Thin pilus PilV adhesins of plasmid R64 recognize specific structures of the lipopolysaccharide molecules of recipient cells

Abstract

IncI1 plasmid R64 encodes a type IV pilus called a thin pilus, which includes PilV adhesins. Seven different sequences for the C-terminal segments of PilV adhesins can be produced by shufflon DNA rearrangement. The expression of the seven PilV adhesins determines the recipient specificity in liquid matings of plasmid R64. Salmonella enterica serovar Typhimurium LT2 was recognized by the PilVA' and PilVB' adhesins, while Escherichia coli K-12 was recognized by the PilVA', PilVC, and PilVC' adhesins. Lipopolysaccharide (LPS) on the surfaces of recipient cells was previously shown to be the specific receptor for the seven PilV adhesins. To identify the specific receptor structures of LPS for various PilV adhesins, R64 liquid matings were carried out with recipient cells consisting of various S. enterica serovar Typhimurium LT2 and E. coli K-12 waa mutants and their derivatives carrying various waa genes of different origins. From the mating experiments, including inhibition experiments, we propose that the GlcNAc(alpha1-2)Glc and Glc(alpha1-2)Gal structures of the LPS core of S. enterica serovar Typhimurium LT2 function as receptors for the PilVB' and PilVC' adhesins, respectively, while the PilVC' receptor in the wild-type LT2 LPS core may be masked. We further propose that the GlcNAc(beta1-7)Hep and Glc(alpha1-2)Glc structures of the LPS core of E. coli K-12 function as receptors for the PilVC and PilVC' adhesins, respectively.

FIG. 1.

(A) Structure of the shufflon. The gene organization of pilU, pilV, the shufflon, and the rci region of plasmid R64 is shown below a restriction map. E, EcoRI; P, PstI; S, SspI; Sp, SphI; V, EcoRV. The panel represents an arrangement (corresponding to pKK010-85) of many isomers of the shufflon subjected to multiple DNA inversions. Open bars A, B, C, and D, shufflon segments; filled triangles, seven sfx sequences; arrows, coding sequences for pilU, pilV and the variable C-terminal segments encoded by it, and rci. (B and C) LPS molecules of S. enterica serovar Typhimurium LT2 (B) and E. coli K-12 (C) (2, 5, 22). Shown are the proposed LPS structures together with the genes encoding the enzymes thought to be responsible for the generation of each linkage. Brackets, O-antigen repeating units. In E. coli K-12 LPS, the terminal GlcNAc residue in brackets was shown to be an incomplete O-antigen moiety (2). The structure of the LPS core shows heterogeneity, as indicated by parentheses. Abbreviations: AbeOAc, O-acetylabecose; Gal, galactose; Glc, glucose; Hep, heptose; KDO, 3-deoxy-d-manno-2-octulosonic acid; Man, mannose; P, phosphate; PPEA, pyrophosphoethanolamine; Rha, rhamnose. The specific receptor structures for the PilVB′, PilVC, and PilVC′ adhesins in the LPS molecules are indicated.

FIG. 2.

Gel electrophoretic analysis of chimeric LPSs. The LPSs from S. enterica serovar Typhimurium LT2 strains harboring the indicated plasmids were analyzed by SDS-PAGE and visualized by silver staining. wt, wild type.

FIG. 3.

Effects of Glc(α1-2)Glc, Glc(α1-2)Gal, and LPS on PilVC′-mediated liquid matings. E. coli K-12 W3110 harboring pKK661 together with pKK641C′ and TN102 were used as donor and recipient strains, respectively. Donor and recipient cultures were diluted 100-fold with LB broth before mixing. As inhibitor molecules, 1 mM Glc(α1-2)Glc (filled triangles), 1 mM Glc(α1-2)Gal (filled squares), and 0.1 mM LPS from E. coli K-12 TN102 (filled circles) were added to the mating mixture. The control mating mixture (open circles) did not contain any inhibitor molecule. The transfer frequency is expressed as a percentage of the number of donor cells and was determined at time intervals.