Roy David Magnuson

Biochemical and genetic characterization of a competence pheromone from B. subtilis.

We have purified and characterized a modified peptide pheromone that accumulates in culture medium as B. subtilis grows to high density. This pheromone is required for the development of genetic competence. When added to cells at low density, the pheromone induces the premature development of competence. The peptide moiety of the pheromone matches nine of the last ten amino acids predicted from a 55 codon open reading frame, comX. comX and comQ, the gene immediately upstream of comX, are required for production of the pheromone. Response to the pheromone requires the comP-comA two-component regulatory system and the oligopeptide permease encoded by spo0K. Spo0K could transport the pheromone into the cell, or function as a receptor, binding the pheromone and sending a transmembrane signal, leading to activation of the ComA transcription factor and induction of competence development.

Hypothetical Functions of Toxin-Antitoxin Systems

Toxin-antitoxin systems are very commonly found both on large, low-copy plasmids, where they increase effective stability ([35][1]), and on bacterial chromosomes, where their function has been the subject of considerable speculation. In this issue of the Journal of Bacteriology, Virginie Tsilibaris

Doc of Prophage P1 Is Inhibited by Its Antitoxin Partner Phd through Fold Complementation

Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an α-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.

Autoregulation of the Plasmid Addiction Operon of Bacteriophage P1

The P1 plasmid addiction operon increases the apparent stability of a plasmid that carries it by killing plasmid-free (cured) segregants. The operon consists of a gene encoding an endotoxin responsible for eath n uring (doc), preceded by a gene encoding a relatively unstable antidote that can revent ost eath (phd). When the copy number of the operon was increased, expression of a lacZ reporter fused to the promoter of the operon decreased, indicating that expression of the operon was stabilized by an autoregulatory circuit. Transcription of the lacZ reporter was repressed about 10-fold when phd, without doc, was expressed from an exogenous promoter. DNase I footprinting showed that Phd binds a perfect 10-base pair palindromic DNA sequence and, at higher concentrations, an adjacent, imperfect palindrome. The palindromic sites are located between the −10 region of the putative promoter and the start codon of phd. Electrophoretic mobility of DNA containing the promoter region was retarded in the presence of Phd and further retarded in the presence of Phd and Doc. When doc was co-expressed with phd, repression of the lacZ fusion was enhanced more than 100-fold. Thus, both products of the addiction operon participate in its autoregulation.

Modular Organization of the Phd Repressor/Antitoxin Protein

The P1 plasmid addiction operon is a compact genetic structure consisting of promoter, operator, antitoxin gene (phd), and toxin gene (doc). The 73-amino-acid antitoxin protein, Phd, has two distinct functions: it represses transcription (by binding to its operator) and it prevents host death (by binding and neutralizing the toxin). Here, we show that the N terminus of Phd is required for repressor but not antitoxin activity. Conversely, the C terminus is required for antitoxin but not repressor activity. Only a quarter of the protein, the resolution limit of this analysis, was required for both activities. We suggest that the plasmid addiction operon is a composite of two evolutionarily separable modules, an operator-repressor module and an antitoxin-toxin module. Consideration of similar antitoxin proteins and their surroundings indicates that modular exchange may contribute to antitoxin and operon diversity.

Characterization of the Phd Repressor-Antitoxin Boundary

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.

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.

Percolation of the Phd Repressor-Operator Interface

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.

Crystallization of Doc and the Phd–Doc toxin–antitoxin complex

The phd/doc addiction system is responsible for the stable inheritance of lysogenic bacteriophage P1 in its plasmidic form in Escherichia coli and is the archetype of a family of bacterial toxin-antitoxin modules. The His66Tyr mutant of Doc (Doc(H66Y)) was crystallized in space group P2(1), with unit-cell parameters a = 53.1, b = 198.0, c = 54.1 A, beta = 93.0 degrees . These crystals diffracted to 2.5 A resolution and probably contained four dimers of Doc in the asymmetric unit. Doc(H66Y) in complex with a 22-amino-acid C-terminal peptide of Phd (Phd(52-73Se)) was crystallized in space group C2, with unit-cell parameters a = 111.1, b = 38.6, c = 63.3 A, beta = 99.3 degrees , and diffracted to 1.9 A resolution. Crystals of the complete wild-type Phd-Doc complex belonged to space group P3(1)21 or P3(2)21, had an elongated unit cell with dimensions a = b = 48.9, c = 354.9 A and diffracted to 2.4 A resolution using synchrotron radiation.

Type II Toxin-Antitoxin Loci: The phd/doc Family

The phd/doc family is one the smallest families of toxin–antitoxin modules and was first discovered as a plasmid addiction module on E. coli bacteriophage P1. The toxin Doc interacts with the ribosome, competes with hygromycin, and inhibits translation. Structurally, Doc resembles Fic domains, which are known to transfer an AMP moiety to the hydroxyphenyl group of a tyrosine in the target protein. Although much of the AMP/ATP binding site of Fic is conserved in Doc, no specific enzymatic activity has yet been linked to Doc. Nevertheless, mutations in the loop corresponding to the active site loop of Fic render Doc inactive. Regulation of the P1 phd/doc operon is understood in terms of a detailed molecular mechanism and involves conditional cooperativity. The N-terminal domain of Phd forms the DNA-binding unit and represents a common DNA-binding fold that is also shared with a number of antitoxins from different TA families, among which YefM is the best studied. Enhancement of the DNA-binding affinity of Phd by Doc stems both from allosteric coupling between the Doc- and DNA-binding sites on Phd and from avidity effects due to Doc-mediated bridging of two Phd dimers bound to the operator site. Activation of the system at high Doc-to-Phd ratios stems from a low-to-high affinity switch in the interaction between Phd and Doc.