Expression of nitrogen fixation genes in foreign hosts. Assembly of nitrogenase Fe protein in Escherichia coli and in yeast.

J Berman,J M Gershoni,A Zamir

doi:10.1016/s0021-9258(18)89011-1

Abstract

In Klebsiella pneumoniae, the nifH gene encodes the Fe protein (Kp2) polypeptide that is assembled into a homodimer responsible for the reduction of nitrogenase. Escherichia coli or the yeast Saccharomyces cerevisiae, transformed with the K. pneumoniae nifH gene in suitable expression vectors, synthesize the Fe protein polypeptide. This study examines the assembly of the nifH gene product into its characteristic dimeric structure in E. coli and in yeast. Immunoblotting methods, as well as 55Fe2- labeling of K. pneumoniae were employed to detect native nitrogenase components in cell lysates. E. coli and yeast transformants contained a protein similar to native Kp2 in its immunoreactivity, apparent molecular weight, and lability in the presence of oxygen or MgATP. While in E. coli the co-introduction of nifH and nifM resulted in enhanced levels of the nifH product, it appears that the nifH gene product alone is sufficient for the assembly of an Fe protein-like structure in foreign prokaryotic and eukaryotic hosts.

Highlights

Escherichia coli and in a lower eukaryote, the yeast Saccharomyces cereuisiae
In Klebsiella pneumoniae,the n i m gene encodes the nents in cell lysates. Using this assay we find that theE. coli
The co-introduction of nifM together with nifH into in suitable expression tein polypeptide. This the niM gene product structure inE. coli and vsiintenuyctdoteoyairstsets,.xcIsamhymnamirtnhaueecnsstoeitzbrhelieosttthitaciesndsgFeimmempbeerrlytiohc--ofEwdi.imtchoeolriuicctasuctshrueascntagunirneeg,nshtuhagengceeesxmttieennngtttihonaftthateshsleeevmneiblflHoyf the ~ i f Hproduct of the Kp2-like product alone is ods, as well as labelingof K . pneumoniae were sufficient for the assembly of an Fe protein-like structure in employed to detect native nitrogenase components in both E. coli and yeast

Summary

Introduction

Escherichia coli and in a lower eukaryote, the yeast Saccharomyces cereuisiae. E. coli, potentially capable of nitrogen fixation when transformed with the entire nif gene cluster (Received for publication, December 10,1984) [6], was used to determine the minimal genetic requirements. Pneumoniae were sufficient for the assembly of an Fe protein-like structure in employed to detect native nitrogenase components in both E. coli and yeast.

Results

Conclusion