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Insights into membrane association of Klebsiella pneumoniae NifL under nitrogen-fixing conditions from mutational analysis.

ABSTRACT: In Klebsiella pneumoniae nitrogen fixation is tightly controlled in response to ammonium and molecular oxygen by the NifL/NifA regulatory system. Under repressing conditions, NifL inhibits the nif-specific transcriptional activator NifA by direct protein-protein interaction, whereas under anaerobic and nitrogen-limited conditions sequestration of reduced NifL to the cytoplasmic membrane impairs inhibition of cytoplasmic NifA by NifL. We report here on a genetic screen to identify amino acids of NifL essential for sequestration to the cytoplasmic membrane under nitrogen-fixing conditions. Overall, 11,500 mutated nifL genes of three independently generated pools were screened for those conferring a Nif(-) phenotype. Based on the respective amino acid changes of nonfunctional derivatives obtained in the screen, and taking structural data into account as well, several point mutations were introduced into nifL by site-directed mutagenesis. The majority of amino acid changes resulting in a significant nif gene inhibition were located in the N-terminal domain (N46D, Q57L, Q64R, N67S, N69S, R80C, and W87G) and the Q-linker (K271E). Further analyses demonstrated that positions N69, R80, and W87 are essential for binding the FAD cofactor, whereas primarily Q64 and N46, but also Q57 and N67, appear to be crucial for direct membrane contact of NifL under oxygen and nitrogen limitation. Based on these findings, we propose that those four amino acids most likely located on the protein surface, as well as the presence of the FAD cofactor, are crucial for the correct overall protein conformation and respective surface charge, allowing NifL sequestration to the cytoplasmic membrane under derepressing conditions.

SUBMITTER: Milenkov M

PROVIDER: S-EPMC3021237 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Insights into membrane association of Klebsiella pneumoniae NifL under nitrogen-fixing conditions from mutational analysis.

Milenkov Maria M Thummer Robert R Glöer Jens J Grötzinger Joachim J Jung Sascha S Schmitz Ruth A RA

Journal of bacteriology 20101105 3

In Klebsiella pneumoniae nitrogen fixation is tightly controlled in response to ammonium and molecular oxygen by the NifL/NifA regulatory system. Under repressing conditions, NifL inhibits the nif-specific transcriptional activator NifA by direct protein-protein interaction, whereas under anaerobic and nitrogen-limited conditions sequestration of reduced NifL to the cytoplasmic membrane impairs inhibition of cytoplasmic NifA by NifL. We report here on a genetic screen to identify amino acids of ...[more]

PMID: 21057007

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Project description:The acceleration of the nitrogen cycle and the nitrogen excess observed in some coastal waters has increased interest into understanding the biochemical and molecular basis of nitrogen metabolism in various microorganisms. To investigate nitrogen metabolism of a novel heterotrophic nitrification and aerobic denitrification bacterium Klebsiella aerogenes strain (B23) under nitrogen-rich conditions, we conducted physiological and transcriptomic high-throughput sequencing analyses on strain B23 cultured on potassium nitrate-free or potassium nitrate-rich media. Overall, K. aerogenes B23 assimilated 82.47% of the nitrate present into cellular nitrogen. Further, 1,195 differentially expressed genes were observed between K. aerogenes B23 cultured on potassium nitrate-free media and those cultured on potassium nitrate-rich media. Gene annotation and metabolic pathway analysis of the transcriptome were performed using a series of bioinformatics tools, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Non-Redundant Protein Database annotation. Accordingly, the nitrogen metabolism pathway of K. aerogenes B23 was analyzed; overall, 39 genes were determined to be involved in this pathway. Differential expression analysis of the genes involved in the nitrogen metabolism pathway demonstrated that, compared to the control, FNR, NarK/14945, fdx, gshA, proB, proA, gapA, argH, artQ, artJ, artM, ArgR, GAT1, prmB, pyrG, glnS, and Ca1 were significantly upregulated in the nitrogen-treated K. aerogenes B23; these genes have been established to be involved in the regulation of nitrate, arginine, glutamate, and ammonia assimilation. Further, norV, norR, and narI were also upregulated in nitrogen-treated K. aerogenes B23; these genes are involved in the regulation of NO metabolism. These differential expression results are important for understanding the regulation process of key nitrogen metabolism enzyme genes in K. aerogenes B23. Therefore, this study establishes a solid foundation for further research into the expression regulation patterns of nitrogen metabolism-associated genes in K. aerogenes B23 under nitrogen-rich conditions; moreover, this research provides essential insight into how K. aerogenes B23 utilizes nutritional elements.

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Insights into membrane association of Klebsiella pneumoniae NifL under nitrogen-fixing conditions from mutational analysis.

Publications

Insights into membrane association of Klebsiella pneumoniae NifL under nitrogen-fixing conditions from mutational analysis.

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