RESEARCH ARTICLE Response to vanadate exposure in Ochrobactrum tritici strains Mariana Cruz Almeida, Rita Branco, Paula V. MoraisID* CEMMPRE, Centre for Mechanical Engineering, Materials and Processes, Department of Life Sciences, University of Coimbra, Coimbra, Portugal * [email protected] a1111111111 a1111111111 Abstract a1111111111 a1111111111 Vanadium is a transition metal that has been added recently to the EU list of Raw Critical a1111111111 Metals. The growing needs of vanadium primarily in the steel industry justify its increasing economic value. However, because mining of vanadium sources (i. e. ores, concentrates and vanadiferous slags) is expanding, so is vanadium environmental contamination. Bio- leaching comes forth as smart strategy to deal with supply demand and environmental con- OPEN ACCESS tamination. It requires organisms that are able to mobilize the metal and at the same time Citation: Almeida MC, Branco R, Morais PV (2020) are resistant to the leachate generated. Here, we investigated the molecular mechanisms Response to vanadate exposure in Ochrobactrum underlying vanadium resistance in Ochrobactrum tritici strains. The highly resistant strain tritici strains. PLoS ONE 15(2): e0229359. https:// doi.org/10.1371/journal.pone.0229359 5bvl1 was able to grow at concentrations > 30 mM vanadate, while the O. tritici type strain only tolerated < 3 mM vanadate concentrations. Screening of O. tritici single mutants (chrA, Editor: Fanis Missirlis, Cinvestav, MEXICO chrC, chrF and recA) growth during vanadate exposure revealed that vanadate resistance Received: November 6, 2019 was associated with chromate resistance mechanisms (in particular ChrA, an efflux pump Accepted: February 4, 2020 and ChrC, a superoxide dismutase). We also showed that sensitivity to vanadate was corre- Published: February 24, 2020 lated with increased accumulation of vanadate intracellularly, while in resistant cells this was not found. Other up-regulated proteins found during vanadate exposure were ABC trans- Copyright: © 2020 Almeida et al. This is an open access article distributed under the terms of the porters for methionine and iron, suggesting that cellular responses to vanadate toxicity may Creative Commons Attribution License, which also induce changes in unspecific transport and chelation of vanadate. permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Data Availability Statement: All relevant data are within the manuscript. Vanadium (V) is a transition metal, which can exist in two oxidation states at neutral pH: V 2 Funding: This work was supported by CEMMPRE (IV) (vanadyl ion, cationic species VO ) and V(V) (vanadate ion, anionic species H2VO4) [1]. and by FundacËão para a Ciência e a Tecnologia With an abundance of 0.013%, vanadium is considered a relatively abundant element on Earth (FCT, https://www.fct.pt/) under the projects UID/ and a very valuable resource for different industrial applications. In fact, 80% of all V produced EMS/00285/2013, PTW-PTDC/AAG-REC/3839/ worldly is being used as an additive for steel industry [2]. 2014, ERA-MIN/0002/2015 and POCI-01-0145- Vanadium primary resources come from ores, concentrates and vanadiferous slags and are FEDER-031820. MCA has a postdoctoral grant within the project Renature - Centro-01-0145- mainly mined in South Africa, China, Russia, and the USA [2]. Recently, the European Union FEDER-000007. The funders had no role in study (EU) has published the third list of Critical Raw Materials where vanadium is listed as critical design, data collection and analysis, decision to for the EU economy. A critical metal is defined as one whose lack of availability during a publish, or preparation of the manuscript. national emergency would affect the economic and defensive capabilities of that country [3]. Competing interests: The authors have declared Because vanadium is being extensively used not only for the traditional steel, automobile that no competing interests exist. and aviation industries but also in the newest vanadium redox flow batteries, its supply PLOS ONE | https://doi.org/10.1371/journal.pone.0229359 February 24, 2020 1 / 14 Bacterial response to vanadate demands will undoubtedly increase further. New technologies are welcome to recover and recirculate vanadium from materials at the end of use. Bioleaching is an accepted strategy that requires organisms able to mobilize the metal and at the same, time resistant to the leachate generated. Besides, the use of organisms able to accumulate vanadium, either intracellularly or on the surface of cell membranes, could enhance the vanadium recovery process. During the last two decades, microbial metal accumulation has received increased attention due to the potential use of microbes as ªmetal living factoriesº, for metal uptake, transforma- tion and accumulation [4, 5]. Metal accumulation can occur in two ways: i) by metabolism- independent passive sorption; ii) by metabolism-dependent active uptake. This energy-driven process dependent on active metabolism normally occurs when an element is absorbed in a faster rate and then retained by an organism (bioaccumulation). Intracellular accumulation is especially important for essential metals. However, non-essential metals can also be taken up due to similar chemistry to essential ones [4]. Vanadium toxicity is comparable to that of lead, mercury and arsenic [6]. Nevertheless, V (III), V(IV) and V(V) species are of biological relevance and nature has evolved several enzy- matic systems using vanadium in their active sites as relevant components for their function including vanadium-dependent haloperoxidases, nitrogenases and vanabins [7]. On the other 3- 3- hand, by mimicking the phosphate ion (PO4 ), vanadate (VO4 ) can form transition-state analogues of phospho-protein intermediaries and therefore can easily substitute for phosphate in enzymes such as phosphatases and kinases [8]. A restricted number of bacteria can employ vanadium in varying biological functions. Vanadium nitrogenases have been reported in bacterial strains belonging to the genus Azoto- bacter and in cyanobacteria of the genera Anabaena and Nostoc [9, 10]. Vanadate-dependent haloperoxidases have been found in bacteria such as Streptomyces and marine cyanobacteria [11]. Several studies have reported that bacteria of the genera Shewanella, Pseudomonas and Geobacter can use vanadate as a primary electron acceptor in respiration [12±15]. Moreover, vanadium oxidation forms as vanadate and vanadyl ions can complex with carboxylate, cate- cholate and hydroxamate ligands present in siderophores [16]. Vanadium resistant microorganisms have been isolated from environmental contaminated sites such as phytoremediation plants (up to 30 mM Na3VO4), V mining-impacted soils [17] and the intestine of the vanadium-rich ascidian Ascidia sydneiensis samea (10 mM Na3VO4) [18±20]. However, the molecular mechanisms underlying vanadium resistance in microorganisms are still poorly understood. Only a study in P. aeruginosa described the importance of an efflux pump, MexGHI-OpmD, for the bacterial resistance to vanadium [21]. However, it is not known whether this is a direct or indirect effect since this pump exerts other cellular roles [21, 22]. This work aimed to determine whether vanadate resistance in Ochrobactrum tritici strains is related to low uptake of the metal or due to the presence of efflux systems (or both). Consid- 3- 3- ering the chemical structure vanadate (VO4 ) and its similarity to arsenate (AsO4 ), it could be supposed that bacteria could use arsenate-detoxifying mechanisms to detoxify vanadate. To examine this possibility, two strains of Ochrobactrum tritici, the type strain SCII24T (resistant to both arsenate and arsenite) and 5bvl1 (less resistant to arsenate and sensitive to arsenite) were used to test the vanadate resistance. Notably, the assays to define the tolerance to vana- date by the arsenite and arsenate resistant strain SCII24T showed a vanadate sensitive profile, which questioning the involvement of the arsenite/arsenate resistance genetic determinants in vanadate resistance. Considering that vanadium and chromium are transition metals, neigh- bors of the fourth period of the periodic table, in this work, we hypothesize that vanadate resis- tance mechanism could be correlated with chromate resistance mechanisms (in particular ChrA efflux pump). Therefore, the bacterial sensitivity to vanadate could be due to PLOS ONE | https://doi.org/10.1371/journal.pone.0229359 February 24, 2020 2 / 14 Bacterial response to vanadate accumulation of vanadate intracellularly, while resistant cells would possess a vanadate export mechanism as a detoxification strategy. Materials and methods Media and growth conditions Bacterial strains used in this study are listed in Table 1, and their resistance to chromate, arse- nate and arsenite, already studied in previous works are specified. Strain 5bvl1 was previously isolated from activated sludge in a chromium-contaminated area [23], while type strain SCII24T was isolated from rhizosphere of wheat [24]. Minimum inhibitory concentration (MIC) assays Minimum inhibitory concentration (MIC) determination was performed in solid R2A plates. Briefly, 20 μl of cellular suspensions (108 CFU/ml), prepared in NaCl 0.9%, were placed onto agar R2A medium plates containing increased concentrations of vanadate (0, 3, 7.5, 15 and 30 mM) and incubated for 24 h at 30 ÊC (until growth was observed in control