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although their expression is required for normal T4P function (
Brown et al.,
2010
). An ongoing analysis of
bfpF
double mutants indicates that BfpE, BfpB,
BfpU, BfpG, and BfpL are absolutely required for pilus assembly, while BFP
can be assembled in the absence of BfpI (unpublished data). However, how
these findings apply to systems that lack a retraction ATPase, such as the T4P
systems of ETEC, is not known.
Subcellular localization
The subcellular localization of T2S and T4P components has been the subject
of some debate and may be of considerable functional importance. Studies with
fluorescent fusion proteins have revealed the critical importance of gene dos-
age effects, while single molecule fluorescence and super-resolution micros-
copy techniques provide deeper insights into the actual distribution of machine
components. In T2S systems, components fused to fluorescent proteins were
observed at the poles when over-expressed, but these same fusion proteins
produced peripheral foci of fluorescence when expressed at wild-type levels
(
Buddelmeijer et al., 2009
;
Lybarger et al., 2009
). Thus, T2S are likely distrib-
uted around the cell and not exclusively at the cell poles.
The T4aPs of
Myxococcus xanthus
and
Pseudomonas aeruginosa
have been
shown by transmission electron microscopy (TEM) and fluorescence micros-
copy to exit the cell primarily at a pole (
Nudleman et al., 2006
;
Bulyha et al.,
2009
;
Cowles and Gitai, 2010
;
Higashi et al., 2011
), although non-polar fibers
have been observed in
P. aeruginosa
(
Cowles and Gitai, 2010
)
.
In these two sys-
tems, the T4P fibers mediate directional movement and their polar localization
may be necessary for this function.
In EPEC, the BFP mediates initial attachment and aggregation into spheri-
cal aggregates. These interacting fibers create an extensive, overlapping, and
complex meshwork and TEM has not yet demonstrated their subcellular origins.
One recent study utilized photo-activated localization microscopy (PALM) to
image single BfpB secretin molecules fused to photo-activated fluorophores and
expressed in the context of the complete
bfp
operon (
Lieberman et al., 2012
).
Such super-resolution techniques have made subdiffraction limit imaging pos-
sible and reveal unprecedented detail of T4P cell biology (
Betzig et al., 2006
;
Hess et al., 2006
). BfpB is localized to one or both of the bacterial cell poles in
20% of cells and is distributed around the cell envelope in the remainder; par-
ticularly when the cells are involved in aggregates, the secretin molecules form
clusters (
Lieberman et al., 2012
). Furthermore, the secretin molecules may be
distributed in a helix around the cell envelope (
Lieberman et al., 2012
), which
may be a common phenotype for bacterial secretion systems (
Aguilar et al.,
2010, 2011
;
Lieberman et al., 2012
). By distributing pili around the cell enve-
lope, EPEC cells may be better able to form aggregates early in infection when
BFP expression is critical (
Zahavi et al., 2011
) and thus confer a considerable
advantage to the pathogen as it first colonizes the gastrointestinal tract, where
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