ENVIRONMENTAL BACTERIA FROM ARGENTINE PAMPAS WITH ABILITY TO DEGRADE GLYPHOSATE

GARAVAGLIA, B.S.; OTTADO, JORGELINA; PIAZZA, AINELÉN; MASOTTI, FIORELLA; GOTTIG, NATALIA

Rosario

Congreso; SAMIGE-SAIB-2020; 2020

The broad-spectrum systemic herbicide Glyphosate (GLY), N (phosphonomethyl)glycine, is a phosphonate (Phn) characterized by a chemically stable carbon to phosphorus (C?P) bond. GLY based herbicides (GBH) have become the most widely used worldwide, and consequently, their residues have got widespread occurrence in different environments. Moreover, GLY has been recently classified as ?probably carcinogenic to humans? by the International Agency for Research on Cancer (IARC). Therefore, the importance of studying mechanisms of Phn biodegradation is self-evident. In our laboratory, we have isolated and characterized 62 bacterial strains able to grow in a minimal culture medium (MS1) with GLY as the only phosphorous (Pi) source from an Argentine Pampas location that massively uses GBH, and identified. Bacteria from the genera Achromobacter, Acinetobacter, Agrobacterium, Ochrobactrum, Pantoea and Pseudomonas were found and growth curves of one representative strain from the 13 different bacterial species isolated were obtained. Particularly, Agrobacterium tumefaciens CHLDO, Achromobacter denitrificans SOS5, Achromobacter insolitus SOR2, Achromobacter xylosoxidans SOS3, and Ochrobactrum haematophilum SR were able to grow in 1.5 mM of GBH, pure GLY or aminomethylphosphonic acid (AMPA), GLY main degradation subproduct, within 100 hours. To analyze the ability of all these bacterial strains to consume GLY and AMPA, supernatants of grown cultures were analyzed by TLC at 48 and 72 h. In GBH, the highest GLY degradation was observed for O. haematophilum SR, A. tumefaciens CHLDO and Achromobacter spp., showing GLY degradation rates at 72 h of 55%, 42%, 17-26%, respectively. In MS1+GLY, A. tumefaciens CHLDO showed the best performance; and in MS1+AMPA, besides the five previously studied bacteria, Pantoea ananatis CAS4 was included. All of them were able to consume AMPA reaching values between 45 and 70%. Finally, A. tumefaciens strain CHLDO was chosen for genome sequencing and further analysis. Particularly, Phosphonate Utilization genes (phn genes), involved in phosphonate catabolism and thus GLY degradation, via C-P lyase pathway, were identified. Hence, phn genes comprise 15 open reading frames, designated as phnFGHIJKLOCDE2E1-duf1045phnMN within almost four transcriptional units, were identified. In order to evaluate the capacity of GBH to activate transcription of the different phn genes in A. tumefaciens CHLDO compared to phosphate grown cells, the expression of all genes from phn cluster was analyzed by qRT-PCR assays. Out of the four predicted transcriptional units, genes from three of them (phnF, phnGIJKLO and phnCDE2E1) showed increased expression in the presence of GBH, meanwhile, the last operon duf1045phnMphnN showed no significant changes in transcripts levels. Overall, the advantage of deciphering molecular determinants of phosphonate degradation gives promising tools for bioremediation techniques applicable to GLY-contaminated environments.