Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea

Abstract

The ammonia-oxidizing archaea have recently been recognized as a significant component of many microbial communities in the biosphere. Although the overall stoichiometry of archaeal chemoautotrophic growth via ammonia (NH(3)) oxidation to nitrite (NO(2)(-)) is superficially similar to the ammonia-oxidizing bacteria, genome sequence analyses point to a completely unique biochemistry. The only genomic signature linking the bacterial and archaeal biochemistries of NH(3) oxidation is a highly divergent homolog of the ammonia monooxygenase (AMO). Although the presumptive product of the putative AMO is hydroxylamine (NH(2)OH), the absence of genes encoding a recognizable ammonia-oxidizing bacteria-like hydroxylamine oxidoreductase complex necessitates either a novel enzyme for the oxidation of NH(2)OH or an initial oxidation product other than NH(2)OH. We now show through combined physiological and stable isotope tracer analyses that NH(2)OH is both produced and consumed during the oxidation of NH(3) to NO(2)(-) by Nitrosopumilus maritimus, that consumption is coupled to energy conversion, and that NH(2)OH is the most probable product of the archaeal AMO homolog. Thus, despite their deep phylogenetic divergence, initial oxidation of NH(3) by bacteria and archaea appears mechanistically similar. They however diverge biochemically at the point of oxidation of NH(2)OH, the archaea possibly catalyzing NH(2)OH oxidation using a novel enzyme complex.

Fig. 1.

Accumulation of NO₂⁻ by N. maritimus. Determinations in the presence of 200 µM NH₄⁺ (dashed lines, squares), 200 µM NH₂OH (solid lines, diamonds), 50 µM NH₂OH (solid lines, triangles), 1 mM NH₂OH (solid lines, circles), and no substrate (dashed lines, crosses). Inset shows accumulation of NO₂⁻ after 20 h of incubation. Data are means of triplicates, with variation less than 10%. The experiment was repeated at least three times and produced similar results. Error bars represent SEM.

Fig. 2.

Stoichiometry of NH₂OH oxidation in N. maritimus. O₂ uptake (solid squares), NH₂OH consumed (open circles), NO₂⁻ produced (gray triangles); Data are means of triplicates, with variation of less than 10%. Incubations were carried out for 8 min while the conditions were at optimum (i.e., the unavoidable degradation of NH₂OH did not interfere with the stoichiometry). The experiment was repeated at least three times and produced similar results. Error bars represent SEM.

Fig. 3.

ATP formation by N. maritimus. (A) ATP and (B) NO₂⁻ concentration in N. maritimus cells incubated in presence of either no substrate (white bars), 200 µM NH₄⁺ (gray bars), or 200 µM NH₂OH (black bars). Concentration of ATP and NO₂⁻ in heat-killed cells and cells treated with 100 µM CCCP for 20 min is also shown. The NO₂⁻ trace amounts detected in the controls are likely due to leaching of residual traces of NO₂⁻ in the nylon filters used to harvest N. maritimus cells. Error bars represent the SEM.

Fig. 4.

Kinetics of N. maritimus NH₂OH consumption and accumulation of ¹⁵N in a stable isotope tracer experiment. N. maritimus cells were incubated in the presence of 200 µM NH₄Cl and 200 µM NH₂OH. After 60 min (arrow) 20 µM ¹⁵NH₄Cl (99.5% purity) was added to the cell suspension. After 120 min NH₃-oxidation activity was blocked by addition of C₂H₂ (0.1%). (A) Consumption of NH₂OH (dashed lines, filled circles) and accumulation of ¹⁵N in NH₂OH pool (solid lines, filled diamonds). (B) Accumulation of NO₂⁻ (dashed lines, filled circles), ¹⁵N in NO₂⁻ pools (solid lines, filled diamonds).