Abstract: Conjugative transfer of plasmid R388 requires
the coupling protein TrwB for protein and DNA transport,
but their molecular role in transport has not been deciphered.
We investigated the role of residues protruding into
the central channel of the TrwB hexamer by a mutational
analysis. Mutations affecting lysine residues K275, K398,
and K421, and residue S441, all facing the internal channel,
affected transport of both DNA and the relaxase protein
in vivo. The ATPase activity of the purified soluble variants
was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour
in vivo. Alteration of residues located at the cytoplasmic
or the inner membrane interface resulted in lower activity
in vivo and in vitro, while variants affecting residues in the
central region of the channel showed increased DNA and
protein transfer efficiency and higher ATPase activity, especially
in the absence of TrwA. In fact, these variants could
catalyze DNA transfer in the absence of TrwA under conditions
in which the wild-type system was transfer deficient.
Our results suggest that protein and DNA molecules have
the same molecular requirements for translocation by Type
IV secretion systems, with residues at both ends of the
TrwB channel controlling the opening?closing mechanism,
while residues embedded in the channel would set the pace
for substrate translocation (both protein and DNA) in concert
with TrwA.
Fuente: Mol Genet Genomics (2017) 292:1037-1049
Editorial: Springer
Fecha de publicación: 01/10/2017
Nº de páginas: 13
Tipo de publicación: Artículo de Revista
DOI: 10.1007/s00438-017-1331-3
ISSN: 1617-4615,1617-4623