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In recent decades, there has been a rapid increase in the prevalence of multidrug‐resistant pathogens, posing a challenge to modern antibiotic‐based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co‐inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach. [ABSTRACT FROM AUTHOR]

Toxin‐antitoxin loci regulate adaptive responses to stresses associated with the host environment and drug exposure. Phylogenomic studies have shown that Mycobacterium tuberculosis encodes a naturally expanded type II toxin‐antitoxin system, including ParDE/RelBE superfamily members. Type II toxins are presumably regulated exclusively through protein–protein interactions with type II antitoxins. However, experimental observations in M. tuberculosis indicated that additional control mechanisms regulate RelBE2 type II loci under host‐associated stress conditions. Herein, we describe for the first time a novel antisense RNA, termed asRelE2, that co‐regulates RelE2 production via targeted processing by the Mtb RNase III, Rnc. We find that convergent expression of this coding‐antisense hybrid TA locus, relBE2‐asrelE2, is controlled in a cAMP‐dependent manner by the essential cAMP receptor protein transcription factor, Crp, in response to the host‐associated stresses of low pH and nutrient limitation. Ex vivo survival studies with relE2 and asrelE2 knockout strains showed that RelE2 contributes to Mtb survival in activated macrophages and low pH to nutrient limitation. To our knowledge, this is the first report of a novel tripartite type IIb TA loci and antisense post‐transcriptional regulation of a type II TA loci. [ABSTRACT FROM AUTHOR]

Of the ~80 putative toxin‐antitoxin (TA) modules encoded by the bacterial pathogen Mycobacterium tuberculosis (Mtb), three contain antitoxins essential for bacterial viability. One of these, Rv0060 (DNA ADP‐ribosyl glycohydrolase, DarGMtb), functions along with its cognate toxin Rv0059 (DNA ADP‐ribosyl transferase, DarTMtb), to mediate reversible DNA ADP‐ribosylation (Jankevicius et al., 2016). We demonstrate that DarTMtb‐DarGMtb form a functional TA pair and essentiality of darGMtb is dependent on the presence of darTMtb, but simultaneous deletion of both darTMtb‐darGMtb does not alter viability of Mtb in vitro or in mice. The antitoxin, DarGMtb, forms a cytosolic complex with DNA‐repair proteins that assembles independently of either DarTMtb or interaction with DNA. Depletion of DarGMtb alone is bactericidal, a phenotype that is rescued by expression of an orthologous antitoxin, DarGTaq, from Thermus aquaticus. Partial depletion of DarGMtb triggers a DNA‐damage response and sensitizes Mtb to drugs targeting DNA metabolism and respiration. Induction of the DNA‐damage response is essential for Mtb to survive partial DarGMtb‐depletion and leads to a hypermutable phenotype. [ABSTRACT FROM AUTHOR]

Bacterial toxin-antitoxin systems are important factors implicated in growth inhibition and plasmid maintenance. Type II toxin-antitoxin pairs are regulated at the transcriptional level by the antitoxin itself. Here, we examined how the HigA antitoxin regulates the expression of the Proteus vulgaris higBA toxin-antitoxin operon from the Rts1 plasmid. The HigBA complex adopts a unique architecture suggesting differences in its regulation as compared to classical type II toxin-antitoxin systems. We find that the C-terminus of the HigA antitoxin is required for dimerization and transcriptional repression. Further, the HigA structure reveals that the C terminus is ordered and does not transition between disorder-to-order states upon toxin binding. HigA residue Arg40 recognizes a TpG dinucleotide in higO2, an evolutionary conserved mode of recognition among prokaryotic and eukaryotic transcription factors. Comparison of the HigBA and HigA-higO2 structures reveals the distance between helix-turn-helix motifs of each HigA monomer increases by ~4 Å in order to bind to higO2. Consistent with these data, HigBA binding to each operator is twofold less tight than HigA alone. Together, these data show the HigB toxin does not act as a co-repressor suggesting potential novel regulation in this toxin-antitoxin system., (© 2019 John Wiley & Sons Ltd.)

Members of the genus Shigella carry a large plasmid, pINV, which is essential for virulence. In Shigella flexneri, pINV harbours three toxin-antitoxin (TA) systems, CcdAB, GmvAT and VapBC that promote vertical transmission of the plasmid. Type II TA systems, such as those on pINV, consist of a toxic protein and protein antitoxin. Selective degradation of the antitoxin by proteases leads to the unopposed action of the toxin once genes encoding a TA system have been lost, such as following failure to inherit a plasmid harbouring a TA system. Here, we investigate the role of proteases in the function of the pINV TA systems and demonstrate that Lon, but not ClpP, is required for their activity during plasmid stability. This provides the first evidence that acetyltransferase family TA systems, such as GmvAT, can be regulated by Lon. Interestingly, S. flexneri pINV also harbours two putative partitioning systems, ParAB and StbAB. We show that both systems are functional for plasmid maintenance although their activity is masked by other systems on pINV. Using a model vector based on the pINV replicon, we observe temperature-dependent differences between the two partitioning systems that contribute to our understanding of the maintenance of virulence in Shigella species., (© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

Toxin-antitoxin systems are mediators of diverse activities in bacterial physiology. For the ParE-type toxins, their reported role of gyrase inhibition utilized during plasmid-segregation killing indicates they are toxic. However, their location throughout chromosomes leads to questions about function, including potential non-toxic outcomes. The current study has characterized a ParDE system from the opportunistic human pathogen Pseudomonas aeruginosa (Pa). We identified a protective function for this ParE toxin, PaParE, against effects of quinolone and other antibiotics. However, higher concentrations of PaParE are themselves toxic to cells, indicating the phenotypic outcome can vary based on its concentration. Our assays confirmed PaParE inhibition of gyrase-mediated supercoiling of DNA with an IC 50 value in the low micromolar range, a species-specificity that resulted in more efficacious inhibition of Escherichia coli derived gyrase versus Pa gyrase, and overexpression in the absence of antitoxin yielded an expected filamentous morphology with multi-foci nucleic acid material. Additional data revealed that the PaParE toxin is monomeric and interacts with dimeric PaParD antitoxin with a K D in the lower picomolar range, yielding a heterotetramer. This work provides novel insights into chromosome-encoded ParE function, whereby its expression can impart partial protection to cultures from selected antibiotics., (© 2018 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

Type II toxin-antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a nontoxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res-xre locus from Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli. The 2.2 Å crystal structure of the intact Pseudomonas putida RES-Xre TA complex reveals an unusual 2:4 stoichiometry in which a central RES toxin dimer binds two Xre antitoxin dimers. The antitoxin dimers each expose two helix-turn-helix DNA-binding domains of the Cro repressor type, suggesting the TA complex is capable of binding the upstream promoter sequence on DNA. The toxin core domain shows structural similarity to ADP-ribosylating enzymes such as diphtheria toxin but has an atypical NAD + -binding pocket suggesting an alternative function. We show that activation of the toxin in vivo causes a depletion of intracellular NAD + levels eventually leading to inhibition of cell growth in E. coli and inhibition of global macromolecular biosynthesis. Both structure and activity are unprecedented among bacterial TA systems, suggesting the functional scope of bacterial TA toxins is much wider than previously appreciated., (© 2018 John Wiley & Sons Ltd.)

GCN5-related N-acetyltransferase (GNAT) is a huge superfamily of proteins spanning the prokaryotic and eukaryotic domains of life. GNAT proteins usually transfer an acetyl group from acetyl-CoA to a wide variety of substrates ranging from aminoglycoside antibiotics to large macromolecules. Type II toxin-antitoxin (TA) modules are typically bicistronic and widespread in bacterial and archael genomes with diverse cellular functions. Recently, a novel family of type II TA toxins was described, which presents a GNAT-fold and functions by acetylating charged tRNA thereby precluding translation. These GNAT toxins are usually associated with a corresponding ribbon-helix-helix-fold (RHH) antitoxin. In this issue, Qian et al. describes a unique GNAT-RHH TA system, designated KacAT, from a multidrug resistant strain of the pathogen, Klebsiella pneumoniae. As most type II TA loci, kacAT is transcriptionally autoregulated with the KacAT complex binding to the operator site via the N-terminus region of KacA to repress kacAT transcription. The crystal structure of the KacT toxin is also presented giving a structural basis for KacT toxicity. These findings expand our knowledge on this newly discovered family of TA toxins and the potential role that they may play in antibiotic tolerance and persistence of bacterial pathogens., (© 2018 John Wiley & Sons Ltd.)