GNAT toxins evolve toward narrow tRNA target specificities.

Type II toxin-antitoxin (TA) systems are two-gene modules widely distributed among prokaryotes. GNAT toxins associated with the DUF1778 antitoxins represent a large family of type II TAs. GNAT toxins inhibit cell growth by disrupting translation via acetylation of aminoacyl-tRNAs. In this work, we explored the evolutionary trajectory of GNAT toxins. Using LC/MS detection of acetylated aminoacyl-tRNAs combined with ribosome profiling, we systematically investigated the in vivo substrate specificity of an array of diverse GNAT toxins. Our functional data show that the majority of GNAT toxins are specific to Gly-tRNA isoacceptors. However, the phylogenetic analysis shows that the ancestor of GNAT toxins was likely a relaxed specificity enzyme capable of acetylating multiple elongator tRNAs. Together, our data provide a remarkable snapshot of the evolution of substrate specificity.

Auxiliary interfaces support the evolution of specific toxin-antitoxin pairing.

Toxin-antitoxin (TA) systems are a large family of genes implicated in the regulation of bacterial growth and its arrest in response to attacks. These systems encode nonsecreted toxins and antitoxins that specifically pair, even when present in several paralogous copies per genome. Salmonella enterica serovar Typhimurium contains three paralogous TacAT systems that block bacterial translation. We determined the crystal structures of the three TacAT complexes to understand the structural basis of specific TA neutralization and the evolution of such specific pairing. In the present study, we show that alteration of a discrete structural add-on element on the toxin drives specific recognition by their cognate antitoxin underpinning insulation of the three pairs. Similar to other TA families, the region supporting TA-specific pairing is key to neutralization. Our work reveals that additional TA interfaces beside the main neutralization interface increase the safe space for evolution of pairing specificity.

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis.

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology.

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.

A tRNA-Acetylating Toxin and Detoxifying Enzyme in Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems allow bacteria to adapt to changing environments without altering gene expression. Despite being overrepresented in Mycobacterium tuberculosis, their physiological roles remain elusive. We describe a TA system in M. tuberculosis which we named TacAT due to its homology to previously discovered systems in Salmonella. The toxin, TacT, blocks growth by acetylating glycyl-tRNAs and inhibiting translation. Its effects are reversed by the enzyme peptidyl tRNA hydrolase (Pth), which also cleaves peptidyl tRNAs that are prematurely released from stalled ribosomes. Pth is essential in most bacteria and thereby has been proposed as a promising drug target for complex pathogens like M. tuberculosis. Transposon sequencing data suggest that the tacAT operon is nonessential for M. tuberculosis growth in vitro, and premature stop mutations in this TA system present in some clinical isolates suggest that it is also dispensable in vivo. We assessed whether TacT modulates pth essentiality in M. tuberculosis because drugs targeting Pth might prompt resistance if TacAT is disrupted. We show that pth essentiality is unaffected by the absence of tacAT. These results highlight a fundamental aspect of mycobacterial biology and indicate that Pth's essential role hinges on its peptidyl-tRNA hydrolase activity. Our work underscores Pth's potential as a viable target for new antibiotics. IMPORTANCE The global rise in antibiotic-resistant tuberculosis has prompted an urgent search for new drugs. Toxin-antitoxin (TA) systems allow bacteria to adapt rapidly to environmental changes, and Mycobacterium tuberculosis encodes more TA systems than any known pathogen. We have characterized a new TA system in M. tuberculosis: the toxin, TacT, acetylates charged tRNA to block protein synthesis. TacT's effects are reversed by the essential bacterial enzyme peptidyl tRNA hydrolase (Pth), which is currently being explored as an antibiotic target. Pth also cleaves peptidyl tRNAs that are prematurely released from stalled ribosomes. We assessed whether TacT modulates pth essentiality in M. tuberculosis because drugs targeting Pth might prompt resistance if TacT is disrupted. We show that pth essentiality is unaffected by the absence of this TA system, indicating that Pth's essential role hinges on its peptidyl-tRNA hydrolase activity. Our work underscores Pth's potential as a viable target for new antibiotics.