Res. Microbiol. 152 (2001) 671–678  2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923-2508(01)01246-3/FLA

Estimation of the abundance of the cadmium resistance gene cadA in microbial communities in polluted estuary water Cécile Ogera , Thierry Bertheb , Laurent Quilleta, Sylvie Barraya , Jean-François Chiffoleauc , Fabienne Petita∗ a Laboratoire de Microbiologie du Froid, UPRES 2123, Faculté des Sciences, F76821 Mont Saint Aignan cedex, France b UMR CNRS Sysiphe 7619, Université Paris VI, F75005 Paris, France c IFREMER, Centre de Nantes, Dept DEL/PC, B.P. 21105, F44311 Nantes cedex 3, France

Received 13 December 2000; accepted 14 March 2001

Abstract – We describe herein a molecular method for estimating the abundance of the cadA gene, which encodes a Cd2+ /ATPase protein transporter, in bacterial DNA extracted from samples of environmental water. Competitive polymerase chain reaction (cPCR) may be the most appropriate technique for assessing the prevalence of the cadA gene in microbial communities in highly heterogeneous and polluted environments, such as the Seine estuary (France). We describe the development of this method: (i) the choice of two specific primers, based on the sequences encoding the cadmium binding site and the ion channel domains; (ii) the construction of a competitor sequence and assessment of its amplification efficiency; and (iii) the estimation of the copy number of the cadA gene. The cadA content in the bacterial community is expressed as the number of gene copies per ng of total DNA extracted, which is independent of the DNA extraction yield. This molecular procedure was improved to analyze cadA levels in bacterial DNA extracted from estuary water accidentally contaminated with cadmium. Results revealed a subsequent increase in the copy number of the cadA gene in the microbial community.  2001 Éditions scientifiques et médicales Elsevier SAS competitive PCR analysis / cadmium resistance / estuary water / cadA gene / DNA quantification

1. Introduction Contamination of the environment by heavy metals leads to strong selection pressure on microorganisms, resulting in major changes in the structure and diversity of the microbial community [2, 10]. The resulting population is predominantly resistant to the heavy metals, due to an increase in the pre-existing community of resistant bacteria, and the appearance of new resistant microorganisms through bacterial gene transfer [10, 19]. As less than 1% of microorganisms from environmental communities can be cultured [1, 36], molecular biology has proved invaluable in investigations of natural microbial communities. Nowadays, extensive use of molecular biology methods has led to the development of a new approach in microbial ecology, in which many unculturable and new bacteria have

∗ Correspondence and reprints.

E-mail address: [email protected] (F. Petit).

been studied via their DNA [23, 31, 36]. Nevertheless, the major goal for molecular biology in the field of ecology is to obtain results that are qualitatively and quantitatively representative of the original sample [9, 11, 17, 25]. A few quantitative molecular methods have been described, including in situ hybridization with fluorescent probes [1, 23], MPN-PCR (most probable number technique) [7, 22] and competitive polymerization chain reaction (cPCR) [15, 18, 35]. Among them, cPCR may be the most suitable for environmental sample analysis because gene amplification is similarly inhibited by chemicals and humic acid for both the target and competitor DNAs [4, 38]. Thus, although absolute quantification from environmental samples by PCR is now thought to be unattainable, it should be possible to estimate gene abundance if the major factors limiting cPCR are controlled [15, 20, 33, 37, 38]. In this work, we used molecular biology techniques to investigate the cadmium-resistant microbial community of the Seine estuary. Over the last ten years, the water and sediment of the Seine estuary (France)

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have become increasingly contaminated with heavy metals, especially cadmium. The concentration of dissolved cadmium, which is the biologically available chemical form, varies according to the salinity of the water [5]. We focused on the group of bacteria harboring the cadA gene which encodes a Cd2+ efflux pump involved in a cadmium resistance mechanism [30]. The cadA gene is located either on a plasmid or on the chromosome and has been isolated from four Gram-positive bacteria and sequenced [12, 14, 16, 21]. CadA is a Cd2+ /ATPase protein transporter, with the seven domains that characterize the P-class of ATPases [14]. The aim of the present work was to develop a molecular method to investigate whether strong contamination induced changes in the cadA level of bacterial communities in estuary water. We report here the use of competitive PCR as a molecular procedure to detect and to quantify cadA gene in bacterial DNA extracted from an estuary sample. This molecular method was improved so as to estimate the cadA level in a bacterial community from Seine estuary water accidentally contaminated with cadmium.

2. Materials and methods 2.1. Plasmids

pKPY11 corresponds to pT7-5 into which the entire cadAC operon from Staphylococcus aureus was inserted [14]. The competitor plasmid pKSA-1 carries a 1454-bp competitor sequence which is amplified with the same pair of primers as the target DNA. pKSA-1 was constructed as follows ( figure 1): the 302-bp BstEII-SpeI fragment, carried by pKPY11, was removed from the S. aureus cadA gene. The ends of the deleted plasmid were filled in with the Klenow fragment of Escherichia coli DNA polymerase I. The fragment was replaced by blunt-ended PvuIIPvuII fragments, inserted in tandem, taken from the cadA gene, but from outside the 1058-bp target sequence. The competitor sequence obtained was 50% [calculated as follows (368 bp × 2)/(1058 bp − 302 bp)] nonidentical to the target DNA but with a similar G + C content, and a low level of sequence homology in order to prevent heteroduplex formation [18, 35].

Figure 1. Competitor plasmid, pKSA-1. Construction of pKSA-1: A competitor sequence for cadA (1454 bp) was obtained by deleting a 302-bp Bst EII-SpeI fragment from the cadA gene of S. aureus carried on pKPY11, and replacing it with a tandem insertion of both Pvu II-Pvu II fragments of the cadA gene, located outside the 1058-bp target sequence.

2.2. Sampling of estuary water and nucleic acid extraction

Samples of water from the Seine estuary (France) were collected in sterile centrifuge bottles containing 0.1% SDS (final concentration), and 1 mM Na2 EDTA pH 8.0. Bacterial DNA was extracted as previously described [24], with the following modifications. Crude total DNA was purified by elution through Elutip-d columns (Schleicher & Schuell, Dassel, Germany). The eluted DNA was precipitated with two volumes of isopropanol and resuspended in 100 µL H2 O. The concentration and purity of the total DNA preparation was determined by spectrophotometry, by measuring absorbance at 260 and 280 nm (Shimadzu UV-180). 2.3. PCR amplification and quantification of cadA gene

The cadA sequence was amplified by PCR as follows: 20 to 100 ng DNA from estuarine water was

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amplified in 50 µL of the following reaction mixture: 5 µl of 10 × PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl), 200 µM of each dNTP (Boehringer), 2 mM MgCl2 , 2.5 U Taq polymerase (Boehringer), and 0.25 µM of each primer. PCR amplification was carried out in a Perkin Elmer thermocycler (Gene amp 6700) as follows: 5 min at 94◦ C to denature the DNA; 30 cycles of 1-min denaturation at 94◦ C, 1-min 30 annealing, and 2-min extension at 72◦ C; and a final extension step of 5 min at 72◦ C. During the first ten cycles, the annealing temperature was progressively stepped down from 60◦ C to 50◦ C [13]. Quantification of cadA gene from DNA extracted from estuarine bacteria by competitive PCR (cPCR) was performed as previously described [38]. A set of standard samples containing 50 fg of competitor sequence (pCad-c), carried by pKSA-1, was mixed with a serial dilution (0.5 fg to 5 pg) of the target sequence (pCad), carried by pKPY11. In the same way, a known amount of competitor DNA, depending on the intensity of the cadA signal, was added to 100 ng of DNA extracted from estuary water (Cad-s) during PCR amplification [38]. The PCR products (10 µL) were analyzed by electrophoresis in an agarose gel (1% w/v, 0.5 × TAE). Gels were run at 50 V/cm for 45 min, stained with ethidium bromide and viewed under UV light. PCR products were quantified from digitized gel images (AlphaImagerTM 1220). 2.4. Restriction analysis and Southern hybridization of PCR products

PCR products were digested with SpeI. Southern blot analysis was performed as previously described [28]. The cadA probe, corresponding to a 1058-bp DNA sequence from the cadA gene of S. aureus, was labeled by incorporating digoxygenindUTP during cadA gene amplification from pKPY11, using cad1 and cad2 primers, according to standard protocols (Boehringer). 2.5. Determination of dissolved cadmium

The concentration of dissolved cadmium was determined by electrothermal atomic absorption with Zeeman correction after prior concentration by APDC-DDDC/Freon [6]. Quality assurance procedures were applied, as described elsewhere [5].

Figure 2. Selection of specific primers for amplifying cadA. (A) Alignment of Cd2+ binding domains of CadA from S. aureus (accession n◦ J04551 and n◦ P37386), Bacillus firmus (accession n◦ M90750), L. monocytogenes (accession n◦ L28104) and Lactococcus lactis (accession n◦ U78967) and similar protein motifs involving the Hg2+ binding site from E. coli (accession n◦ J01730), Shigella flexneri (accession n◦ P04129), Bacillus megaterium (accession n◦ Y09907), and S. aureus (accession n◦ P08663). The amino acids corresponding to the cad1 primer ( figure 1A) are shaded in gray, and identical amino acids are indicated by asterisks (∗). (B) Alignment of ion channel domain of CadA and four other P-type ATPases isolated from E. coli (accession n◦ PWECBK), Enterococcus faecalis (accession n◦ A29576), the yeast S. cerevisiae (accession n◦ NP015289), and Homo sapiens (accession n◦ U78967). The amino acids corresponding to the cad2 primer are shaded in gray, and identitical amino acids are indicated by asterisks (∗). (C) Choice of specific primers: alignment of the corresponding sequences of the bacterial cadA gene domains, the selected and the deduced sequences for cad1 (cadmium binding site) and cad2 (ion channel site) primers. Degenerate nucleotides are shaded in gray. R = A, T; Y = C, T; H = A, T, C.

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3. Results 3.1. Primer 3.1.1. Primer design

To amplify the cadA sequence, the cad1 primer was designed to correspond to the Cd2+ binding domain, which encodes the conserved paired cysteine residues that are specifically involved in cation binding [14, 30]. This highly conserved region of the CadA protein ( figure 2A) was unlikely to hybridize to the most similar known sequence currently identified, that of mercury reductase [14, 16, 29]. The second primer corresponded to the Cd2+ channel domain of the CadA protein and its specificity was checked by comparison with the closest similar motifs of H+ ATPase [14, 16] ( figure 2B). These two primers were chosen based on codon usage and the alignment of DNA sequences corresponding to the cadmium binding and ion channel sites of cadA genes. We checked that primer degeneracy was not so high as to cause PCR bias [34], by calculating degeneracy scores for each primer after alignment of the corresponding nucleotide sequences of the known cadA genes ( figure 2C). 3.1.2. Primer validation

PCR products of the expected size (1058 bp) were amplified with cad1 and cad2 primers from both DNA extracted from estuarine samples and pKPY11, which carries the S. aureus cadA gene, used as a positive control ( figure 3A). Two restriction fragments of the expected sizes (474 bp and 583 bp) were obtained when PCR products obtained from pKPY11 were digested with the SpeI restriction enzyme ( figure 3B). Restriction and Southern blot analysis showed that only a fraction of the PCR products from the estuarine sample were digested by SpeI ( figure 3C), and suggested that putative cadA genes other than the S. aureus cadA carried by pKPY11 were also amplified. The absence of the SpeI site in amplified cadA fragments indicated the heterogeneity of the PCR products. 3.2. Quantification of the number of copies of the cadA gene by competitive PCR from DNA extracted from estuary water

Competitive PCR (cPCR), in which a known amount of competitor DNA is added to the sample during PCR amplification, was used to quantify cadA

Figure 3. cadA gene amplification from DNA extracted from estuary water. (A) Agarose gel (1% w/v) showing the cadA sequence amplified with primers cad1 and cad2, 1058-bp in size. Lanes: 1, molecular weight ladder (Boehringer); 2, negative control (deionized water); 3, negative control (100 ng of E. coli DNA); 4, 100 ng of DNA extracted from estuarine sample; 5, positive control (50 fg of pKPY11). (B) SpeI digestion of PCR products, analyzed by gel electrophoresis. Lanes: 1, digestion products of DNA amplified from pKPY11; 2, from DNA extracted from estuary water; 3, 100bp size ladder (Sigma). (C) Corresponding Southern blot, probed with cadA labeled with digoxygenin UTP. Lanes: 1, pKPY11; 2, DNA from estuarine sample.

Figure 4. Efficiency of amplification of competitor and cadA sequences. Amplification rate for 50 ng of both pKPY11 ( ) and pKSA-1 ( ). Two or three replicates were performed for each experiment. The relative amounts of PCR product were determined by using the integrated density values (pixel) for the corresponding bands in an ethidium bromide-stained agarose gel (alpha-ImagerTM 1220).

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mixture (dNTPs, enzyme, primers and MgCl2 ) [20]. Therefore, to prevent cPCR bias in our experimental conditions, the number of amplification cycles did not exceed 30. Standard samples and 100 ng of DNA extracted from estuary water (Cad-s), supplemented with 50 fg of competitor (pCad-c) were analyzed by cPCR [see Section 2] ( figure 5A). To determine the unknown target concentration we plotted the Cad-s/pCad-c ratio on a calibration curve: log (pCad/pCad-c) = f (copy number of cadA), where the cadA copy number was calculated, assuming for pKPY11, which contains a single copy of cadA, a mass of 5.96 × 10−18 g ( figure 5B). The cadA copy number (Q) in the DNA extracted from environmental samples was expressed per ng of extracted nucleic acid analyzed by PCR or per mL of estuary water (Q × D) where D is the dilution factor. To determine the detection limit of cadA gene, a serial dilution of a known number of copies of cadA, carried by plasmid pKPY11, were analyzed by cPCR. This showed that cadA could be detected and hence quantified if the amount of PCR analyzed DNA (100 ng) contained at least 16 copies of cadA (data not shown).

Figure 5. Estimation of copy number of cadA gene by competitive PCR. (A) Estimation of cadA copy number in bacterial DNA extracted from an environmental sample. A variety of concentrations of pKPY11 (pCad) and 100 ng of DNA (Cad-s) extracted from estuary water were coamplified with 50 fg of competitor, pKSA-1 (pCad-c). PCR products were analyzed by electrophoresis and stained with ethidium bromide. Concentration of pKPY11: Lanes: 2, 5 pg; 3, 0.5 pg; 4, 50 fg; 5, 5 fg; 6, 0.5 fg. Lane 7, DNA from estuary water (environmental sample). Lane 1, molecular weight ladder (Sigma). (B) The corresponding calibration curve log (pCad/pCad-c) = f (copy pCad) was constructed after determination of the relative amounts of PCR products by using integrated density values (pixel) for the corresponding bands in an ethidium bromide-stained agarose gel (alpha-ImagerTM 1220). The ratio Cad-s/ pCad-c was plotted on the curve and the number of copies of cadA amplified in bacterial DNA extracted from environmental sample was deduced assuming a mass of 5.96 × 10−18 g for pKPY11, which carries a single copy of the target sequence.

from DNA extracted from estuary water [see Materials and methods]. As recommended by Lee et al. [15], we checked that the two sequences were amplified with similar efficiency before using the competitor ( figure 4). A plateau phase was reached after 30 cycles, showing that PCR amplification should not be limited by the components of the reaction

3.3. cadA level in DNA of bacterial community from polluted samples

Following accidental cadmium contamination of the estuary in June 1996 a high concentration of dissolved cadmium in the water was detected (table I). We sought to determine whether this contamination would have some effect on the cadA level in total DNA of microbial communities living on the edge of the industrial site. Nucleic acids extracted from water samples were analyzed by cPCR and we observed that the number of copies of the cadA gene per ng of DNA increased 25-fold in total DNA of the bacterial community from the contaminated site (table I). This increase was not observed at the same site one year later, nor at the closest upstream and downstream unpolluted sites. These results suggested that transient but severe cadmium contamination of the water induced an increase in the cadA level in total DNA. 4. Discussion The molecular biological investigation of microbial communities in estuary environments is a challenge due to physico-chemical gradients and their

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Table I. cadA level of bacterial DNA extracted from estuarine water contaminated with cadmium.

I Dissolved cadmium (nM) cadA level (copy number/ng of DNA)

0.82a* 838,9a*

II 0.13b 35.5b

0.20a nda

III 0.18b 35.5b

0.18a nda

0.18b ndb

Dissolved cadmium (nM) and cadA level (copy number/ng of DNA) in bacterial DNA extracted from estuarine samples: a contaminated site (I), and the two closest sites downstream (II) and upstream (III); (a) July 1996; (b) September 1997. Nd (not detectable). (*) Sampling for determination of dissolved cadmium was carried out 15 days before sampling for estimation of cadA content of bacterial DNA.

spatio-temporal variations in estuary water. In particular, there are wide variations in the concentration of suspended particulate matter (approximately 0.1– 10 g/L), ionic strength, pH, chemical pollutants, and humic compounds [5, 8]. We previously described a protocol for the extraction of DNA that can be used to analyze estuary water samples [24]. Its efficiency has been improved, and the contribution of nitrifying bacteria to the oxygen budget of the Seine estuary has been investigated by molecular quantification of the genus Nitrobacter [4]. We extended the study of the estuarine microbial community to cadmium-resistant bacteria, by quantifying cadA, a gene conferring resistance to Cd2+ . Several genetic systems involved in metal resistance have been characterized. The cadA gene was selected because it is highly specific for cadmium, although cadA may also confer resistance to zinc [30]. The primers cad1 and cad2 enabled PCR amplification of the cadA gene from estuary samples. As SpeI is a restriction enzyme known to specifically digest the cadA gene of S. aureus, but not the cadA genes of other organisms, the results of our restriction analysis suggest that the sequences of cadA genes isolated from the Seine estuary are rather heterogeneous. All cadA genes reported to date are in bacteria with DNA of low G + C content that form part of the Firmicutes group: S. aureus, Bacillus firmus, Listeria monocytogenes), and Lactococcus lactis [12, 14, 16, 21]. The sequences of the PCR products amplified by cad1 and cad2 primers from DNA extracted from culturable bacteria in estuary water or directly from the water correspond to the cadmiumtransporting ATPase (data not shown). Competitive PCR is particularly suitable for estimating the copy number of this gene because it allows comparison of different samples of water, taken in an estuary, having different chemical compositions [15]. As the cadmium resistance gene cadA was carried on a plasmid in S. aureus, L. lactis, and L. mono-

cytogenes [14, 16, 29], it is not possible to quantify accurately the number of bacteria bearing the cadA gene. It is therefore preferable to estimate the copy number of cadA per ng of DNA (Q), giving the level of cadA in total DNA extracted. Thus, this mode of expression is independent of the yield of DNA extraction, and should be more appropriate for comparing environmental samples, especially estuarine water. In similar studies, the yield of DNA extraction was assumed to be constant [15] or else its constancy had been checked before molecular quantification by cPCR. To accurately estimate cadA copy number per mL of sample, we improved this method by taking into account an extraction efficiency factor. This was estimated by adding a constant amount of bacterial marker before each nucleic acid extraction [24]. This enabled us to estimate the extraction efficiency by calculating the ratio of marker DNA finally extracted to the initial amount added. However, this method is time-consuming and not suitable for simultaneously analyzing large numbers of samples from the same study. Thus, although it was not possible to determine the absolute number of copies of cadA originally present in the samples analyzed, the proposed method enabled the comparison of the abundance of the cadA gene in estuary water samples, taken at different times, either from a high turbidity zone or from the river. Exposure of a bacterial community to high concentrations of heavy metals causes a rapid increase in the frequency of resistance transfer and a dramatic decrease in bacterial diversity [3, 26, 27]. The bacterial community may adapt to toxic changes in the environment in several ways: (i) selective expansion of the pre-existing resistant bacteria; (ii) induction of genes involved in resistance to pollutants; or (iii) acquisition of resistance genes by genetic transfer [2, 10, 19]. Moreover, some metabolic genes can be used as func-

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tional molecular markers for assessing changes in the diversity of the population [4, 32]. In this study we observed that exposure to a toxic concentration of dissolved cadmium is accompanied by an increase in the copy number of the cadA gene in the bacterial community. We suggest that this is due to the bacteria associated with suspended matter derived from sediments located around the source of the contamination. The quantitative method proposed here may be used to study the effects of cadmium contamination on the bacterial community by monitoring cadA levels in bacterial DNA extracted from estuary water.

[7]

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[10]

[11]

Acknowledgements We thank Stéphanie Craquelin who first actively took part in this study, Mr A. Ficht (SNS, Rouen, France) for sampling, Prof. M. Mergeay (Flemish Institute for Technological Research, Belgium) and Dr. T. Valleys for helpful discussions and suggestions for the manuscript, and Dr. P. Cossart (Institut Pasteur, Paris, France) for kindly providing pKPY11. The first author holds a research grant from the Region Haute-Normandie. This work was supported by the Seine-Aval Scientific Research Program, grants from the government, Region Haute Normandie, Agence de l’Eau, and industries in the Haute-Normandie region (France).

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the results allows to support either a unifactorial or bi-factorial solution (the authors choose to ..... is a good tool to use in our area. However, to establish preva-.

Estimation of the Inhomogeneity Field for the ...
Sled et al. [13] describe the N3 approach to inhomogeneity field correction. ... This report discusses the methods, datasets, and results obtained from running the.

Marine subsidies alter the diet and abundance of ...
We analyzed eight years of survey data on all ... For example, while the Gulf of California ..... University (Department of Biology, Graduate College, Boritski.

The Estimation of the Growth and Redistribution ...
Sep 1, 2008 - Ct,v = Ct,u +Cu,v and It,v = It,u +Iu,v . (2.4). However, this property is not satisfied by any of the decomposition procedures pre- sented in the last section. Therefore, to deal with this issue, one has to choose ..... 1998 and 2001 a

Noticing the Abundance -
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