Ammonium excretion properties of azotobacter vinelandii glna conditional-letal mutant strains

AMBROSIO R; ORTIZ MÁRQUEZ JC; CURATTI L

Cordoba

Congreso; XI Congreso Anual de la Sociedad Argentina de Microbiología General; 2015

ASOCIACION CIVIL DE MICROBIOLOGIA GENERAL

AMMONIUM EXCRETION PROPERTIES OF Azotobacter vinelandii glnA CONDITIONAL-LETAL MUTANT STRAINSEXCRECION DE AMONIO EN CEPAS DE Azotobacter vinelandii CON UNA MUTACION Rafael Ambrosio, Juan Cesar Ortiz-Marquez, Leonardo CurattiInstituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET), Mar del Plata, Argentina. Fundación para Investigaciones Biológicas AplicadasCurrent demand of food and bioenergy pushes agriculture to relay on extensive use of fertilizers, pesticides and other chemical products, to improve crop productivity. However, extensive use of these agrochemicals is harmful to the environment, often times is also to human?s health, and represents a significant share of agricultural costs. Thus, we are interested in developing N-biofertilizing bacteria. We have isolated Azotobacter vinelandii strains with deletion in nifL that are impaired in nif-genes expression switch-off and produced an excess of ammonium that is released into the medium. We also constructed A. vinelandii strains with a single mutation in the gene encoding for glutamine synthetase, glnA-D49S, which slows down diazotrophic growth and also excretes ammonium. Double mutant strains, show a dramatic increase in the initial rate of ammonium release into the medium but failed to sustain the production probably because of a major impairment of protein synthesis.In this work we advanced in the isolation of mutant strains with attenuated glutamine synthetase (GS) activity that, by means of ensuring a basal level of protein synthesis, might extend the phase of ammonium release into the medium. As a first approach we challenged A. vinelandii cells with different concentrations of the GS inhibitor L-methionine sulfoximine and confirmed the hypothesis that different levels of GS inhibition impacts on the amplitude of the maximum ammonium excretion and also on the extension of the excretion phase. To stably introduce a tunable genetic control of GS activity, we prepared vectors based on the Escherichia coli lacI-lacZYA operon to be used for IPTG control of gene expression in A. vinelandii. This bacterium is unable to use lactose as a sole source of carbon and lacks a transport system for the disaccharide and its analogs. Thus, we introduce the lacZYA in the A. vinelandii chromosome. Then we prepare vectors that allowed substitution of E. coli lacIq (lac repressor) and lacO (lac operator)-bearing promoters for A. vinelandii native promoters of target genes. As positive control, we confirmed fine tuning of the expression of nifB in both wt and lacZYA+ A. vinelandii, although with a different saturation kinetics. We concluded that passive diffusion of IPTG in the wt background would broaden the range for tuning target gene expression. When using glnA as a target gene, as expected, we obtained conditional lethal strains, since A. vinelandii only assimilates ammonium into amino acids by this enzyme. Cells cultivated overnight in the presence of ammonium and 0.3 mM IPTG and then for 6 h in the absence of ammonium and IPTG (for depletion of GS) were transferred to fresh medium lacking ammonium and containing different concentrations of IPTG. While bacteria incubated in the absence of IPTG presented the highest levels of ammonium excretion, 10 μM IPTG produced a lower maximum level and no ammonium excretion was observed at 0.1 or 10 mM IPTG.