Involvement of GroEL in nif gene regulation and nitrogenase assembly

Abstract

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...

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis

“…It was, therefore, concluded that the nifS gene product, together with the products of several other nif genes, is required for the activities of all three nitrogenase systems inAzotobacter vinelandii, perhaps by stabilizing the nitrogenases against oxidative inactivation (14). Interestingly, it was reported that when K pneumoniae nif genes were introduced into E. coli, the assembly of the nitrogenases required the E. coligroEL gene product, which is a major molecular chaperone involved in many cellular functions (9). Recently, the nifS gene product from Azotobacter vinelandii has been overproduced and purified (36).…”

Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms

“…In a portion of the nodules induced by the groEL1 mutant, the endosymbionts differentiated to elongated bacteroids; however, the nodules were not functional and nitrogen fixation activity was not detected. GroEL is required for nitrogenase assembly in other bacteria (22). Thus, likewise, in the absence of GroEL1, subunits of nitrogenase may not form an active enzyme complex.…”

The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

“…Unfortunately, commercially available E. coli GroES antibodies showed extensive nonspecific crossreactivity with these solutions and were therefore not helpful in this regard. Since the early 1990s, when GroEL research was in its infancy, several reports have appeared suggesting that GroEL might have something to do with nitrogenase regulation and assembly (40)(41)(42)(43)(44). For example, 35 S pulse-chase experiments with Klebsiella pneumoniae suggested transient binding of newly synthesized Fe protein and MoFe protein polypeptides to GroEL.…”