Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party

Published online 25 April 2016 Nucleic Acids Research, 2016, Vol. 44, No. 11 5457–5469 doi: 10.1093/nar/gkw317 Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party Elisa Maiques1,† , Nuria Quiles-Puchalt2,3,† , Jorge Donderis1 , J. Rafael Ciges-Tomas1 , Christian Alite1 , Janine Z. Bowring3 , Suzanne Humphrey3 , José R. Penadés3,* and Alberto Marina1,* 1 Instituto de Biomedicina de Valencia (IBV-CSIC) and CIBER de Enfermedades Raras (CIBERER), 46010 Valencia, Spain, 2 Departamento de Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidad CEU Cardenal Herrera, 46113 Moncada, Valencia, Spain and 3 Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK Received July 10, 2015; Revised April 12, 2016; Accepted April 13, 2016 We have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions. INTRODUCTION Staphylococcal pathogenicity islands (SaPIs) are mobile genetic elements that carry and disseminate virulence genes in Staphylococcus aureus (1–3). They reside passively in the host chromosome under the control of Stl, a global SaPI- encoded repressor. Following infection by a helper phage, or induction of a resident prophage, SaPIs excise, replicate autonomously and are packaged in phage-like particles composed of phage virion proteins (4,5), leading to very high frequencies of inter- and intrageneric transfers (6,7). To initiate the SaPI cycle, a specific phage-encoded protein binds to the SaPI-encoded repressor Stl, acting as an antirepressor (8,9). Both the trimeric and the dimeric phage-encoded Dut proteins are the antirepressor proteins for a subset of SaPIs, including SaPIbov1, SaPIbov5 or SaPIov1 (8–10). The fact that the trimeric Duts were one of the SaPI inducers aroused our curiosity. Why viruses encode an enzyme already present in their prospective eukaryotic or prokaryotic host cells is an intriguing question. As with our model, in which Duts were involved in the transfer of different SaPIs, others have also proposed that virus-encoded Duts could be moonlighting proteins with different regulatory functions (11). Our laboratories have recently focused on the elucidation of the mechanisms by which Duts perform their regulatory role. In response to this question, and surprisingly for a metabolic enzyme, a comparison of trimeric Dut sequences from various staphylococcal phages revealed high sequence similarity, except for a nonconserved central region, that we defined as motif VI (8) (Supplementary Figure S1A). This motif is highly divergent among S. aureus phage enzymes but, importantly, is not required for enzyme activity (12) and is absent in some functionally related Duts from other species (Supplementary Figure S1B). However, our results analyzing the Dut protein from phage 80␣ (Dut80␣) revealed that motif VI is essential for interaction with the SaPI-encoded Stl repressor, determining the affinity with which the Dut proteins bind to the Stl repressor (8,9). * To whom correspondence should be addressed. Tel: +34 96 339 17 54; Fax: +34 96 369 08 00; Email: Correspondence may also be addressed to José R Penadés Tel: +44 141 330 8770; Fax: +44 141 330 4297; Email: † These authors contributed equally to the paper as first authors.  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT 5458 Nucleic Acids Research, 2016, Vol. 44, No. 11 MATERIALS AND METHODS Bacterial strains and growth conditions The bacterial strains used in these studies are listed in Supplementary Table S1. The procedures for preparation and analysis of phage lysates, in addition to transduction and transformation of S. aureus, were performed essentially as previously described (16,17). DNA methods General DNA manipulations were performed using standard procedures. The oligonucleotides used in this study are listed in Supplementary Table S2. The labeling of the probes and DNA hybridization were performed according to the protocol supplied with the PCR-DIG DNA-labeling and Chemiluminescent Detection Kit (Roche). Plasmid construction The plasmid constructs expressing the different Dut proteins were reported previously (Supplementary Table S3) or were prepared by cloning PCR products obtained with the oligonucleotide primers listed in Supplementary Table S2. All clones were sequenced by the Institute Core Sequencing facility. Dut proteins were expressed in S. aureus under inducing conditions from the Pcad promoter in the expression vector pCN51, as previously described (8). The gene encoding the Stl from SaPIbov1 was cloned in the expression vector pETNKI-hisSUMO3-LIC (kindly supplied by Patrick Celie, NKI Protein facility). This vector contains 6His-tag for affinity purification and SUMO protein to increase solubility. The His-SUMO3 tag can be removed using the enzyme SUMO Protease 2 (SENP2). The ligation-independent cloning (LIC) system was used to clone the insert (18). To amplify the stl gene the StlM1SUMO-FW and Stl-N267SUMO-RV primers (Supplementary Table S2) were used and genomic DNA from S. aureus strain JP3603 was used as the template. The resulting vector, pETNKI-Stl, was sequenced for verification at the IBV Core Sequencing Facility. Protein expression and purification The expression of His-tagged wild-type (WT) and mutant Dut proteins were done in E. coli BL21 (DE3) (Novagen) strain transformed with the corresponding gene cloned in pET-28a plasmid (Novagen) (Supplementary Table S3) (...truncated)