Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

Abstract

In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N 2 -fixation (nif) genes in response to molecular O 2 and ammonium. We investigated the influence of membrane-bound oxidoreductases on nif-regulation by biochemical analysis of purified NifL and by monitoring NifA-mediated expression of nifH¢-¢lacZ reporter fusions in different mutant backgrounds. NifL-bound FAD-cofactor was reduced by NADH only in the presence of a redox-mediator or inside-out vesicles derived from anaerobically grown K. pneumoniae cells, indicating that in vivo NifL is reduced by electrons derived from membrane-bound oxidoreductases of the anaerobic respiratory chain. This mechanism is further supported by three lines of evidence: First, K. pneumoniae strains carrying null mutations of fdnG or nuoCD showed significantly reduced nif-induction under derepressing conditions, indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase-N or NADH:ubiquinone oxidoreductase. The same effect was observed in a heterologous Escherichia coli system carrying a ndh null allele (coding for NADH dehydrogenaseII). Second, studying nif-induction in K. pneumoniae revealed that during anaerobic growth in glycerol, under nitrogen-limitation, the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nif-induction. The final line of evidence is that reduced quinone derivatives, dimethylnaphthoquinol and menadiol, are able to transfer electrons to the FAD-moiety of purified NifL. On the basis of these data, we postulate that under anaerobic and nitrogen-limited conditions, NifL inhibition of NifA activity is relieved by reduction of the FAD-cofactor by electrons derived from the reduced quinone pool, generated by anaerobic respiration, that favours membrane association of NifL. We further hypothesize that the quinol/quinone ratio is important for providing the signal to NifL.

Molecular Basis of Transcriptional Antiactivation

“…Inhibition of Ti plasmid conjugative transfer by TraM joins a growing list of prokaryotic regulatory systems involving an activator-antiactivator complex, including NifA-NifL regulation of nitrogen fixation in Azotobacter vinelandii (33) and Klebsiella pneumoniae (34), ComK-MecA-ClpC regulation of competence in Bacillus subtilis (35), GcvA-GcvR regulation of oxidative cleavage of glycine (36), and CRP-CytR regulation of many operons in E. coli (37). In the cases of NifA-NifL and ComKMecA, the antiactivator binds to and titrates the activator, thus preventing DNA binding and transcriptional activation (33)(34)(35).…”

“…In the cases of NifA-NifL and ComKMecA, the antiactivator binds to and titrates the activator, thus preventing DNA binding and transcriptional activation (33)(34)(35). However in the cases of GcvA-GcvR and CRP-CytR, the antiactivator and the activator bind promoter DNA in tandem, and the interactions between the antiactivator and the activator at the promoter inhibit transcriptional activation (36,37).…”

Flavin Redox Switching of Protein Functions

“…The reduction potential for NifL (-196 mV, pH 7) is accessible to a number of potential electron donors (41). In Klebsiella pneumoniae, the respiratory quinone pool is thought to be a source of electron donors for NifL (23,73). In order for NifL to be reduced by the quinone pool, NifL is proposed to shuttle between the membrane and the cytoplasm in K. pneumoniae (73).…”

Insights into Membrane Association of Klebsiella pneumoniae NifL under Nitrogen-Fixing Conditions from Mutational Analysis

“…However, upon anaerobiosis and simultaneous absence of combined nitrogen, the flavoprotein NifL is sequestered to the cytoplasmic membrane (21,54). This membrane sequestration of the negative regulator NifL, allowing cytoplasmic NifA to induce nif gene expression by activating the alternative 54 RNA-polymerase (16,41), is the key mechanism for regulating nitrogen fixation in K. pneumoniae in response to the environmental signals (12,21). In previous studies we obtained evidence that membrane sequestration of NifL under nitrogen-fixing conditions is achieved by the reduction of the N-terminally bound FAD cofactor by electrons derived from the reduced menaquinone pool (11,12,55).…”

“…This membrane sequestration of the negative regulator NifL, allowing cytoplasmic NifA to induce nif gene expression by activating the alternative 54 RNA-polymerase (16,41), is the key mechanism for regulating nitrogen fixation in K. pneumoniae in response to the environmental signals (12,21). In previous studies we obtained evidence that membrane sequestration of NifL under nitrogen-fixing conditions is achieved by the reduction of the N-terminally bound FAD cofactor by electrons derived from the reduced menaquinone pool (11,12,55). Using partial anaerobic respiratory chains of Wolinella succinogenes reconstituted in liposomes further demonstrated that under anaerobic conditions electrons are directly transferred from the menaquinol pool onto NifL-bound FAD without the requirement for any further K. pneumoniae specific protein (e.g., receptor proteins or NifL-specific oxidoreductases).…”