A RelE/ParE superfamily toxin in Vibrio parahaemolyticus has DNA nicking endonuclease activity

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Highlights

  • The toxin Vp1843 in V. parahaemolyticus belongs to the RelE/ParE superfamily.

  • The toxin Vp1843, unlike RelE/ParE toxins, has a DNA nicking endonuclease activity.

  • Lys37 and Pro45 in Vp1843 play a critical role in its DNA nicking activity.

  • The DNA nicking activity is assigned as a third activity in the RelE/ParE toxins.

Abstract

Type II toxins in toxin-antitoxin (TA) systems fold into a similar fold and belong to the RelE/ParE superfamily. However, they display two distinct biochemical activities: RelE toxins are mRNA interferases, while ParE toxins are DNA gyrase (Gyr) inhibitors. Previously, we found a TA system, vp1842/vp1843, on the Vibrio parahaemolyticus genome whose toxin Vp1843 belongs to the RelE/ParE toxin superfamily. Vp1843, unlike RelE toxins, has neither protein synthesis inhibitory activity nor ribonuclease activity. In this study, we examined the inhibitory potency of Vp1843 with Escherichia coli Gyr. The result showed that Vp1843, unlike other ParE toxins, had little Gyr inhibitory activity, but rather converted supercoiled DNA to open-circular DNA. Analysis showed further that Vp1843 cleaves a single strand in DNA, and that the antitoxin Vp1842 neutralized the nicking endonuclease activity of Vp1843. Mutations of Lys37 and Pro45 in Vp1843 abolished its nicking activity, suggesting that they play a crucial role in nicking endonuclease activity. To our knowledge, Vp1843 is the first toxin with DNA nicking endonuclease activity among the RelE/ParE toxin superfamily.

Graphical abstract

RelE and ParE family toxins belong to the RelE/ParE superfamily, despite distinct biological activities; RelE toxins are a protein synthesis inhibitor by cleaving mRNA, while ParE toxins have inhibitory activity toward Gyr. We found that the Vibrio parahaemolyticus toxin, Vp1843, unlike RelE and ParE toxins, exhibited a DNA nicking endonuclease activity. The present result may assign DNA nicking endonuclease activity of Vp1843 as a third biological activity in the RelE/ParE superfamily toxins.

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Introduction

Toxin-antitoxin (TA) systems, which were initially characterized as plasmid-borne mediators of plasmid stability, have in recent years been identified within the chromosomes of numerous bacterial species [1], [2], [3], [4]. In the well-known type II TA systems, they are composed of two closely linked genes organized in an operon that encodes a stable toxin and its cognate labile antitoxin [5]. In a steady state, antitoxins neutralize the effects of toxins by direct protein-protein interactions [6]. In addition, antitoxins and TA complexes bind to their promoter DNAs within their own operons and negatively regulate their own transcription [7]. Under conditions of environmental stress, such as amino acid and carbon source limitation, labile antitoxins are selectively degraded by ATP-dependent proteases such as Lon, ClpXP, or ClpAP, leading to rapid growth arrest and cell death due to the cellular effects of toxins [8], [9]. Phylogenetic studies have found that type II toxins, such as RelE and ParE, belong to the RelE/ParE superfamily, and fold into a similar fold, despite the fact that they display two distinct biological activities [10]. Namely, the RelE type toxins, such as YafQ and YoeB, stall the ribosome by cleaving mRNA in a translation-dependent fashion [11]. They are hence referred to as mRNA interferases. Recent structural studies of the ribosome in complex with RelE or YoeB provided insight into the structural basis for ribosome-dependent ribonuclease activity and suggested catalytic residues in individual toxins [12], [13], [14]. On the other hand, ParE was originally found on plasmid RK2 with ParD being the antitoxin, and DNA gyrase (Gyr) was identified as its target molecule [15]. Subsequently, several ParE genes have been found on bacterial chromosomal DNA, and biochemical studies reported that some ParE toxins strongly poison Gyr, although the molecular mechanism by which they inhibit Gyr is still an area of debate [16], [17], [18].

Vibrio parahaemolyticus, a seafood enteropathogen in coastal countries, causes acute gastroenteritis in humans [19]. In a previous study [20], we found that gene clusters, vp1829/vp1830 and vp1842/vp1843, in the V. parahaemolyticus genome database have sequence homology to that encoding the Escherichia coli TA protein, DinJ/YafQ. Expression of the putative toxin gene, vp1843, in E. coli strongly inhibited cell growth, while co-production of the putative antitoxin gene, vp1842, neutralized this effect. In contrast, the expressions of vp1830 in the presence of 0.2% arabinose had no influence on cell growth. These results suggested that vp1842/vp1843 serve as a TA system in V. parahaemolyticus. It was further found that Vp1843, unlike the E. coli homologue YafQ, has neither protein synthesis inhibitory activity nor ribonuclease activity. Rather, the expression of vp1843 in E. coli resulted in a morphological change in the cells, while co-expression with vp1842 had no influence on the cell shape [20].

During the course of the previous study, we found that Vp1843 has sequence homology not only to E. coli YafQ, but also to Caulobacter crescentus ParE [21]. To gain more insight into the biological properties of Vp1842/Vp1843, the inhibitory potency of Vp1843 was investigated with E. coli Gyr. We found that it, unlike other ParE toxins, had little Gyr inhibitory activity. Rather, it exhibited DNA nicking endonuclease activity. Mutational analysis suggested that Lys37 and Pro45 in Vp1843 play a crucial role in the nicking activity. The present result may lead to assigning DNA nicking endonuclease activity of Vp1843 as a third biological activity in the RelE/ParE superfamily toxins.

Section snippets

Materials

Restriction endonucleases and DNA-modifying enzymes were purchased from MBI Fermentas. The E. coli Gyr and relaxed plasmids, pBR322 and pUC19, were supplied by New England Biolabs. Ciprofloxacin (CFX) was obtained as hydrochlorides from Enzo Life Sciences. The plasmid vectors pET-15b, pET-22b, pACYC-Duet1, and pBAD/Myc-HisA used for expression of vp1842/vp1843 in E. coli cells were from Novagen and Invitrogen, respectively. The plasmids pEXP5-CALML3 used for nicking activity were from

Gyr inhibitory activity

Vp1843 has sequence homology not only to E. coli YafQ, but also to C. crescentus ParE, as shown in Supplementary Fig. S1. Both YafQ and ParE toxins belong to the RelE/ParE superfamily, despite distinct biological activities; YafQ is a protein synthesis inhibitor that cleaves mRNA [14], while ParE has inhibitory activity toward Gyr [15]. Since Vp1843 exhibited neither protein synthesis inhibitory activity nor ribonuclease activity [20], we tested whether Vp1843 could have any inhibitory activity

Discussion

A large number of DNA nicking endonucleases have been found in a variety of organisms and were investigated extensively from both structural and functional points of view [24], [25]. Some nicking enzymes and engineered nicking enzymes have become invaluable tools in diagnostic probes for cancer cells and in genome modifications [24], [25]. These nicking enzymes have been grouped into main three classes, PD-D/EXK, HNH and GIY-YIG, based on the amino acid residues (amino acid motifs) comprising

Conflict of interest

None declared.

Acknowledgements

We are grateful to Dr. T. Ueda (Kyushu University) for his active interest and helpful discussions. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 26660090 to M.K.). J. Zhang was sponsored by CSC (China Scholarship Council, No. 201308310430).

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