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Journal of Bacteriology, January 2004, p . 240-243, Vol . 186,
No . 1
Identification of Residues of the Kid Toxin Involved in Autoregulation of the
parD System
Marc Lemonnier, Sandra Santos-Sierra,
Consolación Pardo-Abarrio, and Ramón Díaz-Orejas*
Centro de Investigaciones Biológicas (CSIC), 28040 Madrid, Spain
Received 27 August 2003/ Accepted 30 September 2003
The toxin-antitoxin system parD (kis kid) of plasmid R1 is
coregulated by the coordinated action of its two gene products . Here
we describe the isolation and the in vivo characterization of three
single-amino-acid changes in the Kid toxin, G4E, C74Y, and E91K,
that affect the coregulatory activity but preserve the toxicity
of the protein .
parD (kis kid) is a toxin-antitoxin (TA) module found in plasmid
R1 (1, 2) . As many other TA modules (3,
5), parD is organized as an operon, with the
gene of the antitoxin, kis (killing suppressor),
preceding the gene of the toxin, kid (killing determinant) .
Efficient repression of the parD operon requires the concerted
action of the Kid toxin (110 amino acids) and the Kis antitoxin (85
amino acids) (9) . The Kis antitoxin alone poorly represses
the parD promoter, and this activity can be allocated to specific
residues at the amino-terminal region of the protein (9,
14) . Moreover, Kis and Kid form a stable complex
that both neutralizes the toxin and represses the expression of the
parD operon (11) . The crystal structure of
the dimeric Kid toxin has been determined at high resolution, and a
collection of amino acid residues involved in toxicity has been
allocated within this structure (4,
13) . However, information on residues implicated in the
coregulatory activity of Kid has not been available . In this work, we
searched for Kid mutants affected in their coregulatory activity by
using two complementary genetic approaches that were previously
described in our work on kis mutations involved in
autoregulation (14) .
The first, "restrictive" approach consisted of the mutagenesis of
the pAB24 plasmid, which contains the wild-type parD operon,
and the consecutive screening for Kid mutants unable to corepress the
parD promoter . A possible limitation of this approach was that
Kid mutations that affected the interactions between Kis and Kid
required for the neutralization of Kid toxicity should lead to growth
inhibition and therefore could be counterselected . The second,
"permissive" approach relieved this constraint: instead of pAB24, we
used pB24, a mutated version of pAB24 carrying the kid85
mutation . This mutation abolishes the toxicity of the Kid protein but
maintains its activity as a corepressor (13),
thereby permitting the eventual isolation of kid mutations
which lead to derepression by disrupting the interactions between Kis
and Kid .
Thus, the pB24 or pAB24 plasmid was mutagenized in vitro with
hydroxylamine as described previously (6) and this DNA was
used to transform the Escherichia coli CSH16 strain ( lac
supE) containing pOM34, a mini-R1 recombinant in which the
lacZ gene is transcriptionally fused to the parD promoter
(9) . In this background, parD mutants that
failed to repress in trans the parD promoter elicited
ß-galactosidase synthesis and led to the formation of red
transformants on MacConkey agar plates supplemented with lactose
(Fig . 1A) . Plasmids from red colonies were isolated
and retransformed in the same strain, and those reproducing the
mutant phenotype were kept for further analysis . The sequences of the
complete parD operon in each of the mutant plasmids were
determined and led to the identification of 11 different mutations in
the kid gene (Fig . 1B) . As expected from the
mutagenic action of hydroxylamine, the changes isolated were C-G/A-T
transitions and introduced missense or nonsense mutations . The latter
led to truncated proteins that were not toxic (data not shown), in
agreement with previous data that showed the relevance of the
carboxyl end of the protein in toxicity (4,
12) .
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FIG . 1 . Kid mutations that affect the autoregulation of the parD
system . (A) CSH16 cells carrying the reporter of parD expression
plasmid pOM34 and the mutated derivatives of plasmid pB24 (Tetr
kis74 kid85) or pAB24 (Tetr kis+
kid+) were streaked on MacConkey agar plates supplemented
with lactose, tetracycline (10 µg per ml), and ampicillin (50 µg per
ml) . The plates were photographed after a 12-h incubation at 30°C . (B)
Localization of the mutations . Mutations that were isolated by both
approaches, i.e., that were found in pAB24 and also in pB24, are
included in a grey box . For clarity, their localization is indicated
only on the pAB24 sketch . Mutations that were isolated only in pAB24 or
pB24 are indicated above and below the corresponding plasmid
representation, respectively . Asterisks denote mutations that were
described in a previous work as mutations that affect both the toxicity
and autoregulatory activities of Kid (13).
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Therefore, we concentrated our analysis on the more informative
missense mutations . kid4 (which leads to a G4E amino acid change
in Kid), kid29 (T29I), kid70 (G70D), kid74 (C74Y),
kid79 (T79A), kid87 (G87R), kid91 (E91K), and
kid94 (P94L) . With the exception of G4E and T29I, the changes
were clustered at the carboxy-terminal third of the Kid protein in a
20-amino-acid region bounded by residues 74 to 94 . In a previous
search for nontoxic mutants of Kid, the changes T29I, P94L, and G70S
(the last affecting the same residue as G70D of our current study)
were shown to affect both the toxic and the autoregulatory activities
of Kid (13) . Therefore, their isolation in our
present approach was theoretically expected, as internal controls of
nonspecific disruption of the autoregulatory activity of Kid . These
three mutations will not be considered further in this work . Among
the remaining mutations, we noticed that the C74Y, T79A, and
G87R changes were isolated in the permissive approach, using pB24
(Fig . 1B) . This suggested either that the observed
deregulation phenotype could be the consequence of a synergistic
effect between these mutations and the mutations originally present
in pB24 (kis74 and kid85) or that the nontoxic kid85
mutation present in pB24 could prevent an eventual negative effect on
growth due to the additional mutations .
To test these hypotheses, each of the kid74, kid79, and
kid87 mutations was introduced separately in pAB24 by using a
site-directed mutagenesis kit (Promega) . The resultant plasmids were
used to transform the MLM373 strain [(lac,
pro) supE thi)] (13), which also
contained the pMLM132 plasmid, a mini-F derivative that bears the
lacZ gene under the control of the parD promoter (13).
parD expression levels were monitored in ß-galactosidase
assays that were carried out as described previously (8) . The
Kid changes T79A and G87R did not significantly affect the autoregulatory
activity of the protein (Fig . 2A), confirming that the
deregulation phenotypes associated with the kid79 and kid87
mutations were due to interactions with the kid85 or kis74
mutation present in pB24 . In contrast, the Kid C74Y change provoked a
fivefold increase in parD promoter activity (Fig.
2A), confirming that the single C74Y change was
sufficient to lead to deregulation . Furthermore, growth was severely
inhibited in cells that contained the pAB24 plasmid bearing the
kid74 mutation but not in those that contained the other mutant
or wild-type pAB24 plasmid (Fig . 2B) . Growth
inhibition was not observed when the kid74 mutation was
present in pB24 (data not shown; see also Fig . 1A) . Therefore,
this indicates that the C74Y change has been counterselected by
the pAB24 restrictive approach and that the negative effect of a Kid
C74Y change is suppressed by the mutations present in pB24 . In
addition, these experiments confirmed that the kid4(G4E) and
kid91(E91K) mutations lead to deregulation of parD expression,
as revealed by the respective six- and threefold increases in
ß-galactosidase activity (Fig . 2A) .
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FIG . 2 . Effect of mutations on parD autoregulation and cell
growth . (A) Autoregulation . MLM373 cells containing the parDp-lacZ
fusion carrier plasmid pMLM132 and the derivatives of the pAB24 plasmid
carrying the different mutations were grown to mid-exponential phase in
Luria broth (LB) containing tetracycline (10 µg per ml) and
chloramphenicol (20 µg per ml) at 37°C . ß-Galactosidase activity was
measured from aliquots of the cultures as described previously (8) .
Error bars represent the standard deviations calculated from three
independent experiments . For the negative control (rightmost lane),
pBR322 was used instead of pAB24 . (B) Growth of MLM373 cells containing
the different pAB24 mutant plasmids . Overnight cultures at 37°C in LB
broth containing tetracycline were diluted in the same medium to an A600
of 0.02 to 0.04 and were grown at 37°C with aeration . Samples were
removed at the times indicated to measure optical density at a
wavelength of 600 nm . The graphic shows a typical pattern that was
reproduced in three independent experiments.
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To determine if the mutations kid4(G4E), kid74(C74Y), and kid91(E91K),
which affected Kid coregulatory functions, would also influence
its toxic activity, the kid genes carrying single mutations
were isolated and cloned under the control of a T7 RNA polymerase-dependent
promoter (15) in the pET3d-his vector (Stratagene) to
yield the series of pET3d-hiskid mutant plasmids . As a
control, the "nontoxic" kid85 mutant allele was also included
in the analysis . Expression of the cloned genes was induced in the
BL21(DE3) strain (Stratagene) by adding isopropyl-ß-D-thiogalactopyranoside
(IPTG) to the medium . In addition, the strains contained the
pMLM126 plasmid, a replication-thermosensitive pSC101 derivative in
which the kis antitoxin gene was placed under the control of
the arabinose-inducible araBAD promoter . Following induction
with IPTG, inhibition of cell growth was observed in cells in which
the wild-type kid gene was expressed but not in those that
expressed the kid85 mutant, as expected (Fig . 3,
compare panels B and C) . Moreover, the three deregulated kid
mutants tested (producing Kid mutants G4E, C74Y, and E91K) showed a
toxic phenotype (Fig . 3B and C) . The phenotype was
comparable to the one displayed by the wild-type Kid protein in the
case of G4E but slightly less marked in the case of C74Y and
significantly more severe in the case of E91K (Fig . 3C) .
In fact, background levels of E91K protein obtained in the absence of
IPTG induction were sufficient to dramatically affect cell viability
(Fig . 3B) . When overproduction of the Kis antitoxin
was induced with arabinose to neutralize the toxic effect of Kid, a
neat recovery of viability was observed in cells that produced either
the wild-type Kid protein or the mutant protein G4E or C74Y (Fig .
3, compare panels C and D) . Under the same conditions,
the E91K mutant protein was resistant to neutralization by Kis .
However, neutralization of E91K toxicity was observed when Kis was
overproduced in the absence of kid induction (Fig.
3, compare panels A and B) . This suggested that the
hypertoxic phenotype of the E91K mutant needed a large excess of Kis
antitoxin cellular levels to be neutralized . This is consistent with
the isolation of the kid91(E91K) mutation in the pAB24
plasmid, because the posttranscriptional control of parD
expression ensures that the antitoxin is produced at higher levels
than the toxin (2, 10) .
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FIG . 3 . Analysis of toxicity of the Kid mutants and neutralization by
the Kis antitoxin . BL21(DE3) cells carrying the kis overproducer
plasmid pMLM126 and the different pET3d-hiskid wild-type or
mutant plasmids were grown exponentially to an A600 of
0.4 in Luria broth (LB) medium containing ampicillin (50 µg per ml),
chloramphenicol (20 µg per ml), and arabinose (0.2%) . The cultures were
washed twice in LB medium, and serial dilutions (10-1 steps
from left to right) of the different cultures were spotted on plates
containing LB medium solidified with agar and supplemented with the same
antibiotics . In addition, arabinose (0.2%) was present in the plates
shown in the panels A and D, and IPTG (0.1 mM) was included in the
plates shown in panels C and D . The plates were placed at 30°C, and the
photographs were taken after 24 h of incubation.
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To summarize, we found three single-amino-acid changes in the Kid
toxin, G4E, C74Y, and E91K, that were able to affect the coregulatory
activity of Kid without causing a loss of the toxic activity of the
protein . The G4E change severely impairs the coregulation activity
but maintains both the toxicity of the protein and its capacity to
interact with the Kis antitoxin to form a nontoxic complex .
Therefore, this change may affect Kid-Kis interactions in a way that
the complex is proficient in neutralization of toxicity but deficient
in properly interacting with the parD operator sequences
and/or host factors required for parD regulation . A future
biochemical characterization of this mutant protein will help to
address this issue . Likewise, the Kid C74Y protein is efficiently
neutralized by a supply of Kis in a trans configuration .
However, introducing the kid74(C74Y) mutation in a wild-type
parD operon leads to deregulation of parD expression
and severe cell growth inhibition, suggesting that the Kis-Kid C74Y
complex may assemble in a first step and then be altered, upon
binding to the parD operator sequences or to other proteins of
the regulatory complex, in such a way that autoregulation is impaired
and unneutralized toxic Kid C74Y protein becomes exposed to its
target .
This hypothesis is supported by the recent structural data on the
closely related MazE-MazF TA complex, which argues in favor of a
rearrangement of the complex upon DNA binding (7) . Strikingly,
the C74 residue is bordered by two residues, R73 and D75, that
are essential for the toxic activity of Kid (13) . Moreover,
the region of the homologous MazF toxin that corresponds to the
R73-D75 region of Kid is involved in contacts with the carboxy-terminal
region of the MazE antitoxin, suggesting that the C74 residue
might be crucial for Kis-Kid interactions required for neutralization
as well as for autoregulation . Finally, the E91K mutant protein is
neutralized by the Kis antitoxin, provided that the latter is present
in excess . Perhaps the most striking feature of this mutant is that,
compared with the wild-type toxin, its dramatic cytotoxic effect
requires very low levels of expression . It might be that the E91K
change favors a more efficient interaction of the Kid toxin with its
target, in a manner that is less efficiently competed by the Kis
antitoxin . Support for this hypothesis has been provided by the
structural analyses of the Kid toxin and of the MazE-MazF complexes,
which suggest that the target may compete with the C-terminal region
of the antitoxin to bind to the toxin (7,
4) . Addressing this issue awaits further structural
and functional information on toxin-target complexes, as well as on
autoregulation complexes bound to DNA .
This research was supported by grants from the European Union (grant
QLK2-CT-2000-00634), the Ministerio de Educación y Cultura, Spain
(grant BIO99-0859-CO3-01), the "Programa de Grupos Estratégicos de la
Comunidad de Madrid," 2000-2003, and by the Spanish REIP Network of
the "Fondo de Investigaciones Sanitarias."
* Corresponding author . Mailing address: Centro de
Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain .
Phone: 3491-8373112 . Fax: 3491-5630432 . E-mail: ramondiaz@cib.csic.es.
Present address: Institut für Biochemische Pharmakologie, Universität
Innsbruck, A-6020 Innsbruck, Austria .
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