Towards smart N-biofertilizers: controlling bacterial population and fertilizing properties by metabolic engineering of Azotobacter vinelandii

CURATTI L; AMBROSIO R

San Luis

Congreso; XIIIV Congreso Anual de la Sociedad Argentina de Microbiología General; 2018

ASOCIACION CIVIL DE MICROBIOLOGIA GENERAL

The growing demand for food to support population increases has generated a strong dependence on fertilizers. Excessive use of nitrogen fertilizers leads to environmental impacts such as eutrophication of fresh water and air pollution. Agricultural promotion of biological N2-fixation appears as an alternative. However, it could only be exploited in a few symbiotic crops (legumes), displaying a strong specificity of the symbionts. On the other hand, free-living diazotrophs would bypass this specificity constraint, but normally only genetically modified strains display strong N-biofertilizing properties. To contribute to develop genetically modified N-biofertilizers that could be safely released to the field in the future, we prepared conditional lethal strains of Azotobacter vinelandii. These strains express the glutamine synthetase (GS) gene under an IPTG inducible promoter (trcP-glnA). Mutant cells cannot survive long in the absence of the inducer. However, cells can accumulate variable levels of GS as a function of the inducer?s accumulation. Most of the GS accumulates as an inactive covalently-modified enzyme. Upon shifting to non-inducing medium, cells gradually activated the GS by reversion of the covalent modification and the life span of the population was somehow proportional to the previous induction intensity. Thus, at low levels of induction cells stopped dividing after a few generations and started to release ammonium as a consequence of failure to assimilate it into amino acids. Conversely, GS overloaded cells, kept producing biomass at the expense of the accumulated GS for an extended life-span. When cell division split the GS pool below a threshold level, cells started to release ammonium, and finally the bacterial population also declined. The bacterial population at which a critical GS level was reached finally determined the overall ammonium that was produced. When these strains were inoculated into cultures of the alga Chlorella sp, we were able to confirm that bacteria with low levels of GS promoted algal growth at the expense of N2 from the air sooner than bacteria bearing higher levels of GS. However, after some generations, the N-fertilizing property became more prominent for the GS-loaded bacteria. It was also confirm that after the algal fertilization, the bacterial population tended to decline.Work is in progress to assess these prototypes of smart-biofertilizers in plants.