Metabolic regulation network adjusting the rates of nitrogen fixation and assimilation in Azotobacter vinelndii

ORTIZ MÁRQUEZ JC; CURATTI L

Mar del Plata

Congreso; VIII Congreso de Microbiologia General; 2012

Sociedad Argentina de Microbiologia General

Biological nitrogen fixation (BNF) is the natural conversion of nitrogen from the air into ammonium that display diazotrofic bacteria and archaea.  An very important part of research on BNF is focused on the posibility of replacing part of the nitrogen fertilizers demand of crops by associations with diazotrofic bacteria, either symbiotic or associative. While symbiotic associations appeared to be more efficient at channeling products of nitrogen fixation to plants, the use to be highly strain (and/or cultivar) specific, limiting a broader application. On the other hand, associative or free-living nitrogen fixers normally excreted limited amounts of ammonium into their surroundings. We are interested in the improvement of free-leaving strains for their capacity of ammonium excretion towards the development of bacterial nitrogen-biofertilizers by metabolic engineering. We have already showed that Azotobacter vinelandii strains with mutations in the nifL gene are impaired in sensing ammonium cellular status, express nitrogenase and accessory nif genes constitutively and excrete ammonium to the culture medium. These strains were shown to be a prototype for the diazotrophic culture of eukaryotic microalgae in artificial symbiotic culture (Ortiz Marquez et al 2012 AEM 78, 2345).  This work was aimed at further improvement of strains by genetic engineering of ammonium assimilation.  In A. vinelandii ammonium assimilation takes place exclusively by the glutamine synthetase (GS)- glutamate synthase (GOGAT) cycle. Thus, GS null mutants have not been isolated up-to-date. It has been shown that D50 participates of ammonium binding and GS affinity towards this substrate in Escherichia coli GS. To attenuate the metabolic flux of ammonium towards its assimilation into amino acids in A. vinelandii, were identified by sequence alignment D49 as a candidate active site amino acid and constructed the corresponding D49A and other mutant strains. These strains presented 30% GS activity and increased ammonium release to the medium. However, when introduced into the nifL-null genetic background, these mutant GS allele abolished the ammonium overproducing trait of nifL mutants. Further experiments showed that an inhibitor of GS activity (MSX) produced a decrease of nitrogenase genes expression. We also observed that GS activity is reduced to 25% in nifL mutant strains and remained insensitive to ammonium loaded in the medium and considerably less sensitive to partial inhibition by MSX. These results might provide strong genetic confirmation for a suspected metabolic regulation network that adjusts the rates of nitrogen fixation and assimilation in A. vinelndii.