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The Pseudomonas Community in Metal-Contaminated Sediments As Revealed by Quantitative PCR: a Link with Metal Bioavailability

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The Pseudomonas Community in Metal-Contaminated Sediments As Revealed by Quantitative PCR: a Link with Metal Bioavailability Research in Microbiology 165 (2014) 647e656 www.elsevier.com/locate/resmic Original article The Pseudomonas community in metal-contaminated sediments as revealed by quantitative PCR: a link with metal bioavailability Stephanie Roosa a, Corinne Vander Wauven c, Gabriel Billon b, Sandra Matthijs c, Ruddy Wattiez a, David C. Gillan a,* a Proteomics and Microbiology Lab, Research Institute for Biosciences, Universite de Mons, 20 Place du Parc, B-7000 Mons, Belgium b Geosystemes Lab, UFR de Chimie, Lillee1 University, Sciences and Technologies, 59655 Villeneuve d'Ascq, France c Institut de Recherches Microbiologiques JMW, 1 Av. E. Gryzon, 1070 Bruxelles, Belgium Received 21 July 2014; accepted 21 July 2014 Available online 4 August 2014 Abstract Pseudomonas bacteria are ubiquitous Gram-negative and aerobic microorganisms that are known to harbor metal resistance mechanisms such as efflux pumps and intracellular redox enzymes. Specific Pseudomonas bacteria have been quantified in some metal-contaminated environ- ments, but the entire Pseudomonas population has been poorly investigated under these conditions, and the link with metal bioavailability was not previously examined. In the present study, quantitative PCR and cell cultivation were used to monitor and characterize the Pseudomonas population at 4 different sediment sites contaminated with various levels of metals. At the same time, total metals and metal bioavailability (as estimated using an HCl 1 M extraction) were measured. It was found that the total level of Pseudomonas, as determined by qPCR using two different genes (oprI and the 16S rRNA gene), was positively and significantly correlated with total and HCl-extractable Cu, Co, Ni, Pb and Zn, with high correlation coefficients (>0.8). Metal-contaminated sediments featured isolates of the Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas lutea and Pseudomonas aeruginosa groups, with other bacterial genera such as Mycobacterium, Klebsiella and Methylobacterium. It is concluded that Pseudomonas bacteria do proliferate in metal-contaminated sediments, but are still part of a complex community. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Pseudomonas; Sediments; Metals; Bioavailability; Resistance; Quantitative PCR 1. Introduction to antibiotics [6], leading to the expansion of nosocomial diseases [7]. The ubiquity of the genus is a direct consequence Pseudomonas bacteria are ubiquitous Gram-negative and of its high adaptability. For instance, Pseudomonas bacteria aerobic microorganisms that are member of the g-Proteobac- are able to survive in hydrocarbon-, PCB- and PAH- teria. These bacteria are found in numerous natural environ- contaminated environments due to oxidative degradation ments, including soils, sediments and water [1,2], and have processes and biosurfactant production [8,9]. Pseudomonas been found in association with various plants and animals [3]. spp. are also known to degrade various pesticides [10] and Pseudomonas bacteria are also responsible for different human harbor metal resistance mechanisms such as efflux pumps and infections [4e6] and some species have developed resistance intracellular redox enzymes [11e13]. It is therefore not sur- prising to find specific Pseudomonas bacteria in metal- contaminated rivers and sediments [14e16] and these apti- * Corresponding author. tudes to react with metals have even led to proposing their use E-mail addresses: [email protected] (S. Roosa), cvdwauve@ as bioremediation tools [13,17,18]. ulb.ac.be (C.V. Wauven), [email protected] (G. Billon), slmatthi@ ulb.ac.be (S. Matthijs), [email protected] (R. Wattiez), david. Metals are frequent contaminants of aquatic sediments [email protected] (D.C. Gillan). [19,20]. However, the total concentration of metals in http://dx.doi.org/10.1016/j.resmic.2014.07.011 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. 648 S. Roosa et al. / Research in Microbiology 165 (2014) 647e656 sediments is not indicative of any biological effect, as metals with reference sites [14]. Unfortunately, metal bioavailability are not always bioavailable [21e26]. For a unicellular or- was not assessed. ganism, a compound may be defined as bioavailable when it is The aim of the present research was to use quantitative able to cross the cellular envelope at a given time [27]. Once PCR with specific primers to estimate the biomass of the inside the cell, metals may have various effects according to whole Pseudomonas community in 4 aquatic sediments their concentration and chemical properties [28]. When con- showing contrasted metal bioavailability. At the same time, centration levels are too high, bacteria usually react by the total metal levels and bioavailable metals were estimated in expression of specific metal resistance systems such as P-type the sediments using, respectively, strong acids and 1 M HCl ATPases, metallothioneins, RND efflux pumps and/or CDF treatment. We also used cell cultivation to test the metal transporters [13,28,29]. It is therefore expected that bacteria resistance capacities of some Pseudomonas isolates obtained displaying the most efficient resistance systems are selected in at the most contaminated station. metal-contaminated environments where metals are bioavailable. 2. Materials and methods As a large panel of metal resistance systems was observed in many Pseudomonas species [13], and given the ubiquity of 2.1. Description of the sites and sediment sampling the genus and its numerous species, it is anticipated that Pseudomonas bacteria will have an advantage in sedimentary Four sites were considered in this study: Station 130 environments where metals are bioavailable, an advantage that (St130) in seawater and MetalEurop (MET), Ferin (FER) and may lead to higher biomasses. However, to the best of our R^aches (RAC) in freshwater (Fig. 1). Station 130 is situated in knowledge, the biomasses of Pseudomonas and metal the North Sea, on the Belgian Continental Plate. It presents bioavailability were never assessed in parallel in natural sed- low concentrations of metals in comparison to the other iments. Pseudomonads have been quantified in the environ- freshwater sites [36]. The MetalEurop site is the most metal- ment using most-probable-number (MPN) enumeration [30], contaminated site of the present study. It is located in front fluorescent in situ hybridization [31] and quantitative PCR of a smelter in the River Deuˆle (northern France). This plant (qPCR) [14,32e35], but most of these previous studies were was closed in 2003 after decades of accidental discharges into not performed in metal-contaminated sediments or did not the river that started in 1893; today, the contamination level of assess metal bioavailability [32e35]. In a study restricted to the site still remains many orders of magnitude higher than Pseudomonas aeruginosa living in a metal-contaminated river, background values [37,38].TheR^aches site is mildly the quantification of three genes (a Mn-containing superoxide contaminated and Ferin is also a control site, supposedly un- dismutase SodA, a heat shock protein HtpX and a metal- contaminated; these two sites are situated in the same river lothionein) was performed and significantly higher copy basin as MetalEurop. All sites selected in this study have a numbers were found at the more contaminated sites compared similar granulometry, oxygen penetration depth, organic Fig. 1. Map of the sites and their coordinates: St130: Station 130; FER: Ferin; RAC: R^aches; MET: MetalEurop. S. Roosa et al. / Research in Microbiology 165 (2014) 647e656 649 matter and pH (all sediments are muddy with a mean grain the molar concentration of AVS exceeds that of the metals size < 50 mm and a POC content between 1 and 3%). (i.e., the metal/AVS ratio is less than unity), they exist pre- Sediment sampling at St130 was performed using a Rein- dominantly as insoluble metal sulfides, which presumably are eck corer (Ø ¼ 15 cm) onboard the Zeeleeuw research vessel not biologically available [44]. For AVS measurement [45], (June 2011; depth: ±11 m). For the freshwater sites, sediments wet sediments (1 g, anoxic) were mixed with 6 M HCl for 1 h were collected in the middle of the river using a Plexiglass at 25 C. The H2S gas that was produced was then trapped in a tube (Ø ¼ 7 cm) fixed to a stainless steel bar (June 2011; 1 M NaOH solution. The sulfide concentration was measured depth: ±3 m). For all sites, three separate cores were obtained with a sulfide-specific electrode (Orion). Results were found to (n ¼ 3) and for each core, the wateresediment interface be reproducible with an analytical precision 15%. To (0e1 cm) and an anoxic layer (4e6 cm) were immediately calculate the TI, concentrations of bioavailable Cd, Co, Cu, isolated in sterile plastic vessels. Samples were transported to Ni, Pb and Zn were used. the lab in dry ice and divided into two parts: one part was stored at À80 C for biomolecular analyses and the other part 2.3. Quantification of Pseudomonas by quantitative PCR at À20 C for chemical analyses. For bacterial cultures, sub- samples of sediments were brought back to the laboratory at Before DNA extraction, sediments (24 samples) were 4 C and directly used. washed using three different buffers in order to remove a maximum of PCR inhibitors, as described in [46]. For each 2.2. Quantification of metals sample, DNA was then extracted from 750 mg of sediment using three PowerSoil DNA extraction columns (Mobio®)in  2.2.1. Total metals (TE) parallel (3 250 mg). DNA extracted from these three col- umns was then pooled and a final purification step was per- Weighed amounts of dried and sieved (63 mm) sediments ® were digested with concentrated acids as described elsewhere formed with a Qiaquick purification kit (Qiagen ) in order to [39]. Total metal concentrations were determined using increase the quality of the DNA extracts (260/280 and 260/230 inductively coupled plasma mass spectrometry (ICP-MS, ratios above 1.8) and ensure reliable and reproducible qPCR Thermo Elemental, X7 series). Only 12 metals or semi-metals quantification.
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