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

MARÍA SOLEDAD MALORI ; PATRICIA LORENA ABDIAN ; ANGELES ZORREGUIETA ; JULIO JAVIER CARAMELO

Buenos Aires

Congreso; SAIB 2017; 2017

SAIB

In natural environments microbes live in multicellularstructures called biofilms, in which cells are embedded in a matrix ofself-produced biopolymers. The extracellular matrix determines the immediateconditions of life of biofilm cells, and also provides adhesion to surfaces andmechanical stability. Despite the importance of the matrix in the biofilm modeof life, very little is known about the mechanisms leading to matrix assemblyand the extracellular proteins involved in this process. We have recentlycharacterized the RapA lectin secreted by Rhizobium leguminosarum, which has aprofound impact in the organization of the biofilm matrix. The RapA lectininteracts specifically with the acidic exopolysaccharides (EPS/CPS) produced byR. leguminosarum in a calcium-dependent manner. The protein is composed of twoCHDL domains that are similar to the extracellular domains of eukaryotic cadherins.Aiming to obtain a tool to study the development of the matrix during biofilmformation, we dissect the protein in its two halves, and study the propertiesof the individual CHDL domains. The domains were amplified by PCR usingspecific primers, cloned as His tag fusions and purified from the solublefraction of Escherichia coli BL21(DE3) induced cells. The purified domains wereanalyzed by CD spectroscopy with the addition of calcium ions, and in afunctional test by means of a binding inhibition assay (BIA) with the EPS. Ourresults show that the lectin activity is confined to the carboxy terminal CHDLdomain of RapA, which contains the calcium binding site and is able to bind tothe EPS, although with less affinity than the entire RapA lectin.