Mutational Analysis of Plasmid R64 Thin Pilus Prepilin: the Entire Prepilin Sequence Is Required for Processing by Type IV Prepilin Peptidase

The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the pilS product, and a minor component, one of the seven pilV products. The pilS product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of the pilU product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with the pilV product. To reveal the relationship between the structure and function of the pilS product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant pilS products were analyzed. Four pilS mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type pilS gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the pilS product requires the entire prepilin sequence.Type IV pili are flexible, rod-like, polarly inserted surface appendages protruding from the cell surface of gram-negative bacteria including Pseudomonas aeruginosa, Bacteroides nodosus, Neisseria gonorrhoeae, Moraxella bovis, Vibrio cholerae, and enteropathogenic and enterotoxigenic Escherichia coli (9, 19, 20, 23, 27, 32). Type IV pili promote the attachment of bacterial pathogens to receptors of host cells during colonization, and they mediate the bacterial locomotion called twitching motility of P. aeruginosa (35) and the social gliding motility of Myxococcus xanthus (36). In addition, they act as receptors for pilus-specific bacteriophage (6).Type IV pili are polymers of type IV pilin subunits (23, 27), which are produced from type IV prepilins by the function of prepilin peptidases (18). In many cases, the N-terminal amino acid of mature pilin is phenylalanine and is N-methylated. In P. aeruginosa, both processing of prepilin and N-methylation of mature pilin are catalyzed by a single bifunctional enzyme, the PilD protein (28). Among all type IV pilins, the N-terminal region including the cleavage site is highly conserved. Particularly, the C-terminal amino acid of the prepeptide is invariantly glycine, and the fifth amino acid of mature pilin is always glutamic acid. The C-terminal one-third of mature pilin forms a disulfide loop between two conserved cysteine residues (21, 25).During bacterial conjugation, the donor cells harboring self-transmissible plasmids synthesize sex pili encoded by the genes on the plasmids (6). Sex pili of donor cells create a specific contact with recipient cells, leading to the formation of a mating pair. IncI1 plasmids such as R64 and ColIb-P9 form two types of sex pili, a thick rigid pilus and a thin flexible one (1, 2). Thick rigid pili are required for both surface and liquid mating, while thin flexible pili are required only for liquid mating. Cells producing R64 thin pili become sensitive to bacteriophages Iα and PR64FS, which adsorb to the shaft and tip of IncI1 thin pilus, respectively (4, 5).DNA sequence analysis of the R64 pil region responsible for thin-pilus formation revealed that the pil region consists of 14 genes, pilI through pilV, and that several pil products contain amino acid sequence homology with proteins involved in type IV pilus biogenesis (11) (Fig. ​(Fig.1A).1A). Thus, the R64 thin pilus was shown to belong to the type IV family, specifically group IVB, of pili. Open in a separate windowFIG. 1(A) Organization of the tra-pil region of plasmid R64. The horizontal bold line represents a restriction map. B, BglII; E, EcoRI; H, HindIII. The open bar above the map represents the extent of movement of the EcoRI site through DNA rearrangement of the shufflon. Below the map, the open reading frames are represented by open bars. tra, transfer; pil, formation of thin pilus; shf, shufflon; rci, recombinase for the shufflon. DNA regions of pKK641 and pKK692 are indicated above the map. The cross on pKK641 marks the location of the pilS1 mutation. (B) Amino acid sequence of the PilS protein. The downward arrow indicates the type IV prepilin cleavage site. The conserved glycine, glutamic acid, and two cysteine residues are indicated by the outline letters.R64 and ColIb-P9 thin pili were sedimented by ultracentrifugation from the culture medium, in which E. coli cells harboring R64- and ColIb-P9-derived plasmids had grown, and purified by CsCl density gradient centrifugation (13, 37). In negatively stained thin-pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. R64 and ColIb-P9 thin pili consist of a major 19-kDa pilin protein, the product of the pilS gene, and a minor 45-kDa protein, the product of the pilV gene. The amino acid sequence of the pilS product contains residues characteristic of a type IV prepilin, although its prepeptide is unusually long (Fig. ​(Fig.1B).1B). The pilS product is first synthesized as a 22-kDa prepilin and then cleaved between Gly²³ and Trp²⁴ to produce a 19-kDa protein via the function of the pilU product, prepilin peptidase. The N-terminal amino group of the processed PilS protein appears to be modified. The C-terminal segments of the pilV gene are under the control of shufflon DNA rearrangement mediated by the rci product (15, 16). The shufflon determines the recipient specificity in liquid mating by converting seven C-terminal segments of the pilV product (13, 14). The pilV product also carries a type IV prepilin cleavage site. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was shown and suggested to play an important role in liquid mating (37).Recently, the three-dimensional structure of the N. gonorrhoeae pilin was determined by X-ray crystallography (21). The monomer structure was an α-β-roll fold with an 85-Å N-terminal α-helical spine. The gross monomer structure resembles a ladle with the N-terminal half of the α-helical spine forming the handle. From the monomer structure, a model of fiber structure with a parameter of five turns per helix, 41-Å pitch, and 60-Å diameter (34) was proposed. In the model, the N-terminal α helices gather in the center of the fiber, forming a core of coiled α helices banded by a β sheet. Slight similarities including two conserved cysteine residues are noted between the amino acid sequences of R64 and N. gonorrhoeae pilins, suggesting that the two proteins fold similarly and then assemble to form similar fibers.In N. gonorrhoeae, P. aeruginosa, and V. cholerae, amino acid substitutions were introduced into the prepeptide and highly conserved N-terminal regions of prepilin genes (3, 22, 26). The mutant genes were analyzed with respect to processing, secretion, and function. The importance of the conserved glycine in the prepeptide and some hydrophobic amino acids in the N-terminal region has been established.This work was performed to reveal the relationship between the structure and function of the pilS product. Thirty-two missense and deletion mutations were introduced throughout the entire sequence of the pilS product by PCR and site-directed mutagenesis. The characteristics of the mutant pilS products were analyzed in terms of processing, secretion, and assembly to active thin pili with the pilV product. The activities of the thin pili composed of the mutant pilS genes were determined as the transfer frequency in liquid mating and the sensitivity to IncI1-specific phages.