Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice

Abstract

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.

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

“…Construction of strain 1507B, a nifL deletion non-polar mutant, was similar to strain 1507 previously described (Desnoues et al, 2003), except that it carries the KIXX cassette in the same orientation as the nifA gene. A nifL polar mutant, designated 1507A, was constructed for this work, as follows.…”

“…Nitrogenase activity was determined with bacterial suspensions incubated at an OD 600 of 0?1, in N-free minimal lactate medium, at 30 uC, under an argon atmosphere containing 1 % oxygen and 10 % acetylene, according to the derepression protocol described by Desnoues et al (2003). Nitrogenase specific activity is expressed as nmol ethylene min 21 (mg protein) 21 .…”

“…Plasmids and bacterial strains are listed in Table 1. P. stutzeri was grown in minimal lactate medium or in LB medium at 30 uC as described by Desnoues et al (2003). The yeast strain used for the two-hybird analysis was Saccharomyces cerevisiae PJ69-4A harbouring three reporter genes, lacZ, ADE2 and HIS3, under the control of the GAL promoter (James et al, 1996).…”

“…A 30 kb DNA region containing the nitrogen fixation (nif and rnf) genes has been previously characterized and the regulatory nifLA region mapped within the main nif cluster. By using different lacZ fusions, it was observed that nifA controlled the expression of other nif (and rnf) operons and that chromosomal nifLA-lacZ fusion expression was strongly reduced in the presence of oxygen and ammonia (Desnoues et al, 2003). As NtrC and RpoN were also found to control nifLA expression (Desnoues et al, 2003), this suggested that the regulation circuitry in P. stutzeri resembled more the situation in Klebsiella pneumoniae than in Azotobacter species, where nifLA expression is not impaired in the presence of ammonia (Blanco et al, 1993), although P. stutzeri is phylogenetically closer to Azotobacter (Rediers et al, 2004) than to Klebsiella.…”

“…It is now established that several strains of Pseudomonas stutzeri can fix nitrogen (Vermeiren et al, 1999;Rediers et al, 2004). P. stutzeri A1501, isolated from rice, fixes nitrogen in the free-living state, under microaerobic conditions in media devoid of ammonia (You et al, 1991;Lin et al, 2000;Desnoues et al, 2003). A 30 kb DNA region containing the nitrogen fixation (nif and rnf) genes has been previously characterized and the regulatory nifLA region mapped within the main nif cluster.…”