Pseudomonas stutzeri is a
facultative anaerobic bacterium with the capability of denitrification.
In searching for regulators that control the expression of this trait
in
response to oxygen withdrawal, we have found an unprecedented
multiplicity
of four genes encoding transcription factors of the FNR family. The fnrA
gene
encodes a genuine FNR-type regulator, which is expressed constitutively
and controls the cytochrome cbb3-type
terminal
oxidase (the cco operon), cytochrome c peroxidase (the ccp
gene) and the oxygen-independent coproporphyrinogen III oxidase (the hemN
gene), in addition to its previously demonstrated role in arginine
catabolism
(the arc operon). The fnr homologues dnrD, dnrE
and dnrS encode regulators of a new subgroup within the FNR
family.
Their main distinctive feature is the lack of cysteine residues for
complexing
the [4Fe-4S] centre of redox-active FNR-type regulators. However, they
form a phylogenetic lineage separate from the FixK branch of FNR
proteins,
which also lack this cysteine signature. We have studied the expression
of the dnr genes under aerobic, oxygen-limited and denitrifying
conditions. DnrD is a key regulator of denitrification by
selective
activation of the genes for cytochrome cd1
nitrite reductase and NO reductase. The dnrD gene is part of
the
30 kb region carrying denitrification genes of P. stutzeri.
Transcription
of dnrD was activated in O2-limited
cells and
particularly strongly in denitrifying cells, but was not under the
control
of FnrA. In response to denitrifying growth conditions, dnrD
was
transcribed as part of an operon together with genes downstream and
upstream
of dnrD. dnrS was found about 9 kb upstream of dnrD,
next to the nrdD gene for anaerobic ribonucleotide reductase.
The
transcription of dnrS required FnrA in O2-limited
cells. Mutation of dnrS affected nrdD and the
expression
of ferredoxin I as an element of the oxidative stress response. The dnrE
gene is part of the nar region encoding functions for
respiratory
nitrate reduction. We found the highest amount of dnrE transcripts
in aerobically nitrate-challenged cells. The gene was transcribed from
two promoters, P1 and P2, of which promoter P1 was under the control of
the nitrate response regulator NarL. The multiplicity of FNR factors in
P. stutzeri underlines the versatility of the FNR scaffold to
serve
for transcriptional regulation directed at anaerobic or
nitrate-activated
metabolic processes.