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Proteomics-based identification of outer-membrane proteins responsible for import of macromolecules in Sphingomonas sp. A1: alginate-binding flagellin on the cell surface.

A nonmotile gram-negative bacterium, Sphingomonas sp. A1, directly incorporates macromolecules such as alginate through a "super-channel" consisting of a pit formed on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Here, we demonstrate the proteomics-based identification of cell-surface proteins involved in the formation of the pit and/or import of alginate. Cell-surface proteins were prepared from the outer membrane released as vesicles during the conversion of intact cells to spheroplasts. Seven proteins (p1-p7) with acidic isoelectric points were inducibly expressed in the outer membrane of strain A1 cells grown on alginate and showed significant identity with bacterial cell-surface proteins (p1-p4, TonB-dependent outer-membrane transporter; p5 and p6, flagellin; and p7, lipoprotein). Each mutant with a disruption of the p1-p4 or p6 gene showed significant growth retardation in the alginate medium. Flagellin homologues (p5 and p6) were further analyzed because strain A1 forms no flagellum. p5 was found to be uniformly distributed on the cell surface by immunogold-labeling electron microscopy and to exhibit alginate binding with a nanomolar dissociation constant by a surface plasmon resonance sensor. The cell surface of the p6 gene disruptant differed from that of the wild-type strain A1 in that pit formation was incomplete and cell-surface structures shifted from pleats to networks. These results suggest that, distinct from bacterial flagellins constituting a helical filament of flagella, strain A1 cell-surface flagellin homologues function as receptors for alginate and/or regulators of cell-surface structures.