Parst Is a Widespread Toxin–Antitoxin Module That Targets Nucleotide Metabolism

ParST is a widespread toxin–antitoxin module that targets nucleotide metabolism Frank J. Piscottaa, Philip D. Jeffreyb, and A. James Linka,b,c,1 aDepartment of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544; bDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; and cDepartment of Chemistry, Princeton University, Princeton, NJ 08544 Edited by Marlene Belfort, University at Albany, Albany, NY, and approved December 4, 2018 (received for review August 27, 2018) Toxin–antitoxin (TA) systems interfere with essential cellular pro- I toxins are typified by an RNA-level TA interaction, where an cesses and are implicated in bacterial lifestyle adaptations such as antisense RNA binds toxin mRNA to inhibit its translation. Type persistence and the biofilm formation. Here, we present structural, II TA systems function via protein–protein interactions, where biochemical, and functional data on an uncharacterized TA system, binding of the antitoxin to the toxin inhibits its activity (10). Of the COG5654–COG5642 pair. Bioinformatic analysis showed that the two earliest known TA systems mentioned above, hok/sok is this TA pair is found in 2,942 of the 16,286 distinct bacterial species an example of a type I system, while ccdAB is type II (11, 12). in the RefSeq database. We solved a structure of the toxin bound Upon translation, type I toxins are small, hydrophobic peptides to a fragment of the antitoxin to 1.50 Å. This structure suggested that lead to cell lysis through disruption of the plasma mem- that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin brane. Type II toxins, however, function through a variety of specifically modifies phosphoribosyl pyrophosphate synthetase mechanisms. A large group of these are endoribonucleases that (Prs), an essential enzyme in nucleotide biosynthesis conserved cleave RNA in either a ribosome-dependent or independent in all organisms. We propose renaming the toxin ParT for Prs manner. The well-studied HigB, RelE, and MazF toxins, among ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT many others, all fall into this group (13–15). Beyond these, is a unique example of an intracellular protein mART in bacteria however, there is much variation. HipA phosphorylates glutamyl- and is the smallest known mART. This work demonstrates that TA tRNA synthetase (GltX), inactivating it, while CcdB engages in systems can induce bacteriostasis through interference with nucle- gyrase-mediated double-stranded DNA cleavage (16, 17). An- otide biosynthesis. other toxin, FicT, also interferes with DNA gyrase, but rather by inactivation through adenylation (18). More recently, DarT was toxin–antitoxin system | ADP-ribosylation | posttranslational modification found to ADP-ribosylate single-stranded DNA (19). The con- tinued discovery of new toxins like DarT emphasizes the role of ur understanding of toxin–antitoxin (TA) systems has pro- TA systems in cell biology. Ogressed significantly since their identification nearly 35 y Type II TA systems are quite prevalent throughout bacterial ago. In the pioneering work, ccdAB was shown to enhance the genomes. In a comprehensive analysis by Makarova et al. (20) in stability of a mini-F plasmid in Escherichia coli by killing 2009, of the 750 reference genomes searched, putative type II daughter cells lacking the plasmid and also established that the TA systems were found in 631, with on average 10 loci in each hit ccdA genetic element could provide an antidote for the post- (6,797 total). The number of occurrences in a single genome can segregational killing (PSK) of ccdB (1). An investigation into the be much larger, however; the deadly pathogen Mycobacterium E. coli R1 plasmid revealed a similar system hok/sok that also tuberculosis has over 88 TA loci in its genome, while the soil improved plasmid stability, where again one element, sok, could bacterium and plant symbiont Sinorhizobium meliloti contains neutralize the PSK activity of the other, hok (2). Results such as an astounding 211 predicted type II loci (21, 22). Makarova these led to the hypothesis that the role of such genetic regions et al. proposed the existence of 19 new putative type II TA pairs, was to ensure the inheritance of plasmids during cell division. It was later discovered, however, that these systems resided not Significance only in plasmids but also on bacterial chromosomes and that, while not conferring replicon stability, they may play an impor- – tant role in regulating cell growth (3–5). Further investigations Toxin antitoxin (TA) systems are pairs of genes found throughout into bacterial TA systems have also implicated them in biofilm bacteria that function in DNA maintenance and bacterial sur- vival. The toxins in these systems function by inactivating growth and persister formation. The toxin gene mqsR was first critical cell processes like protein synthesis. This work describes shown to be up-regulated in E. coli biofilms and later confirmed a TA system found in 17% of all sequenced bacteria. A high- to promote biofilm formation through regulation of quorum resolution crystal structure and an array of biochemical tests sensing (6, 7). One of the most ubiquitous TA systems, hipBA, revealed that this toxin targets an essential enzyme in nucle- was the first to be implicated in persister formation; the well- otide biosynthesis, a unique way in which TA systems can halt characterized mutant hipA7 was shown to increase E. coli per- bacterial growth. sistence up to 1,000-fold, due to a weakened interaction with its antitoxin (8). Various other toxins have also been implicated in Author contributions: F.J.P. and A.J.L. designed research; F.J.P., P.D.J., and A.J.L. per- biofilm and persister formation, including RelE, YafQ, and formed research; F.J.P., P.D.J., and A.J.L. analyzed data; and F.J.P. and A.J.L. wrote MazF (9). As the importance of biofilms and persisters in clinical the paper. settings grows, the study of TA systems provides an avenue for The authors declare no conflict of interest. better understanding the mechanisms driving these processes, as This article is a PNAS Direct Submission. well as a possible path to intervention. Published under the PNAS license. Although most downstream effects of these toxins are gener- Data deposition: The atomic coordinates and structure factors have been deposited in the ally similar (bacteriostasis or cell death), the methods by which Protein Data Bank, www.wwpdb.org (PDB ID codes 6D0I and 6D0H). these systems work are remarkably diverse. TA systems are 1To whom correspondence should be addressed. Email: [email protected]. broadly grouped into six classes, type I through VI, based on the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nature of the TA interaction, although type I and II comprise 1073/pnas.1814633116/-/DCSupplemental. most known TA systems and have been extensively studied. Type Published online December 31, 2018. 826–834 | PNAS | January 15, 2019 | vol. 116 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1814633116 Downloaded by guest on October 2, 2021 including 12 new toxins and antitoxins, again highlighting the cludes the structure of an RES domain protein. Solving this structure variety that exists within the type II class. To organize the rapidly allowed us to determine that the toxin is a mono-ADP-ribosyl- increasing amount of data available, the TA database was cre- transferase (mART). We further show, both in vivo and in vitro that ated in 2010 and is an invaluable resource in parsing what we the toxin ADP-ribosylates phosphoribosyl pyrophosphate synthetase know about these systems (23). (Prs), an essential enzyme involved in nucleotide biosynthesis. This Of the 19 predicted TA systems, one stood out to us in par- report expands the set of bacteriostatic mechanisms employed by ticular. The COG5654 (RES domain)-COG5642 TA family is TA systems to include protein-modifying ADP-ribosylation. one of five putative TA systems from this paper where both components were uncharacterized. We had encountered this Results pair in genome mining for natural products studied by our lab- Sphingobium sp. YBL2 Bears Multiple COG5654–COG5642 TA Systems. oratory and were intrigued by how poorly understood it was Our investigation into Sphingobium sp. YBL2 began when a considering how widespread it appeared to be; it was present in genome-mining algorithm developed in-house for discovering 250 of the 750 genomes analyzed by Makarova et al. Following new lasso peptide producers identified this organism as having its identification in 2009, we found only one study that examined two lasso peptide gene clusters of interest (24). It was immedi- the putative COG5654–COG5642 TA pair, a genetic study on ately evident that one of these gene clusters differed from the S. meliloti. This organism has 211 putative TA loci as mentioned expected architecture, containing within it genes encoding two above, with roughly 100 of these on the organism’s two mega- hypothetical proteins, yblI and yblJ (Fig. 1A). A BLAST of these plasmids. The authors demonstrated through a series of genetic sequences revealed them to be a COG5654–COG5642 putative deletions that only four of these were actively functioning as TA TA pair, first identified in a computational study in 2009 and systems, including a COG5654–COG5642 locus, which con- later confirmed to function as a TA pair in S. meliloti (20, 22). To firmed the hypothesis put forth in Makarova’s work (22). our knowledge, this is the last published work on this TA family. Here, we report a detailed physiological, structural, and bio- COG5654 toxins contain a roughly 150-aa RES domain, chemical study of the COG5654–COG5642 TA system, starting named for a highly conserved Arg-Glu-Ser motif. In addition, the with a computational approach that demonstrates its prevalence domain also features two strongly conserved Tyr and His resi- in bacterial genomes.

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