Stability of EcoRI restriction-modification enzymes in vivo differentiates the EcoRI restriction-modification system from other postsegregational cell killing systems

Abstract

Certain type II restriction modification gene systems can kill host cells when these gene systems are eliminated from the host cells. Such ability to cause postsegregational killing of host cells is the feature of bacterial addiction modules, each of which consists of toxin and antitoxin genes. With these addiction modules, the differential stability of toxin and antitoxin molecules in cells plays an essential role in the execution of postsegregational killing. We here examined in vivo stability of the EcoRI restriction enzyme (toxin) and modification enzyme (antitoxin), the gene system of which has previously been shown to cause postsegregational host killing in Escherichia coli. Using two different methods, namely, quantitative Western blot analysis and pulse-chase immunoprecipitation analysis, we demonstrated that both the EcoRI restriction enzyme and modification enzyme are as stable as bulk cellular proteins and that there is no marked difference in their stability. The numbers of EcoRI restriction and modification enzyme molecules present in a host cell during the steady-state growth were estimated. We monitored changes in cellular levels of the EcoRI restriction and modification enzymes during the postsegregational killing. Results from these analyses together suggest that the EcoRI gene system does not rely on differential stability between the toxin and the antitoxin molecules for execution of postsegregational cell killing. Our results provide insights into the mechanism of postsegregational killing by restriction-modification systems, which seems to be distinct from mechanisms of postsegregational killing by other bacterial addiction modules.

FIG. 1.

Change in cellular levels of EcoRI R and M proteins after inhibition of protein synthesis. Two cultures of MC1061(pMB4) were grown at 30°C to early log phase (OD₆₀₀ of 0.2). One culture was shifted to 42°C while the other culture was kept at 30°C. After a 30-min incubation at the respective temperature, spectinomycin was added to the two cultures to inhibit protein synthesis. Samples were taken just before the addition of spectinomycin (time zero) and 0.5, 1, 1.5, 2.5, and 3.5 h later. The samples were processed and examined for amounts of the EcoRI R and M proteins by Western blot analysis as described in Materials and Methods. (A). Change in optical density at 600 nm of the two cultures incubated at 30°C (open circle) or 42°C (filled circle) after blockage of protein synthesis. (B) Levels of EcoRI RM proteins in cells at each sampling point as determined by Western blot analysis. Samples containing the same amounts (50 μg) of cellular proteins were analyzed in each lane. Chemiluminescent signals visualized on film are shown. (C and D) Changes in levels of the EcoRI R protein (open circle) and M protein (filled circle) in cells incubated at 30°C or 42°C after blockage of protein synthesis. The amount of the EcoRI R or M proteins at each sampling point was normalized to the amount at time zero.

FIG. 2.

Estimation of numbers of EcoRI R and M protein molecules per cell. BIK5607(pIK172) and BIK5607(pMB4) were grown at 30°C to log phase. Equal amounts (28 μg) of cellular proteins prepared from independent cultures of each strain were analyzed by Western blotting. Different amounts of purified His-tagged EcoRI R and M proteins mixed with cellular proteins prepared from MC1061 were used as the standards. A sample containing 28 μg of cellular proteins from MC1061 was also included in the Western blot analysis as a negative control. Chemiluminescent signals were visualized on film.

FIG. 3.

Change in cellular levels of EcoRI R and M proteins after induction of plasmid loss by temperature shift. BIK5607(pIK172) and BIK5607(pIK173) were grown at 30°C to log phase. Plasmid loss was induced by temperature shift to 42°C. The cultures were then incubated at 42°C and diluted whenever their optical density at 600 nm reached about 0.3. Samples were taken just before the temperature shift (time zero) and 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 h later. The samples were examined for amounts of the EcoRI R and M proteins by Western blot analysis as described in Materials and Methods. (A) Growth curves of BIK5607(pIK172) (restriction positive) (open circle) and BIK5607(pIK173) (restriction negative) (filled triangle). Optical densities at 600 nm at indicated time points were normalized to the value at time zero using dilution factors. (B) Change in viable cell counts. Viable cells were counted for BIK5607(pIK172) (open circle) and BIK5607(pIK173) (filled triangle) on LB agar. Viable cell numbers at indicated time points were normalized to the values at time zero using dilution factors. (C) Levels of EcoRI R and M proteins in BIK5607(pIK172) at each sampling point as determined by Western blot analysis. The sample taken just before the temperature shift (time zero) was diluted with a sample containing cellular proteins from the EcoRI r⁻ m⁻ strain MC1061 so that the total amount of protein in each dilution was kept the same. (D and E) Change in cellular levels of EcoRI R (open circle) and M (filled circle) proteins after temperatureshift. Cellular amounts of EcoRI R and M proteins were estimated from the standard curve generated with the standards. Results from quantitative analysis of the signals shown in panel C (experiment 1) are presented in panel D. Results from an independent experiment (experiment 2) are shown in panel E. Samples containing 51 μg of cellular proteins were analyzed in experiment 1, while samples containing a smaller amount (24 μg) were analyzed in experiment 2.

FIG. 4.

Pulse-chase analysis of stability of EcoRI RM proteins. MC1061(pMAN-RM) and MC1061(pMAN885EH) (vector only, as control) were grown at 37°C in the supplemented M9 minimal medium containing glycerol, chloramphenicol, and all of the 20 amino acids except methionine and cysteine. Expression of the EcoRI RM genes was induced by adding arabinose to their log-phase cultures. After 35 min of incubation at 37°C, the cultures were labeled with a mixture of [³⁵S]methionine and [³⁵S]cysteine for 5 min and then chased with an excess of unlabeled methionine and cysteine. Samples were taken after 3 min of incubation (time zero), and additional samples were taken after 1 and 3 h of incubation at 37°C. The labeled EcoRI R and M proteins in the samples were separately immunoprecipitated with the anti-EcoRI R or anti-EcoRI M antibody and, after SDS-PAGE, visualized by autoradiography (exposure time, 24 h for R and 64 h for M) as described in Materials and Methods.