Identification of a NifL-like protein in a diazotroph of the beta-subgroup of the Proteobacteria, Azoarcus sp. strain BH72.

Abstract

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 gamma-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 beta-subgroup of the PROTEOBACTERIA: Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O(2) 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 O(2) levels, results which were corroborated by GUS (beta-glucuronidase) assays using a transcriptional nifL::gusA fusion. N(2) 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 O(2), suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O(2) and combined nitrogen as NifL in bacteria of the gamma-PROTEOBACTERIA: