Mercury Reduction and Methyl Mercury Degradation by the Soil Bacterium Xanthobacter autotrophicus Py2

Abstract

Two previously uncharacterized potential broad-spectrum mercury (Hg) resistance operons (mer) are present on the chromosome of the soil Alphaproteobacteria Xanthobacter autotrophicus Py2. These operons, mer1 and mer2, contain two features which are commonly found in mer operons in the genomes of soil and marine Alphaproteobacteria, but are not present in previously characterized mer operons: a gene for the mercuric reductase (MerA) that encodes an alkylmercury lyase domain typical of those found on the MerB protein, and the presence of an additional gene, which we are calling merK, with homology to glutathione reductase. Here, we demonstrate that Py2 is resistant to 0.2 μM inorganic mercury [Hg(II)] and 0.05 μM methylmercury (MeHg). Py2 is capable of converting MeHg and Hg(II) to elemental mercury [Hg(0)], and reduction of Hg(II) is induced by incubation in sub toxic concentrations of Hg(II). Transcription of the merA genes increased with Hg(II) treatment, and in both operons merK resides on the same polycistronic mRNA as merA. We propose the use of Py2 as a model system for studying the contribution of mer to Hg mobility in soil and marine ecosystems.

FIG 1

Arrangement of potential mer operons in Xanthobacter autotrophicus Py2. R, merR; T, merT; P, merP; A, merA; K, potential glutathione reductase. Locus tags are indicated above the genes.

FIG 2

MICs of Hg(II) and MeHg in Py2. (A) Hg(II) MICs. The 0 and 0.1 μM Hg(II) growth curves are overlapping. (B) MeHg MICs. The 0, 0.02, and 0.05 μM MeHg growth curves overlap. Error bars represent the standard deviations of the means from three independent cultures.

FIG 3

Loss of Hg(II) in the presence of Py2. (A) Loss of Hg(II) by growing cultures of Py2. (B) Loss of Hg(II) by Py2 suspended in buffer. Pretreated cells were exposed to 0.02 μM Hg(II) in growth media for 1 h prior to the assay. Mock treated cells were incubated for 1 h in media with no Hg(II).

FIG 4

Capture of Hg(0) in Py2 cultures exposed to Hg(II) or MeHg. (A) Data from experiments conducted with Hg(II). (B) Data from experiments conducted with MeHg. Gray bars represent Hg present in the reaction vessel, and white bars represent Hg present in the trapping solution. Error bars represent the standard deviations of the means for three independent reactors and traps.

FIG 5

Gel electrophoresis of reverse transcription-PCR (RT-PCR) products from intragenic regions of the Py2 mer operon. Three independent cultures (1, 2, and 3) were split into two aliquots, one of which (Hg) was exposed to 0.1 μM Hg(II) and one of which was not exposed Hg(II) (No Hg). After preparing mRNA, half of each sample was treated with reverse transcriptase (RT), and half was not (No-RT). gDNA indicates the use of Py2 genomic DNA as a template.

FIG 6

Gel electrophoresis of RT-PCR products from intergenic regions the Py2 mer operon. (A) Intergenic region between merA₁ and merK₁. (B) Intergenic region between merA₂ and merK₂. The template cDNA and no-RT samples were identical to the Hg(II)-exposed samples in Fig. 6. gDNA indicates the use of Py2 genomic DNA as a template. The rightmost lane is the 2-log ladder from New England BioLabs.