DNA-cytosine methyltransferase (M.EcoDcm), M32307; CKO_00981, hypothetical protein CKO_00981 [Citrobacter koseri ATCC BAA-895], YP_001452564.

The thermosensitivity of the L80P mutant enzyme might come from its inefficient expression (transcription or translation) or its instability. The N terminus of M.EcoRII is reported to regulate transcription of the M.EcoRII gene by binding to the promoter (62, 63). The predicted helix-turn-helix motif 20 amino acid residues upstream of L80P is likely to be responsible for this regulation (28). Therefore, at the high temperatures, the L80P mutation could make M.EcoRII a superrepressor with stronger binding to the regulatory cis element, leading to more severe autorepression than the wild-type. This would lead to decreased methylation activity.

However, this is unlikely to be the sole cause of thermosensitivity because the thermosensitivity was seen in vitro (Fig. 4) and under the condition of inhibition of de novo protein synthesis (Fig. 6). Against the superrepressor hypothesis is the finding that the mutant M.EcoRII (L80P) was not dominant over the wild-type with respect to the thermosensitivity (N. Handa and I. Kobayashi, unpublished observation). The possibility that L80P mutation interferes with translation of the M.EcoRII protein is also unlikely for the above reasons.

Our results (Fig. 7) indicate that the L80P mutation significantly decreases the stability of M.EcoRII and causes its faster degradation and disappearance. This instability is likely to be the cause of the thermosensitivity. Although the effect was clear at the higher temperature (42°C), this elevated instability is likely to contribute to the elevated capacity of plasmid maintenance observed for prolonged growth at the lower temperature (30°C) (Fig. 3). The mutant M.EcoRII antitoxin will decrease faster than the wild-type antitoxin and allow earlier exposure of chromosomal recognition sites to lethal attack by the restriction endonuclease molecules not yet diluted.

We also noted that even the wild-type M.EcoRII protein decreased faster than the bulk of cellular proteins after inhibition of protein synthesis (Fig. 7). This is in contrast to the stability of R.EcoRI and M.EcoRI after inhibition of protein synthesis (26) (Although a similar thermosensitive methyltransferase in the EcoRI RM system has been identified [25, 51], the mutation point has not been determined yet to our knowledge.). Although one might expect that exposure of unprotected restriction sites on the newly replicated chromosomes is sufficient for lethal chromosome cleavage, the lethality would be modified by such instability of the methyltransferase and many factors. These may include the number of restriction sites along the host genome, the amounts of the restriction and modification proteins resulting from their expression, and availability of the recombination repair (19).

In the case of the classical proteic toxin-antitoxin systems, the antitoxin is unstable because of degradation by a specific protease. This instability is necessary for the stabilization effect through postsegregational killing because the stabilization effect is abolished in bacterial mutants defective in the protease (17, 37, 68). We might imagine that the L80 region could represent a determinant of susceptibility to protease(s) as do regions in the antitoxins CcdA and MazE (27, 41). Is instability of the L80P thermosensitive mutant a consequence of becoming a better target of proteases in vivo? To answer this question, we examined the effects of mutations affecting proteases (lon, clpP/X/A, and hslVU). Under the conditions examined, we identified no single protease mutation that restored the thermosensitivity of growth to cells harboring the plasmid with the EcoRII gene complex with the L80P mutation (S. Ohno, N. Handa, and I. Kobayashi, unpublished observation). However, it is still possible that the mutant became a target for more than one protease.

Although further experiments are necessary to define molecular mechanisms, our present results uncover further similarity between the RM systems and the classical proteic postsegregational killing systems and will lead to a more unified view of death-related programs in the prokaryotes.

 
We thank the investigators listed in Table 1 for the gifts of bacterial strains and plasmids. We thank Ken Ishikawa for valuable discussion.

This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) (grants 1770001 and 19790316), the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (17049008 and 19037006), and the Naito foundation to N.H. and by grants from the National Project on Protein Structural and Functional Analyses (Protein 3000), the 21st century COE project Genome language (MEXT in Japan), and Grants-in-Aid for Scientific Research (15370099 and 17310113 from JSPS) to I.K. M.W. is a postdoctoral fellow of the 21st century COE project, Genome language.

 
* Corresponding author. Mailing address: Department of Medical Genome Sciences, Graduate School of Frontier Science and Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81 3 5449 5326. Fax: 81 3 5449 5422. E-mail: ikobaya{at}ims.u-tokyo.ac.jp

Published ahead of print on 11 January 2008.

S.O. and N.H. contributed equally to this work.

Present address: Laboratory of Gene Expression and Regulation, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Present address: Department of Biochemistry, Graduate School of Medicine, Tohoku University, Sendai, Japan.

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Journal of Bacteriology, March 2008, p. 2039-2049, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01319-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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