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Journal of Bacteriology, January 2005, p. 765-770, Vol. 187, No. 2
0021-9193/05/$08.00+0     doi:10.1128/JB.187.2.765-770.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of the Phd Repressor-Antitoxin Boundary

James Estle McKinley and Roy David Magnuson*

Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, Alabama

Received 5 August 2004/ Accepted 6 October 2004


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ABSTRACT
 
The P1 plasmid addiction operon (a classic toxin-antitoxin system) encodes Phd, an unstable 73-amino-acid repressor-antitoxin protein, and Doc, a stable toxin. It was previously shown by deletion analysis that the N terminus of Phd was required for repressor activity and that the C terminus was required for antitoxin activity. Since only a quarter of the protein or less was required for both activities, it was hypothesized that Phd might have a modular organization. To further test the modular hypothesis, we constructed and characterized a set of 30 point mutations in the third and fourth quarters of Phd. Four mutations (PhdA36H, V37A, I38A, and F44A) had major defects in repressor activity. Five mutations (PhdD53A, D53R, E55A, F56A, and F60A) had major defects in antitoxin activity. As predicted by the modular hypothesis, point mutations affecting each activity belonged to disjoint, rather than overlapping, sets and were separated rather than interspersed within the linear sequence. A final deletion experiment demonstrated that the C-terminal 24 amino acid residues of Phd (preceded by a methionine) retained full antitoxin activity.

Plasmid stability. Bacteriophage P1 is transmitted horizontally as a viral particle and vertically as a low-copy-number plasmid. Replication, partition, and addiction functions all contribute to the remarkable stability of the plasmid prophage (2, 24). The P1 addiction operon is a toxin-antitoxin system that increases plasmid stability by arresting plasmid-free daughter cells (24). The operon encodes an unstable 73-amino-acid antitoxin, Phd, which prevents host death while the plasmid is maintained, and a stable toxin, Doc, which mediates cell death on curing of the plasmid. Phd antitoxin is less stable than toxin, due to the action of the host-encoded ClpXP protease (25). While the plasmid is retained, the antitoxin is maintained in sufficient quantities to neutralize the toxin. After plasmid loss, the antitoxin continues to be degraded but is not replenished by new protein synthesis. The falling level of antitoxin relative to toxin eventually reveals the toxin and arrests the plasmid-free cell. Poisoning of the plasmid-free daughter cells leaves more resources for plasmid-containing cells (7) and may thus produce a benefit to the parasitic plasmid and a detriment to the host bacterium.

Phenomena. Analogous toxin-antitoxin systems are common on large plasmids and bacterial chromosomes and have been implicated in diverse phenomena, including plasmid stability (4, 24, 30), plasmid competition (12, 32), antibiotic sensitivity (39), and programmed cell death (1) and growth control (9). If expression of a toxin-antitoxin system is blocked by any mechanism (for example, by superrepression, by starvation, by loss of the module, by antibiotics, or even by the activation of other toxin-antitoxin modules [19]), then the continuing proteolysis of the antitoxin may unveil the toxin and thus arrest the cell. Although toxin-antitoxin systems share a number of organizational and regulatory features, at the molecular level they are a diverse group comprising more than five families with little or no significant amino acid homology between families (16, 40).

Modular and mosaic antitoxins. Phd is a multifunctional protein. It not only binds and neutralizes Doc (29), it also binds to operator sequences in the promoter region and represses transcription of the operon (28). Previously, a deletion analysis of Phd showed that the N terminus was required for repressor but not antitoxin activity and that the C terminus was required for antitoxin but not repressor activity (Fig. 1A). Only the third quarter of the protein, the resolution limit of the deletion analysis, was needed for both repressor and antitoxin activity, indicating that the protein was fairly modular (40). Genetic studies indicate that other heterologous antitoxins, such as MazE (27, 44) and CcdB (3), may have a similar organization in which the N-terminal regions are specialized for transcriptional regulation and the C-terminal regions are specialized for the neutralization of the toxin. Several toxin-antitoxin systems, including Axe/Txe and YefM/YoeB, appear to be chimeric in that their antitoxins are similar to Phd but their toxins are not similar to Doc (8, 17, 18, 35). In these cases, the chimeric boundary appears to map in the vicinity of the repressor-antitoxin boundary (40). Based on the deletion analysis of Phd and on the existence of these chimeric toxin-antitoxin systems, it was hypothesized that the toxin-antitoxin systems have a modular structure and that modular exchange has contributed to the diversity of the toxin-antitoxin systems (40).



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  		FIG. 1. (A) Deletion analysis of Phd. Previous deletion analysis had shown that the N terminus of Phd was required for repressor but not antitoxin activity and that the C terminus of Phd was required for antitoxin but not repressor activity. Only the third quarter of the protein, the resolution limit of the previous deletion analysis, was needed for both repressor and antitoxin activities (40). The transition or overlap region is underlined and shown in boldface type. The minimal functional segments from the previous study (regular type) and from this study (boldface type) are indicated below the sequence. The combined results indicate that repressor and antitoxin domains have no more than five amino acids in common. (B) Conservation in the C-terminal half of Phd, as shown in an alignment of the C-terminal half of bacteriophage P1 Phd and a homolog from S. enterica serovar Typhimurium (St Phd). Identities are shown in boldface type. (C) Possible patterns of mutations. The positions of mutations affecting antitoxin activity and of those affecting repressor activity might be identical, overlapping, interspersed, or separated. (D) Observed pattern of mutations. The position of each point mutation with a significant defect in antitoxin activity is indicated above the sequence with bold vertical marks. The nature of each point mutation tested is indicated below the sequence. The position of each mutation with a significant defect in repression is indicated below the sequence and substitution lines. Major defects in repression are indicated with bold vertical marks, and minor defects in repression are indicated with regular vertical marks. No point mutations significantly affected both activities.

Present study. The present study was designed to test this modular hypothesis and to locate the predicted repressor-antitoxin boundary. Since the N-terminal three-fourths of Phd were sufficient for repressor activity and the C-terminal half of Phd was sufficient for antitoxin activity, it was clear that the putative boundary or overlap region was located in the third quarter of the protein (Fig. 1A). To obtain a more detailed map of the structural and functional requirements within this region, we performed alanine scanning mutagenesis (13) in the third quarter of the protein. In essence, each amino acid side chain was lopped off, one at a time, to yield an alanine residue. Since the fourth quarter made minor quantitative contributions to repressor activity and was essential for antitoxin activity (40), we also constructed a number of mutations at conserved positions (Fig. 1B) in the fourth quarter of Phd in order to identify additional positions that might affect antitoxin activity or repressor activity or both. Alanine substitutions (13) and charge reversal mutations in the third and fourth quarters of Phd were generated by site-directed mutagenesis and then tested for both repressor and antitoxin activities (Fig. 1D).

Construction and validation of point mutations. Mutations were constructed by an inverse PCR method (34), a versatile technique that can be used to generate point mutations, deletions, or short insertions (26, 36, 42). In all cases pRDM034, a plasmid in which phd is under the control of the isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible Ptac promoter (Table 1) was used as a template and the PCR was catalyzed with Pfu Turbo (Stratagene) DNA polymerase, under the conditions recommended by the manufacturer. Each inverse PCR contained a forward oligonucleotide primer and a corresponding reverse primer. Mutant codons were introduced into the 5' end of the forward primer. For point mutations, the 5' end of the reverse primer abutted the 5' end of the forward primer but hybridized to the opposite strand of the template and was extended in the opposite direction. Prior to primers being used in a PCR, T4 polynucleotide kinase was used to phosphorylate the oligonucleotide primers in order to permit the circular self-ligation of the resulting PCR products. The PCR products were analyzed by electrophoresis in a 1% agarose gel in 1x Tris-acetate-EDTA to confirm the presence and size of the expected PCR product, purified by ethanol precipitation, and then ligated overnight at 20°C with T4 DNA ligase (New England Biolabs). The reaction mixtures were then treated with DpnI, a methylation-dependent restriction enzyme, in order to cleave the parental plasmid template and thus enrich for the desired mutated progeny plasmids (42). The resulting DNA was then transformed into Escherichia coli by calcium chloride transformation (11). For each construct, four transformants were colony purified and saved as freezer stocks. Plasmids from each transformant were purified and then characterized by restriction digestion and dideoxynucleotide sequencing. A single sequence-verified plasmid construct was selected for use in all further experiments. We were unable to construct PhdA36D. In its place we describe the properties of an aberrant PhdA36H construct.


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  		TABLE 1. Phages, plasmids, and bacterial strains

Patterns. Potentially, the amino acid positions in the third and fourth quarters affecting repressor activity and those affecting antitoxin activity might be identical, overlapping, interspersed, or separated, as illustrated in Fig. 1C. An identical or strongly overlapping pattern might indicate the presence of a structural core essential for both activities. Conversely, a separated pattern might be indicative of a simple butt joint between two structurally and functionally independent modules. An interspersed pattern might be expected if, for example, different sides of a single alpha-helix were involved in different functions. To distinguish between these possibilities, we generated a set of point mutations in the third and fourth quarters of Phd, as indicated in Fig. 1D, and then tested each point mutation for both repressor and antitoxin activities in order to determine whether the affected amino acid was important for the repressor activity, antitoxin activity, both activities, or neither activity.

Repressor and antitoxin assays. Repressor activity was indicated by the ability to repress expression of a lacZ reporter fused to the promoter of the P1 addiction operon. Each mutation was tested a minimum of nine times, representing at least three time points in at least three independent experiments. In vivo, Doc mediates cooperative interactions between adjacent Phd-binding sites and thereby dramatically enhances repression. Here, however, we define repressor activity as the intrinsic repressor activity of Phd, in the absence of Doc. For repression assays, no inducer was used. Antitoxin activity, measured in a second strain, was indicated by the ability to form colonies in the presence of an otherwise lethal level of toxin. For antitoxin assays, IPTG was used at a concentration of 50 µM to induce expression, from separate plasmid constructs, of the toxin Doc and the antitoxin Phd, or its variants or its controls.

Repressor mutations. A total of 30 point mutations at 28 amino acid positions in the third and fourth quarters of Phd were tested for altered repressor activity. None of the mutations tested significantly enhanced repressor activity. Four point mutations, PhdA36H, V37A, I38A, and F44A, were observed to have major defects in repressor activity (Table 2). Another five point mutations, PhdV39A, K41A, Y47A, K48A, and A50D, were observed to have minor but statistically significant defects in repressor activity (Table 2). None of these nine repressor mutations caused a defect in antitoxin activity (Table 3), indicating that they were specifically defective in the repressor function. Six of these nine mutations, including all four of the stronger mutations, were located at well-conserved, hydrophobic residues within the third quarter of Phd. These mutations might be important for repressor-operator recognition by Phd, for the dimerization of Phd, or both. Although deletion mutations indicated that the fourth quarter of Phd makes a quantitative contribution to repression (40), none of the point mutations in the fourth quarter of Phd showed a significant defect in repression, indicating that the contribution of the fourth quarter to repressor activity is poorly localized or involves poorly conserved amino acid residues, or both. The apparent repressor-antitoxin joint maps to an area of predicted alpha-helical structure (40). It is possible that the C-terminal quarter of Phd might contribute to the formation of the putative alpha-helix and, thus, contribute quantitatively to repressor activity without containing any amino acid residues that are specifically required for repressor activity. If this were true, then swapping the C-terminal region with a heterologous segment of high alpha-helical propensity might produce an effective repressor protein.


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  		TABLE 2. Repressor activity of Phd mutationsa


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  		TABLE 3. Antitoxin activities of Phd mutationsa

Antitoxin mutations. All 30 point mutations were tested for antitoxin activity. Alanine mutations at four positions were observed to specifically abolish antitoxin activity (Table 3) without affecting repressor activity (Table 2). Two of these mutations, PhdF56A and F60A, involved phenylalanine, a highly hydrophobic and aromatic side chain. The other two, PhdD53A and E55A, involved a negatively charged side chain. To further investigate the role of these negatively charged side chains, additional charge reversal mutations were constructed and tested. PhdD53R, like PhdD53A, was defective in antitoxin activity. Interestingly, although PhdE55A was defective for antitoxin activity, the corresponding charge reversal mutation, PhdE55R, was not defective, indicating that E55, or alternatively R55, might engage in a hydrogen bond rather than an ionic bond with the cognate toxin. Secondary structure predictions indicate that this region of Phd is likely to form an alpha-helix (40). Three of the four positions implicated in toxin recognition, D53, F56, and F60, map to the same side of the hypothetical alpha-helix. Remarkably, the antitoxin portion of the protein is not well conserved. Only 6 out of the 24 C-terminal residues are conserved between Phd and its homolog from Salmonella enterica serovar Typhimurium. Mutations in three out of these six residues did not disrupt antitoxin activity. A mutation at F60, one of the few nonconserved residues in this region that were tested, abolished antitoxin activity. Possibly, other nonconserved residues will also be shown to be important for antitoxin activity.

Diversifying selection. It is interesting that the region involved in what is arguably the most essential interaction in this system is poorly conserved. Since neither toxin nor antitoxin is evolutionarily fixed, variation of the toxin-antitoxin interface is certainly possible. Could it also be advantageous? We propose three nonexclusive circumstances that might result in selection for diversity. (i) Since toxin-antitoxin systems with overlapping specificities interfere with each other (14, 24), the divergence of the relevant specificities might permit the development and exploitation of a new ecological niche. Consistent with this idea, it is not unusual for a single bacterium to have several different or well-diverged toxin-antitoxin systems. (ii) Horizontal transfer to a new host might require adaptation to fit more smoothly into the local context (15, 43). (iii) The toxin-antitoxin system is a genetic parasite whose interests may differ from the interests of the host bacterium. The evasion of host defenses (which might, in some cases, include domesticated toxin-antitoxin systems) might also contribute to the rapid diversification of a toxin-antitoxin system.

Modularity of the Phd repressor-antitoxin. As predicted by the modular hypothesis, point mutations affecting repressor activity were separated, within the linear sequence, from those affecting antitoxin activity. No point mutation tested had significant effects on both activities. Phd{Delta}(2-49) is a 25-amino-acid peptide consisting of a methionine residue followed by the 24 C-terminal residues of Phd. Remarkably, Phd{Delta}(2-49) (MAALDAEFASLFDTLDSTNKELVNR; positions shown to be important for antitoxin activity are shown in bold) retained full antitoxin activity (Table 3), indicating that all necessary determinants for the antitoxin activity must reside within the C-terminal one-third of the protein. The reciprocal deletion mutation, Phd{Delta}(50-73), had neither repressor nor antitoxin activity, but a slightly longer deletion mutation, Phd{Delta}(55-73), retained at least partial repressor activity (40). Thus, the combined deletion experiments indicate that the repressor and antitoxin domains have an overlap of five amino acid residues (AALDA) or fewer (Fig. 1A), while experiments with point mutations indicate that the two domains are separated by two amino acid residues (AL) or fewer (Fig. 1D). The results are most consistent with the hypothesis that repressor and antitoxin domains are joined with a tight butt joint.

Modular organization. The modular structure of the toxin-antitoxin systems may simply be the natural consequence of an evolutionary history of illegitimate recombination which, in turn, may be a consequence of an evolutionary scenario that favors diversity (40). Both legitimate and illegitimate recombination can produce a combinatorial increase in the underlying positional diversity. Successive rounds of illegitimate recombination, followed by the selection of the functional recombinants with novel specificities and the subsequent deletion of nonfunctional sequences (23, 40), should tend to produce toxin-antitoxin systems that are well-ordered and modular at the individual level and that are diverse and chimeric at the population level. In this case, well-ordered means that the genetic distance between interacting components, such as operator and repressor or antitoxin and toxin, is minimized. Modular means that the toxin-antitoxin system is composed of two evolutionarily independent modules, namely, an operator repressor module and an antitoxin-toxin module. Thus, modular also means that essential repressor and antitoxin functions will map to different and nonoverlapping genetic segments or domains. Chimeric means that modular exchange will contribute to toxin-antitoxin diversity and that the resulting mosaic boundaries will coincide with the repressor-antitoxin boundaries. Diverse means that different specificities will be retained and preserved and that, consequently, the positions that govern protein-ligand specificity will not be especially well-conserved.

Conclusion. We suggest that toxin-antitoxin systems tend to be well-ordered, modular, chimeric, and diverse. Selection for diversity and ordinary genetic processes (such as point mutation, horizontal gene transfer, and homologous and, especially, nonhomologous recombination) are sufficient to produce these typical features. Similar considerations and conclusions may also apply to colicins (20, 37, 38, 41) and to restriction-modification systems (21, 22, 33).


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ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grant 1 R15 GM67668-01.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, University of Alabama in Huntsville, Wilson Hall, Room 258, 301 Sparkman Dr., Huntsville, AL 35758. Phone: (256) 824-6094. Fax: (256) 824-6305. E-mail: magnusr{at}email.uah.edu. Back


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Journal of Bacteriology, January 2005, p. 765-770, Vol. 187, No. 2
0021-9193/05/$08.00+0     doi:10.1128/JB.187.2.765-770.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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