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then processed by the BfpP prepilin peptidase (
Zhang et al., 1994
;
Ramer
et al., 2002
), which cleaves pre-bundlin and the pilin-like proteins at a specific
N-terminal cleavage site, removing a small leader sequence and adding a
methyl group to the nascent N-terminal leucine residue. Simultaneously with
pre-bundlin processing by BfpP at the cytoplasmic face of the IM, the globular
head domain of bundlin is stabilized by DsbA, a periplasmic oxidoreductase
that is not a component of the BFP system. DsbA catalyzes the formation of a
disulfide bond in the C-terminus of bundlin critical for pilus assembly (
Zhang
and Donnenberg, 1996
). The simultaneous and independent action of BfpP and
DsbA on pre-bundlin indicates that prior to incorporation into the pili, pilins are
integral transmembrane proteins.
T4P fibers are analogous to the pseudopilus complex of the T2S, except
that they extend through the secretin into the extracellular space. T4P fibers
are composed of polymerized pilin proteins and incorporated into a 3-start left-
handed helix with the N-terminal alpha-helices of each subunit buried in the
filament core and the C-terminal globular domains exposed on the surface of
the pilus (
Parge et al., 1995
;
Ramboarina et al., 2005
). As with T2S, how each
bundlin monomer is incorporated into the pilus is unknown. However, as BfpC
is a homolog of GspL (
Yamagata et al., 2012
), it may recruit processed bundlin
to the IM assembly complex. Like the minor pseudopilins of T2S, the pilin-
like proteins BfpI, BfpJ, and BfpK may form a complex together (
Ramer et al.,
2002
), as has been demonstrated for their T2S homologs. Pilin-like proteins
have been found as integral components in T4P fibers of
Neisseria gonorrheae
(
Helaine et al., 2007
) and
Pseudomonas aeruginosa
(
Giltner et al., 2010
). As
yet unpublished data indicate that BfpI is incorporated into BFP.
BfpE is a polytopic IM protein and the equivalent of GspF. However, in con-
trast to its counterpart, evidence suggests that BfpE may have four transmem-
brane segments, with two periplasmic domains, a small cytoplasmic domain,
and both termini in the cytoplasm (
Blank and Donnenberg, 2001
). The exten-
sion ATPase BfpD provides the energy necessary to extract each pilin molecule
and incorporate it into the pilus (
Sohel et al., 1996
;
Stone et al., 1996
;
Milgotina
et al., 2011
). In a molecular model of the extraction of
P. aeruginosa
major
pilin monomers from a membrane, the hydrolysis of as many as eight ATP mol-
ecules were required (
Lemkul and Bevan, 2011
). The ATPase activity of BfpD
is slightly increased in the presence of the cytoplasmic N-termini of BfpE and
BfpC (
Yamagata et al., 2012
). Another protein absolutely required for pilus bio-
genesis, BfpL, is also found in the IM (
Ramer et al., 2002
) and associates with
the periplasmic face of BfpC (
De Masi et al., 2012
).
Once assembled, the pilus must be extruded through the membrane as more
bundlin monomers are incorporated into the structure at its base. The secretin,
BfpB, plays a crucial role in pilus biogenesis, as without a functional secretin
multimer T4P biogenesis fails, a phenotype that manifests itself in EPEC as the
loss of autoaggregation in
bfpB
and other mutants (
Anantha et al., 2000
;
Durand
et al., 2003
). Here, secretins serve as the exit pore for pilus fibers as they extend
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