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Journal of Bacteriology, April 2001, p. 2516-2526, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2516-2526.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nitric Oxide Signaling and Transcriptional Control
of Denitrification Genes in Pseudomonas stutzeri
Kai-Uwe
Vollack and
Walter G.
Zumft*
Lehrstuhl für Mikrobiologie der
Universität Karlsruhe, D-76128 Karlsruhe, Germany
Received 3 November 2000/Accepted 30 January 2001
The expression of denitrification by a facultatively anaerobic
bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide
(NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus of
Pseudomonas stutzeri. Transcriptional kinetics of
structural genes were monitored by Northern blot analysis in a 60-min
time frame after cells were exposed to an N oxide signal. To
differentiate the inducer role of NO from that of nitrite, mRNA
kinetics were monitored under anoxic conditions in a nirF
strain, where NO generation from nitrite is prevented because of a
defect in heme D1 biosynthesis. NO-triggered responses were
monitored from the nirSTB operon (encoding cytochrome cd1 nitrite reductase), the norCB
operon (encoding NO reductase), nosZ (encoding nitrous
oxide reductase), and nosR (encoding a putative regulator).
Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about
250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell
the transient appearance of nosZ and nosR
transcripts. Constant levels of transcripts of both genes were observed
in an anoxic cell sparged with N2O. NO at 250 nM stimulated
in this cell type the expression of nos genes severalfold.
The transcription factor DnrD, a member of the FNR-CRP family, was
found to be part of the NO-triggered signal transduction pathway.
However, overexpression of dnrD in an engineered strain did
not result in NirS synthesis, indicating a need for activation of DnrD.
NO modified the transcriptional pattern of the dnrD operon
by inducing the transcription of dnrN and dnrO,
located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB
operon towards nitrite. Our data establish NO and DnrD as key elements
in the regulatory network of denitrification in P. stutzeri. The NO response adds to the previously identified
nitrate-nitrite response mediated by the NarXL two-component system for
the expression of respiratory nitrate reductase encoded by the
narGHJI operon.
*
Corresponding author. Mailing address: Lehrstuhl
für Mikrobiologie, Universität Karlsruhe, PF 6980, D-76128
Karlsruhe, Germany. Phone: 49-721-608 3473. Fax: 49-721-608 8932. E-mail: dj03{at}rz.uni-karlsruhe.de.
Journal of Bacteriology, April 2001, p. 2516-2526, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2516-2526.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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