1%
pond
Distribution
Microbial
of
Great
Jackie
of
Diversity
Sippewissett
Cytophagales
Aislabie
Course,
Salt
1999.
in
Marsh.
a tidal
S
diluting
colonies phase
gliding
Thin Plates
distilled
or
seawater
pond
supplemented
Bacteria Direct
or
Materials
mussels
combination
distribution to
of
belonging
Great
Cloning
Flexibacter
almost
fermentative.
shaped
Flexibacter
soil,
The
Introduction
primers
1998).
extract mud.
the
DNA
The
Abstract
tidal
cultured
which
flavobacterium-cytophaga
spreading Cytophagales
microscopy
fresh
distribution
were
mud
Sippewissett
isolation
was
All
pond
gliding
always
Within
water
and
as
onto
capable
medium
supplied
and
specific
to
and
to
are
carbon
strains
isolates
extracted
incubated
and
berries.
in
are
mud. are
of
filamentous
sequencing
of
the
with
seawater
Because
and
Methods
colonies
with
marine this
of
colonies
sterile
phylogenetic
Cytop
marine
best
were
frequently
of
to
Cytophagales
gelatin,
contained:
for
of
as
Dilutions
and of
700
gliding
either
division
the
degrading
division
Salt
grew
a
the
Cytophagafermentans, Cytophagales
from
carbon
known
yellow,
ha
at
nitrogen
purified
ml
of
flavobacterium-cytophaga
medium
facultatively
in
seawater
water
gales,
Marsh
presence
of
room
were
chitin,
tryptone-yeast
their
seawater.
on
sterile
bacteria.
bacteria
berries,
isolated
1
is
of
0.5
members
6S
for
sourcel6S
low
and
biomacromolecules orange
one
homogenised
and
removed temperature
source
gliding
(Banin
group
by
(Seitz
in
rDNA agar
g/l
with
their
seawater
of
were
isolation of
pond
from
nutrient
K 2 HPO 4
particular
either
in
sewage
For
from
gliding,
Most
and
the
anaerobic
or
tryptone-yeast
were
at
of
ability
et
a
extracted
1997).
motility
extract,
red
chitin mud
rDNA
environmental
tidal
spread
a
from
xylan.
major
the
at.
natural
and
final
streaking
are
under
isolated
seawater
pigmentation.
techniques
treatment
washed
1
rod-shaped C.
1993)
genera
and
to
pond
The
then
gliding
g/l
containing
isolation
lineages
salmonicolor
concentration
their
in
cellulose,
from
obligately
degrade
onto
branch
NH 4 C1,
and
water.
were
aerobic
clones
the
plating
revealed
containing
berries,
from
Cytophaga,
extract
inocula
colonies
agar
berries
modified
bacteria,
low
extracts
samples
were
isolated
of
to
plates
Gliding
of
To biomacromolecules
plates 16 obtained
or berry
and The
onto
bacteria
plates
filamentous
the
berry
as
aerobic,
nutrient
g/l the
xylan
from from
and
applied
investigate
of
They
are
anaerobic genera
Cytophagales.
berries
and
marine
carbon
were
agar
ecosystems
directly
NH 4 C1
rinse
0.2-0.5%
in
presence
Flavobacterium,
bacteria
C.
rinse
thin
with
a
were
as
the
(Hugenholtz
a
examined
are
agarovorans,
tidal
(Difco),
to
amplified
seawater
water
carbon
but
Cytophaga
tidal
and
agar
was
source
bacteria.
water
was
tryptone-yeast
edge
from
most
gliding,
mud,
conditions. belonging
pond
further
(w/v).
of
spreading,
some
(Difco).
examined.
omitted.
including
and
pond,
bacteria
300
source, or
such
of
berries
similar
or
water,
using
of
tidal
agar
The
using
The
et
pond
the
rod-
ml
by
and
the
and
the
are
all
a!.
as
a to
Sequence analysis
yeast
for
gut
from
Four
Results
using
expected
31 mussel
DNA Extraction
Microbial
FISH
enrichment
An
specific
incubated
served medium
Enrichment
Whole Cultures
constructed
were
Sequence
and
the
Cytophagales
1,
strain
DNA
500
containing plates.
]6S
chitin,
(w/v) Isolates
9aF
growth.
(94°C,
contents
rRNA-targeted
forward
pond
isolates
30.
extract,
rDNA
16SrDNA
was
the
analysis
and
confirmed
35
was
cellfluoresecent
potassium
gut
as
xylan,
confirmed
The
size
Growth
for
supplemented
information
were
to
was
Diversity
1.5
with
TOPO
the
mud
data
and
extracted
source
None using
extracted
sequence
cultures
tryptone-yeast
using
or
cultures
and
of enrich
PCR
were
the
primer
mm;
reverse
amplified
and
screened
analysis
and
pond
shaking
and
was
(Figure
gliding
Bacterial
all
the
of
hydroxide.
under
TA by
Cytopha
of
neighbour
amplification
sent
laminarin
55°C,
the
course
oligonucleotide
that
counterstained
analysed
isolates may
the
for
for
from
water
from
31
inocula.
analysis
MoBio
from
primer
gel
for
eubacterial
with
at
2; of 9aF
bacteria in
fermentative
for
isolates
using
fermentative
cloning
anaerobic
a
isolates 1
room
DNA
situ
gales
Table electrophoresis
Sequencing
tidal
extract.
sequence
cultures
notes.
mm;
isolate
source
were
the
except
specific
joining.
0.2-0.5%
was
Their
Soil
1
using
ofpure
Tubes
the
492R.
hybridization
production
from
temperature.
pond
72°C,
cycling
produced
were
1).
was
unsuccessful.
kit
determined
universal
kit.
of
17
13,
reverse
using
35,
ability
with
programs
Attempts
analysis.
conditions
probe
PCR
environmental
gliding
for
was
(Invitrogen).
gliding
were
mud, cultures
obtained,
DNA
gliding
used
1
(w/v) kit
30
utilised
mm)
parameters
the
DAPI
the
of
products
to
(Applied
flexirubin
using
of
primer
and
filled
Flavo
water,
eubacterial
(FISH).
poor
was
on
bacteria.
chitin,
bacteria
grow
Prepman by
flavobacterium-cytophaga
flexirubin
x
to
from
bacteria
the
All
30,
35
two
using
was
comparison
isolates
isolate
amplified
the
to
quality,
labelled
1
carbohydrates
and
on
Clones
of
492R
(72°C,
were
ARB
belonged
samples
were
cellulose
Biosystems).
exclude
from
pigments.
determined
1kb
The
the
tryptone-yeast
the
primers
berries
were
by
gliding
method
cloned
and
as
and
were
however
isolates
berry
12
samples
methods
DNA
flooding
with
containing
using
follows:
with
oxygen.
mm)
established
or
a
to
to
and
used
8F
bacteria
phylogenetic
1
into
rinse
xylan
rhodamine
6S
from
cellulose
substrate-free
ladder
the
the
determine
Purified
grew
on
and
x
the
were
the
colonies
rDNA
described
Escherichia for
1.
extract,
(94°C,
forward
water
All
Cytophagales.
the
seawater
bacterium
inserts
and
1
contents
PCR
branch 492R
on
from
strains
prepared
size
in
MicroSeq
tubes
and
DNA
pond
tryptone
sequence
12
which
and
with
seawater
cellulose,
whether
products
tree
mussel
whereas
marker.
primer
of
chitin
in
mm)
control
13,
of
plates
were
of
two
mud
coli
did
from
the
was
20%
the
for
the
is
17
a x
the
evidence
the
amplify Molecular
Cytophagales
to
rarely
colonies
Cytophagales
strategy
common,
(Figure
berry
grew used
tidal
environment
Discussion
Studies
Sphingo
with
marine
maritimus
mud
A
29 their
gut
bacteria
flavobacteria-cytophaga DNA
observed.
Most
observations
stained pond
30-50% FISH
belonged
hybridise
the
specificity
four
l6S
belonged
contents.
as
pond
on
Flay
clone
similarity
observed rinse
mud
C. of
extracted
analysis
2).
rDNA
carbon
DNA
for
clones
bacterium
of
for
low
origin.
that
belonging
the
with
of
despite
probes
probe.
to
latercula
These
and
of
to
in when
water
obtaining
29
the
the
nutrient-seawater
cells
the
and
to
exhibit
of
(Manz
were
which
the
extracted
situ
the
phylogenetic
was
source,
the DAPI,
obtained
in
lack
to
the
the suggests
Isolates cells
and
Cytophagales
results
the
provide cellulose,
a
appeared
known
enrichment and
Great
spp.
Flay
bacterial
psychrophile
few
made
to
produced
probes
of
Cytophagales.
high
et
amplified
are
from
gliding
Cytophagales in
the
mud,
and
specificity
hybridised
gram-positive al.
from
indicate
probe
the
using
Sippewissett
specific
present
organisms
a
for
that
13
numbers
Cytophagales.
a
used
1996;
powerful chitin
to
tree
a
diversity enrichment
enriched
clone
enrichments
and
behaviour
(Figure
environmental
cultures
be
fermentative
plates,
the
(results
marine
when
from
is
that
Polaribacterfilamentus,
showing
in small,
Hugenholtz forward
or
of
35
of
319a
obtained.
to
DNA
of
from
xylan
determined
these
nature.
tool pre-enrichment
the
extreme
colonies
used
bacteria
3).
from reveal
Salt
grouped
Cytophagales
with
not
the
aquarium.
some
culture
F
may
tidal
was
for
the
The
the primer,
primers
with
Sequence
is
shown).
primer
Marsh,
Flay
for
high
the
Cytophagales
Furthermore,
supplied
Amongst
the
samples
the
environment.
et
positions
were
importance.
exclude
not
pond
isolates
(Bruns
indicative
cellulose
FISH
with
(Table
mud
a?., growing
numbers
study
probe
prevalance
indicates
can
successfully
and
Cytophagales
rod-shaped
Mud
mud,
was
1998).
of
observed
when
as
&
the
hybridises
be
analysis
grouped
the
the
subsequently
2).
of
samples
of
carbon
Berthe-Corti,
(Figure
or
inferred of
obtained
with
bacteria
whereas
water
The
of filamentous
Only
fish
population clone
isolates can
isolation
Alternatively
that
chitin
biomacromolecules
gliding
In
bacteria
of
in
xylan
31
but be
this
extracted
pathogen
with source.
indicating
may these
one
Cytophagales
and
enrichment
9a
to
2
from
were
29
(results
in enriched
for
isolate
few
13,
investigation
bacteria
F
of
members
a
of
sequenced
be
berries
Cytophagales
nature
as
that
samples
of
primer
four
clustered
1998).
the
30
this
bacteria
&
Approximately
filaments
isolated
a
many
carbon
bacteria
from
selection
worthwhile
Flexibacter
30
hybridised not
and
clones
b).
that
clones
study.
however
from
were
using
cultures
Further
used
grouped
shown).
of
of
contain
35
mussel
Similar
in
it
source
were
only
were
from
with
of
that
were
mud
the
the
not
tidal
Of
the and
and
also
to
of
the
of a
Microbiology
a
of Manz
FEMS
forming
Seitz
Hugenholtz
on
180: cultures
oligonucleotide Bruns,
macroscopic
Course,
Banin
References
tools
I
Tom
Acknowledgements those
opportunity
the
cloned In
one
some
suite
thank
the
summary,
the
Great
4765-4774.
was
W,
AP,
Schmidt
cloned
cases
Microbiology
of
emerging
A.;
U.
from
my
phylum
1997.
macroscopic
of
Amann
1
a
Sippewissett
Nielsen
6S
seawter
Berthe-Corti
group
P.;
1997.
less
Cytophagales.
to
this
previously
the
142:
aggregates
rRNA-specific
for
isolate
Goebel
probes.
than
evidence
R,
phylogenetic
environment.
cytophaga-flavobacter-bacteroides
members
1097-1106.
increasing
Identification
TH,
sediment
Ludwig
Ecology
200
aggregates
further
B.M.;
Salt
Microbiology
from
Overmam
L.
base
in
indicates
The
Marsh.
for
W,
1998.
oligonucleotide
Great
12: this
strains
my
suspensions
Pace
pairs)
view
The
their
Vancanneyrt
lack
225-236.
in
understanding
environment.
of
N.R.
In
Bacteria
use
that
J.
Sippewissett
Great
must
help such
of
of
144:
the
situ
1993.
of
bacterial
Cytophagales
1998.
sequence
with
as
be
with
2783-2790.
bacteria
pre-erichment
Sippewissett
detection
isolated
M, C.
taken
probes
Physiology
this
Impact
fermentans,
fluorescently
Schleifer
of,
diversity.
Salt
into
information
project,
forming
and
thus
designed
of
in
are
of
consideration.
Marsh.
bacteria
confidence
Salt
far
techniques
the
culture-independent
K-H,
distributed
of
and
which
Journal
are
labelled
Marsh,
the
purple
to
natural
obtained
Scott
1996.
not
Microbial
investigate
in
are
“berries”-
the
of to
may
continuous-flow
in
Dawson
Massachusetts.
sulfur
Application
rRNA-directed
more
use
environment.
Bacteriology.
tidal
same
however
increase
molecular
Diversity
similar
bacteria
bacteria
ponds
as
studies
and
purple
those
of
the
Dr
(in
to of
Table c
b
a
Table
29 Xylan
27 similarity
33 26
Laminarin
tryptone-yeast
Clone
Chitin
Cellulose
Facultative
Growth
l6Sr
Isolated
Isolate#
Colony
means
Source
means means
DNA
2:
1:
#
no
not
growth
pigmentation
on:
on:
(%)
growth
done.
sequence
aerobes
Mud
Mud
Water
Mud
Source
known
Sequence
Gliding
extract
organisms
bacteria
similarity
Flavobacterium
(96%)
yellow
xylan
berries
+
+
+
+
+
13
isolated
of
Uncultured
Actinomycete
Chioroplast
Cytophagales
similarity)
Closest
clones
from
Laminarin
Yellow Berries
ND
ND
organism
+
+
+
obtained +
-
a
17
tidal
eubacterium
(98%)
(93%)
(97%)
pond
match
from
(95%)
Cytophaga
yellow-
orange
mud
cellulose +
-
+
+ +
30
(98%)
the
on
BLAST
tidal
Flexibacter
(9
xylan
yellow
mud
pond + - - -
-
35
1%)
search
with (%
Figure
Figure
Figure
3.
2.
1.
the
Phylogenetic
FISH
and
Cyt
Cytophagales
op
isolates
stained
ha
of
gales.
an
(13,
enrichment
with
tree
isolates
30
of
DAPI
and
selected
(a)
35)
culture
(a)
13
and
Cytophagales
and
and
mud
from
(b)
the
clone
17.
tidal
probe
29
pond
showing
to
flay
known
mud
that
the
grown
bacteria
is
relationship
specific
on
xylan
for of
4
4
S 1 14
I
;
‘ill -
:atc*t
• -e
I t ep
S
—
I
• I
t
S
‘4 t
4
t -‘
PC • Bacteriodes
uncultivated rumen bacteria Cophaga lanollca — WH99 r WH7O WH69 WH71 — CytFerme, Cytophaga fermentans, 1463 WH59 WH72 WH58 WH96 — WH65 WH91 F WH67 WH92 WH68 WH66 — WH94 AneMari2, “Anaerofiexus marltimus, 1386
- ..._w’Capnocytophaga
FixMari6, Flexibacter maritlmus, 1270 CytMarln, Cytophaga marina, 1240 strainl3, Berry Isolate strain35, Sippewissett tidal pond lsolat PbrFllam, Polaribacter filamentus, 1438 CytUilgl, Cytophaga uliglnosa, 1505 CytMari2, Cytophaga marmot lava , 1236 CytLytI2, Cytophaga lytlca, 1483 CytLate2, Cytophaga latercula , 1238 straln3o, Sippewissett tidal pond isolat 541Psyl 3, Marine psychrophlle 4, 1393 - CytMarl3, Cytophaga marmot lava, 1463 Cytophaga johnsonae
Chryseobacterium et a!.
Blattabacterium
SpgMizut, Flavobacterium mizutaii, 1245 FlaYabuu, Sphingobacterium spiritlvorum, 1250 SpgMuit2, Sphingobacterium muitivorum, 1231 mudclone29, Sippewlssett tidal pond clone l58Comit, “Candidatus comitans”, 1500 SpHea.hlngobacterIum heparinum, 1519
I WHO4 WHO3 .3...... Rhodothermus ChfAuran, Chloroflexus aurantlacus, 1413 — FusNecro, Fusobacterium necrophorum, 1467 SpcLitor, Spirochaeta litoralis, 1514
n-In