Affiliation:
1. Department of Microbiology, Wing Hall, Cornell University, Ithaca, New York 14853-8101
Abstract
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
25 articles.
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