are
1.0
mature
berries
with
berry
ribbed
hypothesis
indicated
hybridization
minus),
following
Cloning
the
aggregates
Abstract
Hector
Group
macroscopic
Introduction
bacterial
the
may
The
mussels
berry.
and
II
The
Castro,
mussels
sulfate
in
and
the
Report:
be
phylogenetic
the
focus
subsequent
that
found
goal
sequencing
presence
derived
The
supported
salt
reducing
mussels
(1
Yoshiko
strongly
of
of
in slime
-
marsh
Berries
our
4
the
selected
present
from
mm
incubation of
research
present
groups:
are
matrix
the
Fujita,
of
may
green
suggested
diam.)
involved
16S
presence
in
shallow
ingest
that
the
sulfur study
Katie
rDNA
was
purple
purple
in
produced and
berries
contributes
that
the
the
were
the
in
of
was
pools
bacteria.
Patterson,
cytophaga.
extracted
the
the
inlet
these
or
sulfur
bacterial
“berries”
to
observed
and
pink
formation
in
sulfate-reducing
by
investigate
or
molluscs
the
to
bacteria
1
the
to
Field
pond
aggregates
and
from
determine
components
the
Sippewisset
from
Enrichments
expelling
Erik
is
observations
aggregation
of
the
(most
play
the
necessary
the
the
Zinser
berries
Sippewisset
found
The
berries,
how
berries
a
bacteria
closely
berry-like
role
of
Salt
Group
and
results
and/or
the
suggested
lying
for
of
in
in
Marsh.
or
whole macroscopic
berries,
the
and
the related
the
the
II
berry
of
on
why
Salt
pink
bacterial
Microbial
initial
salt
development
cytophagas,
laboratory
cell
and
We
the
precursors.
Marsh.
but
they
to
marsh
material.
fluorescent
in
aimed
formation
presence
Thiorhodococcus
reject
purple
the
species are
Diversity
led
The
July
simulations
top
formed.
and
to
them
of
The
to
or
Atlantic
identify
of
layer
berries
in
a 27,
of
the
also
in
pink
the
the
as
the
situ
1998 1998
presence
the
checkerboard
length
cytophaga,
least
Cloning
of phototrophs
(Banin,
using
the
unsuccessful;
slime.
observed
phototrophs,
berries.
characterization,
In
2.
answer
1. of
rod-shaped
berries
order
1997 sediments
How
What
the
an
polymorphism
Attempts
Previous
1997).
following and
of
the
approach
Seitz
Microbial
to
/why
many
bacterial
was
and
purple
address
and
following
sequencing
hybridization.
embedded
bacteria
the
within
et
highly
are
“symbionts”.
Using
small
sulfate
al.
to
students
microscopy,
authors
sulfur
phylogenetic
including the
species
Diversity
disintegrate
these
(1993)
(RFLP), selected
conserved.
rod-shaped
berries
berries
and
questions:
reducing
in
of
bacteria
questions
speculated
in
a
are
16S
reported
RFLP
the
slime
enrichment
Banin
as
Course,
formed?
shallow
DGGE
field
present
rDNA
the
inoculum,
groups: Microbial
bacteria.
and
bacteria,
The
matrix
and
in
slime
an
observations
also
that
that
cytophaga.
(denaturing
Banin
the
checkerboard
extracted
pools
in
approach
DGGE
compared cultivation,
the
the
it
purple
slime
with
which
Diversity
he
Scanning along
was
dominant
in
again
berries?
successfully
proteolytic
analyses
2
the
around
composed
from
encompassing
appeared
sulfur
with
and
gradient
investigated
marsh.
different
hybridization
microscopy,
Course
electron
laboratory
the
some
species
bacteria,
the
indicated
berries
to
enzymes
of
enriched
The
gel
coccoid
spirochetes
berries
have
stabilize
complex
microscopy
in
electrophoresis),
the enrichment
Group
goals
sulfate
simulations
suggested
the
and
that
attempted
results
composition
purple
using and
for
berries
of
molecular
the
the
II
polysaccharides.
both
reducing
glucoronidases
and our
Microbial
confirmed
aggregate.
restriction
confirmed
species
sulfur
cultivation,
the
research
were
diatoms,
to
was
purple
characterize
presence
and
of
techniques
bacteria.
bacteria,
developed.
purple
composition
the
sulfur
Diversity
the
fragment
the
were
They
July
within
berries,
molecular
were
During
of
presence
sulfur
27,
to
also
at
the
the
1998 1998 of
and
enrichment
in
and
bacteria,
between
Seitz
2.0
references
Section
to
berries
applied following
1.1
major
employed.
laboratory
as
three
the
for
apply
obtain
Enrichments
Organization
et
berries
iron
role
Microscopic
other
and
al.
The
5. to
This
individual
the
additional
pure
sections.
the
(1993),
are
experiments:
Section
in
experiments
reducers.
from
purple
The
various
types
year
are
the
berries.
listed
cultures
results
the
described.
formation
our
Banin
berries
of
sulfur
6
of
characterization
techniques
analysis
Section
in
berry-derived
includes
enrichments,
No
the
group
Section
Section
of
of
to
(1997),
for
growth
phototrophs,
Report
our
these
simulate
from
of
2
The
sought
heat-tolerant
and
our
contains
investigations
the
7.
3
applied
three
the
presents
and
characterizations
was
field
conclusions
berries.
enrichments.
using
to
possible
same
techniques.
our
observed
kinds
confirm
observations
and
a
to
summary
group
the
the
respiratory
the
and
the
of
conditions
media
suggest
molecular
investigation
different
and
cytophagas.
bacteria.
the
3
in
suggested
In
In
the
of
recommendations
findings
of
provided
and
Section
addition,
that
the
and
heat-tolerant
the
ponds.
leading
laboratory
characterization
In
1
results
mussels
fermenting
that
6S
of
addition,
of
We
4
for
rRNA
more
the
the
the
several
Group
These
to
therefore
from
the
microscopic
berries
previous
in
berry
and simulations
field
clones
Microbial
the
we
for
bacteria;
II
are
the
bacteria
used
formation
salt
observations
future
data
Microbial
are
attempted
the
various
investigators,
from
marsh
described
sulfate
berries
generated
techniques
for
are
Diversity
are
research.
the
enrichments
cyanobacteria;
conserved
presented
were
may
Diversity
to
berries
reducing
July
as
and
enrich
in
inocula
from
play
course
as
Finally,
the
27,
applied
by
well
in
a
the
1998 1998
phase
some
first
flasks
made
The
incubation.
enrichments
temperature primary
thiaminium
nicotinic
mi/I
NH4C1,
bicarbonate
84
with
microscope
2.1
below.
enrichments,
g/l
was
ethanol
of
Sulfate
rings
dark
either
of
with
stock
Both
the
Berries
the
enrichment
a
20.0
acid,
discrete
cocci,
of
H2/C02
vitamin
ethanol
ethanol
These
secondary
of
dichioride,
for
Na2HCO3,
buffered
the
slide.
were
Reducing
growth
g/l
and
sulfate
5
were
5
ethanol
mg
NaCI,
and
days,
performed
brown
primary they
Crushed
stock:
(20
secondary
headspace,
bottles
Ca-
were
phase
washed
minimal
reducing
but
3.0 are
and
mM)
and
Bacteria
and
D(+)-pantothenate,
2
colony,
100
observed) not
mIll
enrichments
not
g/l
bright
were
5
then
berries
H2/C02
at
and
in
mg
the ml
MgCl*6HO,
and medium
discussed
bacteria.
respectively.
Na2S,
30
sterile
finally
incubated
cyanocobalamine.
Na2SO4
consisting H20,
H2/C02
cocci,
°C.
the
were
consisting
enrichments
H2/C02
1X
(pH
4
moved
were
which
then
further.
The
mg
concentration
(20
enrichment
initally
7.1-7.3)
of
15
Two
subcultured 4-aminobenzoic
second
added
0.5
mM)
4
motile
could
to
primary
mg
of
(80%)
The
30°C
g/l
showed
3
colony
at
pyridoxamine
cell
or
consisted
to
Due
KC1,
was
be
>30
curved
other
H2/C02 several
showed
a
enrichments
of
interpreted
types:
for
bicarbonate
to
into
a
types
°C
0.15
turbidity
trace
hazy,
four
problems
final
rods,
for
secondary
acid,
of:
times
Group
long
turbidity.
g/l
grew
and
element
enrichment
one
undefined
incubation.
dihydrochioride,
0.2
CaC12*2H20,
consistent
1
within
as
thin
Na2SO4
and
into
buffered
day,
mg
in
II
with
g/l
the
both
rods
liquid
then Microbial
solution
D
Hungate
KH2PO4,
Agar
then
3
the
the
(+)
colony
stages
types
enrichments.
(20
that
with
All
crushed
minimal
first
incubator,
moved
enrichments.
shakes -Biotin,
mM).
SL1O,
subsequent
twitched,
tubes
the 30
are
in
week
10
Diversity
(in
0.25
July
the
mill
mg
with
described
to
were
which
medium
10
The
and
and
room
g/l
life of
the
27,
of
mg
The
a
flat
1
an
1998 1998
weeks.
temperature
(0.05%
berries
spread
2.3
noted
agar
pairs,
purple
Erik
2.2
performed.
(TEM)
cycle
Cytophagas
Purple
surfaces.
Zinser’s
by
and
onto
of
were
Enrichments
sulfur
Table
tryptone,
The
analyses
a
cytophaga:
the
positive
two
under
protocol
crushed
Sulfur
Fluorescence
phototrophs
independent
1
likely
In
contains
types
0.05%
of
addition,
oxic
hybridization
these
Phototrophs
for
predominant
with
vegetative
and
of
conditions.
cytophagas
yeast
data
plates:
results
isolates
a
present
project
in
we
microscope
for
situ
extract
enriched
rods,
YPG
of
several
with
hybridization
PSB,
are
report.
in
the
Many
were
the
in
microcysts
described
(yeast
the
purple
which
slide
for
berry,
of
performed
The
different
green
the
a
water
extract
putative
and
is sulfur
key
5
a
isolates.
(FISH)
in
sulfur a
motile
and
passed
(70%)).
large
section
results
colony
+
on
bacteria
spores.
green-sulfur
peptone
FISH
and
dilute
bacteria
coccus
through
obtained
4.
types
Incubations
transmission
probe
(PSB)
complex
Secondary
+
Group
that
with
arose
glucose
a
bacterium
hypodermic
are
(see
forms
enrichments
irregular
II
over
that
were
media
sect.
electron
Microbial
in
and
tight
there
the
sea
perfomed
4).
from
plates.
ovoid
course
aggregates
water)
needle,
are
are
microscopy
the
Diversity
described
at
cells
July
shakes
Washed
of
and
berry,
at
least
and
two
room
27,
often
TYE
on
then
two
were
as
1998 1998
in in
cytophaga
hybridization
None
motile
J
I
H
G F
E
D
C
B
A
Isolate
of
in
the
liquid,
species
isolates
with
TYE
TYE
TYE
YPG
YPG
YPG
YPG
YPG
YPG
YPG Medium
and
from
the
displayed
gliding
flavo
the
Table
berry
FISH
motility
pale
spreading
opaque,
agar pale border
degrader
rough
yellow
yellow
rough,
yellow
round,
very
large
mucoid
all
large
irridescent
round
opaque
rough
translucent
Char.
Colony
1.
with
of
probe
yellow
pink,
degrader
Isolates
mucoid
opaque
opaque
the
translucent
on
pale
dark
center,
these
off-white
common
surfaces.
morph.
(see
agar
from
enrichments.
section
6
Cytophaga
characteristics
Nor
of
length,
thin
rods
curved,
motile
length
rods
4).
cocci
cocci,
phase-bright
diplo-bacilli
cocci,
cocci,
in
irregular
Char.
Cell
did
gliding
chains
Hence
rods
of
the
(?)
some
rods
some
some
no
clumps
var.
Enrichments.
clusters
motile
chains
chains
of
one
in
rods
evid.
Group
we
of
var.
candidate
cytophagas:
were
of
II
10+
unable
Microbial
(J)
thin
show
to
isolate
Diversity
rods,
July
a
positive
non
a
27,
1998 1998
re-cultivate
had
that
insufficient
possible
darkening
enrichment
substances
for
ferrihydrite,
(approximately
and
enrichment
Several
of
one
electron
40
reducing
identical
Pfennig
2.4
the
been
humic
mM
sulfur
40
week
Iron
bottles,
In
mM
that
days
Seven
ferrihydrite
inadvertently
bottles
donor
the
to
was agent
of
substances,
a
Reducing
iron can
in
were
was
ferrihydrite;
slight
that
the
and
the
secondary
later
the
limiting;
and
washed
in
and
reducing
30
and
inoculum,
containing
iron
subcultured
described
used
10
secondary
turn
darkening
the
a
ml
carbon
mM
sulfate
ferrozine
[amorphous
solids
Bacteria
which
as headspace
solids
chemically
omitted
berries
a
enrichments,
2,6-anthroquinone
inocula, (ii)
activity
few
which
for
source.
was
in
non-reduced
enrichments.
40
in
into
may
of
weeks
the
assay
the
were
from
the
mM the
not
Fe(OH) 3 1
in
from
3
and
was
enrichment
serve
reduce
sulfate
other
different
orange
The
a
added
crushed
the indicated
ferrihydrite
into
60
very
the
primarily
the
as
medium
bottles
ml
bottle
anoxic
the ferric
bottles
reducing
berries.
Another
electron
as
little
iron
as
disulfonate
stoppered
types
on
incubation
an
with
the
the
7
also
iron.
were
solids,
a
activity
supernatant sea
electron
with
were
formulation.
slide
electron
presence
of
AQDS,
bacteria,
possible
acceptors
began
water
incubated
secondary
Five
a
bottle);
again
indicating
and
headspace
(AQDS).
was
it
acceptor.
medium.
was
ml
to
acceptor,
observed
the
of
reason
except
rather
incubated
observed,
darken.
in
aliquots
ferrous
and
No
recognized
material
Group
without
enrichments:
reduction,
AQDS
further
of
(iii)
than
for
Instead
that
The
and
after
80/20
iron
The
from
II
at
the 5
except
was
headspace
the
no
Reduced
mM
10
medium
30
Microbial
is
attempts
one
earlier
in
that
lack
sulfide
the
H 2 /C0 2
iron
mM
the
a
was
divided
°C.
the
model
acetate,
for
week.
(i)
a
medium
of
primary
solids,
acetate
medium.
sulfur observed
5
positive
humic
composition
a
growth
was
at
were
mM
slight
Diversity
between
compound
30
It
July
40
added
source
was
is
contained
acetate
as
°C.
made
mM
was
primary
27,
in
After
one
two
as
1998
1998
to was
conducted
pairs
the
16S
3.3
hybridization.
labelled
(position
Two
3.2
DNA
beadbeater
with
Microbial
from
slides,
3.1
3.0
TA
rDNA
Cloning
PCR
segment.
DNA
set
Molecular
ethanol.
the
system
Amplified
cloning
PCR
and
Berries
with
of
1492)
different
using
reaction
sequences
Diversity
extraction
primers
passed
in
reactions
and
digoxigenin
(Promega)
In
the
Further
kit
PCR
for
were
the
the
characterization
sequencing
bacterial
presence
(Invitrogen).
samples
several
amplification
were
program
cases
course
products
in
washed
were
analysis
the
employed:
was
(DIG)
where
times
1
of
were
Genbank
conducted
guide
6S
RNA1
used
with
were
a
rDNA
of
mix
Ten
and
through
PCR
extracted
of
(1998).
the
filtered
to
sized
bacterial
(Bruce
(1)
of
clones
universal
purify
database
sequences
amplication
from
according
phenolichloroformlisoamyl
universal
syringe
by
sterilized
The
using
Paster,
were
the
the
electrophoresis
16S
using
reverse
8 method
berries
DNA.
was
the
needles
to
sent
forward
rDNA
personal
was
the
seawater,
DNA
BLAST.
conducted
for
(position
was
not
is
Microbial
and
with
based
automatic
(position
lysis
achieved,
cloned
comunication).
on
(2)
crushed
Phylogenetic
a
Group
an
protocol
on
by series
1492)
universal
Diversity
agarose
into
cell
alcohol,
comparing
8)
sequencing
the
II
and
of
for
between
Escherichia
lysis
described
Wizard
Microbial
smaller
checkerboard
forward
universal
gel.
and
course
analysis
through
them
two
of
later
PCR
gauges.
a
(position
in
guide
Diversity
coli
microscope
reverse
to
500
was
July
the
precipitation
the
preparation
existing
use
base
using
(1998).
27,
DNA
8)
of
1998
1998 a
aggregates
the
(Figure probes
sequenced
extracted
brown
incubation
bacteria
(SRB)
analysis PCR
and
membrane
3.4
berries
very
310
oligotroph
Odontella
Thiorhodococcus
presence
pink
Checkerboard
base
products
similar
hybridized “berries”
enrichment,
In
3),
Checkerboard
(Banin
Of
(PSB)
(Figure
flocs
from
the
pairs).
in
formed
the
with
sinensis
according
the
1095,
of
1997,
(one
first
signal
amplified
(from
a
SRB,
enrichment,
ten
1997;
the
(mussel
mussel
2).
The
cytophaga
CH
with
base
by
minus
clones
hybridized
PSB
(4)
For
(91%
Hybridization
for
spirochetes,
non-berry
a
Figure
to
clone
(Figure
hybridization
all
mussel
pair
five
crushed
the
the
the
enrichment
with
(94%
sent
the
similarity),
(2)
difference
pond
protocol
pond
similar
second
1).
(100%
the
samples.
2),
or
only
for
incubated
mixed
ponds
similarity),
by
pseudofeces),
the
and
berries,
DIG
berry
sequencing,
with
passing
to
CH
was
similar
(see
given
(CH)
universal
flavobacteria.
culture
in
and
T.
out
forward
(lane
The
the
the
the
conducted
minuss
with
section
(5)
of
two
in
following
two
through
for
DNA
probe
Sippewisset
five 500)
35)
of
the
PSB
six
probes,
clones
primer
9
the
(7)
clones
PSB
clustered
5.2;
was
Microbial
berry-like
clones
to
from
for
pink
first
(1:10
using
a
The
a
DNA
and
lower
were
syringe
samples
the
from
clone
the
were
one
19
sand
Salt
DNA
dil.)
were
cytophaga,
a
SRB
with
FlavofBacteroides
base
related
dilution
minislotfblot
Diversity
than
one
and
aggregates
related
previously
(1:10
Marsh)
was
(Figures
were
most
extracted Group
purple
species,
pairs
pond
five
in
positive
to
dilution),
the
to
used:
1:10
closely
berries.
(3)
were
a
and
berry,
course
II
sulfur
the
3).
freshwater
first
from
from
sequenced
sulfate
from
and
Microbial
apparatus
88%
(1)
chioroplast
for used
CH,
related
five
bacteria
the
guide
and
the
probe
1:100),
In
purple
the
gamma-
similar
reducing
in
the
mussel
berry
pond
but
(8)
berry-like
obligate
the
from
using
(1998).
to
Diversity
and
second
sulfur
DNA
July
(6)
revealed
and
of
first
for
berries,
and
SRB
the
five
a
bacteria
it
27,
the
nylon
CH
The
CH
was
last
1998 1998
phosphotungstic
Caulobacter-
slides,
4.2
contrast
surrounding
coccoid
was
aqueous
polysaccharide
aqueous
were
4.1 4.0
hybridize
we
for
(lanes
aipha-proteobacteria,
Transmission
believe
the
examined
Crystal Microscopy
smeared
and
grids
Berries
37
In
PSB
and
images
solution
solution
to
order
with
suspended
these
coated rod-shaped
them.
40).
berry
Violet
like
on
under
were
capsules
any
from
to
acid
of
of
bacteria
a
The
visualize dii.
Electron
Figure
slide
with
copper
Staining
washed
of
bright
(PTA;
the
in
probes
SRBs,
1:100,
the
bacteria
sterile
plastic
and
(Gerhardt
berry.
to
4
DNA
violet
sulfate.
field
pH
includes the
in
(Figure
be
Microscopy
stained
spirochetes,
the
for
seawater
filtered
7).
important
and
matrix
stained
oil
samples
brown
the
for
et
TEM
carbon.
After
immersion
6a),
using
berry
the
2
al.,
sterile
minutes.
surrounding
berry,
(80%).
dark
and
crystal
1994).
showed
from
partial
(TEM) and
components
the
PSB
TEM
a
purple,
seawater
flavobacteria
Anthony
10
long
the
the
(1000X)
and
violet-stained
The
The
drying,
The
the
pink
samples
berries.
curved
the
the
berry
with
presence
air-dried
dye
of
(80%),
purple
sand
staining
berry
microscopy.
a
the
was
a
cover
suspension
were
flagellated
This
light
(lane
dii.
berry
cytophaga
washed
crushed
berries,
film
sulfur
of
Group
method
result
heavy
slip
1:10,
purple
bacterial
36).
microbiota.
was
was
bacteria,
II
The
was
off
rod
and
along No
between
was
metal
covered
capsule
for
Microbial
clones
put
with
results
(Figure
staining
the
aggregates
prepared
unexpected,
bacterial
with
on
stained
crushed
mussel
a
two
9
layer
the
with
20%
were
and
other
6b).
revealed
Diversity
slide
microscope
for
July
with
a
dil.
(wlv)
(Figure
10
berries
obtained
phase
TEM 1%
TEM
because
did
and
27,
1:10
1%
(wlv)
not
1998
1998
it
on 5),
fluorescence probe
Na 2 SO 4
from
sulfur
FITC
GSulf
Cy3 Flavo
Rhodamine
SRB2 Dye
Probe
The
NaCl,
samples modifications.
and
from
isolates
4.3
Figure
was
then
following
Fluorescent
the
also
the
(Figures
bacteria
Name!
rather
Probing
7
Whole
enrichments
to
berry
were
crushed
shows
1998
applied
detect
throughout
than
fixed
was
(see
Green
8c
probes
sulfate-reducers
CFB
Phylogen.
Microbial
cell
of
Berry
one
sulfate-reducing
to
and
with
In
0.5
the
more
Figures
fluorescent
in
division
samples
of
with
Sulfur Situ
M.
8d),
berry
were
samples
4%
a
the
the
microscope
informative.
Diversity
Group:
either
All
but
paraformaldehyde
Hybridization
morphotypes
8a
used:
cell
was
from
samples
no
and
were
in
ethanol
as
complicated
autofluorescence
situ
bacteria,
the
8b;
noted
course
prepared
slide
5’-cttttargggatttcctctg
5’
5’-tcagtrccagtgtggggg
Sequence:
hybridization
SRB
Both red were
-cgygcgccrctytact
or
observed,
by
before
signal).
handout
(FISH)
H 2 !C0 2
enrichments
cytophagas,
counterstained
of
the
by
for
by
the
11
red
the
washing
3.5
passage
putative
However,
without
a
showed
was
color,
was
autofluorescence
hours.
short
followed,
and
performed
(ethanol
through
several
while
slightly
SRB
with
the
good
The
green
probing
Group
probe.
DAPI
some isolates
wash
hybridization
as
times
a
with
curved
sulfur
54
57
58
Tm
on
hypodermic
electron
of
of
II
cells
buffer
Most
the
(blue
the
in
(
from the the
Microbial
bacteria.
C)
rod.
sterile
berry
following
showed
predominant
enrichments!isolates
cells
signal
donor)
contained
Hyb!Wash
the
with
needle.
and
sea
showed
anaerobic
37
37
37
The
in
Diversity
partial
with
the
several
July
water
all
protocol
0.9
SRB2
All
figures).
Temp
berries.
purple
27,
or
(80%),
M
berry
no
1998 1998
the
Temperature
9).
through pond
assess
5.1
5.0
analysis. the
showed
control
berry
cells
phototroph
The
fluorescence,
lab
A
Field Field
TYE
FISH
and
from
site
with
the
with
The
The
(a
which
no
enrichment
do
Observations
Observations
characterization
known
this
results
the
detectable
berry
collected
enrichment
Gsulf
not
and
which
water
Flavo
culture
form
salinity
pond
cytophaga)
support
probe
flows
and probe
may
samples.
in
at
signal.
would
and
from
other
and
the
showed
were
the
indicate
the
of
in
provided
the
pond
hazy
Laboratory
the
the
and
showed
show
conclusion
Hence,
ponds
measured
environment
The
berry
berry.
a
out
and
brown
differences
positive
a
results
in
no
positive
from
positive
a
to
pond,
the
strong
As
evidence
attempt
in
colony
that
Simulations
of the
salt
the
hybridization
the
surrounding
the
these
signal. we
identification
positive
in
12
adjacent
field;
marsh.
culture
inlet,
to
from
the
have
that
understand
measurements
amount
pH,
these
the
Probing
and
isolated
was
signal,
The
tidal
the
with
anaerobic
the
impure,
berry
were
of
pond
channel
of
both
channel
some
the Group
these
why
an
target
and cytophagas.
pond
SRB
were
are putative
the
it
the
sulfate-reducer
of
two
as
sulfide
makes
II
presented
1
was
yellow
the
is 6S
berries from
the
observed
Microbial
isolates
connected
rRNA
cells
cytophagas
conducted.
tide
sense
were
the
While
swarmer
form
changes from
in
berry.
within
awaits
in
measured
that
Table
Diversity
enrichments
to order
in
July
the
a
an
the
not
from
cells
the
further
(Figure
positive
2.
27,
inlet
to
berry
all
cells.
in
the
from
1998 1998
Berries
bacteria,
berry
berries
indigestible
it
are
hypothesis
simply
we bacteria
organisms material present
demissa).
Channel Inlet
Pond
is
not
devised
possible
are
found
The
therefore
consume
digested
The
The
in
the
(Langdon
present
(Figure
While
the
which
suggests
second
two
first
or
berry
inlet
that
in
berry
unpalatable
27
31-33
31
Temp.
Table
the
hypotheses
by
hypothesis
the
accumulate in
berry
10). the
feeding
feed
would
and
hypothesis
the
the
berry
pond
that
berries
ribbed
2.
Atlantic
on
(°C)
mussels,
formation
water
Newell,
the
form
Site
pond.
into
itself.
to
mussel
as states
addressing
berries
in
that
the
Characterization
the
states
in
they
—6
—6
—6 pH
ribbed
the
1990).
but
This
Mussels
the
mussels.
serves
the
berry
that
inlet
feed.
is
form
are
that
mussel,
mussels
hypothesis
a
mussels,
the
the
common
pond
rather
After
where
to
mussels
independently
in
This
mussels
potential
protect
the
and
is
02
10-12
filter. 10-13
13-14
observing
regurgitated.
scenario
the
Data
13
like
inhabited
inlet
suggests
inhabitant
the
(mg/i)
form
mussels
its
do
all
role for
In
berry
were
bacterial
not
bivalve
the
dissolved
this
further
the
of
mussels
that
by
contribute
that
observed
berries
would
the
reside,
Berry
scenario,
of
If
0.01-0.08
0.2
Atlantic 0.5-1.5
S 2
mussels
the the
this
mussels
molluscs,
components
Group
suggests
(mg/i)
excreting
organic
be
Pond.
bacterial
salt
or
and
hypothesis
expelling
to
excreted
a
mussels
ribbed
play
II
marsh,
precursor
when
berry
and
that
are
Microbial
matter,
in
berry-like
components
that
by
20
30
30
Salinity
mussels
suspension-feeding
the
the
formation.
berry
the
by
would
berry-like
is
making
the
berry
tide
supported,
to
the
mussels
formation.
the
mussels
Diversity
(%)
mussel.
ingest
comes
July
(Geukensia
aggregates,
aggregates
them
“mature”
of
pink
This
are 27,
and
the
the
then
in
not
1998 1998
collected from
pond
water
chamber
chamber
the
were
5.2
pseudofeces.
berry
berries,
production.
rejected
(Bayne
intriguing
berries
components
berry berries
excreted
are
formation
washed
Laboratory
the
established
that
collected
may
pond.
A
The
together
in
and
but
tidal
as
containing
containing
from
by
did
series
the
because
in
pseudofeces.
reject
Newell,
hypothesis
into
the
This
of
The
fact
not
channel.
of
berry
The
the
from
the
of
mussel
the
(Seitz
the
with
Simulations
contain
be
second
mucus
them
berry
slime
laboratory
it
berries.
pond.
water
water
berries.
berry
the
the
1983).
is
two
et
Treatment
known
that
as
are
pond
mussel
berry
matrix
serves
hypothesis
berries.
As
al.,
collected
collected
pond.
replicates pseudofeces.
Maturation
not
the
The
pseudofeces
Glass
1993)
experiments
and
pond,
that
yet
berries,
that
to
mucus
mussels
This
Treatment
4
aggregate
bowls
“mature”--meaning
a
is
from
from
contributes
consisted
per
also
sediment
portion
produced
hypothesis
of
that
or
treatment.
This
allows
the
the
in
the
were
are
berry
were
contributes
collected
the
the
3
of
berry
berry
berry
produced,
theory
of
from
consisted
14 used
material
salt
in
to
conducted
precursors,
for
particles
two
allows
the the
pond
may
pond.
Treatment
marsh
the
the
as
suggests
berries
from
mussel
aggregation
experimental
that
to
nearby
possibility
occur
molluscs
ingested
of and
for
that
pseudofeces
Treatment
may
the
to
a
they
are
the
in
sediment
mussel
determine
that
and
are
after
nearby
Group
pond,
1
a
ingest
formed
possibility
consisted
lack
chamber
by
increase
being
not
the
of
that
the
filter-feeding
in
2
chambers.
and
the
components
by
II
berries
pond.
the
collected
production.
consisted
berries
the
a
in
rejected
if
Microbial
bacterial one
chamber
pink
components
the
containing
mussels
their
of
slime
that
Treatment
a of
are
mussels
are
sand
mussel
mucus the
the
from
Five
as
of
that
mussel
present
deposited
containing
species
molluscs
contribute
pseudofeces
Diversity
berries
a
bacterial
collected
July
mussel
holds
water
treatments
a
is
in
of
nearby
4
a
the
27,
was
in
in
the
is
in
the
in
the
1998
1998
to
the a
strongly
6.0
digested
rapidly
berries
looser
excreted
mussels
(PSB)
berries
can
presence
treatment
Treatments
ensure
incubated
consisted two
designed
Conclusions
likely
berries
and
and
The
from
To
that
(Seitz
(<
Mussels
suggest
were
by
the
of
to
4
of
be
1
for
less
further
green
results
the
the
did
pink
or
day)
1,
determine
PSB
the
a
attributed
et
fed
three
2,
chamber
(2)
discrete
mussel mussels
not
berry al.,
that
in
4,
sulfur
filtered
sand
and
berry
and
the
of
Treatment
and
increase
weeks
1993).
the
the
the
formation
Recommendations
pond
the
is
but
if
bacteria
clumps,
pond
to
5
filled
had
mussels
essential
field
and
the
GSB did
abilty
the
in
rather
and
sufficient
in
treatment
the
water
excreted
not
with
presence
3
observations
in
size.
excreted
but
(GSB).
the
of
of
laboratory.
play
result
were
the
to
water
mussels
additional
spiked
they
berries
berry
The
form
an
food
the
excreted
conditions
of
in
As
were
berries
essential
collected
results
purple
the
PSB
formation
for
with
of
produced
to
to
and
the
Pond
aggregates.
formation
berries.
filter.
produce
clearly
Future
15
and
mussels
enrichment
the
as
during
of
sulfur
would
role
unwanted
from
water
the
GSB,
laboratory
in
by
aggregated
Research
Treatment
in
berries
simulation
bacteria,
the the
filtered
of
the
mussels
facilitate
was
the
suggesting
The
berries.
mussel.
process
cultures
berry
material
formation
changed
under
simulations
Group
aggregates
the
the
in
bacteria.
5
pond.
experiments
(1)
treatment
of
served
The
spiked
The
of
dominant
controlled
that
II
or
an
suspension
every
both
of
two
pseudofeces.
Microbial
color
Treatments
increase
these
differed
the
with
as
pond
The
purple
berries
few
the
3
berries.
of
component
bacteria
suggest
in
conditions,
the mussels
water,
control
the
days
feeding.
in
that
from
sulfur
Diversity
mussels
incubated
size
July
were
pink
they
in
Therefore,
that
they
were
the
very
and
of
order
bacteria
27,
sand
of
were
the
the
the
not
1998
1998
in to
relationship
dark
in
the
berries
berries
using
disaggregation
1998).
texture.
using
of
berries
in
laboratory
the
simulations.
sulfide-rich
berry
the
PSB’s
second
purple
most
molecular
microbial
We
Autoinducer
in
together,
Future
Another
excreted
Berries
maturation
in the
should
observed
probable
hypothesis
berry
between
the
dark
purple
research
of
collected
dark
possible
composition by
in
techniques,
exhibited
the
be
did
order
the
via
activity
number
that
berry
purple
compared
sulfur
berries.
not
concerning
mussels
should
the
topic
to
from
while
result
color
addition
sulfur
phototroph
determine
berries
was
whether
(MPN)
as
This
the
of
further
the
to
are
compared
in
and
detected
future
granules.
berry
determine
the
berry
PSB
may
lacked
not
of
phototroph
enumeration.
berries
pursue
mussels
an
other
medium,
yet
research
of
pond
necessitate
disaggregation.
appropriate
in
sulfur
to
light
“mature”
It
the
bacteria
excreted
if
the
this
and
will
was
16
they
pink
berries
composition
the
is
pond
granules.
autoinducer
berries
be
Application
the
supported
the
are
purple
and
berries
to
disaggregation
by
of
berries,
population
characterization
(Pat
the
indeed
interest
the
that
produced
In
sulfur
aggregates.
Fidopiastis
were
mussels
within
incubation
by
addition,
activity.
Group
although
the
of
to
the
bacteria
full
MPN
distribution
same.
more
by
the
technique. results
are
II
of
the
after
personal
berry.
of
It
they
enumeration
Microbial
in
extensively
sulfur
fully
It
should
mussels
from
the
the
of
is
a
differ
possible
developed
mucus
within
the
6-day
berry
granules,
an
Incubation
project
be
laboratory
originally in Diversity
in
July
determined,
incubation
study
pond
the
the
will
holding
shape
that
report,
most
berries,
in
27,
require
results
the
the
of
terms
and
1998
1998
the of Group II Microbial Diversity 1998 July 27, 1998
Other suspension-feeding organisms in the salt marsh may also produce berries. Future studies should address the role that these organisms play in berry formation.
Finally, additional enrichment conditions for cytophagas should be conducted. For example, cultivation on TYE and YPG plates under oxic and anoxic conditions and at different temperatures could be tried.
17 Group II Microbial Diversity 1998 July 27, 1998 7.0 References
Banin, U. 1997. Identification of the bacteria forming the “berries” purple macroscopic aggregates in Great Sippewissett Salt Marsh. Microbial Diversity, Marine Bioloogical Laboratory, Woods Hole, MA.
Bayne, B. L. and R. C. Newell. 1983. Physiological energetics of marine molluscs. In: Saleuddin, A. S. M. and K. M. Wilbur (eds.). The Mollusca, Physiology, Part 1. Academic Press, New York, 4: 407-5 15.
Gerhardt, P., R. G. B. Murray, W. A. Wood, and N. R. Krieg. 1994. Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, D. C., p. 35. Langdon, C. J. and R. I. E. Newell. 1990. Utilization of detritus and bacteria as food sources by two bivalve suspension-feeders, the oyster Crassostrea virginica and the mussel Geukensia demissa. Marine Ecology Progess Series 58: 299-310.
Seitz, A. P., T. H. Nielsen, and I. Overmann. 1993. Physiology of purple sulfur bacteria forming macroscopic aggregates in Great Sippewissett Salt Marsh, Massachusetts. FEMS Microbiology Ecology 12: 225-236.
18 (% Difference)
I I
Purple sulfur sp. WHO1 (1998)
Purple Sulfur sp. Clone C 13 (1997) Thiorhodococcus minus
Rhabdochroma tium marinum Lamprocystis roseopersicina Chromatium okenii
Thiocystis ge/a tinosa
Chroma tium vinosum
Escherióhia co/i She wanella putrefaciens
FI&OP 1
Notes:
•
• •
•
Dental
Sediment Pink Berry
(Bacteroides
Squid
Cytophaga
All
(see
flock
SRB
All
All Flavo/Bacteroides
samples
samples
Cytophaga
V. Cytophaga
Enterics
Universal
Blank
plaque
Blank
Bruce
Actinomyces
All
gazogenes
All
Rothia
Universal
All
probes
Cyanos
Methylotrophs
sp.
samples
S.
Strept.
has
identified
SRB
Streptococcus
Veillonella
oralis
from
and
samples
for
sp.
have
SRB’s
have
‘S
2
1
sanguis
Checkerboard beriy
-a
related
berry
berry
explanation)
gave
sp.
specific
flavobacteria
last
(but
have
no
bacteria)
a year
‘
not
signal—probably
,_
different
SRB
*
Plaque
the
(last
øø.*
identified
and
specific
year
samples
hybridization
.1-rn
distributions
1
sample
not
p
species
last
all
DIG
berries
may
year
primers
found of
.,
a)..D
have
and
streptococci
a)
had
of
a)
cj.
a)
in
flavobacteria,
methylotrophs
were
both
environmental
the
c
berry)
not
Cytophaga
and
used.
and
other
but
(arrow--
flavobacteria
not
species)
Plaque
samples
Staff
bacteria,
samples
the
—
0
0
—
hard
specific
including
Sediment
Samples
to
see)
species
flavobacteria
June29,
of
I 1998 Checkerboardhybridization 7/24/9 8
13579111315171921232527293133353739
S — a Berry“PurpleSulfur” - ft. ft ft Betas-all Nitrosolobus/Nitrosovibrio Nitrosomonasspp R.: 0 EntericsI some gammas p. ft S Methylotrophicbacteria ANGAlteromonassp. ) __•. Alphas-all ANGShewanella sp. Vibrio gazogenes Berry “Plastid’ SRB2, some deltas SRB, some deltas BerrySRB clones C16/C18 LoGCGram Positive Green Sulfur-all Planctomyces-all a Spirochetes Flavos-all Bacteroides Cytophaga berry ClO Cytophaga berry C9 Cyanobacteria Microcoleussp. Lyngbiasp. Nostocsp. Oscillatoriasp. Phormidiumectocarpi. Synechococcus sp. ————S ._ 545. Universal 135 79111315171921232527293133353739
Key:
1. Silvana-salt pond I 12. Milvaenrich t2+ 02 23. Karin Cyan. Pure 34. /FeYoshiko-H 2. Silvana-salt pond 2 13. Milvaenrich t4+ 02 24. Scott- Begg. Environ 35. Pond Berry 3. John oyster 14. Milvaenrich 02 25. Andreas Deep Sea 36. 2Pksand berry 1/10 t8+ 4. Group I- ED 15. Milva enrichment t6 w/o 0 26. Andreas Deep Sea 37. Pksand berry 1/100 5. Group l--LD 16. Grp IIsediment 27. Andreas [no good) 38. Brown berry 6. Group I-MD 17. Grp IIsediment [No good) 28. Andreas Deep Sea 39. Pink sand 1/10 7. Group 1-GI [No good]] 18. [No good] 29. Yoshiko ThioS 40 Mussel 1/10 8. Group I-P2 19. Patti Sip. Hi Nitrogen plot 30. /FeYoshiko-H Group I-G3 20. Patti Sip. Low Nitrogen plot 31. Grpll--PSB ‘ Group l-B4 21. Patti Sip. Control plot 32. 2Grp IICyto/PS 11. +0Milvaenricht 22. Karin Cyan. Assoc. 33. Gm IISRB 02 FL&URE ‘ 0 C
-‘1
c:z
-t FiGuPE 5 -n Q > rn c5 V 0 1
• • • • ••• • . • •
I FIGURE
10