PURIFICATION AND CHARACTERIZATION OF THIN PILI OF INCI1 PLASMIDS COLIB-P9 AND R64 - FORMATION OF PILV-SPECIFIC CELL AGGREGATES BY TYPE-IV PILI

Thin pill of the closely related IncI1 plasmids ColIb-P9 and R64 are r equired only for liquid mating and belong to the type N family of pill , They were sedimented by ultracentrifugation from culture medium in w hich Escherichia coli cells harboring ColIb-P9- or R64-derived plasmid s had been grown, and then the pill were purified by CsCl density grad ient centrifugation. In negatively stained thin pilus samples, long ro ds with a diameter of 6 nm, characteristic of type IV pill, were obser ved under an electron microscope. Gel electro phoretic analysis of pur ified ColIb-P9 thin pill indicated that thin pill consist of two kinds of proteins, pilin and the PilV protein, Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa pr otein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segm ents of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV p roteins were revealed to comprise a minor component of thin pill, Form ation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pill w as demonstrated and may play an important role in liquid mating.