Role of RapD in the development of the biofilm matrix in Rhizobium leguminosarum bv. viciae 3841.

ZORREGUIETA A.; TARSITANO J.; RUSSO D.M.

San Luis

Congreso; XXIII Congreso Argentino de Microbiologia General SAMIGE 2018; 2018

Asociación Civil de Microbiología General SAMIGE

The ability to form biofilms confers bacteria several advantages to survive in unfavourable environments or colonize new niches. Understanding the process of developing a proper biofilm structure in Rhizobium leguminosarum is crucial to further expand the knowledge of this symbiont?s interaction with the host legumes and the soil particles Capsular (CPS) and extracellular polysaccharides (EPS) are key components of the biofilm developed by R. leguminosarum. Besides, extracellular proteins secreted by the Type I secretion system PrsDE participate in the formation of a mature biofilm structure, processing the chains of the polysaccharides or affecting the adhesive properties. Previous studies have shown that all members of the Rap proteins secreted by PrsDE share at least one EPS/CPS-binding domain called Ra (Rhizobium adhering) and could harbour another specific domain. In particular, RapA2 is an EPS-lectin that consists only in two Ra domains and is involved in the binding to the EPS/CPS and affects both thecompetition to infect the legume host and the biofilm matrix. Other members of the Rap proteins are the EPS-glycanases PlyA and B, which modify the length of the chains of the EPS and the recently identified RapD. RapD harbours one Ra domain with high similarity to Ra2 of RapA2 and a specific domain of unknown function. The aim of this study is to understand the role of RapD during biofilmformation. Using Rhizobium l. bv. viciae 3841 as a model, we generated isogenic strains harbouring a deletion in rapD gene as well as RapD overexpressing variants. We quantified extracellular RapD on the culture supernatant from different growth conditions and strains. Presence of the protein on the cell surface was also quantified in a wild type context and on an overexpressing context. Our findings suggest that almost all RapD secreted by the bacteria in a wild type context is sent to the extracellular media. We tested different aspects of biofilm formation like adhesion to abiotic surfaces, the possible impact on EPS-glycanases, surface characteristics and auto aggregative properties. The absence of RapD promotes bacterial autoaggregation and alters biofilm structures in Y minimal medium whereas swimming type motility was reduced in a RapD overexpressing context both in rich and minimal media. Our studies on the purified recombinant RapD suggest that, unlike RapA2, RapD might be able to form homo multimeric structures. Ongoing studies are focused on determining the polysaccharide binding capabilities as well as the affinity and the conditions under which multimeric structures are formed.