Estimation of the abundance of the cadmium resistance gene cadA in microbial communities in polluted estuary water.
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.
Oger
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Laboratoire de Microbiologie du Froid, UPRES 2123, Faculté des Sciences, Mont Saint Aignan, France.
Berthe
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Quillet
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Barray
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Chiffoleau
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Petit
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D000251:Adenosine Triphosphatases / Q000235:genetics*; D001419:Bacteria / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D002104:Cadmium / Q000494:pharmacology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005618:Fresh Water / Q000382:microbiology*; D018628:Gene Dosage; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014877:Water Pollution, Chemical
D017931:DNA Primers; D004269:DNA, Bacterial; D002104:Cadmium; D000251:Adenosine Triphosphatases; C059771:cadmium translocating ATPase