Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria

The ccd locus contributes to the stability of plasmid F by post-segregational killing of plasmid-free bacteria. The ccdB gene product is a potent cell-killing protein and its activity is negatively regulated by the CcdA protein, in this paper, we show that the CcdA protein is unstable and that the degradation of CcdA is dependent on the Lon protease. Differences in the stability of the killer CcdB protein and its antidote CcdA are the key to post-segregational killing. Because the half-life of active CcdA protein is shorter than that of active CcdB protein, persistence of the CcdB protein leads to the death of plasmid-free bacterial segregants.     

Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.

In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.     

Improved seamless mutagenesis by recombineering using ccdB for counterselection

Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.     

Intricate interactions within the ccd plasmid addiction system.

The ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA, and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA*CcdB*DNA complex with a CcdA:CcdB ratio of one. This (CcdA(2)CcdB(2))(n) complex has multiple DNA-binding sites and spirals around the 120-bp promoter region.     

The Two CcdA Proteins of Bacillus anthracis Differentially Affect Virulence Gene Expression and Sporulation

The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.     

Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA

The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.     

Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system

High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or “leaky” production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacI^q gene have been constructed to permit titration of the LacI protein. When a high-copy plasmid is transformed along with the appropriate lacI^q-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression.     

Construction of a novel zero background prokaryotic expression vector: potential advantages

A novel DNA sequence, derived from the antisense strand of the DNA gyrase inhibitor protein, CcdB, was toxic to E. coli. This protein (~6 kDa) decreased the growth rate of E. coli K12 by three orders of magnitude upon induction. The expressed toxic protein in E. coli K12 was soluble while it was insoluble in induced E. coli BL21. A high efficiency prokaryotic cloning/expression vector was constructed using this toxic gene sequence and gave zero background with ~100% cloning efficiency requiring no dephosphorylation. The toxic gene product also affected the survival of a ccdB resistant cell line, thus indicating a different mechanism of toxicity, other than DNA gyrase inhibition, as compared to the ccdB toxicity.     

Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F

Summary The stability determined by the systems ParD of plasmid R1 and Ccd of plasmid F is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. In this paper we report that CcdA and Kis proteins, the antagonists of the Ccd and ParD systems respectively, share significant sequence homologies at both ends. In Kis, these regions seem to correspond to two different domains. Despite the structural similarities, Kis and CcdA are not interchangeable. In addition we have shown that the cytotoxins of these systems, the Kid and CcdB proteins, do not share structural homologies. In contrast to CcdB, the Kid protein of the ParD system induces RecA-dependent cleavage of the cl repressor of bacteriophage very inefficiently or not at all. The functional implications of these results are discussed.     

A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd,Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.     

The thermodynamic stability of the proteins of the ccd plasmid addiction system

The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was measured at different temperatures combining denaturant and thermally induced unfolding. It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a significantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins. This concentration dependence may be of physiological significance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation.The influence of DNA on the thermal stability of the two proteins was probed. It was found that cognate DNA increases the melting temperature of CcdA. In the presence of non-specific DNA the thermal stability was not changed. The melting temperature of CcdB was not influenced by the applied double-stranded oligonucleotides, neither cognate nor unspecific.     

Structural and Thermodynamic Characterization of Vibrio fischeri CcdB

CcdB_Vfi from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdB_Vfi is a dimer that unfolds to the corresponding monomeric components in a two-state fashion. In the unfolded state, the monomer retains a partial secondary structure. This observation correlates well with the crystal and NMR structures of the protein, which show a dimer with a hydrophobic core crossing the dimer interface. In contrast to its F plasmid homologue, CcdB_Vfi possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdB_Vfi shows a number of non-conservative substitutions compared with the F plasmid protein in both the CcdA and the gyrase binding sites. Although variation in the CcdA interaction site likely determines toxin-antitoxin specificity, substitutions in the gyrase-interacting region may have more profound functional implications.     

The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.