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Regulation of Nitrogen Fixation in Klebsiella J. Mol. Microbiol. Biotechnol. (2002) 4(3): 235–242. JMMB Symposium Regulation of Nitrogen Fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, Transducing Two Environmental Signals to the nif Transcriptional Activator NifA Ruth A. Schmitz*, Kai Klopprogge, and A. vinelandii-NifL appears to occur unspecifically Roman Grabbe in response to the availability of reducing equiva- lents in the cell. Nitrogen status of the cells is Institut fu¨ rMikrobiologie und Genetik, Grisebachstr. transduced towards the NifL/NifA regulatory sys- 8, 37077 Go¨ ttingen, Germany tem by the GlnK protein, a paralogue PII-protein, which appears to interact with the NifL/NifA regula- tory system via direct protein-protein interaction. It is not currently known whether GlnK interacts Abstract with NifL alone or affects the NifL/NifA-complex; moreover the effects appear to be the opposite in The enzymatic reduction of molecular nitrogen to K. pneumoniae and A. vinelandii.Inaddition to ammonia requires high amounts of energy, and these environmental signals, adenine nucleotides the presence of oxygen causes the catalyzing also affect the inhibitory function of NifL; in nitrogenase complex to be irreversible inactivated. the presence of ATP or ADP the inhibitory effect Thus nitrogen-fixing microorganisms tightly con- on NifA activity in vitro is increased. The NifL trol both the synthesis and activity of nitrogenase to proteins from the two organisms differ, however, avoid the unnecessary consumption of energy. In in that stimulation of K. pneumoniae-NifL occurs the free-living diazotrophs Klebsiella pneumoniae only when synthesized under nitrogen excess, and and Azotobacter vinelandii,products of the nitro- is correlated with the ability to hydrolyze ATP. In gen fixation nifLA operon regulate transcription of general, transduction of environmental signals to the other nif operons. NifA activates transcription of the nif regulatory system appears to involve a nif genes by the alternative form of RNA-polymer- 54 conformational change of NifL or the NifL/NifA ase, s -holoenzyme; NifL modulates the activity of complex. However, experimental data suggest that the transcriptional activator NifA in response to the K. pneumoniae and A. vinelandii employ signifi- presence of combined nitrogen and molecular cantly different species-specific mechanisms of oxygen. The translationally-coupled synthesis of signal transduction. the two regulatory proteins, in addition to evidence from studies of NifL/NifA complex formation, imply that the inhibition of NifA activity by NifL occurs via Introduction direct protein-protein interaction in vivo. The in- hibitory function of the negative regulator NifL Biological nitrogen fixation, the enzymatic reduction of appears to lie in the C-terminal domain, whereas molecular nitrogen (N2)toammonia, is strictly limited the N-terminal domain binds FAD as a redox- to prokaryotes. However, within the prokaryotes nitro- sensitive cofactor, which is required for signal gen fixation is found in a large number of species transduction of the internal oxygen status. Recently belonging to the bacterial domain, as well as in several it was shown, that NifL acts as a redox-sensitive methanogenic Archaea (Dean and Jacobson, 1992; regulatory protein, which modulates NifA activity Young, 1992; Lobo and Zinder, 1992; Fischer, 1994). in response to the redox-state of its FAD cofactor, The reduction of molecular nitrogen is catalyzed by the and allows NifA activity only in the absence of nitrogenase enzyme complex, and has a high energy oxygen. In K. pneumoniae, the primary oxygen demand, two ATP molecules are consumed for each sensor appears to be Fnr (fumarate nitrate reduc- electron transferred to the catalytic site (Burgess and tion regulator), which is presumed to transduce the Lowe, 1996; Howard and Rees, 1996; Rees and signal of anaerobiosis towards NifL by activating Howard, 1999). Upon the high energy requirement, in the transcription of gene(s) whose product(s) func- nitrogen fixing cells up to 40% of the ATP is utilized by tion to relieve NifL inhibition through reduction of thenitrogenase resulting in a drop of the energy the FAD cofactor. In contrast, the reduction of charge (Daesch and Mortenson, 1972; Upchurch and Mortenson, 1980). In addition the nitrogenase enzyme complex is highly sensitive to molecular oxygen, and becomes irreversibly inactivated by its presence. *For correspondence. Email [email protected]; Thus, to avoid unnecessary consumption of energy Tel. + 49 (551) 393796; Fax. + 49 (551) 393808. nitrogen-fixing microorganisms tightly control both the # 2002 Horizon Scientific Press Further Reading Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research. 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In all diazotrophic Activity by Direct Protein-Protein Interaction proteobacteria examined, the transcriptional activator NifA is required for expression of the nitrogen fixation The transcriptional activator NifA is composed of three (nif) genes. NifA expression and activity is regulated in domains: an amino (N)-terminal domain, apparently response to the environmental signals of molecular involved in regulation, a central catalytic domain, and a oxygen and combined nitrogen. However, the mechan- carboxy (C)-terminal DNA-binding domain (Drummond isms involved in this control vary in different organisms et al., 1990; Morett and Segovia, 1993). Transcription of (Fischer, 1994; Fischer, 1996; Dixon, 1998; Halbleib nif genes by an alternative RNA polymerase (s54-RNA and Ludden, 2000). In free-living and symbiotic holoenzyme) is generally activated by NifA, which binds diazotrophs belonging to the a and b subgroup of the to an upstream activation sequence (UAS) (Morrett and Proteobacteria (genera Rhizobium, Bradyrhizobium, Buck, 1988) and contacts promoter-bound s54-RNA Azospirillum and Herbaspirillum)NifAactivity is polymerase by means of a DNA loop (Buck et al.,1987). directly
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