Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum.
Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.
Zhang
Y
Y
Departments of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Pohlmann
E L
EL
Ludden
P W
PW
Roberts
G P
GP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003624:Darkness; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012247:Rhodospirillum rubrum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D051922:PII Nitrogen Regulatory Proteins; D000644:Quaternary Ammonium Compounds; D014157:Transcription Factors; C404143:PIID regulatory protein, Bacteria; D009591:Nitrogenase; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C036091:ADP-ribosyl-(dinitrogen reductase) hydrolase; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase