Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli

Anaerobic growth of Pseudomonas aeruginosa on nitrate or arginine requires the anr gene, which codes for a positive control element (ANR) capable of functionally complementing an fnr mutation in Escherichia coli. The anr gene was sequenced; it showed 51% identity with the fnr gene at the amino acid sequence level. Four cysteine residues known to be essential in the FNR protein are conserved in ANR. The anr gene product (deduced Mr 27,129) was visualized by the maxicell method and migrated like a 32 kDa protein in gel electrophoresis under denaturing conditions. An anr mutant of P. aeruginosa constructed by gene replacement was defective in nitrate respiration, arginine deiminase activity, and hydrogen cyanide biosynthesis, underscoring the diverse metabolic functions of ANR during oxygen limitation. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all had a functional analogue of ANR, indicating that similar anaerobic control mechanisms exist in these bacteria.     

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.     

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.     

A dramatic conformational rearrangement is necessary for the activation of DNR from Pseudomonas aeruginosa. Crystal structure of wild‐type DNR

The opportunistic pathogen Pseudomonas aeruginosa can grow in low oxygen, because it is capable of anaerobic respiration using nitrate as a terminal electron acceptor (denitrification). An intermediate of the denitrification pathway is nitric oxide, a compound that may become cytotoxic at high concentration. The intracellular levels of nitric oxide are tightly controlled by regulating the expression of the enzymes responsible for its synthesis and degradation (nitrite and nitric oxide reductases). In this article, we present the crystallographic structure of the wild‐type dissimilative nitrate respiration regulator (DNR), a master regulator controlling expression of the denitrification machinery and a putative target for new therapeutic strategies. Comparison with other structures among the CRP‐FNR class of regulators reveals that DNR has crystallized in a conformation that has never been observed before. In particular, the sensing domain of DNR has undergone a rotation of more than 50° with respect to the other structures. This suggests that DNR may undergo an unexpected and very large conformational rearrangement on activation. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Interruption of the denitrification pathway influences cell growth and magnetosome formation in Magnetospirillum magneticum AMB-1

Aims: Intracellular magnetosome synthesis in magnetotactic bacteria has been proposed to be a process involving functions of a variety of proteins. To learn more about the genetic control that is involved in magnetosome formation, nonmagnetic mutants are screened and characterized. Methods and Results: Conjugation‐mediated transposon mutagenesis was applied to screen for nonmagnetic mutants of Magnetospirillum magneticum AMB‐1 that were unable to respond to the magnetic field. A mutant strain with disruption of a gene locus encoding nitric oxide reductase was obtained. Growth and magnetosome formation under different conditions were further characterized. Conclusions: Interruption of denitrification by inactivating nitric oxide reductase was responsible for the compromised growth and magnetosome formation in the mutant with shorter intracellular chains of magnetite crystals than those of wild‐type cells under anaerobic conditions. Nevertheless, the mutant displayed apparently normal growth in aerobic culture. Significance and Impact of the Study: Efficient denitrification in the absence of oxygen is not only necessary for maintaining cell growth but may also be required to derive sufficient energy to mediate the formation of magnetosome vesicles necessary for the initiation or activation of magnetite formation.     

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.     

The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.     

Regulation of denitrification genes in Neisseria meningitidis by nitric oxide and the repressor NsrR

The human pathogen Neisseria meningitidis is capable of growth using the denitrification of nitrite to nitrous oxide under microaerobic conditions. This process is catalyzed by two reductases: nitrite reductase (encoded by aniA) and nitric oxide (NO) reductase (encoded by norB). Here, we show that in N. meningitidis MC58 norB is regulated by nitric oxide via the product of gene NMB0437 which encodes NsrR. NsrR is a repressor in the absence of NO, but norB expression is derepressed by NO in an NsrR-dependent manner. nsrR-deficient mutants grow by denitrification more rapidly than wild-type N. meningitidis, and this is coincident with the upregulation of both NO reductase and nitrite reductase even under aerobic conditions in the absence of nitrite or NO. The NsrR-dependent repression of aniA (unlike that of norB) is not lifted in the presence of NO. The role of NsrR in the control of expression of aniA is linked to the function of the anaerobic activator protein FNR: analysis of nsrR and fnr single and nsrR fnr double mutants carrying an aniA promoter lacZ fusion indicates that the role of NsrR is to prevent FNR-dependent aniA expression under aerobic conditions, indicating that FNR in N. meningitidis retains considerable activity aerobically.     

Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide.

Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen. Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2. Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance. Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation. NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification). The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate. A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis. Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1.     

Gene structure and expression profile of cytochrome bc nitric oxide reductase from Hydrogenobacter thermophilus TK-6

The genes for a nitric oxide reductase-like cytochrome bc complex were cloned from a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6. The structural genes norC and norB, which encode cytochrome c and cytochrome b subunits of the complex respectively, are probably transcribed as a tricistronic operon with a following gene encoding a putative membrane protein. NorC has, unusually, two hydrophobic transmembrane spans in its N-terminus. Immunoblot analysis showed that expression of NorC was induced by nitrate, nitrite, or sodium nitropurusside, suggesting that the norCB gene product is a denitrification enzyme, nitric oxide reductase. The consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide-sensing regulators of proteobacteria were not found in the norC promoter region, but consensus -35 and -10 sequences were found in this region. These results indicate that strain TK-6 has a nitrogen oxide-sensing regulatory system that differs from proteobacterial systems.