Kid, a small protein of the parD stability system of plasmid R1, is an inhibitor of DNA replication acting at the initiation of DNA synthesis

J. MoL BioL (1995) 247, 568-577

JMB Kid, a Small Protein of the parD Stability System of Plasmid R1, is an Inhibitor of DNA Replication Acting at the Initiation of DNA Synthesis Maria Jesds Ruiz-Echevarria, Guillermo Gim6nez-Gallego Rosario Sabariegos-Jare o and Ramon Diaz-Orejas* Centro de Investigaciones Biol6gicas, C.S.I.C. Veldzquez 144, 28006 Madrid, Spain

*Correspondis~ author

The Kid and Kis proteins are the killer component and the antagonist belonging to parD, a killer stability system of plasmid R1. The Kid and Kis proteins have been purified, the second one as a C-LYT-Kis fusion that conserves the antagonistic activity of the Kis protein, but not its auto-regulatory potential. Kid inhibits in vitro replication of CoE1 to a basal level without altering the superhelicity of the template but it does not substantially affect in vitro replication of P4, a DnaA, DnaB, DnaC and DnaG-independent replicon. Kid inhibits lytic induction of a lambda, prophage, but this inhibition can be neutralized by excess DnaB. In addition, a multicopy dnaB recombinant, but not a multicopy dnaG recombinant, prevents the toxicity associated with this protein. Inhibition of ColE1 replication by Kid in vitro is prevented by the C-LYT-Kis protein. Functional analysis indicates that the antagonistic activity of Kis is independent of its activity as a co-regulator of the parD promoter. It is also shown that C-LYT-KIs and Kid interact, forming a tight complex. These results strongly suggest that the toxicity of the kid protein is due to inhibition of DnaB-dependent DNA replication, and that direct protein-protein interactions are involved in the neutralization of the activity of the killer protein by the antagonist.

Keywords: plasmid R1; parD system; Kid assay; Kis assay; DnaB-dependent DNA replication

Introduction The parD system is a killer stability system of plasmid R1 located in the proximity of the basic replicon of this plasmid. This system is organized as a small operon containing two genes, kid and kis, coding for a killer component and its antagonist, respectively (Bravo et al., 1987). The parD system mediates plasmid stabilization by a mechanism that involves killing plasmid-free segregants due to the action of the Kid protein. The Kis protein neutralizes the lethal activity of the Kid protein (Bravo et al., 1988). In addition, the Kis protein, together with Kid, coordinately represses transcription from the parD Abbreviations used: Brij-58, polyoxyethylene-20-cetyl ether; C-LYT, carboxy-terminal region of the N-acetylmuramoyl-L-alanine amidase from Streptococcus pneumoniae; factor Xa, serine protease; LBT, L-broth agar; LAT, L-broth agar supplemented with 20 ~g/ml thymine; LYS, lysozyme; PEG, polyethylene glycol; BSA, bovine serum albumin; OVA, ovoalbumin. 0022-2836/95/140568-10 $08.00/0

promoter (Ruiz-Echevarria et al., 1991a). This system is perfectly conserved and functional in another incFII plasmid, R100 (peru system: Tsuchimoto et al., 1988). Coordinated repression of the peru promoter by the two protein products of the system, PemA (identical to Kis) and PemB (identical to Kid), has also been reported (Tsuchimoto & Ohtsubo, 1993). It was important to test if Kis and Kid interacted physically. These interactions could modulate the repression of the parD promoter by the concerted action of Kis and Kid, and also the neutralization of the killer activity of Kid by Kis. Indirect evidence indicates that interactions between the Kis and Kid proteins could also influence the stability of the Kis protein (Bravo, 1988). The target of the killer protein of the parD (peru) system is not known. Identification of this target and the development of an in vitro assay for Kid and Kis proteins is important to fully understand the mechanism of action of parD and to further analyze functional interactions between the two proteins. The killer protein of the ccd stability system of ~9 1995AcademicPress Limited

569

Kid Inhibits Initiation of DNA Replication

ptasmid F targets subunit A of the gyrase (Bernard & Couturier, 1992; Miki et al., 1992). The parD and ccd systems have a similar genetic organization, and there is significant homology between the antagonists of both systems; however, the killer components of these systems share no homolog34 and they differ at the functional level, suggesting that they could reach different targets (Ruiz-Echevarria et al., 1991b). This theory is supported by the fact that a gyrA mutant that prevents killing by the lethal component of the ccd system (Bernard & Couturier, 1992) does not prevent the lethal action of the Kid protein (M. J. Ruiz-Echevarria, unpublished results). We now report the purification of the Kid and Kis proteins, the latter as a C-LYT-Kis fusion, and show that they interact, forming a tight complex. On the basis of in vivo and in vitro assays with different replication systems, it is shown that Kid is a potent inhibitor of DNA replication, probably acting at the initiation level and on the DnaB protein. It is also shown that the C-LYT-Kis protein is able to neutralize the inhibition of DNA replication mediated by the killer component, and that the auto-regulatory potential of this protein is not essential for its antagonistic activit}~ It is also shown that C-LYT-Kis and Kid form a strong complex, suggesting that physical interactions between Kis and Kid are important for the neutralization of the lethal action of Kid by Kis.

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Results Purification of a hybrid C-LYT-Kis protein by affinity chromatography The enzyme N-acetylmuramoyl-L-alanine amidase from Streptococcus pneumoniae has a strong binding site for choline in its carboxyl-terminal region (C-LYT) that can also bind tertiary amines with high affinity This allows purification of the protein in a single chromatographic step using a common solid support such as DEAE-Sephacel (Sanz et al., 1988). This property has been successfully used to purify acidic fibroblast growth factor and certain peptides through the construction of hybrid proteins between C-LYT and the protein of interest (Ortega et al., 1992). Purification of the Kis protein of the parD system was attempted following the procedure of Ortega et al. (1992). Plasmid pMJR2 carries the C-LYT coding region (lytA) of plasmid pCE17 fused in frame with the kis gene (Figure 1 and Materials and Methods). In addition, the factor Xa recognition sequence was included at the amino terminus of Kis (Figure 1) in order to release an unmodified Kis protein from the fusion protein after treatment with the factor Xa. Expression of the hybrid lytA-kis gene in this plasmid is under the control of the Ipp constitutive promoter and the lac inducible promoter (Figure 1), allowing

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Kid Inhibits Initiation of DNA Replication

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controlled over-expression of the C-LYT-Kis hybrid protein. Plasmid pMJR2 was established in E. coli strain AB18991ac + (Zazo et al., 1992). After lactose induction, chromatography on DEAE-Sephacel of a crude homogenate of a culture of AB18991ac÷/ pMJR2 was carried out. The protein(s) eluted in a single peak after addition of a buffer containing 143 mM choline chloride (data not shown). SDSPAGE, under reducing conditions, indicated that the eluent contained a single polypeptide of 24 kDa, the expected size of the C-LYT-Kis fusion protein (Figure 2). A small band above the 42.7kDa molecular mass marker can be detected in these preparations. This band could correspond to a contaminating protein with affinity for the DEAE matrix, or to dimers of the C-LYT-Kis fusion stabilized by covalent bond(s). The fact that the introduction of four additional amino acid residues to the C-LYT-Kis fusion (processing site of the Xa factor) shifts this band slightly up (data not shown) favors the last alternative. Attempts to release the Kis protein from C-LYT-Kis fusion through digestion with serine protease (factor Xa) were unsuccessful, even after the addition of 1 M urea to increase the accessibility of factor Xa to its target. Since it has been reported that many proteins fused to a heterologous peptide still conserve their activity (Ortega et al., 1992; Germino & Bastia, 1984), we decided to test whether the C-LYT-Kis fusion protein retains the two known activities of the Kis protein: the ability to neutralize the lethal action of Kid, and the capacity to act as a co-repressor of the parD promoter. The antagonistic activity was tested in vivo by evaluating the ability of the pMJR2 (lytA-kis) recombinant to restore the growth at 42°C of a supF(ts) E. coli strain (OV2) containing plasmid pAB112, a mini-R1 derivative carrying an amber mutation in the kis gene. At 42°C

in a supF(ts) background, a truncated Kis protein is synthesized which is unable to antagonize the lethal activity of Kid, leading to cell death (Bravo et al., 1988). The data in Table 1 indicate that the presence of plasmid pMJR2 in h:mTs to pAB112 suppresses the growth inhibition at 42°C observed either when pAB112 is present alone, or when the vector plasmid pEC17 is present in trans to pAB112. This result indicates that the C-LYT-Kis fusion protein is able to antagonize the lethal action of Kid. The autoregulatory activity of the C-LYT-Kis protein was also tested. For this purpose, we made use of the transcriptional fusion plasmid pOM820, in which the expression of the lacZ gene is under the control of the parD promoter (Ruiz-Echevarrfa et al., 1991a). pBR322 recombinants carrying the wildtype kis and kid genes in trans to pOM820 reduce ~-galactosidase expression from pOM820 to the basal level (Ruiz-Echevarria et al., 1991a). However, the ~-galactosidase activity expressed from plasmid pOM820 in E. coli strain CSH50 was virtually the same either in the absence or in the presence of plasmid pMJR1 (lyt-kis, kid) (data not shown). Since the Kid protein expressed from plasmid pMJR1 is in its wild-type state, the lack of autoregulatory activity must be due to the fact that the Kis protein is fused to the C-LYT peptide. Together, these results suggest that the amino terminus of Kis is important for autoregulation, and that the fusion of the C-LYT peptide to this end interferes with the regulatory activity of Kis, but not with the ability of this protein to neutralize the toxic effects of the Kid protein. Kid and C-LYT-Kis complex

interact and form a

Minicell analysis of proteins expressed by cells containing the pAB17 (kis-17, kid) and the pAB174 plasmids (kis-17, Akid) indicated that Kis-17 is very unstable in the absence of Kid (Bravo, 1988). These preliminary data suggested physical interactions between the Kis and Kid proteins. We therefore attempted to purify a complex of the C-LYT-Kis and Kid proteins from a strain containing an over-producer of both proteins. The purification was performed with extracts prepared from AB18991ac ÷ cells containing plasmid pMJR1 previously induced to overproduce the C-LYT-Kis and Kid proteins (see

Table1 Complementation of kis(am) mutant by a lytA-kis recombinant Viable cells/ml Plasmid in OV2 Relevantgenotype 30°C 42°C pAB112 kis(am), kid 1.0 x 107 <105 pAB112/pMJR2 kis(am),kid/lytA-kis 4.0 x 107 5.0 x 107 pAB112/pEC17 kis(am),kid/lytA 6.3 x 107 <105 A pool of 10 coloniesfroman overnightculture grown at 30°C in LATplates was resuspended in LBT,appropriatelydiluted and platedontoLAT.Plateswereincubatedat 30°Cor 42°C,and colony counting was done after 16 h of incubation.

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Materials and Methods). After the addition of choline, proteins eluted from the column in a single peak (Figure 3A). SDS-PAGE analysis of the different fractions of the peak which were collected detected, in the first fractions, a polypeptide with the same molecular mass as the Kid protein (12 kDa), in addition to another one of 24 kDa, corresponding to the C-LYT-Kis polypeptide (Figure 3B). Two types of data confirmed that the 12 kDa band corresponded to the Kid protein: (1) deletion of the kid gene is associated with loss of the 12 kDa band (Figure 3B), and (2) the sequence of the first six amino acid residues of this short protein (Met-Glu-Arg-Gly-GluIle) is identical to the sequence of the first six amino acid residues of Kid. To determine if the co-elution of the two proteins is due to the formation of a complex between the Kis and Kid proteins, or to independent interaction with the DEAE matrix, we carried out sedimentation analyses of early and late fractions in glycerol gradients (Figure 4) that were enriched with the C-LYT-Kis/Kid proteins or the C-LYT-Kis protein, respectively Free C-LYT-Kis protein (late fractions) sedimented with a velocity corresponding to 50 kDa, which suggests that the fused Kis protein is a dimer in solution. However, analysis of early fractions indicated that the fused Kis protein and the

Figure 4. Glycerol gradient analysis of the C-LYT-Kis protein and of the C-LYT-Kis/Kid complex. The Figure shows a densitometric analysis of SDS-PAGE gels corresponding to the different fractions of the glycerol gradients containing either C-LYT-Kis, a C-LYT-Kis/Kid complex or a mixture of lysozyme (LYS), bovine serum albumin (BSA) and ovoalbumin (OVA). Arrows indicate the position of the peak corresponding to the mixture of 3 protein standards. The molecular mass (MW) corresponding to each of the protein standards is also indicated.

Kid protein form a complex that migrates with a velocity corresponding to that of a globular protein of 70 kDa. These data suggest also that the complex could be formed from two subunits of each protein. Purification of the Kid protein from the C-LYT-Kis/Kid complex Attempts to construct a recombinant in which the expression of Kid was under the control of a regulated promoter failed, probably due to basal expression of this cytotoxin. We therefore attempted purification of the Kid protein to homogeneity through dissociation from the C-LYT-Kis fusion protein. Estimation of the isoelectric points for the Kis and Kid proteins indicated that the Kis protein is acidic (pi=4.45), while Kid is highly basic (pI = 10.62). This suggests that ionic interactions could be important in maintaining the C-LYT-Kis/ Kid complex. However, two types of data indicated that non-ionic interactions are involved in the formation of a complex between the C-LYT-KIs and Kid proteins: (1) attempts to separate the proteins by isoelectrofocusing were unsuccessful (data not shown), and (2) dissociation of the Kid protein from the C-LYT-Kis/Kid complex by reverse phase chromatography required that the protein mixture had been denatured by treatment with 6 M guanidinium chloride. Under these conditions, the proteins eluted in three different peaks (Figure 5A). Analysis of the protein content of these fractions in SDS-PAGE under reducing conditions indicated the presence of one single polypeptide in each peak that

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corresponded to the C-LYT-Kis (first two peaks) or the Kid protein (Figure 5B). The finding that the C-LYT-Kis eluted in two different peaks remains unexplained at present. T h e Kid protein inhibits initiation of D N A replication

We found that Kid prevented induction of a lytic lambda prophage. This was tested using a thermosensitive supF(ts) strain of E. coli, OV2, carrying a lambda lysogen and a multicopy parD recombinant, pMJR112, that contains an amber mutation in the antagonist. These cells lose viability shortly after shifting the cultures from 30°C to 42°C, indicating activation of the killer due to inactivation of the antagonist (Ruiz-Echevarrfa et al., 1991b; Bravo et al., 1987). Lytic induction of lambda prophage was triggered by ultraviolet light irradiation, and amplification of lambda DNA sequences was detected in cultures g r o w n at 30°C, but not in cultures shifted to 42°C (data not shown). These observations strongly suggest that Kid interferes

Figure 6. Effect of the Kid protein on the in vitro replication of plasmid DNA. A, Effect of Kid on ColE1 or P4 DNA replication. Assays were performed as indicated in Materials and Methods, in the presence or absence of 0.3 mg of Kid. Lanes 1, 3, 5 and 7 show the ethidium bromide picture; and lanes 2, 4, 6 and 8 the autoradiogram revealing replication. The presence or absence of Kid in the assays and the substrate used in replication are indicated. B, Effect of increasing concentrations of Kid on ColE1 and P4 replication. Quantification of the replication values was as indicated in Materials and Methods. C, Percentage of replication accumulated 40 rain after initiating the reaction, versus the time of addition of the inhibitor (0.15 mg Kid, or 50 mM EDTA).

with lambda DNA replication, and that the killing of the cells could be mediated by inactivation of an essential protein required for replication of the host c h r o m o s o m e and the lambda DNA. The finding that the killer protein of the parD system did not trigger the SOS response (Ruiz-Echevarri et al., 1991b) suggests that it probably acts on replication proteins involved in initiation of DNA replication. The DnaB or DnaG proteins appear as possible targets for Kid because they are initiation proteins required for oriC and lambda DNA replication. DnaA and DnaC, which are also initiation proteins of the host, are less probable candidates, because they are dispensable for lambda DNA replication. We subsequently tested the effect of purified Kid protein on the in vitro replication of plasmid ColE1, the initiation of which is independent of DnaA but d e p e n d e n t on DnaB, DnaC and DnaG, and on the in vitro replication of phage P4 DNA, a process that is independent of the DnaA, DnaB, DnaC and DnaG proteins (Dfaz-Orejas et al., 1994). It was found that Kid can inhibit ColE1 replication at a basal level, but that it has only a minor effect on P4 replication (Figure 6A and B). Inhibition of ColE1 replication by Kid was not due to relaxation of the template, which indicates that the target is not the DNA gyrase. Further analysis indicated that Kid

573

Kid Inhibits Initiation of DNA Replication

Table 2 Protection of the inhibitory action of Kid by a dnaB or dnaG recombinant Viable cells/ml Strain Relevant genotype 30°C 40°C OV2 supF(ts) 525 578 OV2/pABll2 supF(ts)/kis(am), kid 670 1 OV2/pABll2/pMJR-dnaB supF(ts)/kis(am), kid/dnaB 632 612 OV2/pABll2/pSM1 supF(ts)/ kis(am), kid/ dnaG 730 0 Viable cells were obtained from cultures growingat 30°Cat a cell density of 3 x 108 ceUs/ml.The cultures were diluted to give500 coloniesin 0.1 ml, and cells were plated on LAT-agarplates, which were incubatedat 30°Cor at 42°C.Therelativenumberofcoloniesappearingin eachcasewasdetermined after incubationfor 18 h.

is completely inactive ten minutes after initiating the reaction (Figure 6C). Note that after ten minutes of incubation in the absence of Kid, the number of molecules replicated is below 20% of the control. This result, and the loss of efficiency of Kid at later stages of replication, suggest that the killer protein of parD inhibits DNA replication at an early stage.

Killing mediated by Kid can be prevented by a dnaB recombinant but not by a dnaG recombinant The above data suggest that either DnaB or DnaG could be the target of the killer protein. An in vivo approach to evaluate if inhibition of DnaB or DnaG was responsible for the killing mediated by Kid was to test if increasing the levels of DnaB or DnaG protein in the cells could prevent the lethal action of Kid. Expression of Kid in this experiment was achieved using a supF(ts) background, and a parD mutant carrying an amber mutation in the antagonist: shifting these cultures to high temperature inactivated the antagonist and revealed the lethal effects of Kid. The increase in the DnaB or DnaG levels was achieved by introducing into the cells a multicopy dnaB or dnaG recombinant. Table 2 shows that the multicopy dnaB recombinant, but not the multicopy dnaG recombinant, prevents the lethal action of Kid. Further analysis indicated that an excess of DnaB promoted by a multicopy dnaB recombinant can prevent the inhibition of lambda DNA replication mediated by Kid (data not shown). These results strongly suggest that killing mediated by Kid is due to inhibition of the activity of DnaB, a protein that is an essential component of the host replication machiner}~

C-LYT-Kis neutralizes the inhibition of DNA replication promoted by Kid Inhibition of DNA replication by Kid permitted an assay for the antagonist: neutralization of this inhibition. It was found that C-LYT-Kis was able to neutralize the inhibition of ColE1 replication mediated by Kid (Figure 7A and B), and that it does not have an effect on its own on ColE1 replication. Increasing the amount of C-LYT-Kis above the levels needed to neutralize the effect of Kid leads to an

unexpected stimulation of ColE1 replication. This is due to a combined effect of both proteins, because C-LYT-Kis alone does not stimulate ColE1 replication (Figure 7B). Figure 7C shows that the neutralization is maximally efficient when C-LYT-Kis is added simultaneously to Kid, but can also be detected when this protein is added five to ten minutes after incubation of the replication assays at 30°C in the presence of the killer (Figure 7C). This clearly indicates that the antagonist prevents the killer protein reaching its target.

Discussion In this paper we describe the purification and functional evauation of the two proteins of the parD system of plasmid R1. Kis, the antagonist of this system, was purified as a fusion protein, C-LYT-Kis, by using affinity chromatography Kid, the killer component, was purified taking advantage of its association with C-LYT-Kis. The availability of the purified proteins opened the way for an in vitro assay On the basis of in vivo and in vitro replication assays, we propose here that the killer protein of parD is an inhibitor of DnaB-dependent DNA replication, acting at the initiation stage, h~ vivo assays demonstrate that expression of the killer protein interferes with lytic induction of a lambda prophage at a stage after the inactivation of the cI repressor. This result pointed to interference by the killer protein in the replication of the lambda prophage, and implied that killing of the host cells could be due to interference with an essential component of the chromosome replication machiner3~ In addition, Kid is able to inhibit in vitro replication of plasmid ColE1 to a basal level, but it does not have an effect (or only a minor one) on P4 replication, which is independent of DnaA, DnaB, DnaC and DnaG proteins (DfazOrejas et al., 1994). The inhibitory action of Kid in ColE1 replication seems to occur at the initiation stage, since Kid is maximally active when added at time zero, and its activity decreases when added in the lag period during which assembly of the replication complexes occur. DnaB and DnaG are replication proteins acting at the stages of initiation that are required for ColE1 replication and lambda replication, but that are dispensable for P4 DNA replication. Therefore, either one of them could be a

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possible target for the action of the Kid protein. Two observations strongly suggest that the DnaB protein, not the DnaG protein, is the target of Kid: (1) Kid-mediated reduction of ColE1 replication to basal levels parallels the strong inhibition observed by the inactivation of DnaB protein; on the contrar~ inactivation of DnaG by mutations or DnaG antibodies still allows replication of ColE1 to a substantial level (Staudenbauer et al., 1979; Ortega et al., 1986). This different behavior reflects the fact that DnaB is involved in leading and lagging strand synthesis of ColE1, while DnaG is only strictly required for lagging strand synthesis (Staudenbauer et al., 1979). (2) Kid-mediated killing of the host cells can be prevented by a

multicopy dnaB recombinant, but not by a multicopy dnaG recombinant. It can be argued that the target is the DnaC protein, and that overproduction of DnaB favors DnaB-DnaC interactions that could protect DnaC from Kid. However, this alternative seems improbable, due to the ability of Kid to inactivate lambda replication, which is independent of DnaC but dependent on DnaB. Excess DnaB also prevents the inhibition of the lytic induction of lambda by Kid. An excess of DnaB protects against the action of Kid, due probably to a titration effect. Titration of a killer protein by increasing the levels of its target has been reported for the CcdB killer protein and its target, the DNA gyrase (Miki et al., 1992). The inhibition of DNA replication by Kid can be neutralized by the C-LYT-Kis protein. This behavior parallels the neutralization of the killing activity of Kid by Kis, and indicates that the assay of the killer has the required specificit~ It is clear that the C-LYS-Kis protein can prevent the Kid protein reaching its target, and it cannot be discounted that, as in the ccd system, the antagonist could rejuvenate a target inactivated by the killer protein (Maki et al., 1992). As C-LYTKis has lost its capacity to co-regulate transcription from the parD promoter, the data indicate that this activity is not essential for its anti-Kid activity in vitro. An interesting observation is the fact that the concerted action of Kis and Kid can stimulate ColE1 replication by 280%. This stimulation is not observed when replication is assayed in the presence of C-LYT-Kis alone. This observation has not been further explored in this work, but indicates complex relations between parD and the replication machinery of the host, and deserve careful analysis. In this paper we also describe the capacity of the two proteins of parD to interact and form a strong complex. The formation of a complex of the CcdA and CcdB proteins of the killer stability system ccd of plasmid F (functionally homologous to parD), and the possible role of this complex in autoregulation and neutralization of the lethal action of CcdB, has been reported (Tam & Kline, 1989; Miki et al., 1992). A Kis/Kid complex could explain the coordinated action of the two proteins in the regulation of the parD promoter (Ruiz-Echevarria et al., 1992). C-LYT-Kis is able to interact with Kid, but it is inactive as a co-repressor of the ParD promoter. This clearly indicates the relevance of the amino terminus of the protein for regulation and its dispensability for its anti-killer activit~ The homologies between the amino- and carboxy-terminal regions of the Kis and CcdA proteins (Ruiz-Echevarria et al., 1991b), the phenotypes of kis mutants affected at 5' or 3' ends of the kis gene (Bravo et al.," 1987, 1988), and the localization of the antagonizing activity of CcdA in its carboxy-terminal region (Bernard & Couturier, 1991), favor the existence in Kis of an amino-terminal domain involved in autoregulation, and a carboxyterminal domain involved in neutralization of the killer.

Kid Inhibits Initiation of DNA Replication

Materials and Methods Bacterial strains and plasmids The E. coli K12 derivative strains used in this work were: AB1899 (F-, thr-1, ara-14, leu-6, ([FS3]gtp-proA )62, lacY-l,

tsx-33, Ion-l, supE44, gaIK-2, hisG-4, rfbD-1, rpsL-31, kdgK-51, xyl-5, mtl-1, argE-3, thi-1) made lac* by transduction with phage P1 (Zazo et al., 1992); CSH50 (Alac-pro , rpsL ) (Miller, 1972); OV2 (F-, leu , thyA, ara(am ), lac-125(am), galU-42(am), galE, trp(am), tsx(am), tyrT, supF(ts)A-81) (Donachie et al., 1976); OV2 (lambda into-540) (supplied by M. Vicente); and C600 (leu, thr, thi, SupE-44, lacY-l, tonA-21) (Bacl~nann, 1987). Mini-R1 derived plasmids used were: pAB112, a mini-R1 parD derivative carrying a kis(am) mutation (Bravo et al., 1988) and pOM820, a mini-R1 derivative carrying a parD promoter-lacZ transcriptional fusion (Ruiz-Echevarrfa et al., 1991a). ColE1 plasmid (Bazaral & Helinski, 1968) and DNA of phage P4, obtained from P4 vir-1, an immunity-sensitive P4 phage (Lindquist & Six, 1971 ), were used in the in vitro replication assays, pMJ1724 (lytA-kis, kid), pMJR1 (lytA-fxa-kis) and pMJR2 (lytA-fxa-kis, kid) were different parD recombinants (see plasmid constructions) based on the pEC17 lytA fusion-expression vector (Sanchez-Puelles et al., 1990). pMJR112 is a pBR322-parD recombinant carrying the kis(am) mutation of pAB112. pMJR-dnaB is a recombinant based in the pBR322 replicon, which expresses dnaB at high temperature, pSM1 is a multicopy dnaG recombinant in which expression of dnaG is under the control of the lacI-tac promoter system (supplied by E. Lanka). Media The liquid and solid media used were L-broth agar (LBT) and L-broth agar supplemented with 20 ~g/ml thymine (LAT). When required, ampicillin was added at 50 ~g/ml, and kanamycin at 25 or 50 ~g/ml.

DNA isolation and manipulations DNA extractions, construction of recombinant plasmids, transformations and purification of plasmids were essentially according to Sambrook et al. (1989). P4 DNA was obtained from P4 (vir-1) as described (Strack et al., 1992). The Sanger method was used for DNA sequencing (Sanger et al., 1977).

Plasmid constructions Plasmid pMJ1724 (see Figure 1) was constructed by cloning an NsiI-HindIII fragment from pAB24 (Bravo et al., 1987), carrying the kis gene, with the first two codons deleted (Met and His), and the complete kid gene, between the EcoRI-HindIII sites of pCE17 (Sanchez-Puelles et al., 1990). For this purpose, the NsiI and EcoRI ends of the insert and vector were converted into blunt ends by treatment with phage T4 DNA polymerase and Klenow polymerase, respectively; subsequentl}~ the blunt-ended fragments (insert and vector) were ligated. In pMJ1724 the lytA gene is fused in frame with the third codon of the kis gene (Figure 1). To construct plasmid pMJR1, we introduced, by site-directed mutagenesis, the recognition sequence of the Xa factor (a serine protease) and the second codon of the kis gene between the sequences corresponding to the lytA and kis genes (Figure 1). pMJR2 was derived from pMJ1724 by deletion of the complete kid coding

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sequence, followed by introduction of the recognition sequence of factor Xa and the second codon of the kis gene between the sequences of the lytA and kis genes (Figure 2). pRLM31 (R. McMaken) is a vector based on an R1 mutant mini-plasmid that replicates autocatalytically at high temperature (run-away), and which contains the dnaB gene under the control of the lambda Pr promoter and the lambda ci857 thermosensitive repressor. To construct pMJR-dnaB, the dnaB gene was obtained from pRLM31 as a HindIII-EcoRI fragment, and this fragment, which carries a kanamycin resistance determinant, was inserted directionally between the HindIII and EcoRI sites of pBR322. The recombinant expresses flnaB at high temperature, contains the ampicillin resistance of the vector and the kanamycin resistance of the insert, and is sensitive to tetracycline. This recombinant, in contrast to pRLM31, does not contain the parD system. In vitro replication assays

Type II extracts were prepared from strain C600 as described (Staudenbauer, 1984). Standard reaction mixtures (25 ~1) contained the following components: 25 mM Hepes-KOH (pH 8.0), 10 mM KC1, 0.25 mM EDTA, 20 mM Mg(CH3COOH)2, 1 mM DTT, 4 mM ATP, CTP, GTP and UTP (each 0.1 mM), dATP, dCTP and dGTP (each 25 ~¢I), [32P]dCTP, 10 ~M at a specific activity of 3000 Ci/mmol, 25 mM NAD, 25 mM cAMP, 3 mM creatine phosphate, 0.1 m g / m I creatine kinase, P4 DNA or ColE1 DNA (at 0.3 ~tg/ml), 8 m g / m l E. coil proteins of a 0 to 70% ammonium sulfate fraction and 2.5% (w/v) PEG6000. The mixtures were incubated first for ten minutes at 0°C and then for 40 to 60 minutes at 30°C, unless otherwise indicated. Reactions were stopped with 50 mM EDTA, then the proteins were extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1, byvol.) and once with chloroform/isoamyl alcohol (24:1, v/v), and the DNA was precipitated with ethanol, washed and dried. After resuspension in 15 ml TE buffer (10 mM Tris-HC1 (pH 7.5), 0.1 mM EDTA), the samples were run in an agarose gel that was dried and tested by autoradiography The autoradiographic signal corresponding to plasmid replication was quantified using a phosphorimager (Molecular Dynamics). The values from the different samples are given as the percentage of the untreated control.

Assay for ~-galactosidase activity The ~-galactosidase activity expressed in vivo was determined from exponentially growing cultures essentially as described by Miller (1972).

Synthesis and purification of the fusion protein Culture and induction of AB18991ac*/pMJR1 strains were as described (Zazo et al., 1992). For purification of the fusion protein, we followed the procedure described by Ortega et al. (1992). Brief134 the cell lysate in 50 mM Tris-HC1 (pH 6.5) was collected from the French press directly onto DEAE-Sephacel equilibrated with the same buffer, the mixture was incubated for 60 minutes at 4°C with mild shaking, and the solid phase was packed in a chromatography column after being collected in a wide-pore sintered glass funnel, where it was washed with an excess of 50 mM Tris-HC1 (pH 6.5), 1.5 M NaC1. The column was rinsed with the washing buffer until the eluate absorbance fell to a near background level, and then re-equilibrated with the same buffer containing 100 mM NaC1. Once the eluate absorbance stabilized again at the

576

background level, the retained protein was eluted with 143 mM choline chloride in 50 mM Tris-HCl (pH 6.5).

Separation of the C-LYT-Kis and Kid proteins by reverse phase chromatography and protein characterization The mixture of C-LYT-Kis and Kid proteins obtained after chromatography on DEAE-Sephacel was treated with guanidinium chloride to a concentration of 6 M, and subjected to reverse phase chromatography using a HPLC-C4 column equilibrated in 10 mM trifluoroacetic acid prior to loading. Proteins were eluted with a linear gradient of a mixture of 2-propanol/acetonitrile/water (6:3:1, by vol.) in 4 mM trifluoroacetic acid. The protein samples were dialyzed against a buffer containing 50 mM Tris-HCI (pH 7.4), 0.1 mM EDTA and 6 M guanidium chloride. The concentration of the guanidium chloride was reduced to 3 M in a second dialysis stage, and this compound was removed from the dialysis in the third stage. N-Terminal protein sequencing was carried out in a pulse-liquid Applied Biosystem 477 microsequencer switched on line to an Applied Biosystem 120A phenylthiohydantoin amino acid analyzer. Protein (5 rag) in the HPLC elution buffer was loaded into the glass filter of the microsequencer. SDS-PAGE gels (15% (w/v) acrylamide, 44% (w/v) acrylamide/0.8% (w/v) bis-acrylamide) were run and processed basically as described by Zazo et al. (1992).

Analysis of protein complexes in glycerol gradients Glycerol gradients (15% to 35% (w/v); 4.8 ml) were prepared in buffer A, which contained 50 mM Tris-HC1 (pH 7.6), 500 mM NaCI, 2 mM DTT, 0.1% (w/v) Brij-58 and 0.1 mM EDTA. Samples (60 ml) containing 10 to 12 mg of the protein in buffer A were carefully loaded on top of the gradient and sedimented at 47,000 revs/min and 3°C for 43 hours in a swinging rotor (SW 60Ti; Beckanan). A gradient containing bovine serum albumin (M, 66.2 x 103), ovoalbumin (M, 42.6 x 10-~) and lysozyme (Mr 14.4 x 10~) was prepared and run under the same conditions. At the end of the run, the gradients were pierced at the bottom and fractionated in 15 equal samples. Samples of 25 to 65 ml were analyzed by SDS-PAGE. Densitometry of the stained bands was carried out using a computing densitometer 325S from Molecular Dynamics.

Evaluation of lambda induction by dot-blot hybridization Lambda DNA sequences were detected by dot-blot hybridization (Kafatos et al., 1979) using lambda [3-'P]DNA as the probe. Strain OV2 (lambda iron-540) carrying pMJR112 was used in these experiments. Lytic induction of lambda prophage was achieved by the irradiation with ultraviolet light of cultures growing in LBT at 30°C or 37 to 42°C. At 30°C but not at 37 to 42°C, the killer component of the parD system is neutralized by the antagonist. Therefore, 37 to 42°C allows evaluation of the action of the killer component on the lytic induction of a lambda prophage. After ultraviolet light irradiation (350 to 500 erg/mm2), the cultures were grown protected from the light at 30°C or 37 to 42°C. Samples were taken at 30, 60, 90 and 150 minutes. To release the bacteriophage from the cells, the samples were treated with the same volume of chloroform and then clarified by centrifugation. Sub-

Kid Inhibits Initiation of DNA Replication

sequently, the supernatant was treated with phenol to release lambda DNA from lambda phage particles. Basic procedures to induce lambda prophage by ultraviolet light irradiation and for dot-blot hybridization were as described (Miller, 1972).

Acknowledgements This research was supported by grants BIO88-0294, BIO89-0497 and BIO91-1055 of the Spanish Comision Asesora de Investigaci6n Cientffica y T6cnica (CAICYT) and by the European Human Capital and Mobility Program (Network Ref. CHRX-CT92-0010). We gratefully acknowledge the excellent technical assistance of A. Serrano and C. Pardo, and help with the chromatography from M. Zazo, S. Ortega and J. Varela. It is a pleasure to thank M. Couturier and E. Lanka for strains and plasmids, and G. Ziegelin and M. Schlicht for helping with the glycerol gradient analysis and the purification of proteins. M. J. R.-E. acknowledges economic support from the Comunidad de Madrid. R. S.-J. was supported by the Fundacion Rich. Part of this work was done during a visit by R. D.-O. to the Department of Professor Schuster, at the Max-Planck Institut of Molecular Genetics (Berlin), and supported by an interchange between the Max-Planck Society (Germany) and the Consejo Superior de Investigaciones Cient/ficas (Spain).

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Kid Inhibits Initiation of DNA Replication

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Edited by N. Sternberg (Received 10 A u g u s t 1994; accepted 12 January 1995)