Involvement of GroEL in nif gene regulation and nitrogenase assembly.
Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nif) 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 beta-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter of 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.
Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
D
D
Govezensky
T
T
Greener
G
G
Segal
A
A
Zamir
D001426:Bacterial Proteins / Q000378:metabolism*; D018834:Chaperonin 60; D018833:Chaperonins; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D046911:Macromolecular Substances; D008983:Molybdoferredoxin / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000096:biosynthesis* / Q000235:genetics; D009876:Operon / Q000235:genetics; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D014169:Transformation, Bacterial / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D008983:Molybdoferredoxin; D011506:Proteins; D009591:Nitrogenase; D001616:beta-Galactosidase; D018833:Chaperonins