Transcriptional Analysis Of The Uncharacterized NifA-dependent Genes Within The Nitrogen Fixation Island Of Pseudomonas Stutzeri A1501

Pseudomonas stutzeri A1501, isolated from rice paddy soils in south of China, fixes nitrogen under microaerobic conditions in the free-living state. Recently, the whole genome sequencing and DNA microarray analysis of A1501 have been completed. The genome consists of a single circular chromosome with 4,567,418 base pairs, and about 4,146 protein-encoding genes. All the genetic information specific to nitrogen fixation is clustered in the A1501 genome in a 49-kb nitrogen fixation island consisting of 59 ORFs. For further exploring and studying the expression and regulation of nif genes, we carried out site-directed mutagenesis on a crucial tyrosine residue of the positive regulator protein NifA. And we also identified several uncharacterized NifA-dependent genes within the nitrogen fixation island of Pseudomonas stutzeri A1501, which might be involved in nitrogen fixation.NifA is an activator that tightly regulates the transcription of genes required for nitrogen fixation. Sequence analysis shows that a crucial tyrosine residue in region C6 of NifA protein is highly conserved (Tyr370 in Pseudomonas stutzeri A1501).To assayβ-galactosidase activity, Escherichia coli strains were transformed with the expression vector containing the wild-type NifA (or NifA-Y370C mutant) and the nifH-lacZ translational fusion. In contrast to the wild-type NifA, the NifA-Y370C mutant could neither activate the expression of nifH nor restore the nitrogenase activity of NifA deletion mutant strain A1506.These results revealed that a conserved residue (Y370), located in the region C6 of NifA protein is critical for the transcriptional activation of nif genes in Pseudomonas stutzeri A1501, and the mechanism need further investigation.Based on the analysis of transcriptional profiles, 16 genes of unknown function in the nif island were upregulated under nitrogen fixation conditions.We chose three genes: PST1305,PST1312 and PST1325 for further study. PST1305 shares the same operon with nifBQ, PST1312 is located 100bp downstream of the nifLA operon, and PST1325 encodes a 31.7-kDa protein with high similarity to hypothetical proteins present only in nitrogen-fixing bacteria. The upregulation of these three uncharacterized genes under the nitrogen fixation conditions was confirmed by quantitative Real-Time PCR. The matants were constructed by insertion of homologous plasmid pK18mob, and the nitrogenase activities were measured by acetylene reduction assay. All the mutants grew at the same rates, compared with the wild type A1501. PST1312 mutant displayed the same level of nitrogenase activities. But disruption of PST1305 and PST1325 resulted in significant decreases in nitrogenase activities, by 51.96% and 24.27%, respectively. Our results suggest that these two genes might be required for optimal nitrogenase activity and played their role in some steps of the nitrogen fixation process in P. stutzeri A1501.