Localization of TraC, a protein involved in assembly of the F conjugative pilus

Abstract

TraC is one of the proteins encoded by the F transfer region of the F conjugative plasmid which is required for the assembly of F pilin into the mature F pilus structure. Overproduction of this protein from the plasmid pKAS2, which carries only traC, resulted in the formation of inclusion bodies from which soluble TraC was purified. When small amounts of TraC were produced from pKAS2, the protein was localized to the cytoplasm by using anti-TraC antibodies. Similar analysis of a set of TraC-alkaline phosphatase fusion proteins localized all of these fusion proteins to the cytoplasm. However, when TraC was expressed from the F plasmid, much of it appeared associated with the bacterial membrane fraction. Under these conditions, TraC does not appear to be part of the tip of the F pilus, as neither anti-TraC antibodies nor purified TraC had any effect on the infection of F-containing bacteria by the filamentous bacteriophage f1. These data suggest that TraC is normally associated with the membrane through interactions with other proteins specified by the tra region. This interaction may be via the carboxyl-terminal region of the TraC protein, as a mutant TraC protein containing an Arg-Cys substitution at amino acid 811 exhibits an interaction with the membrane weaker than that of the wild-type protein in the presence of the other Tra proteins.

Tra Proteins Characteristic of F-Like Type IV Secretion Systems Constitute an Interaction Group by Yeast Two-Hybrid Analysis

“…Except for TrbI, the other proteins of this group are or are predicted to be periplasmic (14). TrbI is an inner membrane protein but has a large periplasmic domain (34). Hence, interactions among the six proteins are consistent with their cellular localization.…”

“…Hence, the interaction domain of TrbI for TraH is within the segment from R41 to the C-terminal P128. This region is relatively hydrophilic and extends into the periplasm (34).…”

Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI

“…Recent analysis of a trbI::phoA fusion product also confirms the membrane-spanning potential of TrbI sequences. Schandel et al (24) report that a TrbI amino-terminal sequence permits alkaline phosphatase translocation to the periplasm, although the TrbI::alkaline phosphatase polypeptide remains associated with membrane fractions.…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…An integral membrane association might not be obligatory, however, because VirB4-like TrwK R388 cofractionates with the membrane and the cytosol, and soluble TrwK R388 exhibits ATPase activity in vitro (9). Furthermore, TraC F and TrhC R27 associate with the inner membrane but probably peripherally through interactions with other T4SS subunits (109,234).…”