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Cellular responses to postsegregational killing by restriction-modification genes - PubMed

Cellular responses to postsegregational killing by restriction-modification genes

N Handa et al. J Bacteriol. 2000 Apr.

Abstract

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.

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Figures

FIG. 1
FIG. 1

Cell growth inhibition following loss of the EcoRI RM gene complex. Three E. coli strains, AB1157 (rec+), JC5519 (recB21 recC22), and BIK3686 (recC1002) carrying pIK172 (pSC101Ts, EcoRI r+ m+, Apr) or pIK173 (its r− version) were grown with aeration at 30°C in L broth with antibiotics to an optical density at 660 nm (OD660) of 0.3. Then the antibiotics were removed, and the culture was transferred to 42°C with aeration. The culture was diluted every time its OD660 reached 0.3. Total cells (first column from left) were counted under a microscope. The number of viable cells (second column) was estimated by counting the colonies on L agar without selective antibiotics at 30°C. The number of plasmid-carrying cells (third column) was estimated by counting the colonies on L agar with antibiotics at 30°C. The viable-cell number was divided by the total cell number for each point to calculate viability (fourth column). The number of plasmid-carrying viable cells was divided by the number of viable cells to calculate the fraction of plasmid-carrying cells (fifth column). The numbers at time zero were set to unity in the first to third columns. Solid symbols, cells losing the r+ m+ plasmid; open symbols, cells losing the r− m+ plasmid.

FIG. 2
FIG. 2

Cell morphology following loss of the EcoRI RM gene complex. E. coli strains AB1157 (rec+) and JC5519 (recB21 recC22) carrying pIK172 (pSC101Ts, EcoRI r+ m+, Apr) or pIK173 (its r− version) were aerated at 30°C in L broth with selective antibiotics to an optical density of 0.3 at 660 nm. Then the antibiotics were removed, and the culture was transferred to 42°C with aeration as described for Fig. 1. Cells were harvested at the indicated time intervals after the temperature shift and mixed with the same volume of methanol-HCO2H (2:1). After incubation on ice for 10 min, cells were collected by centrifugation, resuspended in 10 mM Tris-HCl (pH 7.5)–10 mM MgSO4, stained with DAPI, and observed under a microscope.

FIG. 3
FIG. 3

Morphological classification of cells following loss of the EcoRI RM gene complex. E. coli strains AB1157 (rec+) and JC5519 (recB21 recC22) carrying pIK172 (pSC101Ts, EcoRI r+ m+, Apr) or pIK173 (its r− version) were treated as described in the legend to Fig. 2. The cells were classified into four types by visual inspection. A cell was judged to be a filament when it was larger than twice the unit size of the r− cell before the temperature shift. The presence or absence of nuclei means that DAPI staining was positive or negative, respectively. The number at the top of each bar is the number of cells examined. Data for cell types I to IV are shown from top to bottom for each bar.

FIG. 4
FIG. 4

Chromosome cleavage following loss of the EcoRI RM gene complex. Cultures of three E. coli strains, AB1157 (rec+), JC5519 (recB21 recC22), and BIK3686 (recC1002), carrying pIK172 (pSC101Ts, EcoRI r+ m+, Apr) or pIK173 (its r− version) were transferred from 30 to 42°C as described in the legend to Fig. 1. The cells were mixed with 2,4-dinitrophenol to block energy metabolism at the indicated time intervals (in hours) after the temperature shift and were treated as described previously (33). DNA was electrophoresed through 1.0% agarose gel at 15°C in 45 mM Tris-borate–1.25 mM EDTA at 165 V with a pulse time of 50 s for 24 h using hexagonal electrodes in a Pharmacia LKB apparatus. Lane M contains Saccharomyces cerevisiae chromosomes.

FIG. 5
FIG. 5

SOS induction following loss of the EcoRI RM gene complex. Isogenic rec+ and recBC strains with a sfiA::lacZ promoter fusion (BIK3920 and BIK3921) carrying pIK172 (pSC101Ts, EcoRI r+ m+, Apr) or pIK173 (its r− version) were aerated at 30°C in L broth with antibiotics and grown to an optical density at 660 nm (OD660) of 0.3. Then the antibiotics were removed, and the culture was transferred to 42°C with aeration. The culture was diluted every time its OD660 reached 0.3. The beta-galactosidase activity was measured as described previously (41). Enzyme concentrations (in units per milliliter) were calculated from the following formula: 1,000 × (OD420 − 1.75 × OD550)/t× v× OD600, where t is the duration of the reaction in minutes and v is the volume of culture in milliliters. The results plotted were from two independent experiments.

FIG. 6
FIG. 6

Growth inhibition and SOS induction following loss of the EcoRI RM gene complex. An E. coli strain with the sfiA::lacZ promoter fusion (GC3403) carrying pIK172 (pSC101Ts, EcoRI r+ m+, Apr), pIK173 (its r− m+ version), or pIK174 (its r− m− version) was aerated in L broth containing selective antibiotics at 30°C and then streaked on L agar plates lacking selective antibiotics but containing 20 ng of X-Gal/ml and 0.25 mM IPTG (isopropyl-β-

d

-thiogalactopyranoside). The plates were incubated at 30 or 35°C. Essentially the same results were obtained without IPTG.

FIG. 7
FIG. 7

Effect of bacterial mutations on growth inhibition following loss of the EcoRI RM gene complex. Various E. coli strains carrying pIK172 (pSC101Ts, EcoRI r+ m+, Apr) or pIK173 (its r− version) were grown with selective antibiotics to log phase at 30°C and were then streaked on L agar for incubation at 37°C for 18 to 23 h. (i) JC5519 (recB21 recC22) and AB1157 (rec+). (ii) BIK733 (ΔrecA306::Tn10) and AB1157 (rec+). (iii) BIK3686 (recC73 recC1002 argA81::Tn10) and AB1157 (rec+). (iv) BIK1538 (recG258::Tn10 mini-Kan) and AB1157 (rec+). (v) HRS2302 (ruvAB::Cm) and AB1157 (rec+). (vi) HRS1100 (ruvC100::Cm) and AB1157 (rec+). (vii) BIK2565 (recN1502::Tn5) and AB1157 (rec+). (viii) BIK2571 (lexA3) and BIK2574 (lexA+). (ix) JC8679 (recB21 recC22 sbcA23) and JC5519 (recB21 recC22). r+, bacteria losing r+ m+ plasmid; r−, bacteria losing r− m+ plasmid; WT, wild type with respect to the gene in question.

FIG. 8
FIG. 8

A self-recognition hypothesis for the destruction/repair behavior of the Rec/Chi system. (Box) Three genetic elements in the cell and their relationships. The restriction modification systems will attack invading nonself (unmethylated at recognition sites) DNA as well as the chromosomal DNA of their ex-host (in postsegregational killing). The RecBCD exonuclease/recombinase system will destroy invading nonself DNA (without a Chi sequence) but will repair self DNA (with a Chi sequence and a homologous DNA). After double-stranded cleavage by a restriction enzyme (i) (or by some other factor), the RecBCD enzyme enters a duplex DNA and initiates exonucleolytic degradation (ii). This would destroy incoming foreign DNAs (iii). For chromosomal DNA (iv), the enzyme encounters a Chi sequence, which serves as an identification marker for the chromosome. This results in the attenuation of degradation and promotes recombinational repair with the sister chromosome (v). For an incoming DNA with a Chi sequence in the proper configuration (such as those from E. coli and other closely related enteric bacteria) (vi), degradation would stop, and homologous recombination with the chromosome would follow, if not inhibited by the mismatch recognition system (vii).

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