Expression of nitrite and nitric oxide reductases in free-living and plant-associated Agrobacterium tumefaciens C58 cells

Abstract

A number of the bacteria that form associations with plants are denitrifiers. To learn more about how the association with plants affects expression of denitrification genes, the regulation of nitrite and nitric oxide reductases was investigated in Agrobacterium tumefaciens. Analysis of free-living cells revealed that expression of the genes encoding nitrite and nitric oxide reductases, nirK and nor, respectively, requires low-oxygen conditions, nitric oxide, and the transcriptional regulator NnrR. Expression of nor was monitored in plant-associated bacteria using nor-gfp fusion expression. In root association experiments, only a small percentage of the attached cells were fluorescent, even when they were incubated under a nitrogen atmosphere. Inactivation of nirK had no significant effect on the ability of A. tumefaciens to bind to plant roots regardless of the oxygen tension, but it did decrease the occurrence of root-associated fluorescent cells. When wild-type cells containing the gfp fusion were infiltrated into leaves, most cells eventually became fluorescent. The same result was obtained when a nirK mutant was used, suggesting that nitric oxide activated nor expression in the endophytic bacteria. Addition of a nitric oxide synthase inhibitor to block nitric oxide generation by the plant prevented gfp expression in infiltrated nitrite reductase mutants, demonstrating that plant-derived nitric oxide can activate nor expression in infiltrated cells.

FIG. 1.

Schematic genetic maps of the nirK (A) and norC (B) regions, including the various lacZ and gfp fusions used in this study. The large arrows indicate the orientations of the nirK and norC open reading frames. The bent arrows indicate the starts of translation of nirK and norC. The boxes upstream of the open reading frames indicate putative NnrR binding sites.

FIG. 2.

Microscopic analysis of free-living cells of A006 grown under either aerobic or nitrate-amended oxygen-limited conditions. (A) Phase-contrast micrograph of A006 cells grown under aerobic conditions. (B) Fluorescence micrograph of the field of cells shown in panel A. (C) Phase-contrast micrograph of A006 cells grown under nitrate-amended oxygen-limited conditions. (D) Fluorescence micrograph of the field of cells shown in panel C.

FIG. 3.

Micrographs of Arabidopsis root tips incubated with strain A006 after 10 h of incubation under an N₂ atmosphere. (A) Bright-field image of roots incubated with A006. (B) Fluorescence image of the same location on the root shown in panel A. (C) Bright-field image of a root hair magnified (×5) from a specific site in panel A. (D) Fluorescence image of the root hair shown in panel C.

FIG. 4.

Micrographs of plant leaves infiltrated with either C58, A006, or A009. Following infiltration, plants were incubated for 6 h under artificial light. (A) Bright-field image of a leaf infiltrated with C58. (B) Fluorescence image of the region of leaf shown in panel A. (C) Bright-field image of a leaf infiltrated with A006. (D) Fluorescence image of the region of leaf shown in panel C. (E) Bright-field image of a leaf infiltrated with A009. (F) Fluorescence image of the region of leaf shown in panel E. A majority of the infiltrated A006 and A009 cells were fluorescent. (G) Bright-field image of a leaf infiltrated with A009 and 1 mM l-NMMA. (H) Fluorescence image of the region of leaf shown in panel G.