Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nij) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent ,-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter or ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system. Biological nitrogen fixation is carried out by dinitrogenase, a two-component enzyme complex. In molybdenum-dependent nitrogenases, the MoFe protein is responsible for substrate binding and reduction, whereas the Fe protein is the exclusive electron donor for the reaction. The MoFe protein is an OX212 tetramer of approximately 220 kDa assembled with four iron-sulfur clusters and containing two copies of an iron-molybdenum cofactor (FeMoco). The Fe protein is an ct2 dimer assembled with a single iron-sulfur cluster (reviewed in reference 38).In Klebsiella pneumoniae, the genetically best-characterized nitrogen-fixing organism, all the genetic information specifically required for nitrogen fixation resides within a contiguous 24-kb nif gene cluster (1). The 20 nif genes within this cluster are organized in seven or eight transcriptional units that together form a regulon, responding, via the ntr system, nifLA (28) and nijX (17), to the oxygen and fixednitrogen status of the cells. The nifA product serves as a nif-specific transcriptional activator, binding to upstream enhancer sequences of nif promoters (30) and acting in conjunction with RNA polymerase containing RpoN (U(54) as a sigma factor (reviewed in reference 28).The assembly of nitrogenase components is a complex process, involving the formation of oligomeric protein structures, the addition of metal clusters, and the biosynthesis of FeMoco and its association with the apo-MoFe protein. The underlying genetics are no less complex and, in K. pneumoniae, involve at least 10 nif genes. In addition to nifH and niJDK, encoding the structural subunits of the Fe protein (Kp2) and the MoFe protein (Kpl), respectively, this group includes genes involved in FeMoco syn...
When present on a multicopy plasmid, a newly discovered gene (sugE) mapping to 94 min on the Escherichia coli chromosome, suppresses a groEL mutation and mimics the effects of groE overexpression. A groEL mutant of E.coli, transformed with the Klebsiella pneumoniae nif gene cluster, failed to accumulate nitrogenase components [Govezensky et al. (1991) J. Bacteriol., 173, 6339–6346]. Transformation with sugE reversed the mutant phenotype. In wild type K.pneumoniae, transformation with sugE accelerated the rate of nitrogenase biogenesis after nif derepression. In E.coli, transformation with sugE enabled bacteriophage T4 growth in a groEL mutant. A continuous 178 codon open reading frame (ORF) in sugE encloses another, in‐frame, 105 codon ORF similar to a predicted ORF in Proteus vulgaris. In vivo products of both sugE ORFs were observed in transformants expressing the gene from a T7 promoter. In non‐transformed cells, a typical sigma 70‐dependent promoter found upstream of the larger ORF directs sugE transcription during growth at 30 degrees C. At elevated temperatures or in stationary phase cells, another promoter, found within the coding sequence upstream of the smaller ORF, is activated independently of sigma 32. The results suggest that sugE encodes a chaperonin‐related system whose composition might vary with temperature and growth phase.
Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.
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