Determination of physiological, taxonomic, and molecular characteristics of a cultivable arsenic-resistant bacterial community.

A collection of 219 bacterial arsenic-resistant isolates was constituted from neutral arsenic mine drainage sediments. Isolates were grown aerobically or anaerobically during 21 days on solid DR2A medium using agar or gelan gum as gelling agent, with 7 mM As(III) or 20 mM As(V) as selective pressure. Interestingly, the sum of the different incubation conditions used (arsenic form, gelling agent, oxygen pressure) results in an overall increase of the isolate diversity. Isolated strains mainly belonged to Proteobacteria (63%), Actinobacteria (25%), and Bacteroidetes (10%). The most representative genera were Pseudomonas (20%), Acinetobacter (8%), and Serratia (15%) among the Proteobacteria; Rhodococcus (13%) and Microbacterium (5%) among Actinobacteria; and Flavobacterium (13%) among the Bacteroidetes. Isolates were screened for the presence of arsenic-related genes (arsB, ACR3(1), ACR3(2), aioA, arsM, and arrA). In this way, 106 ACR3(1)-, 74 arsB-, 22 aioA-, 14 ACR3(2)-, and one arsM-positive PCR products were obtained and sequenced. Analysis of isolate sensitivity toward metalloids (arsenite, arsenate, and antimonite) revealed correlations between taxonomy, sensitivity, and genotype. Antimonite sensitivity correlated with the presence of ACR3(1) mainly present in Bacteroidetes and Actinobacteria, and arsenite or antimonite resistance correlated with arsB gene presence. The presence of either aioA gene or several different arsenite carrier genes did not ensure a high level of arsenic resistance in the tested conditions.

Assessment of heavy metal bioavailability using Escherichia coli zntAp::lux and copAp::lux-based biosensors.

To determine the amount of metals detectable by bacteria, two plasmids were constructed in which the metal-inducible zntA and copA promoters from Escherichia coli were fused to a promoterless Vibrio fischeri luxCDABE operon. The luminescence response of E. coli bearing these constructs was studied as a function of the concentration of several heavy metals and was shown to be influenced by cell growth phase. The zntAp::lux fusion is induced mainly by salts of cadmium, lead, mercury and zinc, with significant induction by other metal ions, whereas the specificity of copA induction is restricted to copper and silver. In optimized assay conditions, metals could be detected at threshold concentrations ranging from nanomolar to micromolar, with maximal induction observed after only 60-100 min incubation. The ability of these biosensor strains to distinguish bioavailable quantities of metals in a sample makes them good candidates as useful tools to monitor metal contamination in environmental samples.