Impact of copper on the abundance and diversity of sulfate reducing prokaryotes in two chilean marine sediments

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(1)Marine Pollution Bulletin 64 (2012) 2135–2145. Contents lists available at SciVerse ScienceDirect. Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul. Impact of copper on the abundance and diversity of sulfate-reducing prokaryotes in two chilean marine sediments Ludovic Besaury a,e, Baghdad Ouddane b, Juan Pablo Pavissich c, Carole Dubrulle-Brunaud a, Bernardo González d, Laurent Quillet e,⇑ a. Faculté des Sciences, Université de Rouen, CNRS UMR 6143-M2C, Groupe de Microbiologie, Place Emile Blondel, 76821 Mont Saint Aignan Cedex, France UMR 8217, Equipe Chimie Analytique et Marine, Université Lille 1, 59655 Villeneuve d’Ascq Cedex, France Facultad de Ciencias Biológicas, Pontificia Universidad Catolica de Chile, Santiago, Chile d Facultad de Ingeniería y Ciencias, Universidad Adolfo Ibáñez, Santiago, Chile e Laboratoire Universitaire MEBE ‘‘ Microbiologie Environnementale et Biologie Evolutive’’, Université de Rouen, 76821 Mont Saint Aignan Cedex, France b c. a r t i c l e. i n f o. Keywords: Marine sediment Copper contamination Sulfate-reducing prokaryotes Real-time PCR DGGE Phylogenetic analysis. a b s t r a c t We studied the abundance and diversity of the sulfate-reducing prokaryotes (SRPs) in two 30-cm marine chilean sediment cores, one with a long-term exposure to copper-mining residues, the other being a nonexposed reference sediment. The abundance of SRPs was quantified by qPCR of the dissimilatory sulfite reductase gene b-subunit (dsrB) and showed that SRPs are sensitive to high copper concentrations, as the mean number of SRPs all along the contaminated sediment was two orders of magnitude lower than in the reference sediment. SRP diversity was analyzed by using the dsrB-sequences-based PCR-DGGE method and constructing gene libraries for dsrB-sequences. Surprisingly, the diversity was comparable in both sediments, with dsrB sequences belonging to Desulfobacteraceae, Syntrophobacteraceae, and Desulfobulbaceae, SRP families previously described in marine sediments, and to a deep branching dsrAB lineage. The hypothesis of the presence of horizontal transfer of copper resistance genes in the microbial population of the polluted sediment is discussed. Ó 2012 Elsevier Ltd. All rights reserved.. 1. Introduction In ecosystems such as marine and salt marsh sediments, sulfate-reducing prokaryotes (SRPs) are involved in sulfur and carbon cycles (Rabus et al., 2006). They are responsible for more than 50% of the degradation of total organic carbon, and for the biodegradation of pollutants (Perez-Jimenez et al., 2001). They also play an important role in metal sulfide immobilization in sediments containing high concentrations of heavy metals, as sulfate reduction leads to the production of hydrogen sulfide, which can react with heavy-metal ions to form insoluble metal sulfides (Labrenz et al., 2000). Copper is an essential element for all living organisms (Burgess et al., 1999), but at high concentrations copper ions inhibit cell metabolism by chelating sulfhydryl groups, thereby resulting in the denaturation of proteins and inactivation of enzymes (Yeager, 1991; Sani et al., 2001). By using SRPs culture, Utgikar et al. (2003) calculated the characteristic copper toxic concentration below which sulfate-reduc⇑ Corresponding author. Tel.: +33 2 32 76 94 51; fax: +33 2 35 14 66 88. E-mail addresses: ludovic.besaury@etu.univ-rouen.fr (L. Besaury), baghdad.ouddane@univ-lille1.fr (B. Ouddane), jpavissi@nd.edu (J.P. Pavissich), carole.brunaud@unicaen.fr (C. Dubrulle-Brunaud), bernardo.gonzalez@uai.cl (B. González), laurent.quillet@univ-rouen.fr (L. Quillet). 0025-326X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2012.07.042. ing activity will continue, and above which sulfate reduction will stop. The inhibitory effect of copper on the sulfate-reducing activity of SRPs has been studied extensively in anaerobic sludge and cultures (Jalali and Baldwin, 2000; Mori et al., 2000; Utgikar et al., 2003; Karri et al., 2006). In contrast, only two publications (Jin et al., 2007; Pavissich et al., 2010) report the impact of copper on sulfate reduction and diversity of SRPs enriched from contaminated sediments. Different molecular techniques have been developed to quantify SRPs in sediments: Fluorescent In Situ Hybridization (FISH) techniques, based on detection of fluorescent 16S rRNA sequence probes, permit direct determination of microscopic counts (Koizumi et al., 2003; Musman et al., 2005; Gittel et al., 2008). As the microbial sulfate-reducing community constitutes a polyphyletic group, an alternative method has been developed, consisting of molecular quantification of a functional gene present in every SRP, the dissimilatory sulfite reductase gene (dsrAB), which encodes for the key enzyme involved in dissimilatory sulfate respiration (Wagner et al., 1998). Different quantitative PCR approaches have recently been developed using the functional marker gene dsrAB: (i) most Probable Number (MPN)-qPCR (Kjeldsen et al., 2009), (ii) competitive quantitative PCR (cPCR) (Leloup et al., 2004; Kawahara et al., 2008), and (iii) real-time qPCR (Foti et al., 2007; Agrawal and Lal, 2009)..

(2) 2136. L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. The use of the dsrAB gene also allows the diversity of SRPs in environments such as marine sediments to be studied (Leloup et al., 2006; Gittel et al., 2008; Jiang et al., 2009), and novel dsrAB gene sequences harbored by bacteria not yet identified by culture, to be discovered (Dhillon et al., 2003; Zhang et al., 2008; Quillet et al., 2012). Different molecular methods using this functional gene have been also applied: Denaturing Gradient Gel Electrophoresis (DGGE) (Dar et al., 2007; Foti et al., 2007), Single Strand Conformation Polymorphism (Quillet et al., 2012), cloning/sequencing (Jiang et al., 2009; Moreau et al., 2010); and Terminal Restriction Fragment Length Polymorphism (Zhang et al., 2008; Belila et al., 2011). In the coastal zone of Chañaral bay in the North of Chile, copper mining industries have discharged untreated residues for more than 70 years. Between 1938 and 1975, the Chañaral coastline received approximately 150 million tons of residues of raw Cu mining flotation tailings from the mining activities of the copper mine El Salvador. In 1976, the tailing discharge point was moved 10 km north of Chañaral Bay to Caleta Palito releasing, up to 1989, more than 130 million tons of waste (Andrade et al., 2006). Studies of the environmental impact of the El Salvador copper mine on the rocky coastal shores of the Chañaral zone have highlighted the existence of very low levels of diversity of sessile species such as algae and nematodes (Correa et al., 1999, 2000; Lee et al., 2002). Moran et al. (2008) have shown that the impact of copper on total microorganisms is associated with changes in the composition of the bacterial communities. In this study we combined biogeochemical analyses with qPCR, DGGE fingerprinting technique, and PCR-cloning, to determine and compare the abundance of Bacteria (16S rRNA genes) and SRPs (dsrB genes), and analyzed the diversity of SRPs in two 25 cm sediment cores from the Chañaral region, one corresponding to coastal sediments with a long-term exposure to copper-mining residues, the other to coastal sediments not copper-impacted and used as a reference.. 2. Materials and methods 2.1. Study site and sediment sampling Sediment samples were taken in December 2008 from the edge of Canal Palito in contact with the Pacific Ocean (26°15.80 S; 70°40.60 W), and on an intertidal site on the beach of Flamenco (26°34.40 ; 70°41.30 ) (Fig. 1). Three types of cores (10-cm diameter, 30 cm long) were collected: (i) core (A) of 30 cm used for total organic carbon (TOC), granulometry, and SRP molecular analyses (abundance and diversity), (ii) core (B) used for pore water extraction, and (iii) core (C) corresponding to a Perspex tube used to determine the pH and redox potential (denoted Eh in the text). The cores A and B were collected using a hand-driven gouge corer and rapidly transported to the laboratory for analytical procedures. The core A was sliced every 2 cm up to a depth of 10 cm, and the remaining section was cut into 5 cm slices. Subsamples (1 g wet weight) of each section of the core A were used immediately for DNA extraction or frozen at 80 °C until subsequent molecular analysis. The core B was cut into 10 cm slices in a glove box purged with nitrogen in order to prevent change in redox conditions and oxidation of reduced S species. About 50 g of wet sediment subsamples were transferred into an acid-cleaned and nitrogen-purged plastic centrifuge bottle, and centrifuged at 8000 rpm for 30 min. Overlying water was separated from each centrifuge bottle and the remaining pore water was vacuum-filtered through a 0.45 lm polycarbonate membrane in a glove box purged with nitrogen. The collected pore water was. then acidified with 0.5 mL of ultrapure HNO3 for metal analysis. The rest of the sediment was frozen for further analyses. 2.2. Determination of total organic carbon Three sub-samples (around 2 g) of each sediment section were used for pore water extraction. Pore water was extracted using 3000 rotations/mm centrifugation during 15 mn. The concentrations of total organic carbon (TOC) was analyzed with a ‘‘TOC Shimadzu 5050’’ carbon analyser (TOC = total carbon (TC) total inorganic carbon (IC)). TOC Shimadzu 5050 calibration was performed using standard TC and IC solutions of 10, 25, 50, 100 mg/l respectively. TOC values were obtained by injecting 100 ll aliquots into the TOC analyzer. Measurement reproducibility was <5%. 2.3. Determination of other auxiliary parameters The pH and Eh were determined as a function of depth in the sediments by means of a glass microelectrode for pH and a platinum electrode for Eh. These indicator electrodes were both combined with an Ag/AgCl reference electrode with a potential equal to +0.22 V versus a hydrogen normal electrode. The electrodes were introduced inside holes (which were capped with rubber during the sampling operation) that were present all along the Perspex tube and corresponded to fixed sediment depths. The results were read only after reaching the potential stability. Concentrations of dissolved Na and sulfate ions in pore water were determined using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICPAES; Varian VistaPro, axial view). 2.4. Granulometry Three levels on the core were chosen for grain size analysis: samples of the top (0–10 cm), the middle (10–20 cm), and the bottom (20–30 cm). Each sampling sediment (around 100–400 g) was wet-sieved using a 63 lm mesh sieve to separate the fine fraction under fresh water. The >63 lm fraction was dried at 60 °C for 48 h. Then, this fraction was dry-sieved using a stack of 26 stainless steel sieves with mesh sizes between 63 lm and 20 mm. The sediment on each sieve was weighed to 0.1 g accuracy. The percentage of particles was then determined for each size fraction, and modified from the Blott and Pye (2001) scale. 2.5. Total and available trace metal analysis Trace metals were quantified in pore waters and in the solid fraction by using ICP-AES for Cu, Pb and Zn and by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS, Thermo Elemental Series 2) for Cd. For the solid fraction an HCl 1 M treatment was done to evaluate the ‘‘available’’ metals fraction, then a total digestion was done to measure total metal concentrations. Briefly for total mineralization, dried sediments were treated first with 10 mL of a concentrated HF solution at ebullition during 2 h, and then, after evaporation of the acids, with 10 mL of a freshly prepared HNO3/ HCl mixture (1/2 v:v) to eliminate at ebullition the remaining solid grains. The solutions recovered were afterwards diluted in a known volume of ultrapure water (Milli Q plus) and analyzed using ICPAES and ICP-MS. The data quality was controlled with the reference material by using Canadian International Standards (HISS-1, MESS3, PACS-2) with each analysis series. The values obtained with this procedure agreed well with the certified values, the analytical uncertainties did not exceed 5%. The ‘‘available’’ metals fraction were extracted by shaking 1 g of wet sediment with 50 mL of 1 M HCl for 24 h at room temperature (20 °C), followed by centrifugation and filtration through 0.45 lm cellulose acetate membrane. The metals extracted by this procedure are weakly.

(3) L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. 2137. Fig. 1. Map of the area studied and location of sampling stations Palito and Flamenco.. associated with the sediments and thus provide a proxy for potential metal bio-availability (Ankley et al., 1993; Cooper and Morse, 1998; Simpson et al., 2000). 2.6. DNA extraction Total nucleic acids were extracted from 2 g of sediment using the RNAÒ powersoil kit (Mo-Bio, France) according to the manufacturer’s instructions. DNA was purified using All Prep DNA/RNA Mini kit (Qiagen, France) based on the manufacturer’s instructions. Ten microliters of extracted DNA was mixed with loading buffer and electrophoresed on 1% [w/v] agarose gel stained with ethidium bromide. Quantification of extracted DNA was carried out after comparison of the amounts of DNA with those from the Smart Ladder DNA standard (Eurogentec, France). Images of the gels were analyzed with the software Quantum-Capt from the QuantumST4 image acquisition system (Vilber Louma, Deutschland).. at 95 °C), annealing (45 s at 64 °C) and elongation (30 s at 72 °C), and a final extension step (7 min at 72 °C). The primer pair dsr2060F: 50 -CAACATCGTYCAYACCCAGGG-30 (Geets et al., 2006) and dsr4R: 50 -GTGTAGCAGTTACCGCA-30 (Wagner et al., 1998) were used to quantify the dsrB gene, as described by Foti et al. (2007). The PCR was carried out as follows: initial denaturation step (10 min at 95 °C), 40 cycles of denaturation (40 s at 95 °C), annealing (40 s at 55 °C) and elongation (1 min at 72 °C), and a final extension step (7 min at 72 °C). The expected size was 380 bp. Melting curve analyses were carried out between 60 and 100 °C to determine whether there was a detectable primer-dimer contribution to the SYBR I Green fluorescence measurement. The procedures were performed in triplicate. The results are expressed in number of cells per gram of sediment (fresh weight basis). The calculation was carried out assuming that each SRP contains one copy of the dsrB gene (Klein et al., 2001), and that the average number of 16S rRNA gene copies per cell is 3.6 (Klappenbach et al., 2001).. 2.7. Quantification of 16S rRNA and dsrB genes by real-time PCR Quantifications of 16S rRNA and dsrB genes were carried out by real-time PCR (System Chromo4 detector, BIORAD, France) using the GoTaq qPCR Master Mix (Promega). Total bacteria quantification was performed using the primer pair 63F: 50 CAGGCCTAACACATGCAAGTC-30 (Marchesi et al., 1998) and BU16S4: 50 -CTGCTGCCTCCCGTAGG-30 (derived from 341 F, as described by Muyzer et al., 1993) to amplify a 314 bp DNA fragment, as reported by Plassart et al. (2008). The PCR was carried out as follows: initial denaturation step (10 min at 95 °C), 40 cycles of denaturation (40 s. 2.8. PCR amplification of dsrB gene and denaturing gradient gel electrophoresis (DGGE) analysis dsrB gene was amplified as indicated above in a 50 lL reaction mixture containing approximately 20 ng of DNA template. A GCclamp (50 -GCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC30 ) was added to the 50 end of primer dsr2060F. PCR was carried out using the RedGoldStarÒ DNA polymerase (Eurogentec) and a program running on a GeneAmpÒPCR system 9700 thermocycler (Applied Biosciences). PCR conditions are described in 2.7. PCR.

(4) 2138. L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. products were visualized on a 1.5% (w/v) agarose gel prepared in 0.5TAE buffer and stained with ethidium bromide. A gradient of 20–80% [w/v] denaturant agents (100% [w/v] containing 7 M urea and 40% [v/v] formamide) was used. The gels were run at 100 V for 16 h using a Protean II system (Bio-Rad, USA) at 60 °C in 0.5 TAE buffer. They were then stained with SYBR SAFE (Invitrogen, France). Bands of interest were excised and incubated at 4 °C in 40 lL of MiliQ water for 48 h. The excised bands were reamplified with 1 lL of this solution with primers dsr2060F and dsr4R as described above. PCR products were then sent to sequencing (Genoscreen, France). Gel profiles were performed in triplicate. 2.9. Clone library construction Two clone libraries of dsrB gene were built from PCR products (using the same primer set dsr2060f–dsr4R) obtained from the two sediment cores at a depth of 4–6 cm. PCR products were mixed with plasmid PGEM-T-Easy according to the manufacturer’s protocol (Promega, France), and recombinant plasmids were used to transform Escherichia coli DH5 a. Two different libraries were obtained: one from the Flamenco sediment, called VF, and the second one from the Palito edge sediment, called VEP. Sixty-four clones from each library were selected randomly and their inserts were amplified using the primer pair pucF: 50 -TTGGTAACGCCAGGGT-30 and pucR: 50 -ACCATGATTACGCCAAG-30 , which recognize specific sequences surrounding the cloning site. PCR conditions were defined as follows: initial denaturation step (10 min at 95 °C), 30 cycles of denaturation (1 min at 95 °C), annealing (1 min at 57 °C), elongation (1 min at 72 °C), and a final extension step (7 min at 72 °C). The size of amplicons was determined by electrophoresis in a 1.5% (w/v) agarose gel in 0.5TAE buffer and amplicons were directly sequenced. All of the sequenced amplicons were of the expected size. PCR products were then sent to be sequenced (Genoscreen, France). 2.10. Phylogenetic analysis Nucleic acid sequences obtained from DGGE gels and clone libraries were translated into amino acid sequences (http:// www.ncbi.nlm.nih.gov/projects/gorf/). DsrB amino acid sequences were analyzed using the BLASTX program (http://www.ncbi.nlm.nih.gov/projects/blast). DsrB sequences were aligned with DsrB amino acid sequences of reference sulfate-reducing prokaryotes and environmental clones using Clustal X2 (Thompson et al., 1997). A phylogenetic tree was constructed using MEGA4.0 software (Kumar et al., 2004), and distance-based evolutionary trees were constructed using Kimura’s corrected similarity values in the neighbor-joining algorithm of Saitou and Nei (1987). The topography of the branching order within the dendrogram was evaluated by applying the Maximum Parsimony character-based algorithm combined with bootstrap analysis to a round of 1000 samplings. A phylogenetic. tree was built with a total of 96 amino acid residues corresponding to the length of the shortest sequence. The DsrB sequence of Thermodesulfovibrio islandicus (GenBank accession No. AAK83124) was used as out-group to root the tree according to Klein et al. (2001). The dsrB gene fragment nucleotide sequences of the clones obtained in this study were deposited in the GenBank database under the accession Nos. JX068851–JX068878. 2.11. Statistical analysis Statistical analyses of microbial quantification were performed using the Kruskal–Wallis test (Dytham, 1999). The significance level for all comparison and correlation tests was set at a P value of 0.05. We used the Shannon–Weaver index to estimate phylotype diversity (Hill et al., 2003). 3. Results 3.1. Biogeochemical parameters of the two sediments Grain size analysis of the sediments, expressed in lm, is presented in Table 1. Neither sediment contains a fine fraction (<63 lm), and their coarse fraction size distribution is different. The samples of the Flamenco core are composed of around 90% of sandy sediment from top to bottom, with the main grain size mode between 100 and 250 lm, and little gravel (2%). Unlike the Flamenco samples, in the Palito edge sediment the grain size curves are not unimodal. The sediment shows a larger grain size distribution along the core. On the top, the distribution is finer (the main mode is 200–1200 lm), while in the middle and at the bottom of the core, the grain sizes are coarser and the main mode is between 1000 and 2500 lm. Moreover, the sediments at the bottom contain gravel (<10%). In the two sediment cores, pH, sulfate, TOC and NaCl values are quite similar (Table 1) and do not show significant differences. Eh values indicate that reducing conditions are present from the first centimeter of the Palito sediment core, but only from the depth of 2 cm in the Flamenco sediment core. The total and available metal concentrations found in the two sediment cores are described in Table 2. According to the sediment quality criteria for the classification of sediments developed by the Hong Kong SAR Government (ETWB, 2002), the Palito edge sediment is highly contaminated as it contains high levels of copper (e.g., concentrations between 231 and 301 mg kg1) that are much higher than the Upper Chemical Excedance Levels (UCELs) assessed in contaminated marine sediment (Cu = 110 mg kg1). Statistical analyses demonstrate a significant difference between the copper (total and available) concentrations in the two studied sites (Kruskal–Wallis test, P < 0.05). Zn, Cd, and Pb concentrations fall below the Lower Chemical Excedance Levels (LCELs) (Zn = 200, Pb = 75, and Cd = 1.5 mg kg1). Flamenco sediment is not contaminated,. Table 1 Physico-chemical characteristics according to the depth of the sediments of Palito (Pal) and Flamenco (Fla). Values of each parameter are given as mean of triplicates. Depth (cm). 0–2 2–4 4–6 6–8 8–10 10–15 15–20 20–25. Main grain-size (lm). Eh (mV). Fla. Pal. Fla. 100–250 100–250 100–250 100–250 100–250 100–250 100–250 100–250. 200–1200 200–1200 200–1200 200–1200 1000–2000 1000–2000 1000–2500 1000–2500. 67 58 163 178 182 187 193 190. NaCl (g L1). Sulfate (mM). TOC (mg g1). Pal. Fla. Pal. Fla. Pal. Fla. Pal. Fla. Pal. 65 136 175 182 194 192 195 197. 28.4 28.2 28.3 28 28.1 28.2 28.3 28. 25.7 25.8 25.8 25.6 25.7 25.8 25.6 25.7. 26.1 25.9 25.9 25.5 25.2 25 24.8 24.7. 24.4 24.1 24.3 24 23.9 23.5 23.3 23.1. 20 19.8 19.7 18.8 18.9 18.7 17.8 17.5. 21.5 21.2 20.9 19.9 20.1 19.6 19.6 19.8. 7.69 7.68 7.65 7.65 7.66 7.62 7.62 7.60. 7.68 7.68 7.66 7.64 7.65 7.64 7.64 7.64. pH.

(5) Pal. 2.7 ± 0.03 2.6 ± 0.03 2 ± 0.02. Fla. <2 <2 <2 4 ± 0.05 3.6 ± 0.04 2.5 ± 0.03. Pal Fla. 3.5 ± 0.03 3.4 ± 0.04 3.6 ± 0.04 1.25 ± 0.02 1.13 ± 0.01 1.15 ± 0.01. 0.75 ± 0.005 0.86 ± 0.007 0.64 ± 0.007. Fla Fla. 2.6 ± 0.03 3 ± 0.02 3 ± 0.03 15.6 ± 0.14 28.4 ± 0.19 20.4 ± 0.21. Pal Fla. 16.5 ± 0.17 14.2 ± 0.15 17.5 ± 0.16 301 ± 2.21 288 ± 2.12 231 ± 2.17. Pal Fla. 5 ± 0.07 6 ± 0.07 6 ± 0.06 0–10 10–20 20–30. Pal Fla. 21 ± 0.35 20 ± 0.38 23 ± 0.24. Depth (cm). 381 ± 2.52 327 ± 2.47 317 ± 2.38. Pal Fla. 0.95 ± 0.01 0.85 ± 0.01 0.95 ± 0.01. Pal. 11 ± 0.15 23 ± 0.20 7 ± 0.06. Pal. Total Pb Available Cd Total Cd Available Zn Total Zn Available Cu Total Cu. Metals (mg kg1). Table 2 Total and available (heavy metal treatment with HCl 1 M) heavy metal concentration (mg kg1) in sediment samples from Palito (Pal) and Flamenco (Fla). Values are given as mean of triplicates.. 0.66 ± 0.005 0.42 ± 0.005 0.55 ± 0.005. Available Pb. L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. 2139. with metal concentrations under the Lower Chemical Excedance Levels (LCELs) (Cu = 65 mg kg1). 3.2. Quantification of total bacteria and SRPs in the sediment cores The vertical distribution of the bacterial abundance and the relative contribution of SRPs to the total number of bacterial cells throughout the sediment core were investigated by qPCR assays based on the 16S rRNA gene and the dissimilatory sulfite reductase gene (dsrB) b-subunit (Fig. 2). All along the Flamenco sediment core, the total number of bacteria is constant, with a mean of 1.27 109 cells/g of sediment. On the contrary, in the copper-contaminated sediment core, the total bacterial abundance decreases from 1.16 109 to 2.47 108 cells/g between the surface and a depth of 20 cm, and the mean of total bacteria is 7.11 108. We detect the presence of SRPs throughout the two sediments cores. The SRP abundance profiles show a trend comparable to those of total bacteria. SRP abundance clearly decreases from 9.4 106 to 2 106 in the contaminated sediment, with a mean of 5.1 106. In the Flamenco sediment, the profile is constant, with a mean of 3.5 108, except at the surface (2.4 107 cells/g). The total number of SRPs in the top 15 cm of sediment is respectively 3.49 107/cm3 in Palito, and 1.3 109 SRPs/cm3 in Flamenco. The proportion of SRPs to the total bacterial community was calculated assuming similar biases in qPCR Fig. 2(C). The proportion of SRPs in the Palito sediment is weak; it reaches a mean of 1% throughout the sediment core. In the Flamenco sediment, this proportion is high, with a mean of 22%, except at the surface where the percentage is 0.9%. Finally, a significant decrease of the abundance of total bacteria and SRPs according to the rise of copper concentration was observed (respectively P = 0.0054 and P = 0.0011). 3.3. Phylogenetic diversity of SRPs in the two sediment cores The diversity of SRPs in the two chilean sediment cores was investigated by DGGE (Fig. 3) and cloning-sequencing of the partial dsrB gene. The corresponding protein sequences were used to construct a phylogenetic tree (Fig. 4). In each specific sediment, the dsrB gene-based DGGE analysis (Fig. 3(A) and (B) displays similar banding profiles, suggesting that the SRP diversity is conserved, whatever the depth. But comparison between the SRP DGGE profiles obtained from the two sediment cores shows very distinct banding patterns, indicating important differences in the community structure, in particular a low diversity in Palito and a greater diversity in the non-impacted site. Flamenco DGGE results indicate that band no. 1 is the most intense and found at all depths, suggesting that the bacterial species harboring this band have a high relative abundance. For the Palito site, we obtained only three different bands, with two bands (1 and 3) with a more or less similar relative abundance. All these bands correspond to dsrB sequences belonging to the Desulfobacteraceae, with the exception of the DGGE band 4 (Desulfobulbaceae) and band 9 which could not be sequenced. These sequences were used for phylogenetic analysis. To improve the knowledge on SRB diversity we chose to construct two clone libraries from these sediments. We selected the same depth (4–6 cm) but one was from the copper enriched sediment (VF clone library) and the other from the sediment not exposed to copper (VEP). A total of 64 clones for each library were analyzed. The coverage values of the libraries determined as described by Singleton et al. (2001) (0.95 and 0.98 for VEP and VF, respectively) indicated that a sufficient number of clones had been analyzed. Sequences displaying more than 95% amino acid identity with each other were grouped in the same Operational Taxonomic Unit (OUT) and thus, nine and seven were identified for VEP and VF libraries, respec-.

(6) 2140. L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. Fig. 2. Vertical distribution of the abundance of total bacteria and SRPs in the Palito and Flamenco sediments determined along the 25-cm cores depth. (A) Total bacteria and (B) SRPs as inferred from real-time PCR data. Values are given as mean standard deviation of triplicates and expressed in a number of cells per gram of fresh sediment. (C) Depth profile of the relative contribution of SRPs to the total bacterial cells as calculated from the data in (A) and (B)..

(7) L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. A. 1. 2. 3. 4. 5. 6. 7. 7 2 1 3 5. 6. 4. 8 9. B. 1. 2. 3. 4. 5. 6. 2141. maculum species which laterally acquired dsrAB (Zverlov et al., 2005). This cluster corresponds to one OTU and represents 6.3% and 4.3% of the clones from VF and VEP libraries, respectively. Cluster C, belonging to the Syntrophobacteraceae family, is also represented in the two clone libraries with 21.8% and 4.3% (one OTU) of the clones from VF and VEP libraries. Cluster D is only composed of clones from the VEP library (2.2% corresponding to one OTU) which are members of Desulfobulbaceae. Cluster E contains clones found in the two libraries and represents one third of the total clones (represented by 3 OTUs). The clones are not affiliated with any DsrB sequence of cultured SRPs, but are close to dsrB clones extracted from marine and estuarine sediments, some of which have been described as contaminated with heavy metals. No DGGE-DsrB sequence belonging to the cluster E has been found. The Shannon–Weaver indexes were calculated for each sediment core (Shannon index Flamenco = 1.705; Shannon index Palito = 1.731), and confirm that the diversity was comparable between the two sites, and not affected by the rise of copper concentration.. 7. 4. Discussion. 1. 2. 3. Fig. 3. Denaturing gradient gel electrophoresis of dsrB PCR products obtained of total DNA extracted from (A) Flamenco sediment, and (B) Palito sediment (lane 1: DNA marker; 2: 0–2 cm; lane 3: 2–6 cm; lane 4: 6–10 cm; lane 5: 10–15 cm; lane 6 : 15–20 cm; lane 7 : 20–25 cm). Numbers indicate bands that were excised and sequenced.. tively, and included to construct the phylogenetic tree (Fig. 4). These OTUs were distributed in five distinct clusters. Bootstrap values for some clades were not high probably due to that partial sequences were used, decreasing the phylogenetic resolution robustness, as has already been observed for other Dsr phylogenetic analyses (Castro et al., 2002; Harrison et al., 2009). Clusters A, B, C, and D were affiliated to Deltaproteobacteria, while cluster E showed no clear phylogenetic affiliation. No DsrB sequences related to known Gram-positive bacteria were detected in the two libraries. Cluster A contains four OTUs affiliated to the Desulfobacteraceae, corresponding to 43% and 57% of clones from VF and VEP, respectively. It is the most representative cluster, and most of the DGGE band sequences are also related to this family. Cluster B is related to an independent lineage within the ‘‘Deltaproteobacteria’’ where DsrB sequences are affiliated with those from Desulfobacterium anilini, a member of the donor lineage for the gram-positive Desulfoto-. We studied the impact of copper on the microbial community by comparing the abundance and the diversity of SRPs in two sediment cores exposed to contrasting copper levels. Comparison of same-depth layers from the uncontaminated and contaminated cores revealed that the total number of bacteria varied by a factor of between 1.5 and 3, while the total number of SRPs varied by a factor of about 100. In the same way, for the top 15 cm, the total number of SRPs in the Flamenco sediment was two orders of magnitude higher than in the Palito sediment, confirming previous results for non-contaminated marine sediments (Kondo et al., 2004; Leloup et al., 2005; Schippers and Neretin, 2006). The proportion of SRPs to the total bacterial community was between 13% and 29% in the non-polluted sediment, with the exception of the superficial layer. This result is consistent with previous estimates of SRP abundance determined by FISH, CARDFISH, and qPCR, which are mostly in the range of 2–20% in near-surface marine sediments (Lehours et al., 2005; Mußmann et al., 2005; Leloup et al., 2009). The low number of SRPs in the first 2 cm of Flamenco sediment can be explained by a positive redox potential, suggesting a concentration of O2 which would strongly prevent SRP development. It is well documented that physico-chemical parameters such as NaCl, total organic carbon, redox potential, pH, and the sedimentary process can modify the abundance of microorganisms in sediments (Deming and Baross, 1993; Stepanaukas et al., 2003; Yokokawa and Nagata, 2005; Leloup et al., 2005). Concerning all the abiotic parameters involved in the two sediment cores studied, the only differences observed were for granulometry and copper concentrations. The differences found in the abundance of total bacteria and SRPs in the two cores, statistically proved, could be explained by the relationship between grain sizes and bacterial dispersion: the finer the grains, the better the bacterial dispersion, whatever the technique used, as proposed by Schmidt et al. (1998) and Kuwae and Hosokawa, (1999). However, for the two-log difference found in our results, this cannot be the only explanation; Stefanija et al. (2009), for e.g., have concluded that there is no link at all between bacterial dispersion and granulometry. Our hypothesis is that these significant differences can only be explained by taking into account the high copper concentration present in the Palito sediment. Indeed, some studies have shown that changes in microbial biomass can be attributed to any metal effects (Knight et al., 1997; Bouskill et al., 2010). It thus seems that SRPs are sensitive to high.

(8) 2142. L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. Fig. 4. Dsr phylogenetic tree showing the affiliation of Palito and Flamenco clones and DGGE-excised bands with SRPs present in the database. This evolutionary tree was generated by the neighbor-joining method. Sequences of 126 aa length were used to construct the tree. Bootstrap resampling (1000 replicates) of the tree was performed to provide confidence estimates for the inferred topologies. An out-group of the DsrB protein of Thermodesulfovibrio islandicus was included to root the tree. Bootstrap values at branching nodes are shown with more than 50% bootstrap support. The number of clones in each library is indicated in parentheses. Nucleotide sequence accession numbers are given in parentheses. The different phylogenetic clusters are shaded in grey. The bar at the bottom indicates the estimated evolutionary distance.. copper concentrations, even though (i) SRPs display a certain degree of metal tolerance due to sulfide production, which can corre-. spond to an effective metal detoxification mechanism (Baath, 1989), (ii) SRPs can even grow in heavy-metal-contaminated sedi-.

(9) L. Besaury et al. / Marine Pollution Bulletin 64 (2012) 2135–2145. ments presenting concentrations close to or higher than the UCELs assessed in contaminated marine sediment (Martins et al., 2009; Quillet et al., 2012), (iii) some SRPs have developed specific resistance mechanisms such as sequestration or transformation to other chemical species (Valls and de Lorenzo, 2002; Gihring et al., 2011). dsrB genes sequences amplified from these copper-contaminated and non-contaminated sediments revealed that the diversity was comparable, with sequences belonging to Desulfobacteraceae, Syntrophobacteraceae, Desulfobulbaceae, laterally acquired dsrAB bacteria, and a deeply dsrAB branching group. These different SRP families and groups have already been described in diverse marine sediments (Dhillon et al., 2003; Mußmann et al., 2005; Leloup et al., 2006; Kondo et al., 2007; Suzuki et al., 2007; Kaneko et al., 2007; Gittel et al., 2008; Jiang et al., 2009), and some of them have also previously been reported in marine sediments contaminated by metals (Gillan et al., 2005; Moreau et al., 2010; Quillet et al., 2012). The clusters A and E are those most frequently found in the two sediments. DsrAB sequences of Desulfobacteraceae have already been described in enrichments from copper-contaminated sediments (Pavissich et al., 2010). The cluster E corresponds to a deeply dsrAB branching group that was first described by Dhillon et al. (2003) in Guaymas basin sediment and then in different marine-estuarine sediments (Leloup et al., 2006; Zhang et al., 2008; Quillet et al., 2012). As this cluster is present in both clone libraries, it seems not especially linked to a heavy-metal-contaminated marine sediment habitat. As already described by Feris et al. (2003), Gillan et al. (2005), and Quillet et al. (2012), it may be possible that the diversity of the microbial population in the Palito sediment could have been initially affected by the dramatic increase of copper concentrations in the 1940s, but that after 70 years of heavymetal contamination, the diversity was recovered, even though the environment was still contaminated. Interestingly, although Desulfovibrio has been frequently described in marine sediments (Boyle et al., 1999; Perez-Jimenez and Kerkhof, 2005; Haouari et al., 2006; Leloup et al., 2007), reported as being especially active in a heavy-metal-contaminated sediment (Quillet et al., 2012), and already described in environments strongly contaminated by metals, in particular copper (Cabrera et al., 2006; Martins et al., 2009; Moreau et al., 2010; Pavissich et al., 2010), sequences belonging to this family were absent in the Flamenco and Palito sediments. This absence has also been observed in other marine sediments (Dhillon et al., 2003; Leloup et al., 2006; Kaneko et al., 2007; Jiang et al., 2009). The diversity of the dsrB gene studied by DGGE is very low and corresponds only very partially to that observed in the two marine sediments, and determined by cloning/sequencing. A possible explanation for the low abundance of Syntrophobacteraceae, Desulfobulbaceae, and laterally-acquired dsrAB-microorganisms in the sediments analyzed, is that such dsrB sequences are in a copy number lower than the detection threshold of PCR-DGGE analyses, but high enough to obtain dsrB clones, as already described by Steger et al. (2011) and Leloup et al. (2009). It is well known that the DGGE approach presents the advantage that a large number of samples can be analyzed in a short time, but DGGE can lead to an underestimation of the diversity, as described by Muyzer and Smalla (1998). The absence of DGGE bands corresponding to dsrB gene sequences belonging to cluster E, one of the two clusters with high number of clones, can be explained by the difficulty of separating and excising all DGGE bands (especially the faint and diffuse bands) and that some DGGE bands did not give any amplification product after the excision and purification (Delgado et al., 2008; Piñar et al., 2010), as was the case for band no. 9, even after several cloning attempts following solutions proposed by Sekiguchi et al. (2001).. 2143. The low diversity for Desulfobulbaceae found in the non-exposed sediment was also surprising, but in this case the plausible explanation is that the cloning/sequencing approach is also not free from biases, as Rainey et al. (1994) described different cloning efficiencies according to different vectors and primers set used. Our results show a large decrease in the quantity of SRPs in the contaminated sediment compared to the non-polluted sediment, although diversity is maintained. To explain these results, we propose the hypothesis that only bacteria having acquired very good tolerance to high concentrations of metals can grow in these strongly contaminated environments. The capacity of SRPs to produce sulfide, which can react with the metallic ions, the so called ‘‘sulfide protection effect’’ by Temple and Le Roux (1964), cannot alone explain the tolerance to copper. Dıaz-Ravina and Baath (1996) and Jin et al. (2007) have proposed that bacterial adaptation to heavy metal contamination results from genetic changes. Naz et al. (2005) have demonstrated the existence, in some SRPs, of DNA sequences that exhibit strong nucleotide homology with known heavy-metal-translocating ATPases, and this suggests the presence of multiple genetic mechanisms for metal resistance, particularly for cadmium. Karnachuk et al. (2003) and Abicht et al. (2011) have demonstrated that some SRPs have developed specific copper-resistance mechanisms, such as the pco operon that encodes Cu resistance, or the copA gene encoding a putative copper-translocating P-type ATPase. So metal tolerance is correlated with metal exposure, as already described by Utgikar et al. (2001) and Jin et al. (2007), meaning that sensitive species are immediately eliminated upon exposure to high copper concentrations, and that only those SRPs receiving either chromosomal DNA or plasmid DNA encoding copper-resistance genes can thrive in such highly-copper-contaminated environments. Bacterial resistance to copper conferred by plasmids has been described in bacteria found in soils and sediments, such as Pseudomonas (Cha and Cooksey, 1991), Xanthomonas (Lee et al., 1994) or Escherichia coli (Rensing and Grass, 2003). These systems are highly homologous and contain the same genes coding for soluble membrane proteins (Silver, 1996). Chromosomal mobile elements (including transducing phages) also encode copperresistance genes (Wuertz and Mergeay, 1997; Fraser et al., 2007). It has been established that the three gene transfer mechanisms (transformation, transduction and conjugation) have specific biotic and abiotic requirements suggesting different probabilities for their occurrence, in particular in sediment habitats (Ippen-Ihler, 1989; Kokjohn, 1989; Lorenz and Wackernagel, 1994).. 5. Conclusion Finally, the conservation of diversity and the decrease of SRP abundance in the Palito sediment can be understood if we accept the hypothesis that, using mobile elements, copper-resistance genes may be horizontally transferred among bacteria, but that the ability to share and spread those resistance genes between the different SRP species seems to be a slow mechanism in this environment. The study of copper-resistance genes in the two sites will be very helpful in verifying this hypothesis.. Acknowledgements This work was supported by the International Collaborative ECOS-CONICYT program (no. C06B05) and the ECCO French research program ‘‘Misechicui’’. We thank Dilys Moscato for help with the English..

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