REGULARLY ARRANGED PROTEIN ON THE SURFACES OF GRAM‐NEGATIVE BACTERIA

Abstract

SUMMARY Many species of Gram‐negative bacteria carry a layer of regularly arranged sub‐units on the outer surface of their outer membrane. The subunits are arranged tetra‐gonally or hexagonally and have centre‐to‐centre spacings ranging from 4 to 35 nm, depending on the bacterial species. The regularly arranged layer has been detected by electron microscopy in whole cells, cell walls, outer membranes and assemblies of isolated subunits. The regular arrays can be seen in intact cells by shadowing or freeze‐etching and, occasionally, in thin sections or after negative‐staining. Freeze‐etching and negative‐staining have been used to show the regular arrays in isolated cell walls and outer membranes. Negative‐staining is used in the examination of assembled isolated subunits. Optical diffraction of electron micrographs provides more detailed information of the fine structure of the subunits in the regular array. The regularly arranged surface layer can be removed by protein perturbants, by chelation of divalent cations with EDTA and EGTA, by cation substitution or by acidification. The two surface subunits which have so far been purified have been found to be acidic proteins with molecular weights of 67000 (Acimtobacter 199A) and 140000 (Spirillum serpens). In Acinetobacter 199A the surface protein is attached to the protein of the outer membrane through a salt bridge involving Ca2+ or Mg2+. Evidence exists that there may be a similar mode of attachment in other species. Isolated surface proteins from Acinetobacter 199A and from Spirillum spp. have the ability to reassemble into the same pattern as that seen on the bacterial surface, either in isolation or in the presence of cell‐wall fragments to act as nucleating agents. Self‐assembly of Acinetobacter 199A a‐protein requires chloride ions. Acinetobacter 199A a‐protein can only be incorporated onto the bacterial surface if an intact lipopolysaccharide membrane is formed first to receive the intrinsic membrane proteins to which the a‐protein attaches. Impairment of lipopolysaccharide synthesis by bacitracin prevents incorporation of a‐protein and other membrane proteins into the outer membrane. Continuing incorporation of pre‐formed radioactive a‐protein into the outer membrane in Acimtobacter 199A can be detected for 10 min after transference from radioactive to non‐radioactive growth medium. Spirillum metamorphum, Flexibacter sp. and Acinetobacter 199A synthesize more surface protein than is required to cover the cell surface. The excess is secreted into the growth medium. The regularly arranged surface protein of Acinetobacter 199A provides partial protection against isolated lysosomal proteinases from polymorphonuclear leucocytes. Spirillum spp. is protected from Bdellovibrio invasion by the surface protein. Secreted a‐protein from Acimtobacter 199A has phospholipase A, activity. No phospholipase activity can be detected when the a‐protein is bound to the bacterial surface.