Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations

Abstract

The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.

Figure 1

Diagram representing an idealized cross-section of a Prochlorococcus cell highlighting mechanisms likely responsible for arsenic entry into the cell and detoxification. (1) Arsenate likely enters the cell through the inorganic high affinity phosphate transporters PstCABS. (2) Once arsenate is inside the cytoplasm it is reduced by the arsenate reductase ArsC to the (III) oxidation state, arsenite. (3) Arsenite can then bind to the trans-acting ArsR-repressive regulator, (4) arsenite can be recognized by the arsenite efflux transporter ACR3 which shuttles the toxin out of the cell, or (5) the arsenite can be methylated by the putative arsenite S-adenosylmethyltransferase, ArsM. Monomethylarsonic acid (MMA) can undergo a series of oxidation and methylation steps to further methylate the species to dimethylarsinic acid (DMA); further methylation into more compound organoarsenicals may also be possible, but cannot be confirmed without laboratory analysis.

Figure 2

Phylogeny of arsenite S-adenosylmethyltransferases (arsM). The phylogenetic tree was constructed using the maximum-likelihood program RAxML. The statistical significance of the branch pattern was estimated by conducting a 100 bootstrap replications of the original amino acid alignment; bootstraps ⩾50 shown. The related SAM-dependent mycolic acid cyclopropane synthetase (cmaS) gene was used as an outgroup. Bold sequences represent sequences of genes with confirmed ArsM function (Rosen, 2002; Qin et al., 2006; Yin et al., 2011).

Figure 3

Amino acid pairwise alignment of cyanobacterial sequences to the red algae Cyanidioschyzon merolae sp. 5508 ArsM. Predicted secondary structures generated by the fully automated protein homology/analogy recognition engine Phyre2 (Kelley and Sternberg, 2009) are depicted below the amino acids. Red arrows above amino acid alignment indicate conserved cysteine residues identified to be integral for catalysis of the arsenic methylation reaction in C. merolae (Ajees et al., 2012). Red boxes indicate conserved motifs of regions involved in SAM binding to the protein. Purple loops in predicted secondary structure symbolize alpha helices and teal arrows symbolize predicted beta sheets.

Figure 4

Boxplot of the statistical average annual phosphate concentration at the surface for the GOS metagenome sites analyzed in this study according to ocean basin location. Outliers are represented by stars.

Figure 5

Boxplots of the relative Prochlorococcus-linked arsenic detoxification gene abundance within the GOS metagenomes of the Atlantic, Pacific and Indian basins. The shaded region represents one copy of a gene per genome; the spread of this shading around 1 represents the error associated with the estimation of Prochlorococcus genomes within the basin using single copy core gene abundance. (a) Average across all basins, (b) Atlantic sites, (c) Indian sites and (d) Pacific sites. Outliers are represented by stars.