A tale of two oxidation states: bacterial colonization of arsenic-rich environments

Abstract

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments-including ground and surface waters-from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the beta-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.

Figure 1. Circular Representation of H. arsenicoxydans Genome

Circles display (from the outside inward): (rings 1 and 2) predicted CDSs transcribed in a clockwise/counterclockwise direction, (ring 3) tRNA (green) and rRNA (violetblue), (ring 4) ISs (yellow) and prophagic regions (violetbrown), (ring 5) clusters of genes involved in arsenic (red; numbered as shown in Figure 4)/metal resistance (dark blue), (ring 6) GC deviation (GC_window − average GC of genome, using a 1kb window); (7) GC skew (using a 1kb window). The high GC-rich island described in Figure 2 is shown in red.

Figure 2. Detailed View of a GC-Rich Island

The chromosomal segment extends between positions 1.97 and 2.07 Mb on the H. arsenicoxydans chromosome. Frames display (from top to bottom): (1) %GC along this island; (2) annotated CDSs on the direct (D) and reverse (R) strand: arsIII gene cluster (six genes in red arrows), part of the clc element of plasmid (or phage) origin, initially described in Pseudomonas sp. strain B13 [22] (64 genes in light blue arrows), and phage-related function (DNA repair, integrase) associated with metabolic capabilities, such as formaldelyde oxidation (17 genes in light green arrows), small genes are represented by a line; (3) synteny maps, calculated on a set of selected genomes (RALME, Ralstonia metallidurans CH34; BURXE, Burkholderia xenovorans LB400; AZOSE, Azoarcus sp. EbN1; PSEF5, Pseudomonas fluorescens Pf-5; and XANAC, Xanthomonas campestris 85–10). A line contains the similarity results between H. arsenicoxydans and one given genome. A rectangle represents a putative ortholog between one CDS of the compared genome and the CDS of the H. arsenicoxydans genome opposite. When, for several CDSs colocalized on the H. arsenicoxydans genome, several colocalized orthologs have been identified in the compared genome, the rectangles will be of the same color. Otherwise, the rectangle is white. A group of rectangles of the same color therefore indicates the existence of a synteny between H. arsenicoxydans and the compared genome, using a gap parameters of five genes maximum [63]. Details on correspondences between genes in the synteny (Table S2) show that the light blue section of this island in H. arsenicoxydans is also found at the same chromosomal location in the compared genomes.

Figure 3. Organization of the aox Gene Cluster in H. arsenicoxydans and Various Arsenic-Metabolizing Microorganisms

The aoxAB operon is close to arsenic-resistance genes in H. arsenicoxydans, A. faecalis, X. autotrophicus, N. hamburgensis, and C. phaeobacteroides. In the first three bacteria, these genes are associated with an aoxRS two-component regulatory system. In H. arsenicoxydans, the CDS number of aoxABCD, aoxRS, and arsRCBCH are hear0479–0476, hear0483–0482, and hear0499–0503, respectively. Sequence information of other genes was obtained from GenBank database and their localization on the chromosome or the plasmid is given by nucleotide numbering. The following bacterial genomes were used: Alcaligenes faecalis, Agrobacterium tumefaciens, Rhodoferax ferrireducens, Burkholderia multivorans, Xanthobacter autotrophicus, Roseovarius sp217, Nitrobacter hamburgensis, Chlorobium phaerobacteroides, Chloroflexus aurentiacus, Thermus thermophilus HB8, Aeropyrum pernix, Sulfolobus tokodai, Environmental sample 1, and Environmental sample 2.

Figure 4. Organization of Arsenate-Resistance Operons (ars) in H. arsenicoxydans

The three operons identified by complementation of an E. coli ars-deficient strain code for an ArsR regulator, one or two ArsC arsenate reductases, an As[III] extrusion pump, and an ArsH putative flavoprotein. The fourth operon present in the genome lacks the As[III] pump-encoding gene.

Figure 5. Phylogenetic Tree of ArsCa Arsenate Reductases in Various Arsenic-Resistant Microorganisms

The proteins of loci 1 (HEAR3302), 2 (HEAR0500), and 4 (HEAR3207) in cluster with reductases present in acr3-type transporter operons. In H. arsenicoxydans, two of them are, however, associated with an ArsB-type transporter. Protein sequences involved in arsenate reduction were retrieved from the National Center for Biotechnology Information GenBank database (http://www.ncbi.nlm.nih.gov/entrez) and phylogenetic trees were reconstructed from multiple sequence alignments using the neighbor-joining algorithm implemented in ClustalX. The following sequences were used as references: Roseovarius nubinhibens, Xanthobacter autotrophicus, Rhodospirillum rubrum, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Nitrobacter winogradskyi, Nitrobacter hamburgensis, Xanthobacter autotrophicus, Acidovorax sp., Comamonas sp., Rubrivivax gelatinosus, Delftia acidovorans, Azotobacter vinelandii, Rubrivivax gelatinosus, Burkholderia multivorans, Ralstonia metallidurans, Shigella flexneri, Shigella flexneri, Polaromonas naphthalenivorans, Comamonas testosteroni, Burkholderia vietnamiensis, Burkholderia pseudomallei, Burkholderia mallei, Azoarcus sp., Methylobacillus flagellatus, Alcaligenes faecalis, Rhodoferax ferrireducens, Pseudomonas syringae, Pseudomonas putida, Pseudomonas aeruginosa, Shewanella oneidensis, Wolinella succinogenes, Corynebacterium efficiens, Corynebacterium efficiens, Alkalilimnicola ehrlichei, and Chlorobium phaeobacteroides.

Figure 6. Effect of Metal and Metalloid Concentration on Swimming Properties in H. arsenicoxydans

Motility assays were performed in the presence of an increasing concentration of As[III], Co[II], or Fe[III]. The level of motility of ULPAs wild-type strain and of its aoxAB knockout derivative was evaluated as the diameter of the swimming ring expressed in millimeters. The results are the mean value of three independent experiments.

Figure 7. Chemotaxis and EPS Synthesis in H. arsenicoxydans in Response to Arsenic

(A) Chemoattraction to As[III]. Left: low melting agar with chemotaxis buffer, right: low melting agar with chemotaxis buffer supplemented with 2 mM As[III]. Bright ring of cells around agarose plugs is indicative of chemotaxis. (B) Transmission electron microscopy (TEM) picture of H. arsenicoxydans grown in As-enriched medium. Circles represent the X-ray spot of analysis, while I and II are the energy dispersive X-Ray spectroscopy corresponding values. Cl and K peaks show organic constituents and Cu labels represent peaks due to supporting grid. Arsenic content is 16.5 % weight as As₂O₃ in I. and 0.0% weight in II; both including microgrid C-coating quantification.