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Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

The first member of the pleuromutilin (PLM) class suitable for systemic antibacterial chemotherapy in humans recently entered clinical use, underscoring the need to better understand mechanisms of PLM resistance in disease-causing bacterial genera. Of the proteins reported to mediate PLM resistance in staphylococci, the least-well studied to date is Sal(A), a putative ABC-F NTPase that—by analogy to other proteins of this type—may act to protect the ribosome from PLMs. Here, we establish the importance of Sal proteins as a common source of PLM resistance across multiple species of staphylococci. Sal(A) is revealed as but one member of a larger group of Sal-type ABC-F proteins that vary considerably in their ability to mediate resistance to PLMs and other antibiotics. We find that specific sal genes are intrinsic to particular staphylococcal species, and show that this gene family is likely ancestral to the genus Staphylococcus. Finally, we solve the cryo-EM structure of a representative Sal-type protein (Sal(B)) in complex with the staphylococcal 70S ribosome, revealing that Sal-type proteins bind into the E site to mediate target protection, likely by displacing PLMs and other antibiotics via an allosteric mechanism.

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.

Bacteria experience a wide range of natural challenges during their life cycles, to which they must respond and adapt to live. Under stressed conditions such as amino acid starvation, bacteria slow down their growth mechanism by producing small alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp. Accumulation of (p)ppGpp results in a comprehensive alteration in cellular metabolism. The alarmone (p)ppGpp is produced and degraded by enzymes belonging to the RelA/SpoT Homologue (RSH) protein family, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli. The members of the RSH protein family can be classified into long multi-domain RSHs and short single-domain RSHs. Long RSH enzymes such as RelA and SpoT, carry both the (p)ppGpp hydrolysis (HD) domain and (p)ppGpp synthesis (SYNTH) domain in the N-terminal domain enzymatic region (NTD), in combination with additional domains in the C-terminal domain regulatory region (CTD). Short single-domain RSHs can be divided into small alarmone hydrolases (SAHs) carrying the HD domain, and small alarmone synthetases (SASs) that only have the SYNTH domain. At the beginning of my PhD, I studied the diversity of RSH proteins across the tree of life. I identified 35,615 RSH proteins from analyses of 24,072 genomes. I used large-scale phylogenetic analyses to classify the RSH proteins into 13 long RSHs, 11 SAHs and 30 SASs subfamilies. To address why bacteria often carry multiple SASs in the same genome and predict new functions, I developed a computational tool called FlaGs – standing for Flanking Genes – to analyse the conservation of genomic neighbourhoods in large datasets. I also developed a web-based version of FlaGs, called webFlaGs, that is publicly accessible and is used by biologists all over the world. The application of FlaGs to SAS RSHs led to the discovery that multiple SAS subfamilies are encoded in conserved and frequently, Number in series missing in publication.

Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system which uses a holin membrane protein in tandem with a cell wall editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall editing enzymes employed was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS). Potential mechanisms for secretion across the outer membrane are discussed., Special Issue: Protein Secretion and Transport

Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (Flanking Genes) takes a list of NCBI protein accessions as input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. The web version of FlaGs (webFlaGs) can optionally include a BLASTP search against a reduced RefSeq database to generate an input accession list and analyse neighbourhood conservation within the same run.

Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (Flanking Genes) takes a list of NCBI protein accessions as input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. The web version of FlaGs (webFlaGs) can optionally include a BLASTP search against a reduced RefSeq database to generate an input accession list and analyse neighbourhood conservation within the same run.

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

Toxin-antitoxin (TA) systems are a large group of evolutionary unrelated small genetic modules, typically consisting of two genes that encode a toxin whose overexpression leads to growth arrest and an antitoxin that neutralises the toxin. They are present in bacteria, archaea as well as in genomes of temperate phage, and involved in important biological functions including genomic element stabilization and defence against phages. Previously, using the prediction by our developed bioinformatics tool FlaGs – that can perform conservation of gene neighbourhood analyses at long evolutionary distance – we discovered a novel class of proteinaceous TA systems related to RelA-SpoT Homolog (RSH) protein family of housekeeping stress-response enzymes. Next, by combining phylogenetic analyses of a diverse set of genomes across the tree of life and predictions from FlaGs, we have discovered a hyperpromiscuous antitoxin domain Panacea, that is widely distributed and capable of neutralising dozens of distinct toxic domains. We asked if Panacea is unique in its hyperpromiscuity. To answer this question, we performed a bioinformatic analysis across the tree of prokaryotic life using our developed tool NetFlax standing for Network-FlaGs for toxins and antitoxins. NetFlax can predict TA-like arrangements and group them into homologous clusters of toxins and antitoxins in an unsupervised manner. In the present study, we have identified 43 clusters that are predicted to be toxins and 34 clusters of predicted antitoxins using the NetFlax pipeline. 21 of these 77 predicted clusters are homologous to previously discovered toxins and antitoxins, which validates the NetFlax strategy. Together, the interconnected clusters constitute a core network of TA-like pairs across all microbial life, where the hyperpromiscuous clusters serve as hubs. The NetFlax network indicates the presence of novel hyperpromiscuous toxin and antitoxin domains still to be explored. The interactive version of the net