Characterization of Genes Essential for Symbiotic Nitrogen Fixation From Bradyrhizobium japonicum Strain I110 View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

1987

AUTHORS

John D. Noti , Allen C. Yun , Otto Folkerts , Istvan Torok , Aladar A. Szalay

ABSTRACT

A total of 96 independent Tn5 insertions within a 39 kilobase pair (kb) segment of chromosomal DNA containing the three structural genes for nitrogenase, (nifH, nifD, and nifK) from Bradyrhizobium japonicum strain I110 were obtained in Escherichia coli and transferred to the wild-type strain by marker exchange. Individual transconjugants containing a Tn5 insertion were inoculated onto Glycine max cv. Wilkin (soybean) and analyzed for their effect on symbiotic nitrogen fixation. In addition to the three structural genes, genes essential for nitrogen fixation (fix genes) were located in three separate regions: 1) 9 kb upstream of the nifDK operon (fix region I); 2) 1.5 kb downstream of the nifDK operon (fix region II); 3) 4.5 kb upstream of nifH (fix region III). All of the fix::Tn5 insertion strains formed nodules which contained low or undetectable levels of nitrogenase activity. Bacteroids isolated from these nodules had approximately the same levels of the nifDK and nifH transcripts as those detectable from nodules formed by the wild-type strain. Western blot analysis of bacteroid proteins from nodules formed by the fix::Tn5 mutants or the wild-type strain showed the presence of similar levels of the nitrogenase protein subunits. DNA fragments containing either the nifD or nifH promoter and 5′-structural gene sequences from Bradyrhizobium japonicum strain I110 were fused in-frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of -galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild-type and Fix mutants generated by transposon Tn5 mutagenesis. No nif positive regulatory mutants were identified from among this array of Fix mutants. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Fix region III was characterized further by DNA sequence analysis and was shown to contain three open reading frames (ORF). One ORF corresponds to the nifB gene. The coding sequence of the nifB gene consists of 1494 nucleotides and is preceded by putative promoter (5′ACGG-8bp-TTGCT 3′) and upstream activator (5′TGT-4bp-T-5bp-ACA 3′) sequences. The second ORF is upstream of nifB and consists of a coding sequence 1356 nucleotides long that is preceded by a putative promoter sequence (5′CIGG-9bp-TTGCT 3′). The third ORF is 831 nucleotides long and immediately follows nifB. More... »

PAGES

202-207

Book

TITLE

Molecular genetics of plant-microbe interactions

ISBN

978-94-010-8496-3
978-94-009-4482-4

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/978-94-009-4482-4_50

DOI

http://dx.doi.org/10.1007/978-94-009-4482-4_50

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1033790761


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