A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA.

Post-transcriptional regulation plays important roles to fine-tune gene expression in bacteria. In particular, regulation of type I toxin-antitoxin (TA) systems is achieved through sophisticated mechanisms involving toxin mRNA folding. Here, we set up a genetic approach to decipher the molecular underpinnings behind the regulation of a type I TA in Helicobacter pylori. We used the lethality induced by chromosomal inactivation of the antitoxin to select mutations that suppress toxicity. We found that single point mutations are sufficient to allow cell survival. Mutations located either in the 5' untranslated region or within the open reading frame of the toxin hamper its translation by stabilizing stem-loop structures that sequester the Shine-Dalgarno sequence. We propose that these short hairpins correspond to metastable structures that are transiently formed during transcription to avoid premature toxin expression. This work uncovers the co-transcriptional inhibition of translation as an additional layer of TA regulation in bacteria.

FASTBAC-Seq: Functional Analysis of Toxin-Antitoxin Systems in Bacteria by Deep Sequencing.

As the number of bacterial genomes and transcriptomes increases, so does the number of newly identified toxin-antitoxin (TA) systems. However, their functional characterization remains challenging, often requiring the use of overexpression vectors that can lead to misinterpretations of in vivo results. To fill this gap, we developed a systematic approach called FASTBAC-Seq (Functional AnalysiS of Toxin-Antitoxin Systems in BACteria by Deep Sequencing). Combining life/death phenotypic selection with next-generation sequencing, FASTBAC-Seq allows the rapid identification of loss-of-function (toxicity) mutations in toxin-encoding genes belonging to TA loci with nucleotide resolution. Here, we present the setup used on the first-time application of FASBACT-Seq to characterize a member of the aapA/IsoA family of type I TA systems hosted on the chromosome of the major human gastric pathogen Helicobacter pylori. We propose FASBACT-Seq as a powerful tool for the functional characterization of TA systems that can in addition uncover key elements for the understanding of gene expression regulation in bacteria.

Type I Toxin-Antitoxin Systems: Regulating Toxin Expression via Shine-Dalgarno Sequence Sequestration and Small RNA Binding.

Toxin-antitoxin (TA) systems are small genetic loci composed of two adjacent genes: a toxin and an antitoxin that prevents toxin action. Despite their wide distribution in bacterial genomes, the reasons for TA systems being on chromosomes remain enigmatic. In this review, we focus on type I TA systems, composed of a small antisense RNA that plays the role of an antitoxin to control the expression of its toxin counterpart. It does so by direct base-pairing to the toxin-encoding mRNA, thereby inhibiting its translation and/or promoting its degradation. However, in many cases, antitoxin binding is not sufficient to avoid toxicity. Several cis-encoded mRNA elements are also required for repression, acting to uncouple transcription and translation via the sequestration of the ribosome binding site. Therefore, both antisense RNA binding and compact mRNA folding are necessary to tightly control toxin synthesis and allow the presence of these toxin-encoding systems on bacterial chromosomes.

A fungal pathogen secretes plant alkalinizing peptides to increase infection.

Plant infections caused by fungi are often associated with an increase in the pH of the surrounding host tissue(1). Extracellular alkalinization is thought to contribute to fungal pathogenesis, but the underlying mechanisms are poorly understood. Here, we show that the root-infecting fungus Fusarium oxysporum uses a functional homologue of the plant regulatory peptide RALF (rapid alkalinization factor)(2,3) to induce alkalinization and cause disease in plants. An upshift in extracellular pH promotes infectious growth of Fusarium by stimulating phosphorylation of a conserved mitogen-activated protein kinase essential for pathogenicity(4,5). Fungal mutants lacking a functional Fusarium (F)-RALF peptide failed to induce host alkalinization and showed markedly reduced virulence in tomato plants, while eliciting a strong host immune response. Arabidopsis plants lacking the receptor-like kinase FERONIA, which mediates the RALF-triggered alkalinization response(6), displayed enhanced resistance against Fusarium. RALF homologues are found across a number of phylogenetically distant groups of fungi, many of which infect plants. We propose that fungal pathogens use functional homologues of alkalinizing peptides found in their host plants to increase their infectious potential and suppress host immunity.