Characterization of the acil-homoserine lactone degradation by Azospirillum brasilense Az39

GUALPA, JOSE; MORA, V; NIEVAS, SOFÍA; MOLINA, ROMINA; CASSAN, FABRICIO; LOPEZ, GASTON; CONIGLIO, ANAHI

Simposio; IV REUNIÓN CONJUNTA DE SOCIEDADES DE BIOLOGÍA DE LA REPÚBLICA ARGENTINA; 2020

A. brasilense Az39 is a PGPR strain with the ability to promote the plant growth and crop yield under agronomic conditions mainly through its capacity to fix atmospheric nitrogen and to produce several phytohormones. Although the mechanisms related to growth promotion have been studied in detail in this bacterial genus, we know little about the mechanisms that govern the bacteria-bacteria interaction and particularly those related with the quorum phenomenon. In our laboratory, we have confirmed that A. brasilense Az39 does not produce acyl-homoserine lactones (AHL) (quorum sensing) but instead degrades these molecules (quorum quenching) due to an enzymatic activity. The objective of this work was to determine the type of enzyme involved in the degradation of AHLs in Az39 and to verify its cellular location. Initially, we analysed whether the inactivation of AHLs was due to the presence of lactonase-type enzymes and for this purpose we cultured the bacteria in 5 ml of MMAB medium supplemented with 10 µM of AHLs (C6-HSL and C10-HSL) for 16 h at 240 rpm, 30 °C and pH 6.5-7.0. After the incubation time, 1 ml of the culture was taken and 1 M HCl was added until achieving a pH of 2.0, finally, 5 µl samples were taken from each reaction mixture at different time intervals (5, 30 and 60 min) to be analyzed by bioassays with reporter strains. To determine the location of the enzyme, some pre-incubated treatments were with different AHLs were performed. These treatments were: (T1) LB + 10 µM of each AHL; (T2) Az39 filtered supernatant; (T3) Az39 filtered and denatured supernatant; (T4) Az39 denatured (100 °C-30 min) and (T5) Az39. All treatments were analyzed using reporter strains. Finally, for the identification of the gene responsible for the degradation activity of AHLs in Az39, an insertional mutant strain was obtained for a putative gene. Our results did not show re-lactonization of AHL after acid treatment, therefore the participation of lactonases in the inactivation of these intercellular signaling molecules by Az39 was ruled out. In contrast, denaturation tests allowed us to suggest that the enzyme responsible for the acylase activity could be bound to the plasma membrane or released to the periplasm. The mutant strain was developed in a gene annotated by sequence homology as a putative penicillin acylase (EC. 3.5.1.11) and it was unable to degrade synthetic AHLs in vitro. These results confirm that A. brasilense Az39 does not produce AHLs but degrades them through the expression of a gene that codes for a putative acylase-type enzyme with a cellular location.