Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501.

The nitrogen-fixing Pseudomonas stutzeri strain A1501 contains two ammonium transporter genes, amtB1 and amtB2, linked to glnK. Growth of an amtB1-amtB2 double deletion mutant strain was not impaired compared to that of the wild type under any conditions tested, and it was still capable of taking up ammonium ions at nearly wild-type rates. Nitrogenase activity was repressed in wild-type strain A1501 in response to the addition of ammonium, but nitrogenase activity was only partially impaired in the amtB1 and amtB2 double mutant, suggesting that the two AmtB proteins are involved in regulating expression of nitrogenase or its activity in response to ammonium. An interaction between GlnK and AmtB1 or AmtB2 was observed in a yeast two-hybrid assay. Ammonium was excreted by the amtB double mutant strain under nitrogen fixation conditions, particularly when nifA was expressed constitutively. This suggests that AmtB proteins play a role in controlling the internal pool of ammonia within the cell.

Zhang T ，Yan Y ，He S ，Ping S ，Alam KM ，Han Y ，Liu X ，Lu W ，Zhang W ，Chen M ，Xiang W ，Wang X ，Lin M ... -  《-》

Involvement of GlnK, a PII protein, in control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1501.

The nitrogen-fixing, root-associated strain Pseudomonas stutzeri A1501 carries a single gene encoding a protein from the PII family, designated glnK. The glnK gene is co-transcribed with two distantly related copies of amtB genes encoding putative ammonium channels. Transcription of glnK was decreased in the presence of ammonia and was partly dependent on NtrC and RpoN under nitrogen-limiting conditions. Inactivation of glnK led to a mutant strain devoid of nitrogenase activity, auxotrophic for glutamine and unable to deadenylylate glutamine synthetase, while inactivation of amtB1 led to a prototrophic and Nif+ mutant strain. RT-PCR analysis showed that nifA transcription was abolished in the glnK mutant, while glnA remained transcribed. Using the yeast two-hybrid system, an interaction between GlnK and the C-terminal domain of NifL was observed, suggesting GlnK-dependent control of NifA activity by NifL. Introduction of a plasmid that expressed nifA from a constitutive promoter restored nitrogen fixation to the glnK mutant, and nitrogenase activity was observed even in the presence of ammonia. GlnK signalling appears to be a key regulatory element in control of ammonia assimilation, of nifA expression and in modulation of NifA activity by NifL.

He S ，Chen M ，Xie Z ，Yan Y ，Li H ，Fan Y ，Ping S ，Lin M ，Elmerich C ... -  《ARCHIVES OF MICROBIOLOGY》

[Analysis of a new nitrogen fixation gene in Pseudomonas stutzeri A1501].

Fan H ，Yan Y ，Li Y ，Ping S ，Zhang W ，Chen M ，Lin M ，Lu W ... -  《-》

Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri A1501.

Yan Y ，Ping S ，Peng J ，Han Y ，Li L ，Yang J ，Dou Y ，Li Y ，Fan H ，Fan Y ，Li D ，Zhan Y ，Chen M ，Lu W ，Zhang W ，Cheng Q ，Jin Q ，Lin M ... -  《BMC GENOMICS》

Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501.

Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.

Xie Z ，Dou Y ，Ping S ，Chen M ，Wang G ，Elmerich C ，Lin M ... -  《MICROBIOLOGY-SGM》