Table_3_RapD Is a Multimeric Calcium-Binding Protein That Interacts With the Rhizobium leguminosarum Biofilm Exopolysaccharide, Influencing the Polymer Lengths.docx

Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix’s properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.

豌豆根瘤菌（Rhizobium leguminosarum）可合成一类酸性多糖，其中大部分被分泌至胞外环境，称为胞外多糖（exopolysaccharide, EPS），另有部分以荚膜多糖（capsular polysaccharide, CPS）的形式附着于细菌表面。Rap蛋白作为PrsDE型I分泌系统（type I secretion system, TISS）的胞外蛋白底物，至少包含一个Ra/CHDL（类钙粘蛋白，cadherin-like）结构域，可通过Ply糖苷酶（Ply glycanases）裂解多糖，或改变细菌黏附特性参与生物被膜基质（biofilm matrix）的构建。已有研究证实，胞外RapA2（一种单体CPS结合钙凝集素）的缺失或过量表达会改变生物被膜基质的特性。本研究揭示了新型Rap蛋白RapD的功能：该蛋白包含N端Ra/CHDL结构域与功能未知的C端区域，可完全释放至胞外环境，并以依赖PrsDE的方式与其他Rap蛋白共同分泌。此外，在利于EPS生成的培养条件下，生物被膜中RapD的分泌水平显著升高。值得注意的是，对ΔrapA2ΔrapD双突变株所产EPS进行尺寸排阻色谱（size exclusion chromatography）分析发现，其EPS分子的粒径显著小于单突变株与野生型菌株（wild type strain），提示RapA2与RapD均可影响细胞表面的EPS加工过程。生物物理研究证实，钙离子可诱导重组RapD形成正确折叠与多聚化；此外，在EPS存在时，RapD还会发生进一步的构象变化。酶联免疫吸附试验（Enzyme-Linked ImmunoSorbent Assay, ELISA）与结合抑制试验（Binding Inhibition Assays, BIA）结果显示，RapD可特异性结合EPS，且该相互作用可能涉及半乳糖残基（galactose residues）。综合以上结果可知，RapD是一类与生物被膜基质相关的多聚体蛋白，可通过影响根瘤菌生物被膜的主要结构组分——EPS的特性，参与调控根瘤菌生物被膜的形成。