Agrobacterium tumefaciens C58 uses ActR and FnrN to control nirK and nor expression

Abstract

Agrobacterium tumefaciens can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator actR significantly reduced nirK expression and Nir activity but not nnrR expression. Purified ActR bound to the nirK promoter but not the nor or nnrR promoter. A putative ActR binding site was identified in the nirK promoter region using mutational analysis and an in vitro binding assay. A nirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter lacZ fusion. Truncation of the nirK promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of paz, encoding the copper protein pseudoazurin, was highly reduced in the actR or fnrN mutants and that ActR binds to the paz promoter. Inactivation of paz reduced Nir activity by 55%. These results help explain why Nir activity is very low in the actR mutant even though a nirK promoter with mutations in the ActR binding site showed significant expression.

FIG. 1.

The ability of ActR to bind to the promoters of nirK, nor, nnrR, and paz (PnirK, Pnor, PnnrR, and Ppaz, respectively) was tested using EMSAs as described in Materials and Methods. Concentrations of purified ActR are indicated. To phosphorylate ActR, 20 mM acetyl phosphate (acetyl P) together with MgCl₂ was added to purified protein.

FIG. 2.

Binding of ActR to mutated nirK promoters (Pnir sequences). Concentrations of purified ActR are indicated. In the sequences at left, the wild-type sequence is shown at the top with the region to be targeted underlined. In other sequences, mutated bases are underlined twice. Conditions were the same as for the experiments shown in Fig. 1.

FIG. 3.

Expression of nirK-lacZ (•) and nirM4-lacZ (▪). Each mark is the average value of β-galactosidase activities measured in duplicate from at least three independent cultures grown in nitrate-amended medium under microoxic conditions. The standard deviation of all data was lower than 10% of each value of β-galactosidase activity. nirK expression was negligible before an OD₆₀₀ of ∼0.4, so the initial reported measurement is at this OD.

FIG. 4.

Expression of truncated nirK-lacZ fusions. In the table at right, activity is the percentage of the full-length fusion, pAnirKZ (nirKZ), shown at top, and length is relative to the predicted start of translation. Cells for these experiments were grown under O₂-restricted conditions with or without nitrate. Activities were determined in duplicate assays from at least two independently grown cultures. Standard deviations were always less than 5% of the average activity with the exception of the pAnirK3Z (nirK3Z) fusion, where the standard deviation was about 10%. The stippled rectangle indicates the location of the ActR binding site, and the filled rectangle indicates the location of the NnrR binding site.