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Journal of Bacteriology, April 2000, p. 2218-2229, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cellular Responses to Postsegregational Killing by
Restriction-Modification Genes
Naofumi
Handa,
Asao
Ichige,
Kohji
Kusano,
and
Ichizo
Kobayashi*
Department of Molecular Biology, Institute of
Medical Science, University of Tokyo, Shirokanedai, Tokyo 108-8639, Japan
Received 16 August 1999/Accepted 13 January 2000
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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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INTRODUCTION |
A type II restriction enzyme, such
as R.EcoRI, will make a double-stranded break at a specific
sequence on DNA (49). A cognate modification enzyme
(M.EcoRI) can methylate the same sequence and protect it
from restriction cleavage. The genes involved are tightly linked and
form a type II restriction- modification (RM) gene complex. Type II RM
gene complexes will attack unmodified foreign DNA such as bacteriophage
DNA but not the modified DNA of the cells where they reside. They have
been considered to function as bacterial tools against invasion by
foreign DNA.
We found that elimination of type II RM gene complexes from bacterial
cells by a competing genetic element inhibits cell growth (43,
44). Our experiments suggested the following course of events
after a cell has lost a type II RM gene complex. In the descendants of
the cell that have lost the type II RM gene complex, the number of
molecules of the modification enzyme will decrease with each cell
division. Eventually, the capacity of the enzyme to modify the many
sites needed to protect the newly replicated chromosomes from the
remaining pool of restriction enzyme will become inadequate.
Chromosomal DNA will then be cleaved at the unmodified sites, and the
cells will be killed.
This is reminiscent of postsegregational cell killing mechanisms, which
have been shown to contribute to the stable maintenance of plasmids
(11, 12, 21). Indeed, linkage of several type II RM gene
complexes stabilizes plasmids (29, 32, 43, 44, 45). In other
postsegregational killing systems, the differential stabilities of the
killer and the antikiller are considered to be essential. What is
essential for RM systems may be the effective concentration of the two
enzymes. The modification enzyme will need to modify all (or almost
all) of the many chromosomal recognition sites along the chromosome in
order to be effective, i.e., to prevent cell death. The restriction
enzyme would need to cut only one (or a few) of these in order to be
effective, i.e., to kill the cell. Even dilution of the two enzymes to
the same extent might lead to cell death. Therefore, the RM systems may
employ a novel strategy of postsegregational host killing.
Microscopic observation of individual cells demonstrated that cell
death can happen after loss of an RM plasmid (15a). However, this early work employed a bacterial strain that is defective in its
own major recombination repair pathway and, instead, expresses recombination repair and restriction alleviation functions of a
bacteriophage (13, 24). The relevance of those data to
bacterial biology is, therefore, not straightforward.
In the present work, we addressed the question of how bacterial cells
respond to loss of an RM gene complex. We used Escherichia coli strains either wild type or mutant with respect to various recombination and repair functions for that purpose. The cleaved huge
chromosomes were detected by pulsed-field gel electrophoresis. Our
results strongly suggest that, after loss of an RM plasmid, the
bacterial cells die as a result of chromosome cleavage and that the
bacterial RecBCD/Chi/RecA machinery helps the cells to survive by
repairing the cleaved chromosomes.
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MATERIALS AND METHODS |
Bacteria, bacteriophage, and plasmids.
All the bacterial
strains used are derivatives of E. coli K-12 and are listed
in Table 1. The recC1002
mutation was isolated as a suppressor mutation of the recC73
mutation, which produces a null phenotype (54). The
resulting mutant allele with two mutations, recC73 and
recC1002, is often referred to as recC1002. The
recBCD mutant alleles were verified by UV sensitivity and/or plaque size of bacteriophage lambda (with or without Chi)
(54). The recC73 mutant was as sensitive to
postsegregational killing as the recB21 recC22 mutant used
here (N. Handa, A. Ichige, and I. Kobayashi, unpublished data).
Bacteriophage P1 vir from our laboratory collection was used in
transduction. Plasmid pIK172 carries the temperature-sensitive
replication initiator of pSC101 (16) and is EcoRI
r+ m+ and ampicillin resistant
(Apr) (43). pIK173 and pIK174 are its
r
m+ (43) and r m versions (32), respectively. Details of
plasmid construction was published elsewhere (15a).
Media, DNA preparation, and transformation.
E. coli
cells were grown in L broth and were supplemented, if necessary, with
antibiotics at the following concentrations: ampicillin, 50 µg/ml;
methicillin, 200 µg/ml; chloramphenicol, 25 µg/ml; kanamycin, 10 µg/ml; tetracycline, 10 µg/ml. Plasmids were introduced into
E. coli cells by electroporation using a Bio-Rad Gene Pulser.
Other methods are described in the figure
legends.
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RESULTS |
Growth inhibition following loss of the EcoRI RM gene
complex.
The EcoRI RM gene complex was inserted into a
plasmid possessing a thermosensitive replication initiator in order to
analyze the effect of loss of the gene complex on cell growth. With a rec+ bacterial strain (AB1157), the inhibition
of plasmid replication following the shift of a r+ culture
or r culture to the nonpermissive temperature for plasmid
replication stopped the increase in the number of plasmid-carrying
viable cells (Fig. 1, first row, first
column). The shift attenuated the increase in viable cell counts for
the r+ culture but not for the r control
culture (second column), as reported previously for a different genetic
background (15a, 43). Later, the increase in the total cell
number as estimated under a microscope was also inhibited (third
column). The cell viability (viable cell count/total cell count)
dropped after the shift but recovered later (fourth column). As a
result of cell growth inhibition, the fraction of viable cells carrying
plasmids remained higher for the r+ plasmid than for the
r plasmid (fifth column). These features are
qualitatively similar to those observed with classical
postsegregational killing systems for plasmids (11, 12, 21).
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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.
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Cell filamentation and loss of nuclei.
In a search for
evidence of the death of individual cells, we observed cells after DAPI
(4',6'-diamidino-2-phenylindole) staining following loss of the RM
plasmid. Some of the rec+ cells appeared
elongated (Fig. 2, top). Most of these
filaments contained multiple nuclei (DAPI-stained areas), while some
lacked detectable nuclei. The cells were classified into four groups based on two criteria, cell length and nuclear content (Fig.
3, top). Many filaments appeared 6 h
after the temperature shift. The filamentation was dependent on the
r+ genotype (Fig. 3, top right). A small but significant
fraction of the cells appeared anucleated. The r+ dependent
formation of cell filaments was observed even at 30°C (time zero).
These results demonstrate that cell death occurred after the loss of
the type II restriction modification gene complex.
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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.
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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.
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Increased cell death in a recBCD mutant.
We next
looked for the E. coli mutants that enhance these death
symptoms. The recBC mutants, which are defective in
recombination repair of DNA double-stranded breaks, turned out to
belong to this class.
The inhibition of cell growth, as seen in the reduction of the slope of
the viable-cell count curve, was stronger in the
recBC mutant culture (Fig.
1, second row, second column) than in the
rec+ culture. Also, the inhibition lasted longer
in the
recBC mutant
culture than in the
rec+ culture. Total cell counts showed the same
tendency (third column).
Even at a lower temperature, the viability of
the RM-carrying
culture was low and decreased further after the
temperature shift
(fourth column). Because of this extensive cell
death, the difference
in the fraction of plasmid-carrying viable cells
between the r
+ m
+ culture and r
m
+ culture was larger in the
recBC strain than
in the
rec+ strain (fifth
column).
Extensive cell filamentation was observed, with the
recBC
strain undergoing loss of the r
+ plasmid (Fig.
2, bottom;
Fig.
3, bottom). The fraction of cells
lacking nuclei was larger than
in the
rec+ strain (
P < 0.0005
in experiment
1).
Chromosome cleavage and degradation.
We then directly analyzed
the chromosomal DNA in these cells by pulsed-field gel electrophoresis
(Fig. 4). As in our earlier work, smears
composed of relatively small DNA fragments in the central and lower
parts of our pulsed-field gels were taken as evidence of chromosome
degradation (32, 43). Under our gel assay conditions in the
present study, large circular DNAs such as intact bacterial chromosomes
are trapped in the well, whereas huge (>700-kbp) linear DNAs form a
band which migrates just below the well in the upper part of the gel
(10, 33). Chromosomes that had been cleaved at few sites and
that had undergone only weak degradation (and also chromosomes
partially restored from the broken pieces) were detected as bands in
this area.
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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.
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The following are reproducible observations about the huge linear DNAs
(Fig.
4, upper part of the gel) and the smear DNAs
(central and lower
parts of the gel) in our pulsed-field gel electrophoresis
analysis.
In the
rec+ strain (Fig.
4, left), (i) more huge
linear DNAs are detected in r
+ cells than in
r
cells; (ii) r
+, but not in r
cells, there are smear DNAs detected; and (iii) the smear DNAs
increase
over the incubation period after the temperature
shift.
In the
recBC mutant strains (Fig.
4, middle panel), (i)
there are many huge linear DNAs in r
+ cells; (ii) there are
more of these in r
+ cells than in r
(
recBC) cells; (iii) there are more of these in
recBC r
+ cells than in
rec+ r
+ cells; and (iv) there are
hugh linear DNAs even at the time of
temperature shift (time zero) (the
huge linear band at 8 h for
r
+ was shifted to the
right lane (0 h for r
; this is an artifact inherent to
the machine used). Also, (i)
there are many smear DNAs in
r
+ cells; (ii) there are more of these in r
+
cells than in r
(
recBC) cells; and (iii) there
are more of these in
recBC r
+ cells than in
rec+ r
+ cells. Finally, the smear
DNAs increase with time until 6 h after
the
shift.
These results strongly suggest that insufficient ability to repair
broken chromosomes after the temperature shift in the
recBC mutant cells leads to their extensive degradation. We concluded
that
the RecBCD enzyme repairs chromosomal breakage caused by
the
restriction enzyme. The
recBC mutant with the
r
m
+ plasmid showed lower, but still
significant, levels of large
linear DNAs. This restriction-independent
chromosomal cleavage
was again reduced in the
rec+ strain, in agreement with another study
(
40).
SOS induction.
The chromosomal DNA degradation and cell
filamentation suggested that the SOS response had been induced. We used
a chromosomal sfiA::lacZ (note that
sfiA is the same as sulA) gene fusion as a
reporter for SOS induction. In this system beta-galactosidase is
induced in response to DNA breakage. The cells carrying the EcoRI r+ m+ plasmid were found to be
slightly induced for the SOS response at 30°C (Fig.
5). The inhibition of r+ m+ plasmid replication by a temperature shift of a liquid
culture increased SOS induction (Fig. 5). The SOS induction before and after the temperature shift was dependent on the presence of the r+ gene on the plasmid and on the
recBC+ genotype of the host bacteria (Fig. 5).
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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.
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Plate assay for postsegregational killing.
In the next series
of experiments, postsegregational killing was estimated from growth
inhibition on solid media. An E. coli strain carrying a
temperature-sensitive plasmid with the EcoRI r+ m+ gene complex was found to form smaller and fewer
colonies at the semipermisssive temperature (35°C) than the control
strain with an r m+ or r m plasmid (Fig. 6). The
inhibition appeared stronger at 37 (see Fig. 7) and 42°C (data not
shown) than at 35°C. Because of the sfiA::lacZ reporter construct in these
strains, SOS induction should result in a blue color on agar containing
X-Gal (5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside). The bacterial cells carrying this r+ m+ temperature-sensitive plasmid were found to be slightly induced for the
SOS response at 30°C and to be more induced at 35°C (Fig. 6). These
results are qualitatively similar to those obtained in liquid media
(Fig. 5) and sugggest that the plate assay provides a reasonable
measurement of postsegregational killing by RM systems, as shown
previously for other classical postsegregational killing systems on
plasmids (12).
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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--D-thiogalactopyranoside). The plates were
incubated at 30 or 35°C. Essentially the same results were obtained
without IPTG.
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Postsegregational killing in other bacterial mutants.
Using
the plate assay, we examined the effects of several bacterial mutations
on postsegregational killing. The extent of cell killing was inferred
from the differences in colony growth between cells losing the
EcoRI r+ m+ plasmid and those losing
the EcoRI r m+ control plasmid
(Fig. 7; compare the right part and the
left part of each plate). At 30°C, the cells losing the
r+ and r plasmid showed indistinguishable
levels of growth (data not shown). The recBCD mutant showed
strong growth retardation (Fig. 7i) as in the liquid media (Fig. 1,
second row, second column). The RecBCD enzyme degrades DNA as an
exonuclease from a double-stranded break. When the enzyme encounters a
specific sequence called Chi (5-GCTGGTGG), it promotes
recombination of this DNA with a homologous DNA molecule with the help
of the RecA protein (9, 28, 34, 60). The recC1002
recC* mutant was found to be as proficient in recombination as the
recBCD+ strain in an assay with a substrate DNA
lacking Chi, but it is defective in recombination enhancement by Chi
(54). The recC* mutation was found to slightly
increase killing in the plate assay (Fig. 7iii).
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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
( recA306::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.
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In liquid assay, the growth inhibition of the
recC* strain
was not as severe as that of the
recBC strain (Fig.
1, third
row).
The
recC* mutant carrying the r
+ plasmid
showed low viability even at 30°C (Fig.
1, third row,
fourth column),
probably as a result of postsegregational killing.
The cell population
of the
recC* strain that survived postsegregational
killing
showed increased viability in this (Fig.
1, third row,
fourth column)
and other independent experiments, presumbably
because the cells did
not carry the restriction gene any more.
The
recC* mutant
culture with the
EcoRI r
+ m
+ gene
complex had a level of chromosome cleavage that was intermediate
between that of the
recBC mutant and that of the
rec+ strain (Fig.
4, right). The
recC* culture with the r
plasmid contained a
level of chromosome cleavage that was intermediate
between that of the
recBC mutant and that of the
rec+ strain.
The growth inhibition turned out to be severe in other mutants
defective in the RecBCD pathway of recombination, including
mutants of
recA,
ruvAB,
ruvC, and
recG
(Fig.
7ii, v, vi, and iv,
respectively). A less severe defect was
observed in a mutant of
the
recN gene which is defective in
DNA double-strand break repair
(Fig.
7vii).
A lower level of inhibition in the
lexA3 mutant (Fig.
7viii), defective in SOS induction, than in the
recBC mutant
was observed.
This indicates that the RecBCD function contributes in
other ways
to cell survival than just the generation of SOS
signals.
The
sbcA mutations on the Rac prophage activate
RecET-mediated homologous recombination (
27), and this can
repair type II
restriction breaks at least on plasmids (
31).
As expected, an
sbcA mutation partially suppressed the
sensitivity of the
recBC mutant to postsegregational killing
(Fig.
7ix).
The extent of postsegregational killing in these mutants was measured
quantitatively by comparing their colony-forming efficiencies
at a
lower temperature and at a higher, nonpermissive temperature
(Table
2). The results roughly paralleled those
from the above
streak assay. The only and interesting exception is the
recA mutant,
which will be a subject of a future study of
ours. Taken together,
these results suggest that RecBCD-mediated
recombination repair
of restriction breaks contributes to cell survival
after postsegregational
killing by the RM systems.
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DISCUSSION |
Our results here demonstrate that (i) the cells of bacterium
E. coli die after loss of an RM plasmid; (ii) this death is
accompanied by cleavage and degradation of their chromosomes; (iii) a
recBC mutant shows more extensive cell death and accumulates
more huge cleaved chromosomes; (iv) recC*, recA,
ruvAB, ruvC, recG, recN, and lexA mutants also show extensive cell death; and (v) SOS
function is induced in a recBC-dependent manner.
Postsegregational cell killing by RM systems.
The observation
of anucleated cells in an E. coli rec+ strain in
our experiments clearly demonstrates that cell death can occur following the loss of an RM gene complex. This and other observations in the present work and previous works (15a, 26, 26a)
strongly support the postsegregational cell killing concept for RM
systems. The formation of anucleated cells was observed in
postsegregational killing by ccd genes on F plasmids
(21).
Type II RM gene complexes are prevalent on bacterial chromosomes
(
26a,
49). A chromosomally located type II RM gene complex
also resists replacement by a homologous stretch of DNA (Y. Nakayama,
N. Handa, and I. Kobayashi, unpublished data). Therefore,
postsegregational
killing appears to be an intrinsic property of RM
gene complexes
rather than of the plasmids that carry them.
Postsegregational
killing would assure maintenance of RM gene complexes
in their
competition against other genetic elements. This
"selfish-gene
hypothesis" can explain several properties of type II
RM systems
(
1,
25,
26,
32,
45,
50). Their behavior as mobile
genetic elements shaping the bacterial genome supports this hypothesis
(
26a,
45a).
We have been able to detect plasmid stabilization by all type II RM
systems we have examined so far, including
PaeR7I
(
43),
EcoRI (
43), and
EcoRV
(
45). However, postsegregational killing
has not been
detected for type I RM systems (
30,
46; Y. Naito,
N. Handa, and I. Kobayashi, unpublished
data).
Resistance to killing through recombination repair of cleaved
chromosomes.
Mutations in recBC, recA,
recG, recN, ruvAB, and ruvC
increased postsegregational killing by the EcoRI gene
complex. These genes are involved in homologous recombination and
recombinational repair of DNA damage (28, 36). A simple
explanation would be that the RecBCD machinery defends the host against
attack by type II RM systems by repairing the restricted chromosomes.
In support of this, it was found that many anucleated cells and cleaved chromosomes accumulated in the recBC mutant (Fig. 2 to 4).
However, protection by the RecBCD system is incomplete, as shown in
Fig. 1 to 4.
Many experiments in vivo and in vitro have suggested that the Chi
sequence plays an important role in RecBCD-mediated recombinational
repair of DNA (
58). The strain with the
recC1002
mutation promotes
recombination as efficiently as the
recBCD+ strain in a lambda recombination assay,
but the recombination
is not stimulated by the presence of Chi on the
substrate DNA
(
54). The postsegregational killing in the
plate assay was stronger
(Fig.
7), and the level of cleaved chromosomes
was higher (Fig.
4), in the
recC1002 mutant than in the
recBCD+ strain. This observation is consistent
with the idea that Chi
recognition is important for the recombinational
repair of restricted
chromosomes in postsegregational killing as in
other forms of
recombinational repair. Three
recBCD mutants
(
recC2145,
recB2154,
and
recB2155)
showed essentially the same phenotype as this strain
(N. Handa, A. Ichige, and I. Kobayashi, unpublished observation).
A
recD
null mutant, which is insensitive to Chi but hyperrecombinogenic,
is as
resistant to postsegregational killing as the wild-type
strain (N. Handa, A. Ichige, and I. Kobayashi, unpublished observation).
Determination of these mutant sequences would help to elucidate
the
underlying molecular
events.
The presence of the r
+ m
+ plasmid appears to
result in damage to DNA even at a temperature permissive for plasmid
replication,
as suggested by the low level of SOS induction in the
plate and
liquid assays, the low level of chromosome cleavage, and the
presence
of a few filamentous cells. We do not know whether this
reflects
low-frequency plasmid loss at this temperature in the genetic
backgrounds used or insufficient modification activity to protect
chromosomal recognition
sites.
Loss of an RM gene complex will generate unmethylated and
hemimethylated chromosome sites at the same locus after several
rounds
of DNA replication. For recombinational repair of the restriction
break
at the former site, the latter site would be the most appropriate
partner.
Some correlation between the amount of cleaved chromosomes and the
level of cell death was observed, although we cannot exclude
the
possibility that, instead of double-stranded breaks, single-stranded
breaks introduced at hemimethylated sites (
22) represent the
real source of lethal damage. Such a single-stranded break, if
present,
might be converted to a double-stranded break after passage
of the
replication fork, which would then be recognized and repaired
by the
RecBCD enzyme as proposed earlier (
58).
In an earlier study, an
E. coli strain bearing a
temperature-sensitive mutant of the
EcoRI restriction enzyme
displayed induction
of the SOS response and chromosome cleavage
(
17). Strains defective
in SOS induction and recombination
(
recA and
recB mutants) were
no more sensitive to
this in vivo DNA scission than were wild-type
bacteria (
17).
A number of
EcoRI restriction enzyme mutants
are apparently
temperature sensitive and create lesions that are
much more toxic in
recA and
recB mutant cells (
18).
Mutations in the
recN gene are known to confer sensitivity
to ionizing radiation but not to UV light. The sensitivity to ionizing
radiation in these mutants correlates with a deficiency in their
capacity to repair DNA double-stranded breaks. RecN and this repair
capacity are SOS inducible (
47,
51). The necessity for RecN
as a defense against an RM gene system (Fig.
7vii) is consistent
with
our hypothesis that double-stranded breaks are the true source
of
lethal
damage.
A mutation in the
ruvAB,
ruvC, or
recG
gene confers sensitivity to DNA-damaging agents and results in a defect
in homologous
recombination. RuvA and RuvB proteins together and the
RecG protein
by itself promote branch migration of Holliday structures
(
37,
65), and the RuvC protein resolves Holliday structures
(
65).
These enzymes may be involved in the later steps of
recombination
repair of restriction breaks in vivo, although other
possibilities
(
55) cannot be ruled
out.
The severe postsegregational killing in the
recBC mutant was
partially suppressed by an
sbcA mutation (Fig.
7ix), which
activates
RecET-mediated homologous recombination coded by the Rac
prophage.
The suppressive effect is likely to be due to repair of
chromosomal
double-stranded breaks by this homologous recombination
machinery
because RecET- and Red-mediated recombination can repair
double-stranded
breaks by type II restriction enzymes (
56,
61,
62). However,
the possible involvement of other Rac prophage
genes (
24) cannot
be
excluded.
The bacterial cells losing RM genes induced the SOS response in a
restriction-dependent and RecBC-dependent manner. Presumably,
the
helicase activity of RecBCD generated single-stranded DNA
from a
restriction break and this in turn induced the SOS response
(
6,
53). This SOS induction is unlikely to be the only role
of the
RecBCD enzyme in cell defense because the
lexA3 mutation,
which tightly suppresses the SOS response (
42), leads to
less-severe
postsegregational killing than
recBC mutations
(Fig.
7viii). The
role of SOS induction in defense is unlikely to be
restriction
alleviation, which is not effective for
EcoRI
restriction (
64).
Its role might be the induction of RuvAB
and/or RecN (
51,
65)
or induction of stable chromosomal
replication (
3).
A self-recognition hypothesis for the destruction/repair behavior
of the RecBCD system.
Acting at a DNA double-stranded break, the
RecBCD enzyme shows two contrasting activities, exonucleolytic
degradation and recombination. The exonucleolytic activity serves to
destroy invading foreign DNAs after restriction breakage (5,
57). When the RecBCD enzyme encounters Chi, it stops degrading
DNA and promotes recombination of this DNA with a homologous DNA
molecule (9, 28, 34, 60). Regeneration of a collapsed
replication fork was hypothesized to be the role for this unusual
destruction/recombination activity (2, 7).
The ambivalent destruction and recombination action of the
RecBCD/Chi/RecA machinery may be understood in terms of the
interactions
between three genetic elements within a cell, the RM
system, the
invading DNA, and the RecBCD/Chi system as illustrated in
Fig.
8. An RM gene complex will attack
any unmodified recognition site,
whether it be on an invading DNA or on
the chromosome. From the
restriction break, the RecBCD enzyme will
start exonucleolytic
degradation of the DNA. This would destroy alien
DNA. If the enzyme
encounters a Chi sequence, which would serve as an
identification
marker for the genome, degradation would stop, and
recombinational
repair would start. In brief, we suggest that the
bacterial RecBCD
system destroys invading DNAs in collaboration with
the RM gene
complexes but protects its own chromosome from attack by RM
gene
complexes.
View larger version (30K):
[in this window]
[in a new window]
|
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).
|
|
Therefore, RecBCD exonuclease and Chi may be regarded as one
self-recognizing system analogous to RM systems. This self-recognition
concept was proposed (
15,
26) on the basis of the observed
postsegregational host killing by type II RM systems (
43)
and
on the mutational alteration of the sequence specificity of the
RecBCD enzyme (
14). Earlier, homologous recombination and RM
systems were proposed to have evolved under similar pressures
associated with cell invasion by foreign DNAs (
48).
Several other bacterial groups seem to have their own unique pairs of
RecBCD-like enzymes and recognition sequences (
23).
If a
piece of chromosomal DNA from one member were to enter another
member
of the same group sharing the same chromosomal identification
sequence,
Chi or a Chi equivalent in the proper configuration,
a part of the
invading DNA would be incorporated by the Rec machinery
into the
chromosome (Fig.
8) (
8). The final discrimination
between
"self" and "nonself" will take place during the process
of
homologous pairing by the mismatch repair system (
38).
Therefore,
the interactions between the RecBCD/Chi system, RM systems,
and
invading DNAs that we identified might have contributed to the
observed genetic structure of chromosomes in bacterial populations,
i.e., mosaics resulting from homologous replacement within a group
of
bacteria (
39,
59).
| |
ACKNOWLEDGMENTS |
We thank the people listed in Table 1 for gifts of materials and
Steve Kowalczykowski, Tom Bickle, Michael Yarmolinsky, Maurice Fox,
Sota Hiraga, and Kenn Gerdes for discussion.
This work was supported by the Ministry of ESSC of the Japanese
government (class B, DNA repair, genome), Nagase Science Foundation, Takeda Science Foundation, Japan Science Society, and NEDO. N.H. was supported by a JSPS Research Fellowship for Young Scientists.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo 108-8639, Japan. Phone: (81) 3-5449-5326. Fax: (81)
3-5449-5422 and -5645. E-mail:
ikobaya{at}ims.u-tokyo.ac.jp.
Dedicated to Tokio Kogoma.
Present address: Laboratory of Genetics, University of Wisconsin,
Madison, WI 53706.
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Journal of Bacteriology, April 2000, p. 2218-2229, Vol. 182, No. 8
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
