Insight into the RapA lectin and its use in the study of biofilm matrix formation by rhizobia

RUSSO, DANIELA; BERRETTA, MARCELO; CARAMELO, JULIO JAVIER; BENINTENDE, GRACIELA; MALORI, MARIA SOLEDAD; NAVAS, LAURA; ABDIAN, PATRICIA LORENA

Aarhus

Conferencia; Biofilms 8; 2018

Despite the importance of the matrix in the biofilm mode of life, little is known about the mechanisms leading to matrix assembly and the extracellular proteins involved in this process. We have previously characterized the RapA lectin secreted by Rhizobium legumi­nosarum and other rhizobia, and showed it has a profound impact in the organization of the biofilm matrix. The RapA lectin interacts specifically with the acidic polysaccharides (exo- and capsular polysaccharides) produced by R. leguminosarum in a calcium-dependent manner. The protein is solely composed of two homologous CHDL (cadherin-like) do­mains that adopt β-sheet conformation, a common fold in carbohydrate binding proteins. Given the large amount of available sequences, in this work we performed a phylogenetic analysis of CHDL domains in Raps (Rhizobium adhering proteins) secreted by rhizobia as a base to predict their functional properties. Then we dissected the RapA protein in its two halves, and studied the properties of the individual CHDL domains. The domains were PCR-amplified, cloned as 6xHistagfusions and purified from the soluble fraction of Escherichiacoli cell cultures. The individual CHDL domains of RapA were structurally analyzed by circular dichroism (CD) spectroscopy. A functional test by means of a binding inhibition assay (BIA) was carried out with the acidic exopolysaccharide, and also with the lipopolysaccharide (LPS) to determine if RapA could act as an anchor for the capsular polysaccharide to the cell surface. No LPS binding by RapA or by the individual CHDL domains were detected under the assay conditions. However, our results show that the lectin activity is confined to the carboxy terminal CHDL domain (Cter-CHDL), which folds in response to calcium addition and is able to bind to the exopolysaccharide, although with less affinity than the entire RapA lectin. Moreover, the green fluorescent protein (GFP) was fused to RapA and to its Cter-CHDL, and the performance of the purified fusion proteins to target the exopolysaccharide was assessed, showing they could be useful as novel fluorescent probes for acidic exopolysaccharides produced by several rhizobia of agronomical interest.