Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic Aquificae

Abstract

Mercury (Hg) resistance (mer) by the reduction of mercuric to elemental Hg is broadly distributed among the Bacteria and Archaea and plays an important role in Hg detoxification and biogeochemical cycling. MerA is the protein subunit of the homodimeric mercuric reductase (MR) enzyme, the central function of the mer system. MerA sequences in the phylum Aquificae form the deepest-branching lineage in Bayesian phylogenetic reconstructions of all known MerA homologs. We therefore hypothesized that the merA homologs in two thermophilic Aquificae, Hydrogenobaculum sp. strain Y04AAS1 (AAS1) and Hydrogenivirga sp. strain 128-5-R1-1 (R1-1), specified Hg resistance. Results supported this hypothesis, because strains AAS1 and R1-1 (i) were resistant to >10 μM Hg(II), (ii) transformed Hg(II) to Hg(0) during cellular growth, and (iii) possessed Hg-dependent NAD(P)H oxidation activities in crude cell extracts that were optimal at temperatures corresponding with the strains' optimal growth temperatures, 55°C for AAS1 and 70°C for R1-1. While these characteristics all conformed with the mer system paradigm, expression of the Aquificae mer operons was not induced by exposure to Hg(II) as indicated by unity ratios of merA transcripts, normalized to gyrA transcripts for hydrogen-grown AAS1 cultures, and by similar MR specific activities in thiosulfate-grown cultures with and without Hg(II). The Hg(II)-independent expression of mer in the deepest-branching lineage of MerA from bacteria whose natural habitats are Hg-rich geothermal environments suggests that regulated expression of mer was a later innovation likely in environments where microorganisms were intermittently exposed to toxic concentrations of Hg.

Fig 1

(A) Alignment of putative Mer proteins, including MerA, MerT, and MerP from Hydrogenobaculum sp. Y04AAS1 (AAS1) and Hydrogenivirga sp. 128-5-R1-1 (R1-1), with mer sequences from Pseudomonas aeruginosa Tn501 as a reference. Highlighted conserved regions of functional importance (3) are as follows: for MerA, the redox active site, two downstream tyrosines, and the carboxy terminus-vicinal CC pair; for MerT, the conserved C residues in the first membrane-spanning domain and in the cytoplasmic loop between the second and third membrane-spanning domains; and for MerP, the metal binding motif GMTCxxC. The membrane-spanning domains (determined using TMpred [http://www.ch.embnet.org/software/TMPRED_form.html]) are boxed (numbered 1 to 3) in MerT. Gray circles indicate the carboxy termini of the proteins. Numbering indicates position in the Tn501 sequence. (B) mer operon gene order. Arrowed boxes indicate each ORF and the direction of transcription; accession numbers are included above each box. Names of putative gene products and corresponding numbers of amino acids (AA) are given above the boxes. The numbered line below each ORF represents the nucleotide position marking the start of the gene counted from the transcription start nucleotide upstream of the hypothetical proteins in each operon.

Fig 2

²⁰³Hg(II) remaining in media during growth of strains AAS1 (A and C) and R1-1 (B) using S₂O₃²⁻ (A and B) and H₂ (C) as electron donors. Cell density (×) is shown, as is loss of Hg(II) (in μM) from growing cultures (●), heat-killed controls (□), and uninoculated controls (△). (D) Initial rates of Hg(II) loss [μmol Hg(II)/h/10⁶ cells] calculated for strains AAS1 and R1-1 in cultures grown on S₂O₃²⁻ or H₂ and for heat-killed controls. Different letters indicate statistical significance (P < 0.05). All data represent the mean Hg(II) loss of triplicate growing cultures ± 1 standard deviation (SD) after subtracting loss rates of uninoculated controls.

Fig 3

Effect of temperature on MR activities. Specific activities of crude cell extracts were determined for Hydrogenobaculum sp. Y04AAS1 (■) and Hydrogenivirga sp. 128-5-R1-1 (●). Activities of R1-1 extracts are expressed as those measured, multiplied by 10. Previously reported data for T. thermophilus (♢) and Tn501 (△) (51) are included for comparison. Averages of three to five replicate assays ± 1 SD are presented. One unit of MR activity = 1 μmol NAD(P)H oxidized min⁻¹.

Fig 4

merA expression in Hydrogenobaculum sp. Y04AAS1 and Hydrogenivirga sp. 128-5-R1-1. (A) HgCl₂-dependent (1 μM) transcription fold induction in strain AAS1 (▲) grown on H₂ compared with induction of merA of HB27 (⧫) and Tn501 (◇). Fold induction was calculated as described in Materials and Methods, except that merA expression in Tn501 and HB27 was normalized to expression of 16S rRNA genes rather than to gyrA (data from reference 51). (B) Effect of growth in the presence of Hg on MR specific activities in crude cell extracts of strains AAS1 and R1-1. Cell extract activities were determined for cultures grown with (filled column) or without (clear column) 10 μM HgCl₂ in S₂O₃²⁻-amended media. The means ± 1 SD of triplicate determinations are shown. The same letters above columns indicate no significant difference by Student's t test (P > 0.05).