Finbarr Hayes

Regulating Toxin-Antitoxin Expression: Controlled Detonation of Intracellular Molecular Timebombs

Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within.

Emerging Roles of Toxin-Antitoxin Modules in Bacterial Pathogenesis.

Toxin-antitoxin (TA) cassettes are encoded widely by bacteria. The modules typically comprise a protein toxin and protein or RNA antitoxin that sequesters the toxin factor. Toxin activation in response to environmental cues or other stresses promotes a dampening of metabolism, most notably protein translation, which permits survival until conditions improve. Emerging evidence also implicates TAs in bacterial pathogenicity. Bacterial persistence involves entry into a transient semi-dormant state in which cells survive unfavorable conditions including killing by antibiotics, which is a significant clinical problem. TA complexes play a fundamental role in inducing persistence by downregulating cellular metabolism. Bacterial biofilms are important in numerous chronic inflammatory and infectious diseases and cause serious therapeutic problems due to their multidrug tolerance and resistance to host immune system actions. Multiple TAs influence biofilm formation through a network of interactions with other factors that mediate biofilm production and maintenance. Moreover, in view of their emerging contributions to bacterial virulence, TAs are potential targets for novel prophylactic and therapeutic approaches that are required urgently in an era of expanding antibiotic resistance. This review summarizes the emerging evidence that implicates TAs in the virulence profiles of a diverse range of key bacterial pathogens that trigger serious human disease.

Amino acid residues crucial for specificity of toxin–antitoxin interactions in the homologous Axe–Txe and YefM–YoeB complexes

Toxin–antitoxin complexes are ubiquitous in bacteria. The specificity of interactions between toxins and antitoxins from homologous but non‐interacting systems was investigated. Based on molecular modeling, selected amino acid residues were changed to assess which positions were crucial in the specificity of toxin–antitoxin interaction in the related Axe–Txe and YefM–YoeB complexes. No cross‐interactions between wild‐type proteins were detected. However, a single amino acid substitution that converts a Txe‐specific residue to a YoeB‐specific residue reduced, but did not abolish, Txe interaction with the Axe antitoxin. Interestingly, this alteration (Txe‐Asp83Tyr) promoted functional interactions between Txe and the YefM antitoxin. The interactions between Txe‐Asp83Tyr and YefM were sufficiently strong to abolish Txe toxicity and to allow effective corepression by YefM‐Txe‐Asp83Tyr of the promoter from which yefM–yoeB is expressed. We conclude that Asp83 in Txe is crucial for the specificity of toxin–antitoxin interactions in the Axe–Txe complex and that swapping this residue for the equivalent residue in YoeB relaxes the specificity of the toxin–antitoxin interaction.

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli

The ability to assemble DNA sequences de novo through efficient and powerful DNA fabrication methods is one of the foundational technologies of synthetic biology. Gene synthesis, in particular, has been considered the main driver for the emergence of this new scientific discipline. Here we describe RapGene, a rapid gene assembly technique which was successfully tested for the synthesis and cloning of both prokaryotic and eukaryotic genes through a ligation independent approach. The method developed in this study is a complete bacterial gene synthesis platform for the quick, accurate and cost effective fabrication and cloning of gene-length sequences that employ the widely used host Escherichia coli.

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

Abstract Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.

Terminator Operon Reporter : Combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications

Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology.

Probe discovery: Disentangling gene networks.

Cell-wall biogenesis in bacteria involves multiple intersecting gene networks. A powerful approach that allies synthetic lethality with small-molecule discovery has now been used to probe these networks and has revealed that the pathway for D-alanylation of teichoic acids in Staphylococcus aureus is a viable target for new antibacterials.

Breaking and Restoring the Hydrophobic Core of a Centromere-binding Protein

Background: Accurate segregation of antibiotic resistance plasmids requires dedicated centromere-binding proteins. Results: Assembly of the hydrophobic core of the ParG centromere-binding protein encoded by multiresistance plasmid TP228 is governed by a triad of key amino acids. Conclusion: ParG retains functionality with certain substitutions of the core triad. Significance: The study provides valuable insights into how multiresistance plasmids are maintained in bacteria. The ribbon-helix-helix (RHH) superfamily of DNA-binding proteins is dispersed widely in procaryotes. The dimeric RHH fold is generated by interlocking of two monomers into a 2-fold symmetrical structure that comprises four α-helices enwrapping a pair of antiparallel β-strands (ribbon). Residues in the ribbon region are the principal determinants of DNA binding, whereas the RHH hydrophobic core is assembled from amino acids in both the α-helices and ribbon element. The ParG protein encoded by multiresistance plasmid TP228 is a RHH protein that functions dually as a centromere binding factor during segrosome assembly and as a transcriptional repressor. Here we identify residues in the α-helices of ParG that are critical for DNA segregation and in organization of the protein hydrophobic core. A key hydrophobic aromatic amino acid at one position was functionally substitutable by other aromatic residues, but not by non-aromatic hydrophobic amino acids. Nevertheless, intramolecular suppression of the latter by complementary change of a residue that approaches nearby from the partner monomer fully restored activity in vivo and in vitro. The interactions involved in assembling the ParG core may be highly malleable and suggest that RHH proteins are tractable platforms for the rational design of diverse DNA binding factors useful for synthetic biology and other purposes.