Molecular basis for cell surface architecture and evolution of bacterial flagellin by structure and unction analysis of the protein

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Alginate-assimilating Sphingomonas sp. strain Al forms a mouth-like pit through the rearrangement of cell surface pleat molecules and incorporates a macromolecule alginate through the pit The pit functions as a concentrator for extracellular polysaccharides. Two proteins, p5 (40 kDa) and p6 (31 kDa), are inducibly expressed on the cell surface in the presence of alginate, suggesting that they are responsible for the formation of the pit In this study, the structure and function of p5 and p6 have been analyzed.Both of p5 and p6 are similar to bacterial flagellins, although strain Al forms no flagella. A p6-disruptant shows significant growth retardation in the alginate medium, and double disruption of p5 and p6 results in growth failure, indicating that these flagellin homologs might be essential for cell viability. The cell surface of the p6-disruptant differs from that of wild-strain Al in that the formation of the pit is incomplete and the cell surface structure changes from a pleat … More structure to a network one. Surface plasmon resonance biosensor analysis indicated that both proteins specifically bind alginate with high affinity (dissociation constant Kd〓〜 nM). Based on these results, p5 and p6 localized on the cell surface can be identified as a receptor for external alginate.p5 consists of N-/C-terminal α (α1 + α2) domain and central B domain. Since p5 mutant lacking α_1 domain showed no alginate-binding ability, α_1 domain is essential to bind to alginate. In comparison of p5 with the Salmonella flagellated flagellin, the structure of α (α1 + α2) domain is similar each other, indicating that the alginate-binding ability commonly observed in bacterial flagellins is due to a domain conserved. On the other hand, p5 β domain is structurally similar to the finger domain of T4 phage protein gp11 located in the hinge region between the base plate and tail fiber. The binding of the finger domain to the base plate protein gp10 is important for the attachment of the tail fiber on the bacterial cell surface. The finger domain shows an affinity with the other protein, suggesting that p5 β domain functions as an anchor interacting with the strain Al cell surface molecules and contributes to the orientation of alginate-binding a domain to the external milieu. Less