JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, X.
Right arrow Articles by Magnuson, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, X.
Right arrow Articles by Magnuson, R. D.

 Previous Article  |  Next Article 

Journal of Bacteriology, March 2005, p. 1901-1912, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.1901-1912.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Percolation of the Phd Repressor-Operator Interface

Xueyan Zhao and Roy David Magnuson*

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

Received 4 October 2004/ Accepted 6 December 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Transcription of the P1 plasmid addiction operon, a prototypical toxin-antitoxin system, is negatively autoregulated by the products of the operon. The Phd repressor-antitoxin protein binds to 8-bp palindromic Phd-binding sites in the promoter region and thereby represses transcription. The toxin, Doc, mediates cooperative interactions between adjacent Phd-binding sites and thereby enhances repression. Here, we describe a homologous operon from Salmonella enterica serovar Typhimurium which has the same pattern of regulation but an altered repressor-operator specificity. This difference in specificity maps to the seventh amino acid of the repressor and to the symmetric first and eighth positions of the corresponding palindromic repressor-binding sites. Thus, the repressor-operator interface has coevolved so as to retain the interaction while altering the specificity. Within an alignment of homologous repressors, the seventh amino acid of the repressor is highly variable, indicating that evolutionary changes in repressor specificity may be common in this protein family. We suggest that the robust properties of the negative feedback loop, the fuzzy recognition in the operator-repressor interface, and the duplication and divergence of the repressor-binding sites have facilitated the speciation of this repressor-operator interface. These three features may allow the repressor-operator system to percolate within a nearly neutral network of single-step mutations without the necessity of invoking simultaneous mutations, low-fitness intermediates, or other improbable or rate-limiting mechanisms.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Bacteriophage P1 can be transmitted horizontally, as a viral particle, or vertically, as the P1 plasmid prophage (36). The low-copy-number P1 plasmid (61) is remarkably stable (67) due to faithful replication (6), partition (5), resolution (7), and addiction (43) functions. Replication and resolution system functions act to maintain a copy number of approximately one to two separate plasmid molecules per chromosome, while the partition function ensures that each daughter cell gets at least one plasmid copy. Should these systems fail, the P1 plasmid addiction operon, a classic toxin-antitoxin system, acts to arrest the plasmid-free cell. Elimination or arrest of plasmid-free cells may leave more resources for plasmid containing cells (17, 74).

The P1 plasmid addiction operon encodes a 73-amino-acid repressor-antitoxin, Phd, which prevents host death, and a 126-amino-acid toxin, Doc, which is responsible for death on curing (plasmid loss) (43). The Phd antitoxin is both synthesized (43) and degraded at a higher rate than the toxin, Doc. Degradation of Phd is dependent upon the host-encoded, ATP-dependent ClpXP protease (44). While the plasmid is maintained, there is enough antitoxin present to neutralize the toxin. When the plasmid is lost or if expression is halted due to some other cause, the continuing degradation of the antitoxin, in the absence of new synthesis, unveils the toxin and thus arrests the cell.

Transcription of the P1 plasmid addiction operon is inhibited by the protein products of the operon (47). The promoter region of the addiction operon contains two 8-bp palindromic sites that are 13 bp apart, center to center (Fig. 1). Each palindromic site is bound cooperatively by a dimer of Phd (30, 47). The two adjacent sites are filled independently in the absence of Doc but cooperatively in the presence of Doc (48). Thus, Doc mediates cooperative interactions between the adjacent sites and thereby enhances repression (48). Doc does not appear to have any direct contact with the operator DNA. A comparable autoregulatory loop is typically found in analogous toxin-antitoxin systems (1, 23, 25, 26, 32, 38, 39, 42, 45, 46, 51, 57, 58, 59, 66, 68, 69, 71, 73, 77, 78, 81, 82, 83, 86).



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 1. Alignments of nucleotide and protein sequences from the P1 and Salmonella addiction operons. A. Alignment of P1 and Salmonella operator regions. In order to identify possible Salmonella operators, regions upstream of P1 phd and Salmonella phd were compared. The P1 sequence contains two palindromic Phd-binding sites spaced 13 bp apart, from center to center. The Salmonella DNA contains three very similar palindromic sites with the same 13-bp spacing between the centers of adjacent sites. The consensus P1 and Salmonella operators are eight nucleotides long and differ only in the first and last positions. Thus, it appears that the 6-bp core of the operator is conserved and that the promoter specificity may be regulated by the two flanking base pairs. B. Alignment of P1 and Salmonella Phd proteins. The two proteins have approximately 41% amino acid identity. An alternative start for the Salmonella Phd protein would add two amino acids to the N terminus of the protein (see Fig. 3). C. Alignment of P1 and Salmonella nucleotide sequences at the phd/doc boundary. Relative to the P1 sequence, the Salmonella sequence has a 3-bp insertion into the stop codon of phd. The insertion generates a new stop codon at the same position, destroys the original start codon for Doc, but simultaneously generates a new start codon in a slightly different position. Thus, the stop codon for P1 Phd and the start codon for P1 Doc overlap by 1 bp (TAATG), whereas the stop codon for S. enterica serovar Typhimurium Phd and the start codon for S. enterica serovar Typhimurium Doc overlap by 4 bp (ATGA). The overlapping start and stop codons are consistent with the hypothesis that toxin and antitoxin synthesis may be translationally coupled. D. Alignment of P1 and Salmonella Doc proteins. The two proteins have approximately 45% amino acid identity. Relative to the P1 Doc protein, the Salmonella Doc protein has two additional amino acids at the N terminus, due to the insertion discussed above, and six fewer amino acids at the C terminus.

 
Analogous toxin-antitoxin systems are commonly found on low-copy-number plasmids and on bacterial chromosomes (reviewed in references 27, 31, 34, 35, 38, 64, 65, 85, and 87). The chromosomal elements, like the plasmid-borne elements, might be viewed as selfish, self-selecting genetic elements (48, 54, 74). Alternatively, it is possible that they regulate cell growth (19, 31, 52) or cell death (2) in a manner that is ultimately beneficial for the bacterial host. Fulfillment of such different functions might require different patterns of gene regulation.

The interaction between repressor and operator can be retained, while the specificity is altered, if the repressor and operator covary or coevolve. Mechanistically, this coevolution presents special conceptual problems. An alteration in the specificity of the system requires, by definition, one or more mutations in the repressor and one or more corresponding or compensatory mutations in the operator. Thus, the fitness or function of a given operator is contingent upon the sequence of the corresponding repressor. This epistatic relationship corresponds to a rugged (digital) adaptive landscape with many high spots (peaks) and many low spots (valleys) corresponding to compatible and incompatible repressor-operator combinations. The passage of the system from one specificity to a second different specificity requires the simultaneous occurrence of two or more mutations, which is unlikely, passage through a low-fitness intermediate (valley), which is unfavorable and infrequent, or stepwise passage through a series of at least two neutral fitness intermediates with overlapping specificities (a ridge or neutral network), which may be unparsimonious. The general problem is applicable to a variety of microevolutionary and macroevolutionary problems and thus has long been the subject of considerable theoretical interest (4, 22, 29, 40, 84) and has recently become a subject of some experimental interest (15).

Here, we compare and contrast the DNA binding specificities of P1 Phd protein and a homologous Salmonella enterica serovar Typhimurium Phd protein. We map the major specificity determinants in the repressors and the corresponding operators. We present evidence that the alteration of this repressor-operator interface is frequent and is most likely to occur through a stepwise sequence of nearly neutral intermediates with "fuzzy" or overlapping specificities. We suggest that certain common features of DNA-binding proteins, including fuzzy recognition and the approximate independence of individual protein-DNA contacts, may be more easily explained by the evolutionary processes that produced them than by the functions they currently perform.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Microbiology. All Escherichia coli strains used were derived from MC1061 (16). Bacterial strains were grown, manipulated, and stored by standard techniques (55). Bacteria (Table 1) were grown at 30°C in Luria broth (LB) or LB agar (Fisher Scientific) containing 100 mg of ampicillin per liter, 30 mg of kanamycin per liter, and 80 mg of spectinomycin per liter, as indicated, to select for plasmid introduction or maintenance. Isopropyl-ß-D-thiogalactopyranoside (IPTG) was used at a final concentration of 0.1 mM as indicated to induce transcription from the Ptac promoter (24). Liquid cultures were grown with aeration in a shaking water bath or in a roller drum revolving at 30 rpm.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Bacterial strains

 
Molecular biology. Genomic DNA was prepared by standard methods (8). Plasmid DNA was prepared by alkaline lysis with standard protocols (70) or commercial kits (Qiagen). Oligonucleotide primers (Table 2) were used in PCRs (56) with Taq polymerase to amplify DNA segments of interest and to flank those segments with convenient restriction enzyme sites.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Oligonucleotide primers

 
Site-directed mutagenesis. Site-directed mutations in P1 phd, Salmonella enterica serovar Typhimurium phd, and the P1 addiction promoter were generated with primer-encoded mutations (mismatches). PCR fragments and plasmid vectors were digested with restriction enzymes, purified on 1.0% low-melt agarose in 1x Tris Acetate EDTA electrophoresis buffer, ligated with T4 DNA ligase, and then introduced into Escherichia coli by calcium chloride transformation (21), using standard protocols and techniques (70). Transformants were selected and colony purified on LB agar containing the appropriate antibiotics. Plasmid constructs (Table 3) were validated by electrophoretic sizing of restriction fragments and by dideoxynucleotide sequencing (MWG Biotech).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Plasmids

 
Identification and cloning of the Salmonella addiction operon. TBLASTN (3) was used to identify an operon in Salmonella enterica serovar Typhimurium LT2 (53) that encoded a homolog of both Phd and Doc. PCR was used to amplify the addiction operon (including promoter, antidote gene and toxin gene) from Salmonella enterica servoar Typhimurium. Chromosomal DNA isolated from Salmonella enterica servoar Typhimurium was used as a PCR template with primers st104 and st103 (Table 2) to generate a 939-bp PCR product that contains a 9 bp tag with a HindIII site, 205 bp upstream sequence, 587 bp of sequence containing the two slightly overlapping ORFs encoding S. enterica serovar Typhimurium Phd and S. enterica serovar Typhimurium Doc, 129 bp downstream sequence, and a 9-bp tag encoding an EcoRI site. The PCR fragment and the pGB2ts vector were digested with HindIII and EcoRI, gel-purified and then ligated to produce pXZ006. The sequence of the insert, as determined by dideoxynucleotide sequencing, conformed in detail to the published sequence of Salmonella enterica servoar Typhimurium LT2 (53). The Salmonella chromosomal DNA or the pXZ006 plasmid containing the operon was used as the template for all subsequent PCRs.

Single-copy lacZ reporters. The addiction operon promoters were first cloned in a multicopy plasmid vector containing lacZYA (pRS415) or lacZ{alpha} (pRS1553) and validated by dideoxynucleotide sequencing. Expression of lacZ{alpha} is less toxic than expression of lacZ and therefore permits the cloning of a stronger promoter on a multicopy vector (C. M. Pepe and R. W. Simons, personal communication). The promoters were then transferred by homologous recombination to a {lambda} phage ({lambda}RS45 or {lambda}RS468) (Table 4) using standard methods (72) (33). All ß-galactosidase experiments were performed on Escherichia coli strains lysogenized with a recombinant {lambda} phage (Table 1). Various plasmids (expressing LacIq or addiction proteins under Ptac control, or addiction proteins under the control of their natural promoters) were introduced into each lysogen by calcium chloride transformation, as indicated (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 4. Phages

 
ß-Galactosidase assays. ß-Galactosidase assays were performed essentially as described by Miller (55). Cells were grown at 30°C in LB medium supplemented with antibiotics to maintain selection for the resident plasmids. At intervals during exponential growth, the Optical Density of the culture at 600 nm was measured, 0.400 ml samples of the culture were vortexed vigorously with 0.010 ml of toluene to permeabilize the cells, and the permeabilized samples were then stored on ice or frozen until needed. (Alternatively, 0.040 ml samples were mixed with 0.360 ml diluent, vortexed with toluene, and stored). Samples of growth medium were also taken to generate spectrophotometric blanks. Samples were thawed in a 30°C water bath, mixed with 0.800 ml of 1 mg/ml solution of prewarmed ONPG (2-nitrophenyl-ß-D-galactopyranoside) in Z-buffer, incubated at 30°C for a measured interval of time to allow the hydrolysis of the substrate, and then quenched by addition of 0.500 ml of 1 M Na2CO3. The quenched samples were stored on ice and then centrifuged for 3 min at 10,000xg to pellet the cells. The absorbance of the supernatant was measured at 420 nm. ß-Galactosidase specific activity was defined as 1,000 x (absorbance at 420 nm) ÷ [(volume of sample in milliliters) x (optical density at 600 nm) x (minutes of incubation with ONPG)].


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Similar operators, antitoxins, and toxins. In order to learn more about the evolution and function of the P1 addiction operon, we identified, cloned, and characterized a homologous chromosomal operon from Salmonella enterica serovar Typhimurium (Fig. 1) as described in Materials and Methods. Inspection of the sequences indicated that the Salmonella and P1 addiction operons had very similar operators, antitoxins, and toxins. The P1 operator consists of two 8-bp palindromic Phd-binding sites, spaced 13 bp apart, center to center (47) (Fig. 1A). The Salmonella promoter region has three similar palindromic sites, also spaced 13 bp center to center (Fig. 1A). The consensus of the Salmonella palindromes, TTGT · ACAA, and the consensus of the P1 palindromes, GTGT · ACAC, shared the same central six bp core sequence and differed only in the symmetric first and eighth positions (Fig. 1A). The P1 and Salmonella Phd proteins are both 73 amino acids long and have approximately 41% amino acid identity, with no gaps (30 identities in 73 amino acids) (Fig. 1B). The corresponding P1 and Salmonella toxins are similar in length (126 and 122 amino acids, respectively), they align without gaps over most (120 amino acids) of their length, and they have approximately 45% amino acid identity (Fig. 1D). In both operons, the stop codon for the antitoxin and the start codon for the toxin are slightly overlapped, although in this instance there are some intriguing differences in detail and evidence of a small 3-bp insertion or deletion (Fig. 1C).

Autoregulation and cross-regulation of the Salmonella addiction operon. Since the P1 addiction operon is negatively autoregulated by its products, we wondered whether the Salmonella addiction operon was also negatively regulated by its products. This question was especially interesting because in the growth control hypothesis (18), a chromosomal system must be triggered, presumably by reduced expression due to starvation, without actually losing the element. Negative autoregulation would tend to oppose or prevent such a triggering of the system. In order to test for promoter activity and transcriptional regulation, 726 bp of DNA upstream of the Salmonella operon was subcloned and fused to a transcriptional lacZ reporter as described in Materials and Methods.

When the full Salmonella addiction operon was provided in trans, expression of the promoter was repressed 25-fold as indicated by the production of ß-galactosidase from the lacZ fusion (Table 5). This indicated that the Salmonella addiction operon was expressed in Escherichia coli and that the Salmonella addiction operon, like the P1 addiction operon, was negatively autoregulated by one or more of its products. In order to test the specificity of regulation, we compared the ability of the P1 and Salmonella operons to regulate the homologous and heterologous promoters in trans. The Salmonella operon strongly repressed its own promoter (25-fold repression) but had little effect on the heterologous P1 promoter (1.5-fold repression), as shown in Table 5. Similarly, the P1 operon strongly repressed its own promoter (>400-fold repression), but had little or no effect on the Salmonella promoter (1-fold repression) (Table 5). Thus, under physiological conditions, the regulation of the two systems was highly specific.


View this table:
[in this window]
[in a new window]
  		TABLE 5. Specific autoregulation of P1 and Salmonella addiction operons

 
Similar corepression. In the P1 addiction operon, the antitoxin alone is sufficient to repress transcription of the operon, but the toxin can dramatically enhance repression. In order to determine whether the same regulatory pattern was observed in the Salmonella addiction operon, we measured expression of the fusion in the presence of the Salmonella antitoxin alone, antitoxin and toxin, or neither protein. In order to avoid complications of autoregulation on the amounts of protein provided in trans, the antitoxin gene and the antitoxin-toxin pair of genes were removed from their natural context and placed under the control of the heterologous IPTG-inducible Ptac promoter. We observed (Table 6) that in the absence of IPTG, the Salmonella antitoxin was sufficient to repress transcription of the promoter approximately 2.5-fold and that in the presence of the toxin repression was enhanced to more than 65-fold. Thus, the general patterns of regulation of the Salmonella and P1 addiction operons were very similar. In the presence of IPTG, the repression by the Salmonella antitoxin could be boosted from 2.5-fold repression to approximately 29-fold repression (Table 6).


View this table:
[in this window]
[in a new window]
  		TABLE 6. Salmonella antitoxin and toxin collaborate to regulate their promoter

 
Specificity determinants map to the antitoxins. We suspected that differences in promoter specificity would map to the Phd proteins. In fact, under conditions where the P1 Phd protein repressed the P1 promoter (7.8-fold repression) it had no effect on the S. enterica serovar Typhimurium promoter (1.1-fold repression) (Table 7). Conversely, under conditions where the S. enterica serovar Typhimurium Phd protein strongly repressed the S. enterica serovar Typhimurium promoter (26.7-fold repression), it had little effect on the P1 promoter (1.3-fold repression) (Table 8). Thus, the differences in specificity observed under physiological conditions in the presence of both antitoxin and toxin (Table 5) were qualitatively recapitulated in the presence of antitoxin alone (Tables 7 and 8), indicating that the specificity determinant mapped to the antitoxin proteins.


View this table:
[in this window]
[in a new window]
  		TABLE 7. Promoter specificity of P1 Phd and variants

 

View this table:
[in this window]
[in a new window]
  		TABLE 8. Promoter specificity of Salmonella Phd and variants

 
ß-Sheet model for the protein-DNA interaction. Since proposed consensus P1 and Salmonella 8-bp Phd binding sites differed only in the first and last positions, it seemed likely that the promoter specificity of the P1 and Salmonella antitoxins might be governed by one or at least a very few amino acid changes. It had previously been proposed that Phd might be a ß-sheet DNA-binding protein (47) and that the N termini of a Phd dimer might form an antiparallel ß-sheet, similar to those observed in Arc and Mnt, that would sit in the major groove of the DNA and mediate the specific protein-DNA contacts. Secondary-structure predictions of P1 and Salmonella Phd indicate that the N termini of the proteins may adopt a ß-sheet conformation. Consistent with this hypothesis, deletion of the N terminus of Phd abolished the repressor but not the antitoxin activity of P1 Phd (74).

Since the N terminus of Phd had been specifically implicated in transcriptional repression, we compared the Phd proteins to the well-characterized ß-sheet DNA-binding proteins Arc (63) (41) and Mnt (41) (Fig. 2A) and then generated a specific model for the Phd-DNA interaction with this antiparallel ß-sheet motif (Fig. 2B). In the most pleasing model (Fig. 2B) the well-conserved hydrophobic residues at positions 2 and 4 point away from the DNA and towards the protein core, while three key hydrophilic residues (positions 3, 5, and 7) point towards the DNA contact-specific bases in the major groove of the DNA. In the dimer (Fig. 2B), amino acid 5 is proposed to be opposite amino acid 5 in the other strand. Similarly, position 2 in one strand is proposed to be in close proximity to position 4 in the other strand, and similarly for positions 3 and 7. Thus, the amino acids in position 3, 5, and 7 are proposed to contact the DNA, while the hydrophobic amino acids in positions 4 and 6 are proposed to point away from the DNA and to engage in productive contacts with each other.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 2. ß-sheet model for DNA binding by Phd. A. N-terminal alignment. The alignment of P1 Phd and S. enterica serovar Typhimurium Phd with two proteins (Arc and Mnt) that are known to contact DNA through an N-terminal antiparallel ß-sheet structure is shown. The amino acids that are known (Arc and Mnt) (41, 63) or hypothesized (P1 Phd and S. enterica serovar Typhimurium Phd) to contact the DNA are in bold. Residues in these positions that differ from those found in wild-type P1 Phd are underlined. B. Model. We propose that in a Phd dimer, the N termini form an antiparallel ß-sheet, as indicated. Positions 3, 5, and 7, shown in bold, are proposed to contact bases in the major groove of the DNA site. The model is reasonably consistent with general observations and models regarding ß-sheet DNA-binding proteins (76, 79). C. Repressors. If the model is correct, then differences in the DNA-binding specificity in this protein family are likely to map to residues 3, 5, and 7. In the case of P1 and S. enterica serovar Typhimurium Phd proteins, positions 3 and 7 differ, but the amino acid at position 5 is conserved. In order to test the model, single and double mutations at positions 3 and 7, in both P1 Phd and S. enterica serovar Typhimurium Phd, were constructed, as shown.

 
The model predicts that DNA contacts are mediated by the amino acids at positions 3, 5, and 7. If the model is correct, then any differences in DNA-binding specificity should map to one or more of these three residues. Since the P1 and S. enterica serovar Typhimurium Phd proteins have the same amino acid at position 5, this amino acid cannot be responsible for the difference in DNA-binding specificity. Consequently, if the model is correct, then the specificity determinant must map to position 3, position 7, or both positions (Fig. 2).

Specificity determinant maps to position 7. To test this hypothesis, we constructed single and double mutations of P1 Phd and Salmonella Phd at the third and seventh amino acid positions (Fig. 2C) and then tested these constructs for their ability to repress transcription from the P1 and the S. enterica serovar Typhimurium addiction promoters, as indicated by lacZ fusions. In fact, relative to wild-type P1 Phd, P1 PhdR7S showed decreased activity on the P1 promoter (1.3-fold versus 7.8-fold repression) and increased recognition of the Salmonella addiction promoter (7.5-fold versus 1.1-fold repression) (Table 7). Alteration of the third residue of P1 Phd (from S to T) tended to increase the strength of repression but did not alter repressor specificity.

In the reciprocal experiment we observed that, relative to wild-type S. enterica serovar Typhimurium Phd, S. enterica serovar Typhimurium PhdR7S had increased recognition of the P1 promoter (4.2-fold versus 1.3-fold repression) and decreased recognition of the S. enterica serovar Typhimurium promoter (0.9-fold versus 26.7-fold) (Table 8). Alteration of the third residue of S. enterica serovar Typhimurium Phd from T to S tended to decrease repressor activity without altering repressor specificity. Thus, in reciprocal experiments, the alteration or exchange of the seventh amino acid in the repressors resulted in the corresponding alteration or exchange of promoter specificity, indicating that in this instance the seventh amino acid was the primary specificity determinant and was responsible for the different promoter specificities of the P1 and S. enterica serovar Typhimurium Phd proteins. The seventh amino acid is the key specificity determinant that permits specific discrimination between the P1 and Salmonella promoters.

Percolation of the seventh amino acid. Although the seventh amino acid plays a critical role in operator recognition, it is not well conserved. We identified a set of Phd homologs, not necessarily associated with Doc homologs, (Fig. 3), and then looked at the variation of amino acid identities in the seventh position. Position 7 was much more variable than, for example, positions 1, 4, 6, 8, 9, 10, and 12 to 18. In the alignment of Phd proteins, amino acids with positively charged, polar, hydrophobic, and negatively charged side chains were observed at position 7, indicating that repressor-operator covariation may be common in this protein family (Fig. 3).



View larger version (111K):
[in this window]
[in a new window]
  		FIG. 3. Percolation of position 7. Although the amino acid identity at position 7 of Phd is an important determinant of DNA-binding specificity, this position is not well conserved. In an alignment of probable Phd homologs, positively charged (RKH), polar (NQST), hydrophobic (AGLF), and negatively charged amino acids (D) are all observed at position 7 (indicated in bold). In protein-DNA complexes, positive, polar, hydrophobic, and negative amino acids most commonly contact G, A, T and C bases, respectively (50). The position-specific iterated BLAST program (3) was used to identify list of Phd homologs, not necessarily associated with a Doc homolog. The sequence of P1 Phd was used to initiate this program. A low limit (expectation < 0.1) was used for inclusion. At each iteration, sequences were manually selected for the presence of the N-terminal region, and proteins lacking homology in this region were excluded from the analysis. Although the inclusion limit was low, the program converged upon a group of sequences for which the expectation of a chance match was less than 1 E-6. These sequences were then downloaded in their entirety and realigned in Clustal W (80) with double the default gap penalties. Sequences with large loops, extensions, or deletions were removedfrom the alignment. Sequences were then grouped according to the chemical nature of the residue corresponding to the seventh amino acid of P1 Phd. For each amino acid observed at position 7 (relative to P1 Phd), two representative sequences are shown, if available. Alignments were shaded on the BoxShade server (http://www.ch.embnet.org/software/BOX_form.html) running BoxShade version 3.21, written by Kay Hofmann and Michael D. Baron. Identical (black) and conserved (grey) amino acids were shaded if at least half of the amino acids at that position were identical or similar. The output was saved in "new RTF" format and edited in Microsoft Word.

 
In a set of major-groove protein-DNA complexes surveyed by Mandel-Gutfreund and Margalit, the positively charged, polar, hydrophobic, and negatively charged amino acid side chains were observed to interact most commonly with G, A, T, and C bases, respectively (50). These preferential interactions are probabilistic, not absolute (10, 50, 60). In Mandel-Gutfreund and Margalit's data set, the preferred ligand of the positive, polar, hydrophobic and negative amino acids was observed in approximately 74%, 46%, 77%, and 93% of the cases, respectively. Interestingly, the most specific classes of amino acids (hydrophobic and negatively charged) were also the least frequently utilized.

Seventh amino acid of the repressor recognizes the first and eighth base pairs in the palindromic site. Since the consensus P1 and Salmonella palindromes differ only in positions 1 and 8 (Fig. 1A), we suspected that the seventh amino acid of the repressor interacted with the first and eighth base pairs of the palindromic repressor-binding site. However, the P1 and Salmonella promoters differ at many nucleotide positions. In order to determine if the ability of the proteins to discriminate between the promoters could be ascribed solely to this difference in the consensus palindrome, we constructed a set of isogenic promoters, based on the P1 promoter, which contained just a single palindromic binding site. We then constructed a set of variant promoter constructs, differing only in the identity of the nucleotides at the first and last positions of the palindromic site.

As predicted by the model, the Salmonella Phd was able to provide a modest (2.1-fold), but statistically significant repression of the TTGT · ACAA construct, but had little or no effect (1.1-fold) on the GTGT · ACAC palindrome (Table 9). The mutant Salmonella PhdS7R displayed the reciprocal pattern-it failed to repress the promoter containing the TTGT · ACAA palindrome (0.9-fold repression), yet it provided a modest and statistically significant repression of the isogenic promoter containing the GTGT · ACAC palindrome (4.9-fold repression). Thus, the allele-specific interactions indicate that the seventh amino acid of the repressor does in fact discriminate between promoter constructs differing only in the first and eighth base pairs of the single palindromic binding site.


View this table:
[in this window]
[in a new window]
  		TABLE 9. Site specificity of various repressors

 
In the reciprocal experiments we observed that P1 PhdR7S lost the ability to repress the GTGT · ACAC construct (0.9-fold repression) but in this instance failed to exhibit significant repression of the TTGT · ACAA construct (1.1-fold repression), most likely due to a lack of sensitivity in the experimental system (which lacks the corepressor and which has only a single palindromic site). Wild-type P1 Phd repressed the GTGT · ACAC construct (5.6-fold repression) and did not repress the TTGT · ACAA construct (1.0-fold repression), as expected (Table 9).

Fuzzy recognition. The repressors with arginine (R) at position 7 recognized the GTGT · ACAC construct but failed to repress the three other constructs (TTGT · ACAA, CTGT · ACAG, and ATGT · ACAT), indicating that this interaction was highly specific. The repressors with serine (S) at position 7 showed evidence of broadened or fuzzy specificity. Repressors with serine at position 7 tended to recognize the CTGT · ACAG construct as well as the TTGT · ACAA construct (Table 9). In the earlier, more sensitive but less well-controlled experiments, the repressors with serine at position 7 interacted preferentially with the S. enterica serovar Typhimurium promoter, but showed a slight but significant activity on the P1 promoter (Tables 5, 7, and 8).

Factors facilitating the stepwise divergence of specificities. Three circumstances may have facilitated the divergence of repressor-operator specificities in the P1 and S. enterica serovar Typhimurium addiction operons. First, the negative feedback loop present in these systems is inherently robust and will function, by self-adjustment, for a range of affinities and specificities. A modest decrease in affinity will result in a compensatory increase in expression, and thus the reestablishment and retention of homeostatic functions, at a slightly different set point. Second, fuzzy recognition is characteristic of protein-DNA interactions. Although repressors with arginine at position 7 were specific for the operators with G at position 1 (and in the symmetric position 1'), repressors with serine at position 7, had a more relaxed or "fuzzy" specificity and could recognize palindromes in which the first position was T, C, or, to a lesser extent, G. Thus, the serine repressors exhibited a relaxed or "fuzzy" specificity and could recognize some nonconsensus sites. Third, there is considerable evidence in this system for the duplication and divergence of sites. The S. enterica serovar Typhimurium operator has three palindromic sites, while the P1 operator has only two. Thus, one site has clearly been lost or gained during the divergence of the two systems. Given at least two sites, template switching during replication might produce, at a fairly high frequency, expansions or contractions of the operator. There is also considerable evidence for the divergence of the sites within an operator. One of the two P1 sites is imperfect, and similarly, two of the three S. enterica serovar Typhimurium sites are imperfect. The tolerance for such imperfect sites is probably greatly facilitated by the presence of the cooperative interactions between adjacent sites. Given a perfect and an imperfect site, an alteration in the repressor may yield the same outcome—one imperfect site and one perfect site.

Nearly neutral network. It may be possible to alter the repressor-operator specificity, or other protein-ligand interactions, by a series of nearly neutral steps (75) (Fig. 4). This avoids the theoretical difficulties associated with invoking simultaneous mutations or low fitness intermediates. Mechanistically, the robust circuitry, the fuzzy recognition of operators by repressors, and the duplication and divergence of binding sites are three factors that may contribute to the ability of this repressor-operator system to explore genotypic space in a nearly neutral stepwise fashion.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4. Nearly neutral network. We suggest that operator-repressor space can be traversed by a nearly neutral network of single step mutations. Such a network requires a degree of fuzzy recognition between amino acid side chains and DNA bases. The degeneracy of the protein-DNA recognition code is well established (11). In the case of the P1 addiction operon and its closer homologs, the development of a nearly neutral network might be further facilitated by the robust properties of the negative feedback loop, by the presence of oligomeric interactions and by the duplication and divergence of the repressor-binding sites. To the first approximation, amino acid-base contacts appear to be fairly independent (9, 10, 12, 28, 50), however, both quantitative and qualitative exceptions to this generalization (reflecting a high degree of interdependence) have been well documented (14, 49, 62). In this figure, a repressor-operator family with a distinctly different or extensively remodeling repressor-operator interface would generate a parallel or intersecting ridgeline. Although biochemically acceptable, the transition between two such ridges might be evolutionarily difficult. The general tendency toward approximately independent contacts (75) in repressor-operator interactions (and other protein-ligand interactions) may reflect the constraints on the evolutionary process by which the system was generated rather than the structural requirements of the protein-DNA interaction itself.

 
Conclusion. The Salmonella and bacteriophage P1 addiction operons have similar patterns of autoregulation but different specificities. The key specificity determinants in this instance map to the seventh amino acid of the repressors and to the first and last base pairs of the 8-bp palindromic Phd-binding sites. The seventh amino acid is not well conserved, indicating that alterations of specificity are common in this protein family. We propose that the robust properties of the negative feedback loop, the fuzzy recognition in the operator-repressor interface, and the duplication and divergence of the repressor-binding sites have facilitated the speciation of this repressor-operator interface. These three features may allow the repressor-operator system to percolate within a nearly neutral network of single-step mutations without the necessity of invoking simultaneous mutations, low-fitness intermediates, or other improbable or rate-limiting mechanisms.


   ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grant 1 R15 GM67668-01. Xueyan Zhao was supported by a Shearwater Fellowship and by the NSF EPSCoR's Alabama Research Infrastructure Improvement Program (award no. 0091853) for the development of the Alabama Structural Biology Consortium.

Victoria Enchia provided assistance in modeling the Phd-operator interaction, and Shanveta Jordan provided technical assistance in generating the point mutations.


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


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Afif, H., N. Allali, M. Couturier, and L. Van Melderen. 2001. The ratio between CcdA and CcdB modulates the transcriptional repression of the ccd poison-antidote system. Mol. Microbiol. 41:73-82.[CrossRef][Medline]
  2. Aizenman, E., H. Engelberg-Kulka, and G. Glaser. 1996. An Escherichia coli chromosomal "addiction module" regulated by guanosine 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc. Natl. Acad. Sci. USA 93:6059-6063.[Abstract/Free Full Text]
  3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
  4. Arnold, S. J., M. E. Pfrender, and A. G. Jones. 2001. The adaptive landscape as a conceptual bridge between micro- and macroevolution. Genetica 112-113:9-32.
  5. Austin, S., and A. Abeles. 1983. Partition of unit-copy miniplasmids to daughter cells. I. P1 and F miniplasmids contain discrete, interchangeable sequences sufficient to promote equipartition. J. Mol.. Biol. 169:353-372.[CrossRef][Medline]
  6. Austin, S., F. Hart, A. Abeles, and N. Sternberg. 1982. Genetic and physical map of a P1 miniplasmid. J. Bacteriol. 152:63-71.[Abstract/Free Full Text]
  7. Austin, S., M. Ziese, and N. Sternberg. 1981. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25:729-736.[CrossRef][Medline]
  8. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. A. Seidman, J. A. Smith, and K. Struhl. 1987. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
  9. Benos, P. V., M. L. Bulyk, and G. D. Stormo. 2002. Additivity in protein-DNA interactions: how good an approximation is it? Nucleic Acids Res. 30:4442-4451.[Abstract/Free Full Text]
  10. Benos, P. V., A. S. Lapedes, and G. D. Stormo. 2002. Is there a code for protein-DNA recognition? Probab(ilistical)ly. Bioessays 24:466-475.
  11. Benos, P. V., A. S. Lapedes, and G. D. Stormo. 2002. Probabilistic code for DNA recognition by proteins of the EGR family. J. Mol.. Biol. 323:701-727.[CrossRef][Medline]
  12. Berg, O. G., and P. H. von Hippel. 1987. Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters. J. Mol.. Biol. 193:723-750.[CrossRef][Medline]
  13. Brosius, J., and A. Holy. 1984. Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc. Natl. Acad. Sci. USA 81:6929-6933.[Abstract/Free Full Text]
  14. Bulyk, M. L., P. L. Johnson, and G. M. Church. 2002. Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors. Nucleic Acids Res. 30:1255-1261.[Abstract/Free Full Text]
  15. Burch, C. L., and L. Chao. 1999. Evolution by small steps and rugged landscapes in the RNA virus {phi}6. Genetics 151:921-927.[Abstract/Free Full Text]
  16. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol.. Biol. 138:179-207.[CrossRef][Medline]
  17. Chao, L., and B. R. Levin. 1981. Structured habitats and the evolution of anticompetitor toxins in bacteria. Proc. Natl. Acad. Sci. USA 78:6324-6328.[Abstract/Free Full Text]
  18. Christensen, S. K., and K. Gerdes. 2003. RelE toxins from bacteria and Archaea cleave mRNAs on translating ribosomes, which are rescued by tmRNA. Mol. Microbiol. 48:1389-1400.[CrossRef][Medline]
  19. Christensen, S. K., M. Mikkelsen, K. Pedersen, and K. Gerdes. 2001. RelE, a global inhibitor of translation, is activated during nutritional stress. Proc. Natl. Acad. Sci. USA 98:14328-14333.[Abstract/Free Full Text]
  20. Clerget, M. 1991. Site-specific recombination promoted by a short DNA segment of plasmid R1 and by an homologous segment in the terminus region of the Escherichia coli chromosome. New Biol. 3:780-788.[Medline]
  21. Cohen, S. N., A. C. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69:2110-2114.[Abstract/Free Full Text]
  22. Cruzan, M. B. 2001. Adaptive landscapes, p. 5-7. In S. Brenner, J. H. Miller, and W. Broughton (ed.), Encyclopedia of genetics. Academic Press, Inc., New York, N.Y.
  23. Davis, T. L., D. R. Helinski, and R. C. Roberts. 1992. Transcription and autoregulation of the stabilizing functions of broad-host-range plasmid RK2 in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa. Mol. Microbiol. 6:1981-1994.[CrossRef][Medline]
  24. De Boer, H. A., L. J. Comstock, and M. Vasser. 1983. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. USA 80:21-25.[Abstract/Free Full Text]
  25. de Feyter, R., C. Wallace, and D. Lane. 1989. Autoregulation of the ccd operon in the F plasmid. Mol. Gen. Genet. 218:481-486.[CrossRef][Medline]
  26. Deane, S. M., and D. E. Rawlings. 2004. Plasmid evolution and interaction between the plasmid addiction stability systems of two related broad-host-range IncQ-like plasmids. J. Bacteriol. 186:2123-2133.[Abstract/Free Full Text]
  27. Engelberg-Kulka, H., and G. Glaser. 1999. Addiction modules and programmed cell death and antideath in bacterial cultures. Annu. Rev. Microbiol. 53:43-70.[CrossRef][Medline]
  28. Fields, D. S., Y. He, A. Y. Al-Uzri, and G. D. Stormo. 1997. Quantitative specificity of the Mnt repressor. J. Mol.. Biol. 271:178-194.[CrossRef][Medline]
  29. Gavrilets, S., and J. Gravner. 1997. Percolation on the fitness hypercube and the evolution of reproductive isolation. J. Theor. Biol. 184:51-64.[CrossRef][Medline]
  30. Gazit, E., and R. T. Sauer. 1999. Stability and DNA binding of the Phd protein of the phage P1 plasmid addiction system. J. Biol. Chem. 274:2652-2657.[Abstract/Free Full Text]
  31. Gerdes, K. 2000. Toxin-antitoxin modules may regulate synthesis of macromolecules during nutritional stress. J. Bacteriol. 182:561-572.[Free Full Text]
  32. Gotfredsen, M., and K. Gerdes. 1998. The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Mol. Microbiol. 29:1065-1076.[CrossRef][Medline]
  33. Hand, N. J., and T. J. Silhavy. 2000. A practical guide to the construction and use of lac fusions in Escherichia coli. Methods Enzymol. 326:11-35.[Medline]
  34. Hayes, C. S., and R. T. Sauer. 2003. Toxin-antitoxin pairs in bacteria: killers or stress regulators? Cell 112:2-4.[CrossRef][Medline]
  35. Hayes, F. 2003. Toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest. Science 301:1496-1499.[Abstract/Free Full Text]
  36. Ikeda, H., and J. Tomizawa. 1968. Prophage P1, an extrachromosomal replication unit. Cold Spring Harb. Symp. Quant. Biol. 33:791-798.[Medline]
  37. Ireton, K., D. Z. Rudner, K. J. Siranosian, and A. D. Grossman. 1993. Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor. Genes Dev. 7:283-294.[Abstract/Free Full Text]
  38. Jensen, R. B., and K. Gerdes. 1995. Programmed cell death in bacteria: proteic plasmid stabilization systems. Mol. Microbiol. 17:205-210.[CrossRef][Medline]
  39. Johnson, E. P., A. R. Strom, and D. R. Helinski. 1996. Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein. J. Bacteriol. 178:1420-1429.[Abstract/Free Full Text]
  40. Kauffman, S., and S. Levin. 1987. Towards a general theory of adaptive walks on rugged landscapes. J. Theor. Biol. 128:11-45.[Medline]
  41. Knight, K. L., and R. T. Sauer. 1992. Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor. EMBO J. 11:215-223.[Medline]
  42. Lah, J., I. Marianovsky, G. Glaser, H. Engelberg-Kulka, J. Kinne, L. Wyns, and R. Loris. 2003. Recognition of the intrinsically flexible addiction antidote MazE by a dromedary single domain antibody fragment. Structure, thermodynamics of binding, stability, and influence on interactions with DNA. J. Biol. Chem. 278:14101-14111.[Abstract/Free Full Text]
  43. Lehnherr, H., E. Maguin, S. Jafri, and M. B. Yarmolinsky. 1993. Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained. J. Mol.. Biol. 233:414-428.[CrossRef][Medline]
  44. Lehnherr, H., and M. B. Yarmolinsky. 1995. Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli. Proc. Natl. Acad. Sci. USA 92:3274-3277.[Abstract/Free Full Text]
  45. Lemonnier, M., S. Santos-Sierra, C. Pardo-Abarrio, and R. Diaz-Orejas. 2004. Identification of residues of the Kid toxin involved in autoregulation of the parD system. J. Bacteriol. 186:240-243.[Abstract/Free Full Text]
  46. Loris, R., I. Marianovsky, J. Lah, T. Laeremans, H. Engelberg-Kulka, G. Glaser, S. Muyldermans, and L. Wyns. 2003. Crystal structure of the intrinsically flexible addiction antidote MazE. J. Biol. Chem. 278:28252-28257.[Abstract/Free Full Text]
  47. Magnuson, R., H. Lehnherr, G. Mukhopadhyay, and M. B. Yarmolinsky. 1996. Autoregulation of the plasmid addiction operon of bacteriophage P1. J. Biol. Chem. 271:18705-18710.[Abstract/Free Full Text]
  48. Magnuson, R., and M. B. Yarmolinsky. 1998. Corepression of the P1 addiction operon by Phd and Doc. J. Bacteriol. 180:6342-6351.[Abstract/Free Full Text]
  49. Man, T. K., and G. D. Stormo. 2001. Non-independence of Mnt repressor-operator interaction determined by a new quantitative multiple fluorescence relative affinity (QuMFRA) assay. Nucleic Acids Res. 29:2471-2478.[Abstract/Free Full Text]
  50. Mandel-Gutfreund, Y., and H. Margalit. 1998. Quantitative parameters for amino acid-base interaction: implications for prediction of protein-DNA binding sites. Nucleic Acids Res. 26:2306-2312.[Abstract/Free Full Text]
  51. Marianovsky, I., E. Aizenman, H. Engelberg-Kulka, and G. Glaser. 2001. The regulation of the Escherichia coli mazEF promoter involves an unusual alternating palindrome. J. Biol. Chem. 276:5975-5984.[Abstract/Free Full Text]
  52. Masuda, Y., K. Miyakawa, Y. Nishimura, and E. Ohtsubo. 1993. chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100. J. Bacteriol. 175:6850-6856.[Abstract/Free Full Text]
  53. McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S. Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan, H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852-856.[CrossRef][Medline]
  54. McKinley, J. E., and R. D. Magnuson. 2005. Characterization of the Phd repressor/antitoxin boundary. J. Bacteriol. 187:765-770.
  55. Miller, J. H. 1992. A short course in bacterial genetics. a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  56. Mullis, K. B. 1990. Target amplification for DNA analysis by the polymerase chain reaction. Ann. Biol. Clin. (Paris) 48:579-582.[Medline]
  57. Murayama, K., P. Orth, A. B. de la Hoz, J. C. Alonso, and W. Saenger. 2001. Crystal structure of omega transcriptional repressor encoded by Streptococcus pyogenes plasmid pSM19035 at 1.5 A resolution. J. Mol.. Biol. 314:789-796.[CrossRef][Medline]
  58. Oberer, M., H. Lindner, O. Glatter, C. Kratky, and W. Keller. 1999. Thermodynamic properties and DNA binding of the ParD protein from the broad host-range plasmid RK2/RP4 killing system. Biol. Chem. 380:1413-1420.[CrossRef][Medline]
  59. Oberer, M., K. Zangger, S. Prytulla, and W. Keller. 2002. The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins. Biochem. J. 361:41-47.[CrossRef][Medline]
  60. Pabo, C. O., and R. T. Sauer. 1992. Transcription factors: structural families and principles of DNA recognition. Annu. Rev. Biochem. 61:1053-1095.[CrossRef][Medline]
  61. Prentki, P., M. Chandler, and L. Caro. 1977. Replication of prophage P1 during the cell cycle of Escherichia coli. Mol. Gen. Genet. 152:71-76.[CrossRef][Medline]
  62. Raumann, B. E., K. L. Knight, and R. T. Sauer. 1995. Dramatic changes in DNA-binding specificity caused by single residue substitutions in an Arc/Mnt hybrid repressor. Nat. Struct. Biol. 2:1115-1122.[CrossRef][Medline]
  63. Raumann, B. E., M. A. Rould, C. O. Pabo, and R. T. Sauer. 1994. DNA recognition by beta-sheets in the Arc repressor-operator crystal structure. Nature 367:754-757.[CrossRef][Medline]
  64. Rawlings, D. E. 1999. Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2. FEMS Microbiol. Lett. 176:269-277.[CrossRef][Medline]
  65. Rice, K. C., and K. W. Bayles. 2003. Death's toolbox: examining the molecular components of bacterial programmed cell death. Mol. Microbiol. 50:729-738.[CrossRef][Medline]
  66. Roberts, R. C., C. Spangler, and D. R. Helinski. 1993. Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2. J. Biol. Chem. 268:27109-27117.[Abstract/Free Full Text]
  67. Rosner, J. L. 1972. Formation, induction, and curing of bacteriophage P1 lysogens. Virology 48:679-680.[CrossRef][Medline]
  68. Ruiz-Echevarría, M. J., A. Berzal-Herranz, K. Gerdes, and R. Díaz-Orejas. 1991. The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co-ordinated action of the Kis and Kid proteins. Mol. Microbiol. 5:2685-2693.[Medline]
  69. Salmon, M. A., L. Van Melderen, P. Bernard, and M. Couturier. 1994. The antidote and autoregulatory functions of the F plasmid CcdA protein: a genetic and biochemical survey. Mol. Gen. Genet. 244:530-538.[CrossRef][Medline]
  70. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  71. Santos Sierra, S., R. Giraldo, and R. Diaz Orejas. 1998. Functional interactions between chpB and parD, two homologous conditional killer systems found in the Escherichia coli chromosome and in plasmid R1. FEMS Microbiol. Lett. 168:51-58.[CrossRef][Medline]
  72. Simons, R. W., F. Housman, and N. Kleckner. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53:85-96.[CrossRef][Medline]
  73. Smith, A. S., and D. E. Rawlings. 1998. Autoregulation of the pTF-FC2 proteic poison-antidote plasmid addiction system (pas) is essential for plasmid stabilization. J. Bacteriol. 180:5463-5465.[Abstract/Free Full Text]
  74. Smith, J. A., and R. D. Magnuson. 2004. Modular organization of the Phd repressor/antitoxin protein. J. Bacteriol. 186:2692-2698.[Abstract/Free Full Text]
  75. Smith, J. M. 1970. Natural selection and the concept of a protein space. Nature 225:563-564.[CrossRef][Medline]
  76. Suzuki, M. 1995. DNA recognition by a ß-sheet. Protein Eng. 8:1-4.
  77. Tam, J. E., and B. C. Kline. 1989. Control of the ccd operon in plasmid F. J. Bacteriol. 171:2353-2360.[Abstract/Free Full Text]
  78. Tam, J. E., and B. C. Kline. 1989. The F plasmid ccd autorepressor is a complex of CcdA and CcdB proteins. Mol. Gen. Genet. 219:26-32.[CrossRef][Medline]
  79. Tateno, M., K. Yamasaki, N. Amano, J. Kakinuma, H. Koike, M. D. Allen, and M. Suzuki. 1997. DNA recognition by ß-sheets. Biopolymers 44:335-359.[CrossRef][Medline]
  80. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
  81. Tian, Q. B., T. Hayashi, T. Murata, and Y. Terawaki. 1996. Gene product identification and promoter analysis of hig locus of plasmid Rts1. Biochem. Biophys. Res. Commun. 225:679-684.[CrossRef][Medline]
  82. Tian, Q. B., M. Ohnishi, T. Murata, K. Nakayama, Y. Terawaki, and T. Hayashi. 2001. Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1. Plasmid 45:63-74.[CrossRef][Medline]
  83. Tsuchimoto, S., and E. Ohtsubo. 1993. Autoregulation by cooperative binding of the PemI and PemK proteins to the promoter region of the pem operon. Mol. Gen. Genet. 237:81-88.[CrossRef][Medline]
  84. Wright, S. 1932. Evolution in Mendelian populations. Genetics 16:97-159.
  85. Yarmolinsky, M. B. 1995. Programmed cell death in bacterial populations. Science 267:836-837.[Free Full Text]
  86. Zhang, J., Y. Zhang, and M. Inouye. 2003. Characterization of the interactions within the mazEF addiction module of Escherichia coli. J. Biol. Chem. 278:32300-32306.[Abstract/Free Full Text]
  87. Zielenkiewicz, U., and P. Ceglowski. 2001. Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems. Acta Biochim. Pol. 48:1003-1023.[Medline]

Journal of Bacteriology, March 2005, p. 1901-1912, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.1901-1912.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, X.
Right arrow Articles by Magnuson, R. D.