Maintenance forced by a restriction-modification system can be modulated by a region in its modification enzyme not essential for methyltransferase activity

Abstract

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.

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.

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 (Δdcm) carrying pOS30 (left; pHSG415 which has a thermosensitive replication unit with wild-type ecoRIIRM, Cml^r) 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.

FIG. 3.

L80P mutant shows stronger capacity to maintain a plasmid. E. coli strain BNH670 (Δdcm) carrying pOS51 (filled triangle; pACYC184 carrying wild-type ecoRIIR(−)M, Cml^r), pOS54 (filled circle; pACYC184 carrying ecoRIIRM(L80P), Cml^r), or pOS57 (open circle; pACYC184 carrying wild-type ecoRIIRM, Cml^r) 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.

FIG. 4.

In vitro thermosensitivity of DNA methyltransferase activity. Crude cell lysate was prepared from a Δdcm strain (BNH670) harboring pOS41 (wild-type M.EcoRII), pOS43 (L80P mutant of M.EcoRII), pOS45 (ΔN83 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.

FIG. 5.

In vivo thermosensitivity of DNA methyltransferase activity in the L80P mutant. E. coli strain BNH670 (Δdcm) carrying a plasmid with ecoRIIM (wild-type; pOS41), ecoRIIM(L80P)(pOS43), or ecoRIIM (ΔN83; 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.

FIG. 6.

Loss of methyltransferase activity in the L80P mutant in vivo under the condition of protein synthesis inhibition. E. coli strain BNH670 (Δdcm) 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).

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 [Δdcm (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.

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.