Dissection of the Bradyrhizobium japonicum NifA+sigma54 regulon, and identification of a ferredoxin gene (fdxN) for symbiotic nitrogen fixation.
Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+sigma(54) regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN (1/2) mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and sigma(54)-binding sites plus a simple operon definition. One of the newly identified NifA+sigma(54)-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional sigma(54 )promoter with the dinucleotide GA instead of the consensus GC motif at position -12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+sigma(54) targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+sigma(54 )regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processes.
Hauser
Felix
F
Institute of Microbiology, Eidgenössische Technische Hochschule, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
Pessi
Gabriella
G
Friberg
Markus
M
Weber
Christoph
C
Rusca
Nicola
N
Lindemann
Andrea
A
Fischer
Hans-Martin
HM
Hennecke
Hauke
H
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020369:Bradyrhizobium / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005288:Ferredoxins / Q000235:genetics*; D005798:Genes, Bacterial; D016296:Mutagenesis; D009586:Nitrogen Fixation / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54 / Q000235:genetics*; D018085:Regulon; D013025:Glycine max / Q000382:microbiology; D013559:Symbiosis / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D005288:Ferredoxins; C061338:NifA protein, Bacteria; D014157:Transcription Factors; D051877:RNA Polymerase Sigma 54