Site-directed mutagenesis analysis of draTG genes and their downstream region from Azospirillum brasilense Yu62.
draT and draG genes are involved in posttranslational regulation of nitrogenase activity of Azospirillum brasilense Yu62. Both genes and their downstream region were mutagenized by Kmr cassette insertions. Analysis of mutations introduced into the draTG region on the A. brasilense Yu62 chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Analysis of mutations introduced into the draTG downstream region (the mutagenized site is about 2 kb downstream from draG) showed that the mutant had higher nitrogenase activity than the wild strain while growing in nitrogen-free medium and medium with 2 mmol/L ammonium. These results reveal that there is no gene required for nitrogen fixation in this mutagenized region, but it is possible that there are genes which play a role in regulating nitrogen fixation. The results of monitoring the expression of transcriptional nifH-lacZ gene fusion in the mutant YZ4 showed that the transcriptional regulation of nif gene in the mutant YZ4 was the same as that in the wild type.
Ma
L
L
National Laboratories for Agrobiotechnology, China Agricultural University, Beijing, China.
Wu
Y
Y
Wang
J
J
Zhao
Y
Y
Li
J
J
D015806:Azospirillum brasilense / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C110078:draTG protein, Bacteria; D009591:Nitrogenase