Vollack
K-U, Zumft WG
Nitric
oxide signaling and transcriptional control of denitrification genes in
Pseudomonas
stutzeri.
J
Bacteriol 183: 2516-2526 (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 (N20),
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
cd1nitrite
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 N20, 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
N20. 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.