Biology Reference
In-Depth Information
investigated by AFM as demonstrated by a study examining DNA transfer in
E. coli.
32
During conjugation, the F-plasmid of donor cells encodes proteins
to prepare specialized pili that bind to the surface of recipient cells. After
gaining access to the recipient cell's cytoplasm, the pilus involved in the
exchange is depolymerized, bringing the mating pair into intimate contact.
Whether the DNA exchange occurs through the extended pilus or during
contact between the cells is debated. Shu
sharing an extended pilus. After using the AFM tip to sever the extended pilus,
the area was probed with a tip functionalized with an antibody to ssDNA. The
authors assert that the DNA was being transferred through the pilus at the
time of dissection, accounting for the recognition by the functionalized tip. As
underscored by this study, the ability to image in liquid and to manipulate a
localized region of the sample is a distinct advantage of AFM.
In a study by Dennis
et al.
32
imaged mating pairs of
E. coli
et al.
, storage inclusions from the bacterium
Cupriavidus necator
were examined.
78
Owing to the amount of sonication
used during the preparation, a mixed population of inclusions was isolated.
Using surface morphology as selection criteria, the authors examined the
properties of each group and determined that each form presented a different
layer of the storage inclusion. Importantly, a previously unidentiied protein
network about 2-4 nm thick was revealed in the study. Biochemical studies
of the PhaP protein had shown that the protein was associated with storage
inclusions but not until the AFM study was the protein localized to the space
beneath the inclusion envelope.
Touhami and colleagues generated high-resolution AFM images to
characterize the pili of a laboratory strain of
(PA01).
79
In addition,
the authors immobilized PA01 to the cantilever and approached the mica
substrate, giving pili on the bacteria the opportunity to adhere to the mica.
This coniguration permitted the authors to calculate the forces required to
either dissociate the pili from the mica or to break the pili. Although the two
scenarios could not be distinguished in their experiments, the sensitivity of
AFM for force measurements is nevertheless highlighted.
The cell walls of Gram-positive bacteria include a thick peptidoglycan
to which teichoic acids are attached, whereas Gram-negative cell walls have
a much thinner peptidoglycan and a lipopolysaccharide-containing outer
membrane.
80,81
In both instances, these cell wall components obstruct access
to the cytoplasmic membrane in AFM studies. Cell walls can be removed
enzymatically using established protocols to provide access to the various
membrane proteins that reside there.
P. aeruginosa
The resulting spheroplasts are
extremely soft and can be imaged in liquid in a non-destructive way.
11,55
Sullivan
82,83
et al.
used AFM to study live
E. coli
spheroplasts and compare their
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