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Marine Pollution Bulletin 56 (2008) 1168–1176

Relationships between hydrosedimentary processes and occurrenceof mercury-resistant bacteria (merA) in estuary mudflats (Seine, France)

Jean-Baptiste Ramond a, Thierry Berthe a,*, Robert Lafite a, Julien Deloffre a,Baghdad Ouddane b, Fabienne Petit a

a Universite de Rouen – CNRS UMR 6143, Morphodynamique Continentale et Cotiere (M2C), Groupe de Microbiologie,

76821 Mont Saint Aignan Cedex, Franceb Universite de Lille – CNRS UMR PBDS 8110, Laboratoire de Chimie Analytique Marine, 59655 Villeneuve d’Ascq Cedex, France

Abstract

The Seine estuary (France) is one of the world’s macrotidal systems that is most contaminated with heavy metals. To study the mer-cury-resistant bacterial community in such an environment, we have developed a molecular tool, based on competitive PCR, enabling thequantification of Gram-negative merA gene abundance. The occurrence of the Gram-negative merA gene in relation with the topology(erosion/deposit periods) and the mercury contamination of three contrasted mudflats was investigated through a multidisciplinaryapproach and compared with a non-anthropized site (Authie, France). The higher abundance of the Gram-negative merA gene in theSeine estuary mudflats indicates a relationship between the degree of anthropization and the abundance of the merA gene in the mudflatsediments. In the Seine mudflats, the maxima of abundance are always located in fresh sediment deposits. Therefore, the abundance isclosely related with the hydrosedimentary processes, which thus seem to be determining factors in the occurrence of the Gram-negativemerA gene in the surface sediments of the Seine’s mudflat.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Seine estuary; Mercury-resistant bacteria; merA gene; Competitive PCR; Hydrosedimentary processes; Sediments

1. Introduction

The Seine estuary is one of the main estuaries of theNorth Western European continental shelf, providing themost significant fluvial input into the English Channel/LaManche (Chiffoleau et al., 2001). The Seine River and itsestuaries drain a catchment area of 79,000 km2 whichincludes 23% of the French population and 30% of theFrench industrial activity (Guezennec et al., 1999). TheSeine was one of the most contaminated catchment areasin the world in the 1970s, with maximum content recordedat 24 mg kg�1 for Hg. Today, the average level for Hg(1.08 mg kg�1) is much lower, but is still in the upper90% of the global scale distribution and well above the nat-

0025-326X/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.marpolbul.2008.02.022

* Corresponding author. Tel.: +332 35 14 67 81; fax: +332 35 14 66 88.E-mail address: thierry.berthe@univ-rouen.fr (T. Berthe).

ural background value determined in pre-historical depos-its (Meybeck et al., 2007). In the water column of the Seineestuary, heavy metals, mainly associated with suspendedmatter, are deposited on mudflats that are deposit and stor-age zones for fine-grained sediment (Laurier et al., 2003;Lesourd et al., 2003; Deloffre et al., 2005, 2006; Meybecket al., 2007). Thus, the mudflats located in the intertidalzones of this highly anthropized estuary have been shownto be long-term storage areas for a range of contaminantsdischarged or transported into the estuary environment(Niessen et al., 2003; Cundy et al., 2005; Meybeck et al.,2007). The sediments of the intertidal mudflats of the Seineestuary are thus important reactive compartments, actingas a sink for contaminants, especially heavy metals, andwhere mercury speciation is mainly controlled by microbialactivities (Niessen et al., 2003; Cundy et al., 2005; Heyeset al., 2006; Sunderland et al., 2006). These mudflats could

J.-B. Ramond et al. / Marine Pollution Bulletin 56 (2008) 1168–1176 1169

also be a putative source of contamination of the water col-umn during erosion events that mainly depend on the sed-imentary processes on mudflats (Lesourd et al., 2003;Cundy et al., 2005; Deloffre et al., 2005, 2006). In similarlycontaminated estuarine and coastal environments, high lev-els of mercury-resistant (HgR) merA bacteria have been iso-lated (Barkay and Olson, 1986; Rasmussen and Sorensen,1998; Reyes et al., 1999; Ramaiah and De, 2003).

Bacterial resistance to mercury is one of the numerousexamples of the genetic and physiological adaptation ofmicrobial communities exposed to high available concentra-tions of contaminants in their environment (Silver andPhung, 1996; Nies, 1999; Barkay et al., 2003). The specificbacterial mercury-resistance mechanism relies on the reduc-tion of toxic mercuric ion Hg2+ into the volatile and lesstoxic elemental mercury Hg0 by the mercuric reductaseMerA (Barkay et al., 2003). This protein is encoded by themerA gene embodied in the mer operon, frequently dissem-inated in transposons and conjugative plasmids and widelyspread in the total microbial community (Yurieva et al.,1997; Nies, 1999; Barkay et al., 2003; Narita et al., 2004;Schelert et al., 2006). This mer-mediated mercury-resistancemechanism is one of the most widely observed in bacteria,even in archaea (Vetriani et al., 2005; Schelert et al., 2006),whereas for other toxic metals many different genetic sys-tems are encountered that can be transferred only betweenphylogenetically-related bacterial species (Silver and Phung,1996; Nies, 1999; Barkay et al., 2003). Thus, in water, soiland sediment, the merA gene is a relevant model in ecologi-cal studies for assessing relationships between microbialmercury-resistance and bioavailable mercury contamina-tion of the environment (Barkay and Olson, 1986; Nazaretet al., 1994; Ni Chadhain et al., 2006).

The aim of the present study was to investigate the rela-tionships between the hydrosedimentary processes and theoccurrence of the Gram-negative merA gene in highly mer-cury-contaminated mudflat sediments (Cossa et al., 2003;Niessen et al., 2003; Meybeck et al., 2007), since in such mac-rotidal systems the mudflat surface is submitted to constantreworking (Lesourd et al., 2003; Deloffre et al., 2005, 2006).

Therefore, the dynamics and abundance of Gram-nega-tive merA bacteria were investigated in the sediments of anestuary that has been exposed to high mercury concentra-tions for decades (Seine estuary, France) (Meybeck et al.,2007), in comparison with an uncontaminated estuary(Authie, France). As noted by many authors (Kirk et al.,2004; Janssen, 2006; Ritz, 2007), the limitations of culturetechniques in isolating environmental bacteria led us todevelop a cultivation-independent molecular tool, basedon competitive PCR (cPCR), to quantify the specific mer-cury-resistance merA gene from Gram-negative bacteria.In contaminated water and sediment samples, gene quanti-fication by cPCR has proven to be suitable because thecontaminants co-extracted with nucleic acids affect bothtarget and competitor sequence amplification equally (Ber-the et al., 1999; Oger et al., 2001; Kondo et al., 2004;Leloup et al., 2005). This multidisciplinary study was car-

ried out on mudflat sediment cores from two contrastingestuaries and microbiological data were analysed in rela-tion with total mercury concentrations and the topograph-ical evolution of the mudflats (erosion/deposit periods).

2. Materials and methods

2.1. Study sites and sampling

Sediments from the mudflats of two estuaries located inthe northern part of France were investigated (Fig. 1). Pre-vious work defined a representative sampling section oneach mudflat, with regard to hydrologic and sedimento-logic conditions (Lesourd, 2000). The three mudflatssampled in the Seine estuary showed contrasting physico-chemical characteristics (Table 1). The Oissel mudflat(49�200288; 01�050660) is located in the upper estuary,and is immerged by freshwater during high river flow andemerged during low river flow (Deloffre et al., 2005). Twocores were collected during an erosion period and freshsediment deposits were only present in the July 2001 core(Table 1). The Northern intertidal mudflat (49�260823;0�140628) is located in the mixing zone of the Seine estuary,and is subjected to the influence of both the tide and themarine waters (Deloffre et al., 2006). The cores collectedin April 2001 and May 2001 was sampled after an erosionperiod and both presented fresh sediment deposits(Table 1). Samples were collected during two periods inthe subtidal mudflat located in the Bay of Seine: in March2002 (49�260000; 0�000700), during a sedimentation periodmainly caused by mud deposits, and in September 2002(49�250950; 0�000730), during a low flow period when theaccumulated sediment had been reworked by waves andtidal currents (Lesourd et al., 2003). The intertidal mudflatsof the Seine estuary consist of clay and silt deposits(<63 lm) originating from terrigenous material and, inthe middle and marine part of the estuary, mainly marineparticles (Lesourd et al., 2003; Deloffre et al., 2005,2006). An intertidal mudflat from the Authie Bay(50�220N, 01�360E) (Fig. 1b), a non-anthropized site, wastaken as a reference site and was sampled twice in 2003during deposition periods (Table 1). A piston corer wasused to obtain 30-cm-long sediment cores. They wereimmediately cut into 15 2-cm-thick sections. Approxi-mately 25 g of each section (wet weight) was transferredto the laboratory in a sterile polypropylene tube at 4 �C.Subsamples (1 g wet weight) of each section were frozenat �20 �C until subsequent molecular analysis.

2.2. DNA extraction

Total DNA was extracted from 0.5 g sediment samples(wet weight) with a Bio-101 FastDNA Spin Kit in combi-nation with the FastPrep FP 120 bead beating system(Bio-101 Inc., La Jolla CA, USA) according to the manu-facturer’s instructions. Total crude DNA was purifiedthrough Elutip-D columns (Schleicher and Shuell, Dassel,

Fig. 1. Study sites: (a) location of study areas, (b) the Authie Bay and (c) the Seine estuary (Northern intertidal, Oissel freshwater intertidal and subtidalmudflats). Arrows indicate the mudflats studied.

1170 J.-B. Ramond et al. / Marine Pollution Bulletin 56 (2008) 1168–1176

Germany). The concentration of the resulting DNA wasestimated by spectrophotometry using a GeneQuant Prospectrophotometer (Amersham Biosciences, USA).

2.3. PCR amplification

The low degree of merA gene sequence homology led us tochoose a primer set that only targets Gram-negative merA

bacteria (Liebert et al., 1997). A sequence of 1238 pb ofthe merA gene from Gram-negative bacteria was PCRamplified with the merA1 and merA5 primers designed byLiebert et al. (1997), and this primer set was used in ourstudy to develop the cPCR. A new set of primers, whichallows the amplification of merA genes from Gram-positiveand -negative bacteria, has recently been proposed, but theseprimers cannot be used to quantify merA genes by cPCR inDNA extracted from mudflat sediments because of thepresence of non-specific amplicons (Ni Chadhain et al.,2006). A touch-down PCR was carried out in a Perkin Elmerthermocycler (Gene Amp PCR system 6700) as follows:5 min at 94 �C for denaturation, 29 cycles of 1 min at94 �C, 1 min annealing and 2 min at 72 �C; and a final exten-sion for 10 min at 72 �C. During the first 10 cycles theannealing temperature was progressively stepped down(every two cycles) from 68 to 64 �C. Fifteen to hundrednanograms of DNA extracted from sediments was PCRanalysed in the following reaction mixture, 1� PCR buffer(10 mM Tris–HCl, pH 8.3, 50 mM KCl), 200 lM of eachdNTP (Eurogentec), 2.5 mM MgCl2, 0.25 lM of each pri-mer, 3% DMSO (final concentration) and 0.08 U Taq poly-merase (GoldStar DNA polymerase, Eurogentec).

3. Competitive PCR

3.1. Construction of the competitor plasmid

A competitor sequence was obtained by PCR asdescribed by Ross et al. (1995). A first 942 bp fragment,located upstream from the target gene (merA) in the mer

operon of the Tn501 transposon on plasmid pHP45XHg(Fellay et al., 1987) was PCR amplified using hybrid (30mer) primers: Comp1 (50-CGG CGG CAC CTG CGTTGG GGT TCA GCG GCG-30) and Comp2 (50-cag gta

ggg gaa caa ATC GCG GCT CCA CCG-30). The nucleo-tides written in italics correspond to the last 15 nucleotidesof the 30-end of the merA1 and merA5 primers (Liebertet al., 1997) and the other 15 nucleotides hybridize withthe 942 bp sequence. The competitor sequence of 985 bpwas then obtained by PCR amplification of the 942 bpfragment with the merA1 and merA5 primers (Liebertet al., 1997). The competitor plasmid pmerA-c was thenconstructed by cloning the competitor sequence of 985 bpin a plasmid using the T/A Cloning Kit (Promega) andplasmid pGEM�-T Easy Vector System kit (Promega)according to the manufacturer’s instructions.

3.2. Molecular quantification of Gram-negative merA gene

by competitive PCR

cPCR is based on co-amplification of the target DNAsequence (Gram-negative merA gene) and the competitorsequence (pmerA-c). The Gram-negative merA gene wasquantified from environmental samples as described in

Table 1Physico-chemical and hydrosedimentary characteristics of the mudflats

Samplingdate

Samplingconditions

River flow(m3 s�1)

Period Surface depositage (days)

Fresh surfacesedimentdeposit (cm)

Sediment totalmercury (HgT)concentration (ng g�1)b

Oissel intertidal mudflat 05/07/2001 Emerged 358 Erosion Old deposita 7 907.6 ± 527.524/09/2001 Emerged 633 Erosion Old deposita 0 1207.5 ± 670.9

Northern intertidal mudflat 03/04/2001 Emerged 2100 Erosion Old deposita 6 658.0 ± 268.430/05/2001 Emerged 682 Erosion Old deposita 5.4 582.6 ± 157.3

Marine subtidal mudflat 19/03/2002 Submerged 920 Deposit N.A. 12 0.3 ± 0.124/09/2002 Submerged 185 Erosion N.A. 0 N.A.

Reference site (Authie mudflat) 19/05/2003 Submerged 10 Deposit 5 2 0.05 ± 0.0127/11/2003 Submerged 6 Deposit 5 1.4 N.A.

N.A.: not available.a <46 days.b Values are corresponding to the average concentrations on the entire core.

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previous studies (Berthe et al., 1999; Oger et al., 2001;Kondo et al., 2004; Leloup et al., 2005). A set of standardsamples containing 150 fg of competitor pmerA-c wasadded to a serial dilution of pHP45XHg (from 2.1 fg to281 fg). Concomitantly, 150 fg of competitor was alsoadded to the DNA extracted from the estuary sedimentsamples (merA-s) with DNA concentration varying from15 to 100 ng ll�1. The cPCR products (15 ll) were ana-lysed by electrophoresis in agarose gel (1.5% (w/v); 0.5�TAE; 50 V cm�1) stained with ethidium bromide andviewed under UV light (Fig. 2a). The PCR products werequantified from digitized gel images (AlphaImagerTM

1220, Alpha Innotech Corporation, USA). A standardcurve was obtained by plotting the log intensity of DNAamplified from the serial dilutions against the intensity ofthe competitor DNA amplification log (pHP45XHg/pmer-A-c) (Fig. 2c). The amount of target in DNA extractedfrom the sediment was then determined from the log inten-sity ratio of (merA-s/pmerA-c) DNA. In these conditions,the limit for the detection of the merA gene by cPCR wasestimated to be 278 gene copies (data not shown).

3.3. Mercury analysis

Total mercury was measured in dry sediments (withoutany pre-treatment) with an AMA 254 solid phase Hg-Ana-lyzer (Altec Ltd., Prague, Czech Republic) using atomicabsorption spectroscopy. After thermal combustion ofthe dried samples (50–100 mg), decomposition productsflow through a catalyst to a gold amalgamator trap thatcollects and preconcentrates the mercury. The amalgama-tor is subsequently heated to 700 �C, and Hg is releasedand detected by atomic absorption spectrometry. Adetailed description of the method is given elsewhere (Halland Pelchat, 1997).

3.4. Topography

During the microbiological survey, an ALTUS altime-ter was deployed near the sampling sites on the intertidal

mudflats of the Seine estuary (Oissel and Northern mud-flats) and of the Authie Bay. With this instrument it ispossible to determine, at high resolution (±2 mm) andhigh frequency (one burst every 10 min), the periods oferosion or deposition, i.e., changes in mudflat elevation(Bassoullet et al., 2000; Deloffre et al., 2005; Andersenet al., 2006). The objectives of this study were: (i) to inves-tigate the age of the sediment according to depth and (ii)to distinguish the freshly deposited sediment (Deloffreet al., 2007). On the subtidal site, X-ray images of thecores were used to distinguish between the fresh/low con-solidated sediment and the old/consolidated sediment fol-lowing the Lesourd et al. (2003) study.

4. Results

4.1. Molecular quantification of the Gram-negative merA

gene in mudflat sediments by competitive PCR (cPCR)

Molecular quantification of the merA gene in total bac-terial DNA extracted from sediments collected in the estu-arine mudflats was performed by cPCR as described inthe experimental procedures. The primer set merA1 andmerA5 (Liebert et al., 1997) amplifies the target DNAsequence corresponding to the 1238 bp of merA genes,and the competitor sequence of 985 bp; the size differencebetween competitor and target sequences makes it easy todistinguish between the two amplicons on the electropho-resis gel. In these PCR conditions, the amplification effi-ciencies of competitor and target sequences were similarand no formation of heteroduplexes occurred (Fig. 2aand c). To allow comparative analysis of the merA genecontent of the sediment samples, the results are expressedas the copy number of Gram-negative merA genes ng�1 oftotal DNA extracted from these samples. Thus calculated,these values correspond to the relative abundance ofGram-negative merA microorganisms in the total micro-bial community and are independent of the DNA extrac-tion efficiency.

PCR cycles

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Fig. 2. Molecular quantification of Gram-negative merA gene by cPCR. Molecular quantification of the copy number of Gram-negative merA genes inmicrobial DNA extracted from environmental samples; (a) 150 fg of competitor sequence carried by pmerA-c was added to a serial dilution (2.1–281 fg) ofthe target sequence carried by pHp45XHg. Similarly, 150 fg of competitor DNA was added to 4–100 ng of DNA extracted from mudflat sediment samples(merA-s). The PCR products are analysed on agarose gel. DNA masses of pHp45XHg: lane 3, 281 fg; lane 4, 210 fg; lane 5, 158 fg; lane 6, 66 fg; lane 7,28 fg; lane 8, 5 fg; lane 9, 2.1 fg; DNA from sediment samples, lanes 10 and 11. Lane 1, negative control; lane 2, size ladder (Smart Ladder, Eurogentec).(b) Amplification kinetics of pHp45XHg and pmerA-c sequences. Fifty picograms of pHp45XHg (�) and pmerA-c (e) were amplified with merA1 andmerA5 primers. The relative amounts of PCR products were determined from integrated density values (pixels) for the ethidium bromide-stained DNA.Three replicates were performed for each experiment. (c) The relative intensities of the PCR bands were used to construct the calibration curve log(pHp45XHg/pmerA-c). The ratio merA-s/pmerA-c was plotted on the curve and the copy number of Gram-negative merA gene amplified from mudflatsediments was calculated assuming a mass of 7.6 � 10�18 g for pHp45XHg, which carries a single copy of the target sequence.

1172 J.-B. Ramond et al. / Marine Pollution Bulletin 56 (2008) 1168–1176

4.2. Hydrosedimentary processes, total mercuryconcentration and occurrence of the Gram-negative merA

gene in estuarine mudflats

The vertical distribution of the abundance of the merA

gene, total mercury concentration, and the sedimentaryprocesses were investigated in 30-cm-long cores. Asobserved by Cossa et al. (2003), the average Hg concentra-tions in the mudflat sediments of the Seine estuary werehigher in freshwater sediments (from 908 to 1208 ng g�1)and decreased downstream from the mixing zone (from582 to 658 ng g�1) to the subtidal mudflats (0.3 ng g�1)(Table 1). In contrast, the concentration of total mercuryin the Authie mudflats corresponded to the geochemicalbackground (0.05 ± 0.01 ng g�1). The levels of total mer-

cury in the sedimentary columns in the Northern mudflatand subtidal mudflats were homogenous (Fig. 3b and c),whereas in the Oissel mudflat the Hg levels were highestat the deeper levels (between �15 and �30 cm, Fig. 3a).The abundance of the merA gene was monitored by cPCRand corresponds to the proportion of Gram-negative merA

bacteria in the total microbial community (Oger et al.,2001; Leloup et al., 2004). Figs. 3 and 4 represent the dis-tribution of the abundance of Gram-negative merA bacte-ria, the sediment deposit evolution on the mudflat surface,and total Hg concentrations. In the Seine and Authie mud-flats, the Gram-negative merA gene is detected in all thecores studied. Except for the September 2001 core fromthe Oissel mudflat (Fig. 3a) and the September 2002 corefrom the subtidal mudflat (Fig. 3c), the abundance of the

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merA gene copy number.ng-1 total DNA

September 2001

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merA gene copy number.ng-1 total DNA

Total mercury concentration (ng.g-1) Total mercury concentration (ng.g-1)

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Fig. 3. Vertical distribution of the Gram-negative merA gene relative abundance (copy number ng�1 total DNA), total Hg concentrations (ng g�1) andhydrosedimentary processes in the Seine estuary mudflats. (a) Oissel intertidal mudflat, (b) Northern intertidal mudflat and (c) subtidal mudflat. The merA

gene was quantified (e) along 30-cm-long cores. (�) merA gene amplified but not quantified (detection limit: 278 merA copies). (�) No merA gene wasdetected. Values are given as mean ± SD of triplicates. (j) Total Hg concentrations. Fresh sediment deposits are indicated by grey areas. Sampling datesare indicated in bold characters.

J.-B. Ramond et al. / Marine Pollution Bulletin 56 (2008) 1168–1176 1173

merA gene is significantly higher in the Seine estuarymudflats than in any of the cores from the non-anthrop-ized reference site (Fig. 4), although there is a low andconstant presence of the Gram-negative merA gene inthese reference cores, even in the fresh sediment deposits(mean value of 6.7 ± 5.1 gene copies ng�1 of totalextracted DNA along the core depth). These results indi-cate a relationship between the degree of anthropization,and thus the mercury contamination, and the relative

abundance of the Gram-negative merA gene in estuarymudflat sediments.

In the Seine estuary, the highest abundance of theGram-negative merA gene is observed in the Oissel mudfl-ats (Fig. 3a), where the mean mercury concentrationsare the highest detected (Table 1). However, the maximumvalues for merA gene abundance are not observed at thedeeper levels where the maximum total mercury concentra-tions are measured, but in the fresh sediment deposits of

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November 2003May 2003

merA gene copy number.ng-1 total DNA

Total mercury concentration (ng.g-1)

Dep

th (

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merA gene copy number.ng-1 total DNA

Total mercury concentration (ng.g-1)

Fig. 4. Vertical distribution of the Gram-negative merA gene relative abundance (copy number ng�1 total DNA), total Hg concentrations (ng g�1) andhydrosedimentary processes in the Authie mudflats. The merA gene was quantified (e) along 30-cm-long cores collected in May and November 2003.Filled symbols (�) merA gene amplified but not quantified (detection limit: 278 merA copies). (�) No merA gene was detected. Values are given as mean of±SD of triplicates. (j) Total Hg concentrations. Fresh sediment deposits are indicated by grey areas. Sampling dates are indicated in bold characters.

1174 J.-B. Ramond et al. / Marine Pollution Bulletin 56 (2008) 1168–1176

the July 2001 core (430.1 ± 59.2 gene copies ng�1 of totalDNA). Moreover, in the September 2001 core, sampledafter an erosion period, the abundance of Gram-negativemerA gene is low and constant along the depth (mean valueof 13.8 ± 7.9 gene copies ng�1 of total DNA extracted). Inthe Northern mudflat (Fig. 3b), the maxima of Gram-negative merA abundance vary from 203.9 ± 67.9gene copies ng�1 total DNA extracted in April 2001, to216.1 ± 41.2 in May 2001 and are always located in freshsediment deposits. Likewise, in the subtidal mudflats(Fig. 3c), the maximum abundance of 244.9 ± 34.2 merA

gene copies ng�1 of total DNA is observed in the freshsediment deposit (0–12 cm) from the March 2002 core. Incontrast, the September 2002 core, sampled after an ero-sion period, displays a low abundance profile of the merA

gene with an average of 7.8 ± 5.9 gene copies ng�1 of totalDNA along the core. Thus, in the Seine mudflat sediments,the cPCR quantification of the merA gene demonstrates: (i)that merA genes are mostly found in newly deposited sed-iments and thus have probably been delivered as suspendedmatter, and (ii) that no relationship is observed betweentotal Hg and merA abundance.

5. Discussion

In the mercury-contaminated mudflats of the Seine estu-ary, HgR (merA) bacteria could play a significant role in thebiogeochemical cycle of mercury by contributing to theemission of elemental mercury to the atmosphere and thusleading to the detoxification of the ecosystem (Barkayet al., 2003). In order to quantify the merA gene in contam-inated sediments from mudflats, we have developed acPCR approach. Although other quantitative molecularmethods (MPN-PCR and Real-time PCR) are availableto quantify genes in environmental samples (Sharmaet al., 2007), the main advantage of cPCR is that it over-comes the effects of contaminants (heavy metals, humic

acids), which inhibit equally the amplification of targetand competitor sequences. As previously reported, cPCRseems to be well-adapted to the study of natural samples,particularly those from estuarine environments (Mollerand Jansson, 1997; Berthe et al., 1999; Oger et al., 2001;Kondo et al., 2004; Leloup et al., 2004).

The higher abundance of the Gram-negative merA genefrom Seine estuary mudflat sediments when compared tothe reference site (Authie) indicates a relationship betweenthis abundance and the degree of anthropization and mer-cury contamination of the estuary watershed (Billon et al.,2001; Meybeck et al., 2007). Moreover, in the sediments ofthe Seine estuary mudflats, a close relationship betweenGram-negative merA gene abundance and the hydrosedi-mentary processes is shown, with a high abundance ofmerA bacteria in fresh sediment deposits. Thus, Gram-neg-ative merA bacteria are deposited on the Seine estuarymudflats through hydrosedimentary processes. Thesebacteria have probably been selected elsewhere in anotherreservoir of the mercury-contaminated Seine watershedand associated with the particulate matter deposited onthe mudflat surface (Meybeck et al., 2007). But the decreasein merA abundance along the core depth suggests that theseallochtonous bacteria, including their DNA, do not remainon the mercury-contaminated mudflats, as alreadyobserved for other bacteria in estuarine environments(Dupray et al., 1997; Berthe et al., 2008). These resultsare consistent with previous studies which demonstratedthat it is the sedimentary processes that control the con-tamination of estuary mudflats by faecal indicator bacteria,and that these processes also control, to a certain extent,the dynamics of sulphate-reducing microorganisms (dsrAB

genes) (Leloup et al., 2005; Berthe et al., 2008). Therefore,in dynamic environments such as estuarine mudflats, themerA gene abundance is greatly influenced by hydrosedi-mentary processes and should not be directly comparedonly with mercury concentrations. Nevertheless, in the

J.-B. Ramond et al. / Marine Pollution Bulletin 56 (2008) 1168–1176 1175

intertidal mudflat of the Seine estuary, intensive release oftrace metals occurs through early diagenesis during thedecomposition of organic substances in the surface sedi-ment pore water (Mikac et al., 1999; Hines et al., 2000;Ouddane et al., 2004). Moreover, it has been shown thatin deep sediment pore water, trace metal concentrationdecreases due to either coprecipitation of the trace metals,or pyrite (FeS2) adsorption, or formation of organic andinorganic complexes (Ouddane et al., 2004). Thus, it can-not be excluded that the high merA gene abundanceobserved in freshly deposited layers could be related partlyto a response of the bacterial community to an increase ofbioavailable mercury in the sediment pore waters. In con-trast, the low merA gene abundance in the deep sedimentscould result from the limited bioavailability of inorganicmercury, as shown by Cardona-Marek et al. (2007) forthe merA gene expression levels in mercury-contaminatedestuary waters.

In conclusion, we have developed a molecular tool(cPCR) enabling the quantification of Gram-negative merA

gene abundance in contaminated environments. This studyunderlines the complexity of the relationship between theabundance of HgR bacteria and the contamination of anenvironment as dynamic as the macrotidal Seine estuary,which is not only the final receptacle of waters originatingfrom a highly urbanised and industrialised drainage basin,but is also submitted to complex hydrosedimentary pro-cesses (Lesourd et al., 2003; Deloffre et al., 2005, 2006;Meybeck et al., 2007). Thus, even though mercury bioavail-ability does directly interfere with the occurrence of a merAbacterial community, this multidisciplinary work pointsout that hydrosedimentary processes also play a criticalrole in the dynamics of merA Gram-negative bacteria inhighly anthropized estuarine environments.

Acknowledgments

This work was supported by the Seine-Aval and PNE-TOX scientific research programs. The first author holdsa research Grant from the Haute-Normandie RegionalCouncil. We thank Dilys Moscato for help with transla-tion. Michel Meybeck’s helpful comments on the manu-script are gratefully acknowledged.

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