The synthesis of proteins
necessary
for the respiratory reduction of nitrate to dinitrogen is induced in
most
denitrifying bacteria by a shift to anaerobiosis. A homolog of the fnr
gene, which encodes a redox-active transcriptional activator in Escherichia
coli, was isolated from Pseudomonas stutzeri by using the anr
gene of Pseudomonas aeruginosa as the hybridization probe (R.
G.
Sawers, Mol. Microbiol. 5:1469-1481, 1991). The coding region was
located
on a 3-kb SmaI
fragment. An open reading frame
of 735 nucleotides, designated fnrA, had the coding potential
for
a protein of 244 amino acids (Mr = 27,089) with 51.2%
positional
identity to the Fnr protein of E. coli and 86.1% to the Anr
protein
of P. aeruginosa. The fnrA gene gave a singl transcript
of
0.85 kb and complemented nitrate-dependent anaerobic growth of an fnr
deletion mutant of E. coli. An open reading frame immediately
downstream
of fnrA encoded adenine phosphoribosyltransferase (EC 2.4.2.7).
Mutations in fnrA were generated in vitro by insertional
mutagenesis
followed by gene replacement. Gene inactivation was shown by loss of
the
fnrA
transcript and detection of an arginine deiminase (EC 3.5.3.6)-negative
phenotype in the mutants. However, neither the enzymatic activities nor
the levels of anaerobic expression of the respiratory enzymes nitrate
reductase
(EC 1.7.99.4), nitrite reductase (EC 1.9.3.2), NO reductase (EC
1.7.99.7),
and N2O reductase (EC 1.7.99.6) were changed in fnrA
mutants versus the P. stutzeri wild type. A promoter-probe
vector
for Fnr-dependent transcription was activated anaerobically in the fnrA
mutants, suggesting the existence of a second Fnr homolog in the same
bacterium.
The Fnr-binding motifs, apparent in the promoter region of genes
encoding
denitrification components of P. stutzeri, are likely to be
recognized
by this second Fnr homolog. Preliminary evidence indicates also the
presence
of the catabolite activator protein, Crp, in P. stutzeri.