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Research Paper |
Max-Planck-Institute for Terrestrial Microbiology, Group Symbiosis Research, Karl-von-Frisch-Strasse,D-35043 Marburg, Germany1
University of Bremen, Faculty of Biology and Chemistry, Laboratory of General Microbiology, Postfach 330440, D-28334 Bremen, Germany2
Author for correspondence: Barbara Reinhold-Hurek. Tel: +49 421 218 2370. Fax: +49 421 218 4042. e-mail: breinhold{at}uni-bremen.de
NifA, the transcriptional activator of nitrogenase (nif) genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the 
-subgroup of the Proteobacteria. This paper reports a functionally similar NifL-like protein outside this group in Azoarcus sp. strain BH72, a diazotrophic grass endophyte belonging to the ß-subgroup of the Proteobacteria. Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O2 and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an Azoarcus sp. BH72 genomic library was used to select cosmids that complemented a nifA mutation in Azotobacter vinelandii. Sequence analysis of the 3·4 kb genomic region complementing nifA showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of Azotobacter vinelandii. According to Northern blot and reverse transcriptase PCR analysis, the nifLA transcript was more abundant at low combined nitrogen and O2 levels, results which were corroborated by GUS (ß-glucuronidase) assays using a transcriptional nifL::gusA fusion. N2 fixation was abolished in a NifLA- and a NifA- mutant, wild-type fixation being restored by nifLA in trans. The NifLA- mutant also failed to activate nifH::gus expression, indicating that NifA is the obligate transcriptional activator for nifHDK. A nifL mutant was diazotrophic and did not show repression of nifH::gusA by ammonium or O2, suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O2 and combined nitrogen as NifL in bacteria of the -Proteobacteria.
Keywords: nitrogenase, gene expression, transcriptional activator, NifA, NifL
Abbreviations: GUS, ß-glucuronidase
c The GenBank accession number for the sequence determined in this work is AF518560.
a Present address: Albert-Ludwigs-University Freiburg, Plant Biotechnology, Sonnenstrasse 5, D-79104 Freiburg, Germany.
b Present address: Department of Biochemistry, Biozentrum University of Basel, CH-4056 Basel, Switzerland.
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