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Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene.

ABSTRACT: 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 fur 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 (M(r) = 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 single 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), nitrate 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.

SUBMITTER: Cuypers H

PROVIDER: S-EPMC206866 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene.

Cuypers H H Zumft W G WG

Journal of bacteriology 19931101 22

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 fur 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 f ...[more]

PMID: 8226670

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Project description: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 D(1) biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd(1) 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.

Project description:Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.

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Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene.

Publications

Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene.

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