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

Abstract

The preferred way of life of Pseudomonas aeruginosa is aerobic respiration (38). When aeration is reduced or absent and nitrate is present, P. aeruginosa obtains energy from nitrate respiration (14,44). When neither oxygen nor nitrate is available, arginine can be used as the sole energy source for motility and growth (48,62). In this case, the arginine deiminase pathway serves to degrade arginine to ornithine and carbamoylphosphate, which allows regeneration of ATP from ADP without respiration (41,62). Anaerobic growth on arginine requires a rich medium and extended incubation; colonies take 4 to 5 days to form. Under these conditions, arginine is converted quantitatively to omithine and hence does not serve as a carbon source (62). Good aeration inhibits nitrate uptake (21) and prevents induction of the nitrate respiration enzymes (12) and of the arginine deiminase pathway in P. aeruginosa (34). Aerobic conditions favor the utilization of arginine as a carbon source via the succinylarginine pathway (13). The molecular mechanisms involved in the switch from aerobic to anaerobic metabolism in P. aeruginosa are unknown.In Escherichia coli and other enteric bacteria, thefnr gene is essential for the expression of fumarate and nitrate respiration and many other anaerobic processes (26,47,54,57,61). Thefnr-dependent genes and operons have a conserved symmetrical sequence, the FNR box, upstream of their promoters. Mutations in the FNR box interfere with anaerobic induction (8,16,24,54). It is generally accepted that under anaerobic conditions the FNR protein acts as a transcriptional activator by binding to the FNR box; however, a physical interaction between FNR and the FNR box has not been demonstrated (9, 54, 59).We are interested in the regulation of anaerobic metabolism in P. aeruginosa, especially in the induction of the arginine deiminase pathway by oxygen limitation. for this pathway are organized as an operon (arcDABC), which encodes a transmembrane protein having arginine/ ornithine antiporter activity (arcD), arginine deiminase (arcA), catabolic ornithine carbamoyltransferase (arcB), and carbamate kinase (arcC) (5, 6, 9a, 30, 31, 62). Most P. aeruginosa mutants that cannot grow anaerobically on arginine (Arc-phenotype) have a defect in the arcDABC operon (43, 62). Recently, we found a mutant whose Arc-phenotype is due to a mutation lying outside the arc operon (19). This mutation had originally been obtained by van Hartingsveldt et al. (63,64) and designated nirD. It causes inability of P. aeruginosa to grow anaerobically on nitrate (Nar-phenotype) and loss of dissimilatory nitrite reductase activity. Other genes required for nitrate respiration are unlinked to nirD (63,64). Here we show that a chromosomal fragment of P. aeruginosa complements the nirD mutant for anaerobic growth on nitrate and arginine and also restores anaerobic induction of nitrate reductase and arginine deiminase in the mutant. We therefore propose that nirD should be renamed anr (for anaerobic regulation of arginine deiminase and ni...

The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

“…In P. aeruginosa the denitrification pathway is regulated by redox signalling, through a cascade of transcription factors; in particular, the global oxygen-sensing regulator ANR (anaerobic regulation of arginine deaminase and nitrate reduction) (Galimand et al, 1991), a homologue of the Escherichia coli oxygen sensor FNR protein, activates, under anaerobic conditions, the gene coding for the transcription factor DNR (dissimilatory nitrate respiration regulator), which, in the presence of N-oxides, promotes the expression of the nir, the nor and the nos genes (Arai et al, 1995(Arai et al, , 1997(Arai et al, , 1999(Arai et al, , 2003 The DNR transcription factor belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase regulator) superfamily of regulators (Zumft, 2002;Körner et al, 2003). The members of this superfamily are usually homodimers, each monomer being formed by three domains (McKay & Steitz, 1981): (i) an N-terminal sensing domain (also referred to as the effector domain) with the typical fold of the cAMP-binding domain of CRP; (ii) a long dimerization a-helix recruited to form the dimer interface; and (iii) a C-terminal DNA-binding domain that contains a helix-turn-helix (HTH) motif.…”

“…The complete denitrification pathway involves four enzymes: nitrate reductase, nitrite reductase, nitric oxide reductase (NOR) and nitrous oxide reductase, operating sequentially to reduce nitrate to dinitrogen gas via nitrite (NO { 2 ), nitric oxide (NO) and nitrous oxide (N 2 O) (Zumft, 1997). The expression and the activity of the NIR and NOR enzymes are tightly controlled because it is mandatory for the bacteria to keep the concentration of intracellular NO below cytotoxic levels, to limit nitrosative stress.In P. aeruginosa the denitrification pathway is regulated by redox signalling, through a cascade of transcription factors; in particular, the global oxygen-sensing regulator ANR (anaerobic regulation of arginine deaminase and nitrate reduction) (Galimand et al, 1991), a homologue of the Escherichia coli oxygen sensor FNR protein, activates, under anaerobic conditions, the gene coding for the transcription factor DNR (dissimilatory nitrate respiration regulator), which, in the presence of N-oxides, promotes the expression of the nir, the nor and the nos genes (Arai et al, 1995, 1997, 1999, 2003 Previous studies, carried out in P. aeruginosa PAO1 with the nirS, the norC and the nosZ promoters fused to the lacZ reporter gene, showed that the DNR transcription factor responds in vivo to N-oxides (Arai et al, 1999(Arai et al, , 2003. A similar response to NO in vivo has been reported for the DNR homologue NNR in Paracoccus denitrificans (Van Spanning et al, 1999) and, for the same regulator, also in E. coli using the FNR-dependent E. coli melR promoter (Hutchings et al, 2000).…”

Functional Characterization of Crp/Fnr-Type Global Transcriptional Regulators in Desulfovibrio vulgaris Hildenborough

“…Previous studies on several bacterial species suggest that Crp/Fnr regulators are involved in responses to a variety of intracellular or extracellular signals, such as anoxia, carbon monoxide, temperature, nitric oxide, and oxidative and nitrosative stress (1,13,35,42) and control metabolic pathways such as photosynthesis, nitrogen fixation, aromatic compound degradation, and respiration (11,25,31,39).…”

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting

“…A sequence identical to that recognized by the oxygensensitive transcriptional regulator Anr is present within the plcH promoter region (Galimand et al, 1991;Trunk et al, 2010). Anr mainly acts as a regulator of gene expression under micro-oxic and anoxic conditions (Arai et al, 1995;Kawakami et al, 2010;Lu et al, 1999;Ye et al, 1995), and its activity increases as oxygen tensions decrease.…”

“…The histone-like nucleoid-associated proteins MvaT and MvaU were both shown to bind and inhibit the transcription of the plcH ORF in P. aeruginosa (Castang et al, 2008). Several publications have reported that a canonical Anr consensus sequence is positioned within the plcH promoter (Galimand et al, 1991;Trunk et al, 2010), suggesting a role for negative regulation of plcH by Anr. However, this regulatory scheme has not been established.…”