Population structure and abundance of arsenite-oxidizing bacteria along an arsenic pollution gradient in waters of the upper isle River Basin, France

Abstract

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta- and Gammaproteobacteria, depending on arsenic (As) and E(h) levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters.

FIG. 1.

Copy numbers of aoxB genes (gray bars) and 16S rRNA genes (white bars) in waters of the Upper Isle River Basin. Values are means (n = 3) ± standard errors (error bars) and are represented on a logarithmic scale. The aoxB/16S rRNA gene ratios (black curve) represent relative abundances of aoxB genes in the waters. White squares with a black cross, non-As-polluted (<10 μg·liter⁻¹ As) or slightly As-polluted (15 μg·liter⁻¹ As) waters; white squares, moderately (50 to 100 μg·liter⁻¹ As) As(V)-polluted waters; gray squares, moderately (50 to 100 μg·liter⁻¹ As) As(III)-polluted waters; black squares, highly (>1,000 μg·liter⁻¹ As) As(III)-polluted water.

FIG. 2.

UPGMA clustering analysis and DGGE profiles of aoxB gene fragments retrieved from waters of the Upper Isle River Basin. Arrows indicate excised and sequenced (primer aoxBM1-2F-ND) DGGE bands (for phylogenetic affiliation, refer to Fig. 3). As indicated in the legend to Fig. 1, the squares represent the levels of As concentration and speciation in water samples.

FIG. 3.

Neighbor-joining phylogenetic tree of AoxB sequences (134 unambiguous amino acids) deduced from DGGE bands of the waters of the Upper Isle River Basin. The Thermus AoxB sequences (ABB17183 and BAD71923) were used as an outgroup. Circles at the branch nodes represent bootstrap confidence level percentages obtained from 1,000 replicates: large black circles, 95 to 100%; small black circles, 75 to 95%; small white circles, 50 to 75%. The scale bar corresponds to 10 mutations per 100 residues. As indicated in the legend to Fig. 1, the squares represent the levels of As concentration and speciation in water samples. Sequence manipulations and alignment were performed with the BioEdit software program (13). All phylogenetic programs are implemented in the Treecon software program (37).