Sulfur Amino Acid Status Controls Selenium Methylation in Pseudomonas tolaasii: Identification of a Novel Metabolite from Promiscuous Enzyme Reactions

Abstract

Selenium (Se) deficiency affects many millions of people worldwide, and the volatilization of methylated Se species to the atmosphere may prevent Se from entering the food chain. Despite the extent of Se deficiency, little is known about fluxes in volatile Se species and their temporal and spatial variation in the environment, giving rise to uncertainty in atmospheric transport models. To systematically determine fluxes, one can rely on laboratory microcosm experiments to quantify Se volatilization in different conditions. Here, it is demonstrated that the sulfur (S) status of bacteria crucially determines the amount of Se volatilized. Solid-phase microextraction gas chromatography mass spectrometry showed that Pseudomonas tolaasii efficiently and rapidly (92% in 18 h) volatilized Se to dimethyl diselenide and dimethyl selenyl sulfide through promiscuous enzymatic reactions with the S metabolism. However, when the cells were supplemented with cystine (but not methionine), a major proportion of the Se (∼48%) was channeled to thus-far-unknown, nonvolatile Se compounds at the expense of the previously formed dimethyl diselenide and dimethyl selenyl sulfide (accounting for <4% of total Se). Ion chromatography and solid-phase extraction were used to isolate unknowns, and electrospray ionization ion trap mass spectrometry, electrospray ionization quadrupole time-of-flight mass spectrometry, and microprobe nuclear magnetic resonance spectrometry were used to identify the major unknown as a novel Se metabolite, 2-hydroxy-3-(methylselanyl)propanoic acid. Environmental S concentrations often exceed Se concentrations by orders of magnitude. This suggests that in fact S status may be a major control of selenium fluxes to the atmosphere. IMPORTANCE Volatilization from soil to the atmosphere is a major driver for Se deficiency. "Bottom-up" models for atmospheric Se transport are based on laboratory experiments quantifying volatile Se compounds. The high Se and low S concentrations in such studies poorly represent the environment. Here, we show that S amino acid status has in fact a decisive effect on the production of volatile Se species in Pseudomonas tolaasii. When the strain was supplemented with S amino acids, a major proportion of the Se was channeled to thus-far-unknown, nonvolatile Se compounds at the expense of volatile compounds. This hierarchical control of the microbial S amino acid status on Se cycling has been thus far neglected. Understanding these interactions-if they occur in the environment-will help to improve atmospheric Se models and thus predict drivers of Se deficiency.

Keywords: atmospheric selenium; selenium cycling; selenium deficiency; selenium fate; trace element fate.

FIG 1

Conversion of selenium in Met+Cy-supplemented (filled symbols) and control (empty symbols) Pseudomonas cultures in terms of selenium oxyanions (A) (triangles, selenite; circles, selenate), volatile methylated Se (B) (triangles, DMDSe; circles, DMSeS), and unknown metabolites (C) (triangles, metabolite 1; circles, metabolite 2). Note that no selenite was detected in Met+Cy-supplemented cultures during incubation and that in panel A, the filled and empty triangles are overlaid at 0.

FIG 2

Separation of selenium species by IC-ICP-QqQ (mass shift mode using signal ⁷⁸Se¹⁶O⁺) of (A) a mix of standards of methylselenic acid, selenate, and selenite; (B) supernatant of the Pseudomonas culture supplemented with methionine and cystine; (C) SPE eluates of the concentrated supernatant.

FIG 3

Conversion of selenite to volatile methylated species (A), metabolite 1 (B), and metabolite 2 (C) in Pseudomonas cultures at stationary phase (∼18 h) supplemented with methionine alone (Met), cystine alone (Cy), and methionine and cystine (Met+Cy) and without supplementation (control).

FIG 4

Mass-spectrometric analysis of the collected fraction corresponding to unknown1. (A) ESI-ion trap-MS analysis via full mass scan and zoom on selected mass spectra (insets) demonstrating isotopic pattern of one Se atom. (B) ESI-ion trap-MS analysis via MS² fragmentation of m/z 183. (C) ESI-Q-TOF-MS high-resolution mass spectra in negative mode (bars) and calculated mass distribution of [(C₄H₈O₃Se) − H]⁻ (boxes).

FIG 5

Simplified overview of possible reactions in S metabolism, highlighting proposed promiscuous enzymatic conversion of selenite instead of sulfite (arrow; reaction 1) and tentative mechanism of formation of the novel Se metabolite identified (dashed line). 3A2HPA, 3‐(acetyloxy)‐2‐hydroxypropanoic acid; 2H3MSPA, 2‐hydroxy‐3‐(methylsulfanyl)propanoic acid; 2H3MSePA, 2-hydroxy-3-(methylselanyl)propanoic acid. For details on reactions 1 to 5, see the text.

FIG 6

Tentative mechanism of formation of 2‐hydroxy‐3‐(methylselanyl)propanoic acid (2H3MSePA) via 3‐(acetyloxy)‐2‐hydroxypropanoic acid (3A2HPA). Note that the promiscuous enzymatic reaction forming methylselenol is not shown.