Quantitative Proteomic Analysis of Cyanide and Mercury Detoxification by Pseudomonas pseudoalcaligenes CECT 5344

Abstract

The cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT 5344 uses cyanide and different metal-cyanide complexes as the sole nitrogen source. Under cyanotrophic conditions, this strain was able to grow with up to 100 μM mercury, which was accumulated intracellularly. A quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been applied to unravel the molecular basis of the detoxification of both cyanide and mercury by the strain CECT 5344, highlighting the relevance of the cyanide-insensitive alternative oxidase CioAB and the nitrilase NitC in the tolerance and assimilation of cyanide, independently of the presence or absence of mercury. Proteins overrepresented in the presence of cyanide and mercury included mercury transporters, mercuric reductase MerA, transcriptional regulator MerD, arsenate reductase and arsenical resistance proteins, thioredoxin reductase, glutathione S-transferase, proteins related to aliphatic sulfonates metabolism and sulfate transport, hemin import transporter, and phosphate starvation induced protein PhoH, among others. A transcriptional study revealed that from the six putative merR genes present in the genome of the strain CECT 5344 that could be involved in the regulation of mercury resistance/detoxification, only the merR2 gene was significantly induced by mercury under cyanotrophic conditions. A bioinformatic analysis allowed the identification of putative MerR2 binding sites in the promoter regions of the regulatory genes merR5, merR6, arsR, and phoR, and also upstream from the structural genes encoding glutathione S-transferase (fosA and yghU), dithiol oxidoreductase (dsbA), metal resistance chaperone (cpxP), and amino acid/peptide extruder involved in quorum sensing (virD), among others. IMPORTANCE Cyanide, mercury, and arsenic are considered very toxic chemicals that are present in nature as cocontaminants in the liquid residues generated by different industrial activities like mining. Considering the huge amounts of toxic cyanide- and mercury-containing wastes generated at a large scale and the high biotechnological potential of P. pseudoalcaligenes CECT 5344 in the detoxification of cyanide present in these industrial wastes, in this work, proteomic, transcriptional, and bioinformatic approaches were used to characterize the molecular response of this bacterium to cyanide and mercury, highlighting the mechanisms involved in the simultaneous detoxification of both compounds. The results generated could be applied for developing bioremediation strategies to detoxify wastes cocontaminated with cyanide, mercury, and arsenic, such as those generated at a large scale in the mining industry.

Keywords: Pseudomonas; arsenic; biodegradation; cyanide; heavy metals; mercury.

FIG 1

Physiological characterization of P. pseudoalcaligenes CECT 5344 cells grown with cyanide as the sole nitrogen source, in the presence or absence of mercury. Growth (top) and cyanide consumption (bottom) of P. pseudoalcaligenes CECT 5344 cells in media without mercury (blue circles) or with mercury (red symbols) at 75 μM (triangles), 100 μM (squares), and 150 μM (diamonds). Cells were grown in M9 minimal media with 50 mM sodium acetate and 2 mM sodium cyanide as the carbon and nitrogen source, respectively. CECT 5344 cells for proteomic and qRT-PCR analyses were harvested at 7.5 h of growth (time indicated by the arrow).

FIG 2

Effect of mercury on proteins involved in cyanide detoxification/assimilation in the P. pseudoalcaligenes CECT 5344 strain. Data are shown as a heatmap of the log₂ of normalized peptide intensity (log₂ NPI). CN, cyanide-containing media without added mercury; CN + Hg, cyanide-containing media with 75 μM HgCl₂. (1) Protein code according to the Uniprot database under the accession number UP000032841. (2) Gene annotation from GenBank (accession HG916826.1).

FIG 3

Heatmap of P. pseudoalcaligenes CECT 5344 proteins affected by mercury under cyanotrophic conditions. The differential expression of proteins is represented as the log₂ fold change (FC). The fold change has been calculated as the ratio normalized peptide intensity in CN + Hg/normalized peptide intensity in CN. (1) Protein code according to Uniprot database under the accession number UP000032841. (2) Gene annotation from GenBank (accession HG916826.1).

FIG 4

Transcriptional gene expression analysis by qRT-PCR of P. pseudoalcaligenes CECT 5344 mer genes. (A) Differential relative gene expression of merR genes. (B) Differential relative gene expression of other mercury-related genes. Gene expression was analyzed by qRT-PCR from cells grown with 2 mM sodium cyanide as the sole nitrogen source, without or with 75 μM HgCl₂. The differential gene expression is represented as the fold change (FC) of the ratio relative gene expression (CN + Hg/CN). Gene/protein identifiers (IDs) are as follows: merR1 (BN5_0701/W6QQW2), merR2 (BN5_2264/W6QVE0), merR3 (BN5_2322/W6QWL9), merR4 (BN5_3351/W6R6A1), merR5 (BN5_3802/W6RKL7) merR6 (BN5_4473/W6R2T83), merT3 (BN5_4474/W6R4B5), merA (BN5_4477/W6R9E5), merD (BN5_4478/W6R2U3), merE (BN5_4479/W6R4C0), merP1 (BN5_3800/W6R0W8), and merT1 (BN5_3801/W6R273).

FIG 5

Phylogenetic distribution of MerR proteins in bacteria and archaea. The tree was constructed using the Phylogeny.fr platform (65). Sequences were aligned with MUSCLE v3.8.31 with default settings. Ambiguous regions were removed with Gblocks v0.91b. The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program 3.1/3.0 aLRT. The graphical representation and edition of the phylogenetic tree were performed with TreeDyn v198.3. The P. pseudoalcaligenes CECT 5344 MerR sequences are highlighted in red. Protein names (protein/gene IDs) are as follows: MerR1 (W6QQW2/BN5_0701), MerR2 (W6QVE0/BN5_2264), MerR3 (W6QWL9/BN5_2322), MerR4 (W6R6A1/BN5_3351), MerR5 (W6RKL7/BN5_3802), and MerR6 (W6R2T83/BN5_4473). Protein sequences correspond to the following phyla and organisms: Alphaproteobacteria (Hyphb, Hyphomonadaceae bacterium; Brady, Bradyrhizobium sp.), Betaproteobacteria (Burkc, Burkholderia cenocepacia KC-01; Nitroe, Nitrosomonas eutropha C91; Ralse, Ralstonia eutropha JMP134; Cuprim, Cupriavidus metallidurans CH34), Deltaproteobacteria (Desul, Desulfarculus sp.; Lujiv, Lujinxingia vulgaris), Gammaproteobacteria (Pseud, Pseudomonas sp. K-62), Enterobacteria (Salmot, Salmonella enterica subsp. enterica serovar Typhimurium; Eschec, Escherichia coli JJ1897), Firmicutes (Bacic, Bacillus cereus), Actinobacteria (Strl, Streptomyces lividans), Cyanobacteria (Syne, Synechocystis sp. PCC 6803; Nost, Nostoc sp. C3-bin3), Deinococcus-Thermus (Thermt, Thermus thermophilus HB27), and Archaea (Nitros, Nitrososphaera sp.; Archae, Archaeoglobi archaeon).

FIG 6

Hypothetical regulation network of the MerR2 regulon of P. pseudoalcaligenes CECT 5344 (A) and transcriptional qRT-PCR analysis of the MerR2-regulated genes (B). In this regulatory model, two MerR transcriptional regulator genes, namely, merR5 and merR6 (red boxes), are putatively under the control of the master regulator MerR2 (blue oval), as well as the transcriptional regulator arsR gene (yellow box) involved in arsenic resistance, the regulatory phoR gene that codes for the transcriptional regulator involved in phosphate metabolism (green box), and several structural genes (violet boxes), as follows: cpxP, chaperone involved in resistance to metals; nrdB, β-subunit of the ribonucleoside-diphosphate reductase; dsbA, dithiol oxidoreductase (disulfide-forming); yghU, glutathione S-transferase; fosA, glutathione S-transferase; ubiD, 3-octaprenyl-4-hydroxybenzoate carboxy-lyase; and virD, amino acid/peptide export protein. The transcriptional analysis by qRT-PCR shown in B was performed with mRNA from CECT 5344 cells harvested after 10 h of growth, when these genes showed the highest expression.

FIG 7

Overview of P. pseudoalcaligenes CECT 5344 metabolism under cyanotrophic conditions in the presence of mercury.