Structure and function of conjugative (F) pili in Escherichia coli K-12

Abstract

Conjugal DNA transfer enables specialized bacterial cells to donate DNA directly to other cells. All of the proteins required for DNA transfer functions are almost invariably encoded by DNA transfer ({dollar}tra{dollar}) genes contained in a plasmid. Presumably, the {dollar}tra{dollar} gene products assemble into a trans-envelope organelle that mediates DNA transfer. At present, the conjugative pilus is the only structure associated with DNA transfer in Gram-negative bacteria. These extracellular filaments are elaborated by donor cells and are required for the recognition and positioning of suitable recipient cells prior to DNA transfer. The F pili, encoded by genes on the F plasmid, are composed of a single subunit, F pilin.;A lac-tra operon fusion plasmid was constructed, pTG801, containing only those F plasmid genes suggested by genetic studies to be required for F pilus synthesis. Cells containing pTG801 expressed the {dollar}tra{dollar} genes under {dollar}lac{dollar} control and assembled functional F pili. Because pTG801 transformants do not transfer DNA, F pilus function was evaluated using bacteriophage that adsorb to the filament. E. coli strain DH1(pTG801) was sensitive to the spherical RNA bacteriophage QB and the filamentous DNA bacteriophage f1, which bind to the sides and tips of F pili, respectively. However, several observations suggested that pTG801 pili are actually structural variants of F pili, even though pTG801 contains all of the known {dollar}tra{dollar} genes required for F pilus synthesis. Electron microscopy studies using spherical bacteriophage R17 decoration of pili and immunogold labeling of pili confirmed this hypothesis. R17 adsorbs to the sides of normal F pili, but R17 did not adsorb well to pTG801 pili. Two monoclonal antibodies that recognize epitopes at and near the amino terminus of F pilin do not decorate the sides of normal F pili. Apparently, the amino terminus is masked in the structure of the normal filament. However, both antibodies decorated pTG801 pili laterally. Moreover, pTG801 pili were restored to normal by the presence of a second compatible plasmid, pRS31, which must therefore contain at least one as yet unidentified tra gene affecting F pilus structure.