berries.
other
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berries.
microscopy
shows
composition primarily
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of Abstract.
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berries.
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macroscopic
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shown.
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researchers
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have
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used
purple
and bacteria
purple
course.
2002,
Sippewissett
Research
the
the
Sharp
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characterized
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of
needs
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formations
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berries
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physiology
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purple
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of
years
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belong
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berries,
results
bacteria
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of
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also
of
the
decade,
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to
poois
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from
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and
the in ‘‘
PSBs.
interacted berries phototrophs
Cytophaga community.
sampled. clone marine
sequences and rods.
characterization
microscopy. phase
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remains
Chromatiaceae,
they
Introduction
stated
sequencing
they
have
library,
In
microscopy,
on
might
clone.”
Purple
much
However,
1997,
in
In
morphology,
form.
in
caught
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their
(Banin,
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a
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be
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complex
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Udi
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of
aggregates
purple
Seitz
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paper
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researchers’
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they and
Banin
inferred
a,
understood
1997;
to
et
be
further
microbial
of
it
intracellular
that
micro-food
related
sulfur
sequences
these
also
are
the
al.
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cultured
was
extracted
did
Bedard,
of
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first
aggregates
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that
noted
DNA
a
determined
confirming
bacteria
PSBs,
attention
to
berries
mini-project
about
characterized
communities
those
a
from
from
the
1998).
pigments
web
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results,
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and
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what
presence
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species
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of
that
a
a or - 10-
morphotypes are consistently major constituents of the purple berries. It also should be
noted the clone library from this year showed only 1 PSB sequence out of 100 sequences,
while other marine bacteria were present in the clone library. This probably is a product
of incomplete cell lysis in the DNA extraction, which was problematic in these
experiments as well. Without microscopy, it is impossible to distinguish between
bacteria that are truly major components of the system and bacteria that are part of the
tidal pool water column and were incidentally associated with the sampled berry. It is not
surprising that Cytophaga, a common and widespread group of marine microorganisms,
could be detected and cultured from purple berry inoculum. It is also not surprising to
find sulfate-reducers from sampling that included anoxic marsh sediment in the tidal
pool. It was my original hypothesis that SRBs produced sulfide and presented a
“nucleating center” for the PSBs to attach and start building an aggregate. However, the
majority of the cells in the berries seem to be accounted for with the microscopy. The
clusters are PSBs, and the small unicellular rods surrounding them seem to belong to the
‘-proteobacteria as well. Cytophaga probes did not light up any cells in the berries. It is
necessary to do some FISH experiments with probes targeting SRBs determine the presence and distribution of SRBs in the berries. The results of this year’s experimentation suggest that sequences thought to represent major constituents of the berries may actually be incidental marine microorganisms that came in with sampling.
This emphasizes the importance of confirming molecular data with microscopy.
According to microscopy, the berries seem to be made primarily of PSBs, cyanobacteria, and at least one species of small unicellular y-proteobacteria. Identification of the gammas surrounding the PSB clusters could elucidate their role in the berry — the high level of organization in the berries suggests that they may play a specific role in the aggregates. It also will prove useful to do an exhaustive search with SRB-specific FISH
berries.”
bacteria
polycarbonate
the
Origin sections were
throughout FISH,
addition, the for
(3Oum). However, Method
and they
region.
probes,
getting
washing
sample.
dried
were
adapted
the
of
The
There
to
Development:
are
(3Oum)
Atlantic
The
the
Berries/Future
at
slides the
gelatine-coated.
see
this
the
results
expelled
46°C
There
incubation
hybridization
probe
from
were
sections
if
membranes.
hybridization
protocol
that
they
were
mussels
for
from
were
an
a
concentration
do
as
few
about
occur
coated
original
would
modification
time
pseudofeces
not
to
the
a
Directions
changes
were
couple
work.
time
Before
20
in
stick
This
class
increased
and
again
not
the
protocol
minutes.
observed
was
adapted
to
washes,
of
report stay
Conventional
berries
was in
using
by
the
adaptations
of
by increased
the
on
dipping
increased
from FISH
glass
from
mussels
by
FISH
This
the
the
expelling
protocol
and and
-11-
Castro
20
protocol
cryosections
glass
slides. S.
proved
if
it
protocol
from
into
minutes
Behrens
they
cryosectioning
also
to
and
from
is
et
slides
adjust
tiny
0.1%
2
may
shown
are
allowed
al.
to
for
hours
5Ong/
that
clusters
keep
to
suggest
for
at
by
high
for
be
thicker
on
30
all
to proved
themselves,
FISH
to
what
sample
the
gelatine-coated
the
the
minutes.
localized
gelling
work
5
methods
of
that
hours, thickness
sections
probes
sections
they
on
PSBs
to
well
250ng/sample.
purple
seawater
strength
be
called
These
and
to
to
whether
were
in
very
for
attached
a
of
diffuse
the
sulfur
subsequently
certain
thick
slides
the
“immature
used.
changes
important
agarose
filtrate
field
sections
or
into
in
not
in In on - 12-
1998. However, this year in the field, pools that contained berries but did not seem to
contain mussels were observed. This is not a conclusive argument against the
involvement of mussels in berry formation, as the berries may spill over from pooi to
pool, or from pool to mat, or from mat to pool. This “spillover” phenomenon was
observed this year in the field: outside the boundary of a berry-containing pool were
many small berries in the top layer of the sediment. This calls for future research
exploring the origin of the berries. One aspect to explore is the origin of the extracellular
slime matrix that holds the clusters together. Is it of eukaryotic or bacterial origin?
Castro et al. suggested that the mucus that holds them together arises from the mussels.
This needs to be tested using cytological stains, or an array of specific lectins. Results
from Seitz et al. suggest that the matrix is made of polysaccharides, but the origin
remains unknown. It would also be interesting to design some experiments to pursue
growth and development of berries in laboratory mesocosms. In addition, since they
contain at least two types of photoautotrophic microorganisms, experiments
characterizing responses to changing light regimes in species composition, metabolism or
physiology of the berries could elucidate the metabolic processes and physiology of the
aggregates as a whole. Comparative studies with other free-living, planktonic purple
sulfur bacteria would also provide insight into the aggregates. This calls for the identification of the purple sulfur bacteria in the aggregates. Microscopy and
morphology suggest that they are Chromatiaceae, but they should be identified with molecular data.
MBL. here
with I
brainstorm, FISH
also
contributed
as
a keep
Alfred
with
There
were Margy
Acknowledgements
FEMS
forming
Seitz,
Report,
Castro,
Bedard,
Banin,
Literature
thank
great
well
helpful
at
unlimited
microscopy
looking
an
and
A.P.,
was
This
MBL
Spormann Microbiol.
deal
Terry Gentile,
—
U.
H.,
extreme
1998.
D.L.
macroscopic
her
general
a
Microbial
Fujita,
and
a
with
work
in
Nielsen,
Cited
dedicated
and
knowledge
Marsh
great when
fieldwork, Microbial
time
a
his
project
asset
and
connecting
fellow
was
histological
was
Ecol.
Y.,
deal
we
input
on
and
T.H.,
student
Diversity
aggregates
made
always
Patterson,
to
team
microscopy
thought
to
design
student,
Dan
about
12:225-236.
the Diversity
was
making
the
and
possible
of
fieldwork
us
training
Buckley
consistently
willing
success
techniques.
anaerobic
berry
J.
we
Mini-Project
with
and
K.,
was
Overmann.
in
important
just
Mini-Project
and
interpretation
Great
other
and
researchers
by
an
was
to
of
for
hadn’t
and
image
generous
run
amazing
E.
this
phototrophs,
tireless
priceless. Sippewissett
helpful.
resources Sebastian
-
labwork.
Zinser.
observations
out
13
Report,
project.
1995.
seen acquisition
-
into
Report,
who
co-worker
work
loans
of
much.
Terry
Microbial
outside
the Physiology
results.
Jane
was
1997.
She
contributed
He
support,
in
Salt
from
field,
1999.
and
Marsh
is
always
systems
Gibson
also
maintaining
His
incredibly
Marsh,
of
Sebastian whose
Zeiss
collecting
Diversity
and
was
expertise
our
and
of
and
willing
heavily
was
he
of
just
lab
purple
mc,
conversations
patience Massachusetts.
Adam
the
inspired
a
the
innovative
Behrens
and
loads
who
Mini-Project
countless
valuable
and
highest
to
to
molecular
sulfur
outside
take
Martiny
this
provided
patience
and
of
us
also
fun.
time
project.
quality.
bacteria
interest
both
resource
helped
with
berries.
of
were
lab
to
the
to us Purple Berry Oyster Pond Sediment
Figure 1. (a) shows clusters of purple sulfur bacteria tightly associated with each other. (b) shows a purple clump from sediment that was not as organized and contained many other types of organisms. . Figure 2. (a) shows a look down across the middle of an equatorially cute purple berry. (b) shows a closeup of the light purple section containing distinct cell cluster patterns. . Figure 3. SybrGreen-labelled purple berries. 3(a) shows a view of a the surface of an equatorially cut bacteria. 3(b) shows the organized clusters from within the berry. 20’um
Figure 4. Cyanobacterial filaments in a cluster within the purple berries. They are localized separately of other purple sulfur bacteria in the berry. Note the obvious heterocysts (non-chla containing cells). .
. .
bacteria around cluster. the
unicellular as as probe. the well to hybridizes probe small the The PSBs
proteobacteria FISH image the rRNA gamma the with Cy3-labeled
Surrounding 5(b) bacterial clusters rod-shaped small cells. the is are
berry focusing tissue, bacteria. of clusters sulfur purple on the
5. 5(a) Figure a high-magnification shows SybrGreen-stained image of
with Zeiss Image LSM5 (available Browser internet). on the
z-stack full be documents files viewed should the CD, attached and (on
markedly cyanobacteria. information the more There the in much is
clusters of purple bacteria sulfur bacteria, excluding most other and
microscope. evidence The a show images matrix of surrounding the
6. Figure 3-dimensional images confocal scanning the from laser