Enterococcus faecalisPcfC, a Spatially Localized Substrate Receptor for Type IV Secretion of the pCF10 Transfer Intermediate

Abstract

The type IV secretion (T4S) systems are ancestrally related to bacterial conjugation machines and are used by many species of gram-negative and gram-positive bacteria as well as the thermophilic archaeon Sulfolobus (19,31,32,61). Most T4S systems translocate DNA or protein substrates directly to target cells by a cell contact-dependent process, and some alternatively translocate substrates to or from the milieu (5,6,20). Many systems translocate DNA substrates within or between bacterial species, although a large number of systems have been adapted by pathogenic species to deliver DNA or protein substrates to fungal, plant, or human cells (7,17,53). The T4S systems are composed of an envelope-spanning secretion channel and a surface structure such as a pilus for gram-negative bacteria or a protein adhesin for gram-positive bacteria to mediate attachment to target cells (1, 20, 32). These general properties-a wide phylogenetic distribution, functional versatility, and structural diversity-make T4S systems excellent subjects for mechanistic studies exploring the evolution of biological complexity. At this time, however, detailed structurefunction comparisons are not possible due to the paucity of information available for systems other than a few gram-negative models, e.g., the conjugation systems encoded by F, RP4, and R388 plasmids and the A. tumefaciens VirB/D4 T4S system.Here, we initiated studies of the pheromone-inducible conjugation system of the Enterococcus faecalis plasmid pCF10 (25). This 67.6-kb plasmid is a member of the family of sex pheromone plasmids found to date only in the enterococci but with the capacity to mobilize transfer of oriT-containing plasmids to Lactococcus lactis and Streptococcus agalactiae (64). pCF10 carries the tetracycline resistance conjugative transposon Tn925 (22) and codes for pheromone sensing and response functions, conjugation functions, and a surface adhesin termed aggregation substance (AS) which is important both for conjugation and virulence (38,68,69). In addition to its medical importance as a reservoir for antibiotic resistance and other virulence traits, the pCF10 transfer system is an attractive model gram-positive T4S system for mechanistic analysis because T4S machine assembly and function are tightly controlled at the transcriptional level, many features of pheromone-inducible regulation are known, and very high plasmid transfer rates are achieved soon after induction (25,38).Bacterial conjugation can be viewed as three biochemical reactions coupled in time and probably also in space. One or more processing factors termed the DNA transfer and replication (Dtr) proteins initiate transfer by assembling as a relaxosome at the origin of transfer sequence (oriT) to catalyze cleavage of the DNA strand (T strand) destined for transfer (33). For pCF10, the PcfG relaxase and the accessory factor PcfF function together to cleave DNA at the nic site within an IncP-type oriT sequence (18, 64). Next, by a mechanism that is poorly understood, the relaxosome or the processe...

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…Furthermore, all three proteins colocalize at discrete sites at the cell membrane (59). Thus, a working model proposes that PcfF recruits PcfG to oriT, and through interactions with unknown membrane constituents, both factors then mediate the binding of the relaxosome (or the pCF10 transfer intermediate) at the membrane near the T4SS channel (59). A similar mechanism was postulated for MobB R1162 , an accessory factor that stimulates R1162 transfer.…”

“…PcfF and PcfG interact with each other, and both Dtr factors also bind the PcfC T4CP (59). Furthermore, all three proteins colocalize at discrete sites at the cell membrane (59). Thus, a working model proposes that PcfF recruits PcfG to oriT, and through interactions with unknown membrane constituents, both factors then mediate the binding of the relaxosome (or the pCF10 transfer intermediate) at the membrane near the T4SS channel (59).…”

“…In the E. faecalis pCF10 transfer system, the relaxase PcfG requires PcfF to nick at the pCF10 oriT sequence (58). PcfF and PcfG interact with each other, and both Dtr factors also bind the PcfC T4CP (59). Furthermore, all three proteins colocalize at discrete sites at the cell membrane (59).…”

“…Recently, biochemical studies were expanded to include T4CPs of gram-positive conjugation machines, including E. faecalis PcfC pCF10 and Clostridium perfringens TcpA pCW3 (59,251). In general, T4CPs possess three domains, an Nterminal transmembrane (TM) domain, a nucleotide binding domain (NBD), and an all-␣-domain (AAD).…”

“…TrwB R388 and other T4CPs carry conserved Walker nucleoside triphosphate (NTP) binding motifs required for function, and the soluble fragment of TrwB R388 displays a DNA-dependent ATPase activity in vitro. Several T4CPs bind ssDNA and dsDNA substrates nonspecifically in vitro, and TrwB R388 also oligomerizes upon DNA binding in vitro (59,257). Finally, the TM domain of TrwB R388 contributes to hexamer formation and influences nucleotide binding properties (134,135).…”