RESUMO
Toxin-antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.
Assuntos
Antitoxinas/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Domínios Proteicos/genética , Sistemas Toxina-Antitoxina/genética , Proteínas de Bactérias/genética , Burkholderia/genética , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Pentafosfato/genética , Óperon/genética , Prófagos/genéticaRESUMO
HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.
Assuntos
Listeria monocytogenes , Listeria monocytogenes/genética , Proteínas de Ligação ao GTP/genética , Resistência Microbiana a Medicamentos , Ribossomos/genética , Ribossomos/metabolismo , Lincosamidas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismoRESUMO
Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.
Assuntos
Toxinas Bacterianas/farmacologia , GTP Fosfo-Hidrolases/genética , RNA Ribossômico 23S/genética , Sistemas de Secreção Tipo VI/efeitos dos fármacos , ADP-Ribosilação/efeitos dos fármacos , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , NAD/genética , Fator G para Elongação de Peptídeos/genética , Photorhabdus/química , Photorhabdus/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 23S/efeitos dos fármacos , Tioestreptona/química , Tioestreptona/farmacologiaRESUMO
Antitoxins encoded by type II toxin - antitoxin (TA) modules neutralize cognate toxins by direct protein - protein contact and in addition, regulate TA operon transcription by binding to operators in the promoter regions. On top of the simple negative feed-back regulation, canonical type II TA operons are regulated by a mechanism called 'Conditional Cooperativity'(CC). In CC, the cellular toxin:antitoxin (T:A) ratio controls the transcription-rate such that low T:A ratios favour repression and high T:A ratios favour de-repression of TA operon transcription. Here a new molecular mechanism that secures selective synthesis of antitoxin in the presence of excess toxin was unravelled. The hicAB locus of E. coli K-12 encodes HicA mRNase and HicB antitoxin. It was shown that hicAB is transcribed by two promoters, an upstream one that is activated by CRP-cAMP and competence factor Sxy and a downstream one that is autorepressed solely by HicB. Excess HicA destabilizes the HicBâ¢operator complex in vitro and consistently, activates hicAB transcription in vivo. Remarkably, the hicAB transcript synthesized from the HicB-controlled promoter produces HicB but not HicA. Thus, the HicA-mediated derepression of hicAB transcription provides a mechanism that conditionally and selectively stimulates synthesis of HicB antitoxin under conditions of excess HicA toxin.