CHARACTERIZATION OF THE CHDL DOMAINS OF RapA, AN EXTRACELLULAR LECTIN FROM Rhizobium leguminosarum INVOLVED IN BIOFILM MATRIX ASSEMBLY

ABDIAN, PATRICIA LORENA ; MALORI, MARIA SOLEDAD; ZORREGUIETA, ANGELES; CARAMELO, JULIO JAVIER

Ciudad Autonoma de Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS (SAIB); 2017

In natural environments microbes live inmulticellular structures called biofilms, in which cells are embedded in amatrix of self-produced biopolymers. The extracellular matrix determines the immediate conditions of life of biofilm cells, and also provides adhesion to surfaces and mechanical stability. Despite the importance of the matrix in the biofilm mode of life, very little is known about the mechanisms leading to matrix assemblyand the extracellular proteins involved in this process. We have recently characterized the RapA lectin secreted by Rhizobium leguminosarum, which has a profound impact in the organization of the biofilm matrix. The RapA lectin interacts specifically with the acidic exopolysaccharides (EPS/CPS) produced by R. leguminosarum in a calcium-dependent manner. The protein is composed of two CHDL domains that are similar to the extracellular domains of eukaryotic cadherins.  Aiming to obtain a tool to study the development of the matrix during biofilm formation, we dissect the protein in its two halves, and study the properties of the individual CHDL domains. The domains were amplified by PCR using specific primers, cloned as His tag fusions and purified from the soluble fraction of Escherichia coli BL21(DE3) induced cells. The purified domains were analyzed by CD spectroscopy with the addition of calcium ions, and in a functional test by means of a binding inhibition assay (BIA) with the EPS. Our results show that the lectin activity is confined to the carboxy terminal CHDL domain of RapA, which contains the calcium binding site and is able to bind to the EPS with higher affinity than the entire RapA lectin.