Rhizobium etli Produces Nitrous Oxide by Coupling the Assimilatory and Denitrification Pathways

Abstract

More than two-thirds of the powerful greenhouse gas nitrous oxide (N₂O) emissions from soils can be attributed to microbial denitrification and nitrification processes. Bacterial denitrification reactions are catalyzed by the periplasmic (Nap) or membrane-bound (Nar) nitrate reductases, nitrite reductases (NirK/cd ₁Nir), nitric oxide reductases (cNor, qNor/ Cu_ANor), and nitrous oxide reductase (Nos) encoded by nap/nar, nir, nor and nos genes, respectively. Rhizobium etli CFN42, the microsymbiont of common bean, is unable to respire nitrate under anoxic conditions and to perform a complete denitrification pathway. This bacterium lacks the nap, nar and nos genes but contains genes encoding NirK and cNor. In this work, we demonstrated that R. etli is able to grow with nitrate as the sole nitrogen source under aerobic and microoxic conditions. Genetic and functional characterization of a gene located in the R. etli chromosome and annotated as narB demonstrated that growth under aerobic or microoxic conditions with nitrate as nitrogen source as well as nitrate reductase activity requires NarB. In addition to be involved in nitrate assimilation, NarB is also required for NO and N₂O production by NirK and cNor, respectively, in cells grown microoxically with nitrate as the only N source. Furthermore, β-glucuronidase activity from nirK::uidA and norC::uidA fusions, as well as NorC expression and Nir and Nor activities revealed that expression of nor genes under microoxic conditions also depends on nitrate reduction by NarB. Our results suggest that nitrite produced by NarB from assimilatory nitrate reduction is detoxified by NirK and cNor denitrifying enzymes that convert nitrite into NO which in turn is reduced to N₂O, respectively.

Keywords: assimilatory nitrate reductase; denitrification; gene expression; nitrous oxide; soil bacteria.

FIGURE 1

Nitrate-dependent growth of R. etli. Cells were cultured under oxic (squares), microoxic (circles) or anoxic (triangles) conditions, with either NH₄⁺ (white) or NO₃⁻ (black), as sole N source. Results are the mean values and error bars from at least two different cultures assayed in triplicate.

FIGURE 2

Nitrate-dependent aerobic growth and NR activity of a R. etli narB mutant. (A) Growth curves. (B) Extracellular nitrite concentration in the growth medium. (C) MV-NR activity expressed as nmol NO₂⁻ produced mg protein⁻¹ h⁻¹. R. etli WT (), WT + vector (X), narB (), narB + NarB⁺ (), and WT + NarB⁺ () strains were incubated aerobically in minimal medium with NO₃⁻ as sole N-source. Data are expressed as the mean value ± SD from two different cultures assayed in triplicate.

FIGURE 3

Nitrate dependent micro-oxic growth of R. etli narB, nirK, and norC mutants. (A) Growth curves. (B) Extracellular nitrite concentration in the growth medium. R. etli WT (), narB (), narB+ NarB⁺ (), norC (X), and nirK () strains were cultured microoxically in minimal medium with NO₃⁻ as sole N-source. Data are expressed as the mean value and error bars from two different cultures assayed in triplicate.

FIGURE 4

Nitric oxide production capacity (A) and N₂O accumulation (B) by R. etli narB, narB + NarB⁺, nirK, and norC mutants. In (A) cells incubated microoxically with NO₃⁻ were transferred to a reaction chamber with NO₂⁻. In (B) cells were cultured microoxically with NO₃⁻ or NO₂⁻ as the sole N-source. Data are expressed as the mean value and error bars from two different cultures assayed in triplicate.

FIGURE 5

Expression of NorC in R. etli narB mutant. Haem-stained proteins of membranes prepared from R. etli WT (lanes 1 and 2), norC (lane 3), narB (lane 4), narB + NarB⁺ (lane 5), and nirK (lane 6) mutants cultured microoxically with NH₄⁺ (lane 1) or NO₃^- (lanes 2, 3, 4, 5, and 6) as the sole N-source. Each lane contains 25 μg of membrane proteins. Haem-stained c-type cytochromes indentified previously (FixP and FixO) and in this work (NorC) are indicated. Apparent protein molecular masses (kDa) are also shown.