Agrobacterium tumefaciens C58 Uses ActR and FnrN To Control nirK and nor Expression

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 regulatoractR significantly reducednirK expression and Nir activity but notnnrR expression. Purified ActR bound to thenirK promoter but not thenor ornnrR promoter. A putative ActR binding site was identified in thenirK promoter region using mutational analysis and an in vitro binding assay. AnirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoterlacZ fusion. Truncation of thenirK 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 ofpaz , encoding the copper protein pseudoazurin, was highly reduced in theactR orfnrN mutants and that ActR binds to thepaz promoter. Inactivation ofpaz reduced Nir activity by 55%. These results help explain why Nir activity is very low in theactR mutant even though anirK promoter with mutations in the ActR binding site showed significant expression.