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Functional interactions between chpB and parD, two homologous conditional killer systems found in the Escherichia coli chromosome and in plasmid R1

Sandra Santos Sierra , Rafael Giraldo , Ramón Dı́az Orejas
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb13254.x 51-58 First published online: 1 November 1998


parD and chpB are homologous conditional killer systems of plasmid and chromosomal origin, respectively. They are bicistronic operons encoding a killer protein (Kid and ChpBK) and an antidote (Kis and ChpBI). It is shown that the antidote of the chpB system can neutralize the toxin of the parD system. This activity is improved by particular amino acid changes at the amino end of the ChpBI antidote. It is further shown that the chpB system is weakly autoregulated and that the activity of a second promoter, previously identified upstream of the regulated promoter, can modulate the functional interactions between the chpB and parD systems.

Key words
  • parD operon
  • chpB operon
  • Conditional killer system
  • Plasmid stability
  • chBI mutant
  • chpB and parD interaction

1 Introduction

parD (kis, kid) is a conditional killer stability system found in the drug resistance factor R1 [1, 2]. As with other proteic killer gene systems, parD encodes a stable toxin (Kid) and an unstable antidote (Kis) [3]. This system shares homology with two conditional killer systems, chpA (chpAI, chpAK) and chpB (chpBI, chpBK), found in the chromosome of Escherichia coli[4]. In a multiple sequence alignment, the antidotes share 19% identical and 21% conserved residues, whereas the toxins share 18% identical and 19% conserved residues. These values almost double in pair-wise alignments [4]. Kid is an efficient inhibitor of the initiation of DNA replication, targeting, most probably, the main replicative helicase of E. coli, the DnaB protein [5]. The Kis protein is able to form a very stable Kis-Kid complex that seems to be relevant for autoregulation of the system and also for neutralization of the Kid toxin [5]. Previous data indicated that the amino- and carboxy-terminal regions of the parD antidote are involved in the neutralization of the toxin and in the formation of the complex that autoregulates its expression [1, 2].

The study of the interactions between parD and the homologous conditional systems chpA and chpB is biologically significant because it addresses the relationships existent between plasmids and host functions. In addition it can give information on regions relevant in the convergence/divergence of the homologous systems. Furthermore, this analysis opens new ways of assigning functions to particular regions of the proteins under study. We have performed a comparative analysis of chpA and parD[6]. This analysis indicated that the amino end of ChpAI can accommodate amino acid substitutions that lead to the functional convergence of the two systems. The mutations isolated maintain the ability of the ChpAI antidote to neutralize the ChpAK toxin but improve the basal activity of ChpAI to neutralize the toxin of the parD system.

In this communication we extend this analysis to the chpB and parD systems. The chpB mutants obtained indicate also that the amino end of the ChpBI antidote is involved in the functional interaction with the toxins. The relevance of a second upstream chpB promoter, previously identified [4], in the functional interaction of the chpB and parD systems is also shown. The results support the role of the amino end of the antidotes in the convergence/divergence of these systems.

2 Materials and methods

2.1 Bacterial strains and plasmids

Bacteria used in these studies were the E. coli K12 strains CC118 F′ Sure (Δ(ara-leu), araD,ΔlacX74, galE, galK, phoA20, thi1, rpsE, rpoB, argE(am), recA1 [7], MC1000 (araD139,Δ(ara-leu)7697,ΔlacX74, galU, galK, rpsL)[8], CSH50 (F, ara, Δ(lac-pro), strA, thi)[9] and the supF-thermosensitive strain OV2 (F, leu, thyA (deo), ara (am), lac-125 (am), galU42 (am), galE, trp (am), tsx (am), tyrT (supF(ts)A81), ile, his)[10]. The plasmids used were: the multicopy plasmid vector pUC19 [11]; the promoter cloning vector pRS551 [12]; the RSF1010-derived tac promoter, expression vector pMMB67E/H [13]; and the parD plasmids pAB17 (kis17, kid+)[1], pAB1120 (kis74, kid+,ΔcopB)[2] and pUUM10, a pBR322-parD (kis+, kid+) recombinant [6].

2.2 Experimental procedures

DNA isolation and genetic engineering techniques were performed basically as described by Sambrook et al. [14]. Amplification of genomic chpB sequences by PCR was done in a 100-μl mixture containing: 10 ng of genomic DNA prepared from the E. coli K12 strain MC1000; Pfu polymerase (2.5 U) and its assay buffer as supplied by Stratagene; a mixture of the four dNTPs at 200 mM each; 100 pmol of each of the two primers. Following an initial incubation for 5 min at 95°C, the reaction mixture was incubated for 30 cycles sequentially at 95°C (1 min), at a temperature close to but below the annealing temperature of the primers (2 min) and at 72°C (1 min 30 s). Three initial cycles in which the annealing temperature was further reduced were also included in most cases. The PCR reaction was terminated with an incubation at 72°C for 10 min. In vitro mutagenesis with hydroxylamine, a mutagen that induces GC-AT transitions due to the hydroxylation of cytosines, was carried out for 35 min at 70°C [15]. Following extensive dialysis against 10 mM Tris-HCl pH 8.0, 1 mM EDTA, the mutagenized DNA was used to transform the E. coli strain OV2 containing the pAB17 or pAB1120 mutants. DNA sequencing of the chpB recombinant and their mutants was performed, on both strands, by the chain terminator method [16], using an automated DNA sequencer (Perkin Elmer, Abi Prism, 377 DNA sequencer). β-Galactosidase assays were done as described by Miller [9].

2.3 Relevant constructions

A 798-bp chromosomal fragment containing the wild-type sequences of the chpB operon flanked by EcoRI and BamHI targets was obtained by PCR amplification of the chpB sequences present in the E. coli chromosome as indicated above, using the primers 5′-TGGAATTCCCTCACCTTTTGCTTTT-3′ and 5′-CGGGATCCGGTTACTAAGGGTTTT-3′, which introduce, respectively, an EcoRI target upstream and a BamHI target downstream of the chpB operon. This fragment was digested with the EcoRI and BamHI enzymes and cloned directionally in the EcoRI-BamHI sites present in the multicopy pUC19 vector. pUC19 recombinants containing the chpB operon were rescued in the CC118 F′ Sure strain as ampicillin-resistant transformants that were defective in α-complementation (white colonies in X-gal/IPTG plates). As expected, the recombinant plasmids isolated from these transformants contained an EcoRI-BamHI fragment of 0.8 kb. One of these recombinants, pSSB, containing the wild-type chpB sequence, as determined by DNA sequencing analysis, was selected for further studies. A pUC19-chpBI recombinant was also constructed. The chpBI region flanked by EcoRI and HindIII targets was obtained by PCR amplification of chpB sequences using the same EcoRI mutagenic primer as above and the following HindIII mutagenic primer: 5′-AAATAAGCTTTTCTTTACCATATTTCGTC-3′.

Construction of promoter and transcriptional fusions: the lacZ fusion vector pRS551 and a pMMB-tac promoter vector were used, respectively, to fuse the chpB promoter (wild-type and mutant; flanked by EcoRI and BamHI) to the lacZ reporter gene and to place the chpB system (flanked by EcoRI and BamHI targets) under the control of the tac promoter. The relevant chpB sequences introduced in these constructions were amplified by PCR using respectively the following pair of primers: 5′-TGGAATTCCCTCACCTTTTGCTTTT-3′, 5′-CTGTGGATCCATCTTTTTATGGTAATACGCAT-3′; 5′-ATGAATTCTATAACTTAAGGAGGTGCA-3′, 5′-CGGGATCCGGTTAGTAAGGGTTTT-3′.

3 Results

3.1 Complementation of parD mutants by the chpBI wild-type gene

As indicated above chpB and parD are conditional killer systems located in the E. coli chromosome and in plasmid R1/R100. As these systems share a high similarity, we decided to explore the possible interactions between their components. For this purpose we initially tested if components of the chpB system could complement the thermosensitivity associated with the parD mutants pAB17 (kis17, kid) and pAB1120 (kis74, kid). These assays were carried out in the supFts strain OV2. The pUC19-chpB recombinant failed to suppress growth inhibition at 42°C associated with the parD mutants. However, deletion of the toxin component in this construction leads to efficient complementation of the pAB1120 parD mutant (Fig. 1). This reveals a basal ability of the wild-type ChpBI antidote to neutralize the parD toxin that is masked by the ChpBK toxin. Interestingly this complementation was clear with the pAB1120 mutant but was not observed with the pAB17 mutant (see Section 4).


Assays of complementation of the pAB17 and pAB1120 mutants by recombinants encoding the complete chpB system or the chpBI antidote. Complementation is analyzed by testing the growth at 30°C (lower plate) and 42°C (upper plate). OV2 cultures contained the parD mutants pAB1120 (left hand sectors) or pAB17 (right hand sectors) and one of the following plasmids: a pUC29-chpB recombinant (chpB); a pUC19-chpBI recombinant (chpBI); a pUC19 vector (negative control: −); a pBR322-parD recombinant (positive control, +).

3.2 Mutants in the amino end of the antidote complement the parD mutants efficiently

The failure of the wild-type chpB recombinant pSSB to complement the parD mutants opens the possibility of isolating mutants in the chpB system that acquire this capacity. To explore this possibility pSSB was mutagenized in vitro with hydroxylamine and then introduced by transformation in cells containing pAB17 or pAB1120, selecting transformants at 42°C. chpB mutants able to complement the parD mutants were obtained. DNA sequencing of these mutants showed that 17 missense mutations, six of them different, were isolated in the amino end of the antidote (Table 1). In all cases they were GC-AT transitions.

View this table:

chpBI mutants that complement parD mutants (pAB17 or pAB1120) at 42°C

MutantAmino acid (codon) changeFrequency
  • The bases of the codons changed by the mutations are indicated in boldface type. The number of the amino acid indicates its position with respect to the initial methionine. Note that the same mutation was found independently more than once.

The amino acid changes in ChpBI introduced by the mutations are located at positions 9, 11, 15 and 17 from the initiator methionine. These changes affect a region that aligns with mutations previously isolated in the ChpAI antidote that have similar phenotypes ([6] and Fig. 3). To verify if the ChpBI mutants retain the ability to neutralize the ChpBK toxin, these mutants were isolated in OV2 in the absence of the parD mutants. This analysis is required to avoid possible interactions between the two homologous and mutated antidotes that could mask the phenotype of the chpBI mutations. In the absence of the parD mutants, the chpBI mutants retained the ability to neutralize the ChpBK toxin as indicated by two effects: (i) the chpBI mutants could be efficiently transformed in OV2 at 30°C and (ii) the chpBI mutants grew efficiently in this background at 37°C and 42°C (data not shown). It can be concluded that the chpBI mutations enhance the interaction of the ChpBI and the Kid toxin while maintaining the ability to neutralize the ChpBK toxin.


Localization of the changes induced by the chpB mutations. The region in which the chpB mutations are located is shown, as well as the predicted amino acid changes introduced by these. The alignment of Kis, ChpAI and ChpBI proteins, the structural predictions for this region and the mutations isolated in ChpAI have been reported [6]. The amino acid change introduced by the kis17 mutation [1] is also shown. Dots: conserved residues in the alignment; arrow above the sequence: β-sheet; filled rectangle: α-helix; open arrows: chpB transcription starting points; underlined sequences: −10 boxes corresponding to the two identified chpB promoters; inverted arrows: inverted repetitions that define the putative chpB operator; T above the −10 sequence of the second promoter: up-promoter mutation; bases in bold character: chpBI translation initiation sequences.

3.3 A chpB promoter mutation complements the parD mutants

Surprisingly, one of the chpB mutations that complements the parD mutants was a C-T transition that is located in the −10 box (Fig. 3) of a second (upstream) promoter found in chpB[4]. This mutation could modulate its effect altering the level of expression of the chpB operon. In order to test this possibility, the chpB promoter regions (wild-type and mutant) were fused to the lacZ gene using the ColE1-type promoter vector pRS551 (see Section 2). Then the chpB promoter activities were evaluated measuring the level of β-galactosidase. The results (Table 2) indicate that the mutation increased six times the overall transcription activity of the promoter region analyzed. This indicates a correlation between the level of chpB expression and the complementation observed. To evaluate the possible effect of the mutation in autoregulation, the effect on the activities of the wild-type and mutant promoters was measured in the presence of a RSF1010-type recombinant containing the chpB system under the control of the tac promoter. This recombinant was able to complement efficiently the parD mutants. A weak regulation of the wild-type chpB promoter by products was observed (Table 2). However, the mutated promoter was very efficiently regulated by the chpB recombinant.

View this table:

Transcriptional activity of the wild-type and mutant chpB promoters in the presence or absence of a chpB recombinant

Promoter fusionschpB in transβ-Galactosidase
chpBp:lacZ11 500100
chpBp:lacZ+6 70058
chpBp*:lacZ65 300100
  • chpBp indicates the promoter of the chpB operon, and * indicates the mutation identified in the upstream chpB promoter. pRS551 in the vector used to make the fusions of the wild-type and mutated promoters to the lacZ gene. The chpB operon in trans is provided as a pMMB construction in which expression of this operon is placed under the control of the tac promoter. Plasmids were established in CSH50, a lacZ-deficient strain. β-Galactosidase assays were determined as indicated [9] in the presence of 1 mM IPTG to induce a high level of expression of the chpB genes.

4 Discussion

In this work it is shown that a small region at the amino end of the ChpBI antidote can accommodate changes in its sequence which allow an efficient neutralization of the parD toxin. These mutations affect residues that are located in consecutively alternating positions (residues 9, 11, 15 and 17 starting from the amino-terminal methionine of ChpBI). The changes increase either the hydrophobicity of the residue or the length of the lateral chain of the amino acid. Note that a change in the conserved proline at the amino end of the antidotes produces different effects in ChpBI and Kis (expansion of the range of action of the antidote versus attenuation of the action of the antidote). This suggests that the non-conserved residues in this region can modulate a differential effect in parD and chpB. Mutations with the same phenotype were also isolated in the homologous region of the antidote of the chpA system [6]. Secondary structure and solvent accessibility predictions [6] suggest that the amino acid changes are located in a putative and partially exposed loop which is conserved in antidotes of the parD family (Fig. 3). These results are consistent with the involvement of this region in the functional interactions that lead to the neutralization of the parD toxin. The amino end region of the Kis protein is also conserved in the antidote of the ccd system [17], which underlines the relevance of this region and suggests the existence of a common root in the phylogeny of these conditional killer systems.

The wild-type ChpBI antidote is able to neutralize the toxicity of the parD toxin. This is clearly shown by the complementation of the kis74 (pAB1120) mutation by a pUC19-chpBI recombinant. However, this complementation was not observed with the pUC19-chpBI-chpBK recombinant (Fig. 1). A possible explanation is that the deletion of the chpBK gene could leave the ChpBI antidote freely available for interactions with the Kid toxin. Interestingly, neutralization of the toxicity of Kid was observed with the kis74 (pAB1120) mutation but not with the kis17 (pAB17) mutation. This could be due to the differential interactions of the two mutated parD antidotes with Kid. Kis17 is inactivated by heat but it could well remain bound to Kid in such inactive form. If the ChpBI wild-type antidote interacts weakly with Kid, the inactive Kis17 protein can prevent the ChpBI-Kid interaction. In favor of this hypothesis is the high (>95%) recovery of the viability of OV2 cultures containing pAB17 when they were shifted from the restrictive temperature, 42°C, to the permissive one, 30°C [1]: this could be explained by the in situ reactivation of the antidote bound to the toxin. In contrast, the kis74 mutation truncates the parD antidote [2] and this could prevent its interaction with Kid. This would leave the parD toxin freely accessible to the ChpBI antidote, which could explain the complementation observed. This interpretation is also consistent with the fact that some of the chpB mutants complement the kis17 mutation less efficiently than the kis74 mutation (see B8, B14 and B20 in Fig. 2). Further biochemical evaluations are needed to test this hypothesis.


Complementation of the pAB17 and pAB1120 mutants by pUC19-chpB mutants. The growth at 30°C (lower plate) or 42°C (upper plate) is shown of cells containing the parD mutants and either the wild-type chpB recombinant pSSB (−; negative controls), the parD recombinant (+; positive controls) or recombinants of the different chpB mutants shown in Table 1.

Transcriptional analysis indicated that the chpB system is weakly autoregulated. This weak autoregulation of chpB (42% repression by products) is different from the efficient regulation by products observed in the parD system (>95% repression) [18, 19]. We found that an up-promoter mutation in the second upstream chpB promoter leads to efficient complementation of the parD mutants. An increase in the level of transcription would in principle increase the levels of the ChpBI antidote, which could explain the complementation observed. However, the increased overall promoter activity is also accompanied by very efficient autoregulation by products. This indicates that the activity of the second promoter modulates the level of expression of the chpB operon and reveals a complex relationship between the activities of the two promoters and the final level of chpB expression. Further work is needed to clarify this point. In any event these results reveal that the activity of the second chpB promoter plays an important role in the ‘cross-talking’ between the two homologous systems.

In summary, the results obtained show that the amino ends of the antidotes as well as the activities of the chpB promoters play an important role in the regulation of the chpB operon and in the functional interactions between the chpB and parD systems. They also reveal a basal activity of the wild-type ChpBI antidote on the homologous Kid toxin.


This research has been financed by Projects BIO94-0707 (CICYT) and PB94-0127 (DGICYT). The excellent technical assistance of Consuelo Pardo Abarrio and Ana Maria Serrano López is acknowledged. We also acknowledge the contributions of the oligonucleotide synthesis, DNA sequencing and photography services of the CIB.


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