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

MALORI, M.S; RUSSO D.M; CARAMELO J.J; LAURA E. NAVAS; MARCELO F. BERRETTA; GRACIELA B. BENINTENDE; ABDIAN P.L.

Aarhus

Congreso; Biofilms 8; 2018

Despite the importance of the matrix in the biofilm mode of life, little is known about themechanisms leading to matrix assembly and the extracellular proteins involved in this process.We have previously characterized the RapA lectin secreted by Rhizobium leguminosarum andother 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 capsularpolysaccharides) produced by R. leguminosarum in a calcium-dependent manner. The proteinis solely composed of two homologous CHDL (cadherin-like) domains that adopt -sheetconformation, a common fold in carbohydrate binding proteins. Given the large amount ofavailable sequences, in this work we performed a phylogenetic analysis of CHDL domains inRaps (Rhizobium adhering proteins) secreted by rhizobia as a base to predict their functionalproperties. Then we dissected the RapA protein in its two halves, and studied the properties ofthe individual CHDL domains. The domains were PCR-amplified, cloned as 6xHistag fusionsand purified from the soluble fraction of Escherichia coli cell cultures. The individual CHDLdomains of RapA were structurally analyzed by circular dichroism (CD) spectroscopy. Afunctional test by means of a binding inhibition assay (BIA) was carried out with the acidicexopolysaccharide, and also with the lipopolysaccharide (LPS) to determine if RapA could actas an anchor for the capsular polysaccharide to the cell surface. No LPS binding by RapA orby the individual CHDL domains were detected under the assay conditions. However, ourresults 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 theexopolysaccharide, although with less affinity than the entire RapA lectin. Moreover, the greenfluorescent protein (GFP) was fused to RapA and to its Cter-CHDL, and the performance ofthe purified fusion proteins to target the exopolysaccharide was assessed, showing they couldbe useful as novel fluorescent probes for acidic exopolysaccharides produced by severalrhizobia of agronomical interest.