The capacity to fix nitrogen is widely distributed in phyla of Bacteria and Archaea but has long been considered to be absent from the Pseudomonas genus. We report here the complete genome sequencing of nitrogen-fixing root-associated Pseudomonas stutzeri A1501. The genome consists of a single circular chromosome with 4,567,418 bp. Comparative genomics revealed that, among 4,146 protein-encoding genes, 1,977 have orthologs in each of the five other Pseudomonas representative species sequenced to date. The genome contains genes involved in broad utilization of carbon sources, nitrogen fixation, denitrification, degradation of aromatic compounds, biosynthesis of polyhydroxybutyrate, multiple pathways of protection against environmental stress, and other functions that presumably give A1501 an advantage in root colonization. Genetic information on synthesis, maturation, and functioning of nitrogenase is clustered in a 49-kb island, suggesting that this property was acquired by lateral gene transfer. New genes required for the nitrogen fixation process have been identified within the nif island. The genome sequence offers the genetic basis for further study of the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in the interaction with host plants; moreover, it opens up new perspectives for wider application of root-associated diazotrophs in sustainable agriculture.genome sequencing ͉ root-associated diazotroph

The Pseudomonas stutzeri strain A1501 (formerly known as Alcaligenes faecalis) fixes nitrogen under microaerobic conditions in the free-living state and colonizes rice endophytically. The authors characterized a region in strain A1501, corresponding to most of the nif genes and the rnf genes, involved in electron transport to nitrogenase in Rhodobacter capsulatus. The region contained three groups of genes arranged in the same order as in Azotobacter vinelandii: (1) nifB fdx ORF3 nifQ ORF5 ORF6; (2) nifLA-rnfABCDGEF-nifY2/nafY; (3) ORF13 ORF12-nifHDKnifTY ORF1 ORF2-nifEN. Unlike in A. vinelandii, where these genes are not contiguous on the chromosome, but broken into two regions of the genome, the genes characterized here in P. stutzeri are contiguous and present on a 30 kb region in the genome of this organism. Insertion mutagenesis confirmed that most of the nif and the rnf genes in A1501 were essential for nitrogen fixation. Using lacZ fusions it was found that nif and rnf gene expression was under the control of ntrBC, nifLA and rpoN and that the rnf gene products were involved in the regulation of the nitrogen fixation process.