Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes

Abstract

In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK 6 ) or 8-methylmenaquinone (MMK 6 ) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK 6 from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.

Menaquinone as Well as Ubiquinone as a Crucial Component in the Escherichia coli Respiratory Chain

“…MK has been shown to participate in the regulation of nitrogen fixation in Klebsiella pneumoniae. NifL, which acts as a co-repressor in the regulation of expression of nitrogen fixation genes, is reduced by the reduced form of MK under anaerobic conditions and becomes incompetent by its sequestration on the membrane (Thummer et al, 2007). A derivative of MK-9(H 2 ) with modification of a sulfate group at its isoprenoid chain has recently been identified in Mycobacterium tuberculosis and has been shown to act as a negative regulator of virulence in mice, suggesting its involvement in the regulation of host-pathogen interactions (Holsclaw et al, 2008).…”

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).…”

“…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). In considering the mechanism of redox switching in NifL, the focus has been more on conformational changes that occur during the transition from the reduced (noninhibitory) to the oxidized (inhibitory) state.…”

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

“…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). This finding strongly indicates that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae (55). In Azotobacter vinelandii, another diazotrophic bacterium regulating its nif gene expression by a nifLA operon, NifA activity is also regulated in response to ammonium and molecular oxygen by direct protein-protein interaction by its negative regulator NifL (4,23,39,40,47).…”

“…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).…”