Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa was characterized which could not grow anaerobically with nitrate as the terminal electron acceptor or with arginine as the sole energy source. In this anr mutant, nitrate reductase and arginine deiminase were not induced by oxygen limitation. The anr mutation was mapped in the 60-min region of the P. aeruginosa chromosome. A 1.3-kb chromosomal fragment from P. aeruginosa complemented the anr mutation and also restored anaerobic growth of an Escherichia coli fnr deletion mutant on nitrate medium, indicating that the 1.3-kb fragment specifies an FNR-like regulatory protein. The arcDABC operon, which encodes the arginine deiminase pathway enzymes of P. aeruginosa, was rendered virtually noninducible by a deletion or an insertion in the -40 region of the arc promoter. This -40 sequence (TTGAC....ATCAG) strongly resembled the consensus FNR-binding site (TTGAT....ATCAA) of E. coli. The cloned arc operon was expressed at low levels in E. coli; nevertheless, some FNR-dependent anaerobic induction could be observed. An FNR-dependent E. coli promoter containing the consensus FNR-binding site was expressed well in P. aeruginosa and was regulated by oxygen limitation. These findings suggest that P. aeruginosa and E. coli have similar mechanisms of anaerobic control.     

Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr.

The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr+ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.     

Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-1. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S. putrefaciens etrA is able to complement an fnr mutant of E. coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etrA mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S. putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.     

Oxygen-sensitive global regulator, Anr, is involved in the biosynthesis of poly(3-hydroxybutyrate) in Pseudomonas extremaustralis

We analyzed the influence of the redox global regulator Anr on the accumulation of poly(3-hydroxybutyrate) (PHB) in Pseudomonas extremaustralis. Anr regulates a set of genes in the aerobic-anaerobic transition including genes involved in nitrate reduction and arginine fermentation. An anr mutant was constructed using PCR-based strategies. The wild-type strain was able to grow in both microaerobic and anaerobic conditions using nitrate as the terminal electron acceptor while the mutant strain was unable to grow under anaerobic conditions. In bioreactor cultures, PHB content in the wild-type strain was higher in microaerobic and anaerobic cultures compared with highly aerated cultures. The mutant strain showed decreased PHB levels in both aerobic and microaerobic conditions compared with the wild-type strain. Inactivation of anr led to decreased expression of phaC and phaR genes as demonstrated in real-time RT-PCR experiments. Associated with the PHB gene region, two putative binding sites for Anr were found that, in line with the phenotype observed in bioreactor cultures, suggest a role of this regulator in PHB biosynthesis.     

Long-term anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr- and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious ldhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.     

arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger.

In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.     

Identification and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon expression in Klebsiella pneumoniae M5al.

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilatory pathway. The structural genes for assimilatory nitrate and nitrite reductases together with genes necessary for nitrate transport form an operon, nasFEDCBA. Expression of the nasF operon is regulated both by general nitrogen control and also by nitrate or nitrite induction. We have identified a gene, nasR, that is necessary for nitrate and nitrite induction. The nasR gene, located immediately upstream of the nasFEDCBA operon, encodes a 44-kDa protein. The NasR protein shares carboxyl-terminal sequence similarity with the AmiR protein of Pseudomonas aeruginosa, the positive regulator of amiE (aliphatic amidase) gene expression. In addition, we present evidence that the nasF operon is not autogenously regulated.     

The Pseudomonas aeruginosa hemA promoter is regulated by Anr,Dnr, NarL and Integration Host Factor

Pseudomonas aeruginosa forms most of its heme under anaerobic denitrifying conditions. To study the regulation of the hemA gene, which codes for the first enzyme of heme biosynthesis in P. aeruginosa, a lacZ reporter gene fusion was constructed. Expression of lacZ under the control of the hemA promoter was found to be increased by 2.8-fold under anaerobic conditions in the presence of the alternative electron acceptor nitrate, relative to the level observed under aerobic growth conditions. Anaerobic fermentative growth or the presence of nitrite did not affect the lacZ expression. The genes encoding the oxygen sensor protein Anr, the redox regulator Dnr, the nitrate regulator NarL and the DNA-bending Integration Host Factor (IHF) are all required for the cooperative anaerobic induction of the hemA promoter hemAp (1). Potential binding sites for these regulatory proteins were identified by site-directed mutagenesis of the promoter fused to the reporter gene. The mode of regulation of P. aeruginosa hemA differs significantly from that described for the hemA gene of Escherichia coli K-12.     

Arginine biosynthesis in Thermotoga maritima: characterization of the arginine-sensitive N-acetyl-L-glutamate kinase

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of approximately 4, and the 50% inhibitory arginine concentration (I0.5) was shown to increase with temperature, approaching above 65 degrees C the I0.50 observed at 37 degrees C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75 degrees C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the Km for acetylglutamate triggered by the inhibitor, but at 37 degrees C arginine also substantially decreased the Vmax of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.