Department of Molecular Biology, University of Gdask, K
adki 24, 80-822 Gda
sk, Poland1
Centro de Investigaciones Biológicas, C.S.I.C., Velázquez 144, 28006 Madrid, Spain2
Institute of Oceanology, Polish Academy of Sciences, w. Wojciecha 5, 81-347 Gdynia, Poland3
Author for correspondence: Grzegorz Wgrzyn. Tel: +48 58 346 3014. Fax: +48 58 301 0072. e-mail: wegrzyn{at}biotech.univ.gda.pl
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ABSTRACT |
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Keywords: plasmid R1, plasmids, oriJ-based plasmid, inheritance of the replication complex
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INTRODUCTION |
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Previous studies have revealed that Kid inhibits the in vitro replication of a DnaB-dependent replicon, ColE1, at an early stage and that this toxin also prevents the induction of the prophage. Moreover, the overproduction of the DnaB protein (a helicase) results in a considerable increase in the viability of cells expressing the kid gene (Ruiz-Echevarria et al., 1995
). These results led to the original proposal that Kid could target DnaB, thus poisoning the replication complex at the initiation stage (Ruiz-Echevarria et al., 1995
). Alternatively, Kid could interact with another component of the replication complex in a manner that could be disrupted by an excess of DnaB in the cell. However, it remains unknown whether this toxin acts before or after the assembly of the replication complex.
To test at which step Kid inhibits the replication process, we used in vivo systems based on two phage-derived replicons, and oriJ. There are two pathways of replication for both of these replicons. After a round of replication, the replication complex is inherited by one of two daughter plasmid copies and it may function in future rounds of replication; a new replication complex has to be assembled in the second plasmid copy (W
grzyn & Taylor, 1992
; Potrykus et al., 2000
). The heritable replication complex contains
-encoded O and P replication proteins and two bacterial proteins: DnaB and DnaK (a molecular chaperone) (W
grzyn & W
grzyn, 1995
, 2001
; W
grzyn et al., 1995a
, b
, 1996
;
ylicz et al., 1998
; Potrykus et al., 2002
).
It is possible to restrict the replication of plasmids and oriJ solely to the pathway that is based on the heritable replication complex (Fig. 1
). Amino-acid starvation prevents the synthesis of new replication proteins, thus the assembly of new replication complexes is inhibited. Therefore, any replication of the plasmids observed under these conditions is dependent on the activity of the heritable replication complexes (W
grzyn & Taylor, 1992
; W
grzyn et al., 1992
; Potrykus et al., 2000
; Bara
ska et al., 2002
). Amino-acid starvation leads to the rapid relA-dependent synthesis of guanosine 3'-diphosphate 5'-diphosphate (ppGpp; Cashel et al., 1996
). Due to the ppGpp-mediated inhibition of pR promoter activity, and the resulting impairment of the transcriptional activation of ori
, the replication of
plasmids is inhibited in amino-acid-starved wild-type hosts, but it occurs in relA mutants (W
grzyn & Taylor, 1992
; Szalewska-Pa
asz et al., 1994
; Wróbel et al., 1998
; Bara
ska et al., 2002
). Conversely, the replication of oriJ-based plasmids proceeds during amino-acid starvation in both wild-type and relA hosts because an analogue of the
pR promoter is not sensitive to ppGpp (Potrykus et al., 2000
). Therefore, plasmids
and oriJ seem to be very useful tools for in vivo studies on the step(s) of action of Kid in the inhibition of DNA replication.
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METHODS |
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Estimation of plasmid DNA replication.
The replication of plasmid DNA in host cells was investigated as described previously (Herman et al., 1994a ; Szalewska-Pa
asz et al., 1994
). Briefly, 5 ml samples of bacterial cultures were withdrawn at 30 min intervals for 4 h. The samples were centrifuged (4000 g, 5 min), and the bacterial pellets were used for the isolation of plasmid DNA by alkaline lysis (Sambrook et al., 1989
). Following its linearization via a restriction digest, the plasmid DNA was subjected to agarose-gel electrophoresis. (Enyzmes that cut a given plasmid at a unique site were chosen. These were purchased from Fermentas and reactions were performed according to the manufacturers instruction.) After staining the gel with ethidium bromide, the intensity of the plasmid bands, which corresponded to the relative amounts of plasmid DNA, was measured by densitometry, using the UVP E.A.S.Y. densitometry system. To assess whether this assay was quantitative, different volumes of a culture of plasmid-harbouring bacteria were withdrawn and used for plasmid DNA isolation. In addition, an equal amount of an external control (purified DNA from another plasmid) was added to each sample. Following electrophoresis and densitometry, we estimated that the values obtained from the test samples differed from the expected values by <10%.
Measurement of plasmid DNA synthesis.
Plasmid DNA synthesis was measured as described by Herman et al. (1994b ) and Potrykus et al. (2000)
. Briefly, the method was similar to that for the Estimation of plasmid DNA replication, but samples of equal cell mass (1 OD unit) were withdrawn and centrifuged. Following the suspension of the pellets in fresh medium (0·5 ml per sample), [3H]thymidine was added up to 20 µCi ml-1 (740 kBq ml-1). After 5 min of pulse-labelling, NaN3 was added to a concentration of 20 mM. The cells were then lysed, and the plasmid DNA was isolated and linearized with restriction enzymes and separated by agarose-gel electrophoresis. The amounts of plasmid DNA present in the gel were estimated by densitometry. Plasmid bands were then excised from the gel, and the agar blocks were dissolved in 2 M HCl at 60 °C overnight. The radioactivity of each sample was then measured in a scintillation counter.
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RESULTS |
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Since amino-acid starvation inhibits protein synthesis, the induction of kid expression by the addition of IPTG was provoked 10 min before the induction of isoleucine starvation. ColE1-like plasmids have been shown previously to replicate in isoleucine-starved relA mutants, though by a mechanism different from the inheritance of the replication complex (Wróbel & Wgrzyn, 1997
, 1998
). Hence, the putative target for Kid is not protected in ColE1-like plasmids. By monitoring the replication of a ColE1 replicon, we found that the induction of Kid production 10 min before the onset of amino-acid starvation was sufficient to inhibit the replication of plasmid DNA (Fig. 2
). In these experiments, equal volumes of the culture were withdrawn at the times indicated in Figs 24
, from which plasmid DNA was isolated and linearized by a restriction digest. After separation of the DNA by agarose-gel electrophoresis, the amount of plasmid DNA was estimated by densitometry. Thus, any increase in the amount of plasmid DNA present would indicate replication, whereas the constant value in Fig. 2(a)
shows inhibition of plasmid replication. When the replication of
plasmid DNA was investigated under the same conditions, the amount of plasmid DNA increased both in the bacteria devoid of Kid and in the cells producing this protein (Fig. 3
).
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Effects of Kid on the replication of the oriJ-based plasmid in non-starved cells
If the hypothesis that Kid acts as a toxin only before the assembly of the replication complex was true, one should be able to observe some effects of kid expression on the replication of plasmids employing the inheritance of the replication complex mechanism in non-starved cells, i.e. when both pathways of plasmid DNA replication operate (Fig. 1). Indeed, we found that the replication of the oriJ-based plasmid was inhibited by Kid in cells cultivated under standard growth conditions, but that this inhibition was moderate (Fig. 5
). These results are compatible with the tested hypothesis.
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DISCUSSION |
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Four lines of evidence support the hypothesis that the Kid toxin acts before, but not after, the assembly of the replication complex. First, Kid loses its inhibitory potential in the in vitro DNA replication of the ColE1 plasmid when it is added several minutes after the initiation of replication (Ruiz-Echevarria et al., 1995 ). Second, Kid does not inhibit the replication of plasmids
and oriJ in amino-acid-starved cells, i.e. under conditions that only allow replication based on previously assembled heritable replication complexes (Figs 3
and 4
). Under the same conditions, the replication of a ColE1-like plasmid, which does not form a heritable replication complex, was inhibited by the expression of kid (Fig. 2
). Third, oriJ plasmid replication was impaired by Kid in non-starved cells; however, the inhibition of replication was not complete, indicating that a significant number of plasmids could still replicate (Fig. 5
). Fourth, experiments that measured the incorportation of [3H]thymidine into
plasmid DNA in non-starved cells expressing kid revealed that the kinetics of plasmid DNA synthesis agree only with the model when it is assumed that after each round of replication only half of the plasmid molecules enter the next round of replication (Fig. 6
).
Our experiments show that plasmid replication based on newly assembled replication complexes, but not that carried out by heritable replication complexes, is inhibited by the Kid toxin. However, these results do not answer the question as to what is the specific target for Kid. Previous studies have indicated that overexpression of dnaB alleviates some of the effects of Kid (Ruiz-Echevarria et al., 1995
). We have also found that the overproduction of DnaB, but not of DnaC, partially suppresses Kid-mediated inhibition of bacterial growth, and that an excess of DnaB in cells does not influence Kid production in our experimental system (data not shown). Therefore, one possibility is that DnaB may be a target for Kid. However, DnaB-mediated alleviation of Kid toxicity might be indirect; hence, the hypothesis that a factor other than DnaB is a real target for Kid is equally possible. Clearly, additional experiments are necessary to specifically address this problem.
It also remains to be elucidated whether Kid has access to its target (DnaB or another component of the replication complex) once the replication complex has been assembled or whether the target protein becomes resistant to Kid after binding to DNA and/or to other proteins. In fact, the protection of particular target proteins from other proteins has been reported previously. For example, the O protein is rapidly degraded by the ClpP/ClpX protease, but it is protected from this protease after the formation of the
replication complex (W
grzyn et al., 1992
, 1995a
;
ylicz et al., 1998
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 May 2002;
accepted 16 May 2002.