Proteomics-Based Identification of Outer-Membrane Proteins Responsible for Import of Macromolecules inSphingomonassp. A1:  Alginate-Binding Flagellin on the Cell Surface^,

Abstract

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.

Superchannel of Bacteria: Biological Significance and New Horizons

“…This evidence indicates that p5 and p6 are essential to the formation of the pit and the maintenance of cell surface structure and that they participate in the determination of cell destiny. 12) Flagellins are exported from the cytoplasm to the cell surface through the type-III exporter encoded by the flagellum cluster, 16) supporting the notion that the type-III secretion system evolved from the flagellar export apparatus, but the expression of p5 is suppressed in mutant cells with a disrupted peptidoglycan hydrolase gene, suggesting the presence of an alternative pathway to export flagellin to the cell surface and to localize it at the cell envelope. 12) If this is the case, flagellins destined for the outer membrane must cross the inner membrane and periplasm and be assembled in the outer membrane containing GSL, a more hydrophobic lipid molecule than LPS, in their appropriate conformation, possibly in the absence of a biochemical energy supply.…”

“…The two proteins resemble each other in primary structure, have significant homology with flagellin, a component of the flagella of Gram-negative bacteria, and are expressed in the outer membrane in the presence of alginate, although neither flagellation nor motility has been confirmed for strain A1 cells. 12) Both p5 and p6 of strain A1 exhibit high similarity with bacterial flagellins in the N-and C-terminal domains, although little homology is observed in the central domain. 12) Furthermore, part of the central domain (approximately 100 amino acid residues) of p5 and of p6 is missing in comparison with the structures of Escherichia coli and Salmonella typhimurium flagellins, resulting in the formation of shaft-type flagella with no propeller.…”

“…12) Both p5 and p6 of strain A1 exhibit high similarity with bacterial flagellins in the N-and C-terminal domains, although little homology is observed in the central domain. 12) Furthermore, part of the central domain (approximately 100 amino acid residues) of p5 and of p6 is missing in comparison with the structures of Escherichia coli and Salmonella typhimurium flagellins, resulting in the formation of shaft-type flagella with no propeller. Since certain pseudomonades that form a polar flagellum produce flagellins lacking part of the central domain, 15) the deletions observed in p5 and p6 are irrelevant to the fact that strain A1 has no flagella.…”

“…A mutant with disruption of p1, p2, p3, or p4 shows apparent growth retardation on alginate as a carbon source. 12) Bacterial outer membrane transporters homologous to p1-p4 are known to incorporate the ironsiderophore complex using energy generated through transient association with an inner membrane complex (TonB-ExbB-ExbD). 13) Since alginate exhibits an ability The mechanism of the transport of alginate, given as a representative among the macromolecules imported, is described in the text.…”

“…This fact suggests that the two flagellin homologs are involved in alginate signaling as CD44-like receptors, a mammalian transmembrane receptor (K d = nM) for hyaluronan, 17) rather than in the harvesting of alginate on the cell surface. E. coli flagellin can also bind to alginate with a K d on the nM level, 12) indicating that alginate-binding is an inherent property of the protein. Flagellin might first be utilized as a cell surface protein and then undergo a change into a flagellar protein, or vice versa.…”