.
2
1 University
Metabolic
University
Interactions
of
Connecticut
Rd,
Laura
Groton
of
of
Denmark,
K.
Denmark,
Marine
a
Department
Baumgartner 1
Purple
Microbial
CT
Woods
06340,
Biological
BioCentrum-DTU,
Sulfur
Diversity
of
LKB00002Uconn.edu
Hole,
and
Marine
Bacterium
Laboratory
MA
Adam
2001
Sciences,
C.
Building
and
Martiny 2
a
Facultative
1084
301,
Shennecossett
2800
Anaerobe Lyngby,
.
.
interaction
unclear.
highly
relative
(strain
(darker)
of
presumed
surrounded
dissecting
halo Figure
marine
well
pyruvate, Truper
conduct
Purple
the
of
shake
limiting
This
as
•-
ACMOI)
1:
purple to
We
PSB
Satellite
Second,
Purple
2001).
environments
lithotrophically
1)
Sulfur
the
to
between anoxygenic
microscope,
phenomenon
and
from by
examined
tubes,
be
bacterium
appeared
(see more
several
appeared,
sulfur
succinate
PSB.
colonies
Bacteria:
However,
Sippewisset
the
methods
the
and distant
enhanced
These
photosynthesis
displaying
an
different
two
such the
as
their
(Bauld
in
was
unusual
and
(Krassilnikova
small
the
PSB
some
Overview
initial
strains.
and
as
development
satellite
interesting
Salt
primary
finally
(PSB)
et
color microbial
growth
colonies
Appendix
colonies
enhanced
strains
colonies
al.
Marsh,
colony
the
morphs 1987),
are
and
enrichment.
colonies
with
and
Introduction
have
for
and
PSB
and
throughout generally
mats
formation
Cr”?
growth.
consisted An
A), 2
appeared
sulfide
spatial
two
and
more
of
Kondratyeva,
the Unusual
been
colonies
developed
(van
Cod,
red
reasons.
some
The
metabolic
as
distribution
colonies
anaerobic
shown
that
to
Gemerden
of
to
the
the
Massachusetts. white
purple
strains
a
nearest
occur
Co-Culture
arose
tube,
white
electron
in
First,
to
bacterium
1984).
strategy appeared
all
grow
colonies
in
photolithoautotrophs
can
then
of in
disc-shaped
the
but
1993).
because
stages:
the
PSB
donor
utilize
heterotrophically
ACMOI
the
PSB
the
is
of
PSB enrichments
near that
Under
surrounded
highest
white
ACMOI
(Pfenning are
first
the
acetate,
implied
are
grew
ACMOI.
colony
common
media
the
the
colonies
dilution
was
some by
malate,
and
was that
as
a in
•
and
suction.
by
DNA achieved
and
media).
1.5%
three
Plating spectrometrically
supernatant
supernatant,
PSBI glucose,
of ACMOI
assessed
ACMO1) requirements
Liquid culture. the
pH
the
and
Specific
grown
tubes
liquid Appendix
be Sippewisset
were
Sample
ACMOI
component
Second,
removing
the
amplified
anaerobically
change
enriched
addition
phototrophic
Extraction
ACMOI.
agar
days
Satellite
ACMOI following
(the
cultures examined
with
Both
Further
in
Cultures
The
We or
DNA PSBI on lactate,
were
ACMOI and
source the
Shake
by
is
plates,
supernatant
of PSBI
A).
CR
the (pH-indicator,
any
were
from
strains
bacteria
ACMOI of
with
25%
of enrichment
for
growth, was Salt
light
the
of
was
was Mixed
colonies grown
After
of
galactose, shake
medium the
substrates
agar
ACMOI
and
PSBI
PSB. malate,
with was
as
monoculture Enrichments
requirements a
with the in PSB
8F
interested and
extracted supernatant,
Marsh,
were
plated
plated galactose-enriched
were
media
continuous
absorbance
nine
growing
from Amplification
were and
cultures
the
in PSB
also
both
enrichments
and
Enrichment
that
plates,
A
percentages
were
liquid
given
in
and
propionate, on
maintained
optical
small
days
1495
Cape
aerobically
on
the
blue
glucose,
(including enriched
without grown also 1%
(2mM): traditional
resulted
from
various in
extracted
freshwater PSB1.
on
(predominantly CR
tube
broad-spectrum
and
were
agar
several
of at reverse
of inoculum
50%
utilized
light
Cod,
density
some
at
these growth,
in
7.6
medium
ACMOI
the acetate,
malate,
were
and
either
or
(deep
added
shake and
in substrates.
and represent
agar)?
supernatant,
Metabolic
in succinate,
and
MA.
and
microbiological
0.2
the
results
questions.
product
from
ACMOI
CR CR
primers
extracting
colonies
of
isolated malate
carried
Methods
dark
(—1
550nm 0.5 anaerobically
yellow im
and
agar
satellite
3
in
tubes The
(all
each to
medium. medium
butyrate,
the
Third,
the
g)
ACMOI)
4
mL
filter-sterilized
will
conditions.
combinations,
PSBI.
the light
of
sediment preferences
ml dilution culture
was at
as
and
out
102
enrichment with
dark
Concentrated
with
from
at
of
(ACMOI)
therefore
the and
individual
55
vials
First,
percentage colony
well what
6.0). from the
to as
initially
seawater
dilution
formate,
and
Mixed Mo
°C
PSB,
0.1%
to
a
were
For
examine CR
in a
series,
as
containing enrichment
on
sediment
what
assess
annealing
is
standard
base,
Bio was
the
molecular formation?
However,
PSBI,
medium
5%
the
or
seawater
or
not
bromothymol enriched
of
grown
cultures colony
supernatant was
of
Ultra
see total
660
is
dark
fumarate,
is
ACMOI
POB supernatant,
of interaction see
the
(by
and
be the
the
the
it
measured
filter-sterilized
Appendix enrichments
growing
sample
nm
media
incandescent
discussed.
Clean
CR centrifugation) in
Appendix original pink
temperature.
(control)
formations
was
all metabolism
identity
phototrophic
plates analyses.
no
(predominantly
and
the in
These
(PSBI).
enrichments
medium
and CR
growth and
galactose, diluted
volume). from dark
Soil
blue
freshwater
from between
from
aerobically
shake PSBI
medium A of
‘10%
for
is
questions
A).
DNA
for
to ACMOI?
to
an known
included with
was
and
the
in
a
of
bulbs.
assess
assess
ACMOI
Further
series were
shake
PSBI
kits After
one
(see to
.
• •
and concentrations
pH7).
regained
bacteriochlorophyll transmission analysis
which
communication).
rods
Microscopic, ARB
Purple
sequenced
Genomic
sequencing.
2 Products
MI3F were grow
Initial
Cloning,
hours
then
culture, the
Figure
or
using for
are
picked
PSBI
At
clones
and
16S
bacteria Also,
The long
Sulfur
of
at
moving
internal
48
higher
that
DNA
motile
2:
Restriction
PSBI
and 37°C
MI3R
Ribosomal
rDNA
in
amplified
cocci
PSBI
hours
rapidly
electron
Spectral
PSBI
at
were
6
appeared
of
Bacterium
contain
internal
kit
random
runs
magnification
back
(Figure
and
granules
added
revealed Cells
from
at
primers.
with
and
at
a.
grown
displayed
400x
utilized
by separated
37
microscopy,
168
towards
Observations
no
granules
amplified Database
internal
ACMO1
Fragment
have
sulfide Accugenix. and
2), to
oC.
and internal
on
within
rDNA
(PSBI)
peaks
have
This
sulfide, possibly
transferred I000x.
been
PCR
ampicillin-X-gal
chemotaxis
(400-l000x),
the
are
sulfur
was
Results on
on
granules. via
PCR
was
distinct
10
Project
at
evident Length
the
from point
a observed
a The and
extracted similar hours
590,
microscope
Phylogeny
Chromatium
granules,
2%
cloned
product
cells
left
35
sulfide
of
and
to
band databases.
800,
agarose
as
4
The
with
with Polymorphism,
addition
image
of
another
methods,
have
highly the
to
with
Discussion
these
plates
right
from
was
and
patterns
sulfide
the reverse
depleted
many
purple
for
is
slide
a
the
(Jane
refractive
gel image
850 from
then
addition
single
pure
as
all ampicillin-X-Gal
clones
for
and
Invitrogen
of
for
gradients, to
sequences sulfide sulfur
were nm,
direction
48 a
which digested
Gibson,
culture is
an cultures
sulfide-starved
comparison
the
polar
from
hours
was
spheres.
and
of
all
intermediate
sent
bacteria
full
was
2mM
of are
flagellum. a
then
with
TOPA
Sequencing
moving
personal
of
sulfide-enriched
which
at
with
16S
(no
to
was
consumed.
dividing
rotation,
37°C.
plate
Accugenix
S 2
Promega amplified
internal
of
rRNA
isolates
Rsal
conducted
TA
culture,
are
(as
digested
away
concentration,
and
Spectral
Cloning
Fifty
and
and
indicative
Na 2 S,
was
and
granules)
allowed
are
and from
Wizard
using
all
for
clones
Hpall
under
bands.
with
short
of
kit.
high
of
for to
.
shake
done
Isolation
strongly ACMOI
Figure
growth with
enhancement
utilized
as
optical
(sugars, stimulating
detail
supernatant
enhanced
culture.
PSBI apparent
PSB
Metabolic
.11.0
—o
did
.cD
4.ICD
0.
by
various
tubes
3:
ACMO1
slightly
colonies
E
below,
enhanced
enrichments
grown
Enrichments
within
removing
density
Optical
PSB
these
and
0.1
0.2
0.3
fatty
0.4
0.5
0.6
The
PSB
0
colony
Observations
and
PSBI
carbon
and
enhanced
did
characterization
was
the but
axenically
addition
substances
acids,
displayed density
_
appeared,
of
colonies
incubate
growth.
not
ACMOI
(sucking)
the
time
the
isolated
growth.
growth
with
with
additions
contain
and
result
of
enrichments
growth
of
of
PSBI
did forming.
sugars
by
fatty
this
different
Optical 0.2
were
it
while
indicating
more
from
Therefore
in
a
hinted
the
not
any
jim
piece
over study.
the
cultures
acid
were
grown
of
fatty
media
the
form
easily
and
filter-sterilized
ACMOI).
dark.
Density
the
When
concentrations
strain
that
was
additions
of
white
also
that
supernatant.
acid
the
control,
at
PSB
in
the
or alone
some
measured
Light
660
mixed
Substrate
carried
PSB the
ACMOI
large
gained
enrichments
colony
central
5
after
were
This
nm
enrichments
showed
while had
metabolic
microscopy
may
culture
colonies
after
supernatant
2
out,
experiment
enriched
more
and
colony
no
of
Days
after
enrichments
However,
be
2
supernatant
affect
but
enhanced
days.
transferring
an
in
from appears
two
product
that
these
in
shake
obligate
showed
were
with
(microscopically,
the
Sugars
days
them.
will
were
from
the
with
various
shakes
shake-tube.
placed
to
tubes
growth
of
be
greater
from
(Figure
ACMO1
it
phototroph.
a
observed
indicate
and
ACMOI
fatty
Shake
discussed
to
non-motile,
in
supematant
ACMO1)
liquid
carbon
had
in
acids
over
the
degree
3)
constant
tubes
cultures
was
that
no
This in
dark
media
the
the
showed
in
sources
apparent
mixed
However,
PSBI
and
black
of
control,
of
greater
was
with
from
light,
PSBI
and
the no
.
(
were
carbon/energy
small
presence
and
source
carbon/energy
observations
rod
formed
without
2-10
The
halo
ACMOI
The
as
halo
Figure
combined and
m
around
well
in
pure
malate.
of
source.
long the
of absence
4:
was degraded Agarose
as
Glucuronic Agar
sources
Galactose
Glucose Media
Media cirowth Table
the
An
culture
shake
the
hexoses
(depending
streaked
ACMOI
data
mixed
The
colony Gas
1. (incu.
(incu.
of
agar.
of
and
tubes
was
suggested
strain
Conditions
malate.
colony
ACMOI
was
acid
culture.
(see was
aerobically
dark) light)
(see
incubated
(see on
grew
produced
Table
on
shown
growth
Figure
The
Figure
a
that
only
agar that
1). strain
under
on
to
the
conditions),
4).
6 during
on
plate.
5a).
initially
grow
seawater-
strain
seawater-based
clearly
anaerobic
These
Note
degradation on
was
promoted
agar
degraded
the
which
and
bubbles
able
darkened
conditions
and
freshwater-base
was
to
of
agarose
plates
agar
utilize
could
agar
consistent
with
and
both
and
agar
be
as
+
+ +
+
various
produced
CO 2
the
gas
in
as
with
plates
the
sole
or
a
bubbles
sole
earlier
H 2 .
energy
with a
.
galactose
requirement
which
using
agar
concentration
Figure
ACMOI
lower
Figure
(see
GC
is
dilutions.
Since
Bromothymol
6:
5a
with
contributed
ACMOI
and
containing
(left)
Figure
bromothymol
of
the
of
HPLC
and
a
0.5-1% enrichments
strain
certain
5b).
b
(right):
to
blue
was
media
The
is
did
the
salt
required
blue.
was
unsuccessful.
not
production
results
a)
with
with
concentration.
Gas
added
grow
Note
varied various
for
bubbles
show
the
on
growth.
to
of
salt yellow
freshwater-based
measure
a
salt
fatty
in
7
decrease
concentrations.
a
This
concentrations
color
acids.
shake
was
a
indicating
pH-change
of
in
Attempted
tested
ACMOI.
pH
plates,
A
at
minimum
increased
(see
by
high
b)
during
incubating
identification
Shake
it
Figure
densities
pointed
salt
acidity
degradation
dilutions
6).
toward
ACMOI
of
in
of
ACMOI,
the
of
this
a
of
in acid
.
.
trees
shows
PSBI.
corresponding
near
psuedoalteromonads
cytophagas,
that
using
Molecular
perform production development
metabolic
substrates.
Figure
increased
its
0
4-’
.11(0
.0
0. 0
(U
U) 4-’
C
(I)
I
0
(U
C
0
U)
(U
were
metabolic
Cytophaga
to
the
to
all
The
7:
Biolog
Vibrios
ensure
ACMO1
aerobic
Optical
0.00
0.03
0.06
0.09
0.12
0.15
0.18
of 0.21
neighbor-joining sent
properties
Analysis
restriction of
growth
the
chromatiaceae,
was
which
reducing
plates
properties
for
to
and
accurate
products
density
was
fermentans.
respiration
Figure
sequencing
observed
on
rhodobacters
are
were
grown
under
(Figures
fragment
galactose
of
equivalents.
also
8).
positioning.
together,
?
ACMOI
under
also
algorithm
on
aerobic
or
after
The
known
rhodobacters,
The
-C’
(Figure
Optical
is
9-11,
various
used
length
anaerobic
at
compared
simply
sequence
are
4
Chromatium
560
each
days
to
and
products
from for
Therefore,
common
polymorphism
nm
8).
utilize
substrates
C,
Density
a
aerotolerant.
Substrate
anaerobic
sequence
of
indicating
these
more
A
conditions
8
vibrios, to
from incubation
phylogenetic
agar
noted
various
in
sequences
complete
it
RFLP-groups
ACMOI
mat
is
to
degradation
was
conditions.
different
spirochaetes,
not
as
further
yielded
environments,
(see
fatty
indicating
PS#,
carefully
clear
also
investigation
analysis
may
Figure
acids.
investigate
with
17
whether
fell
fell
products.
densities
be
Unfortunately,
distinct
examined
either
into
numbers
and
7).
into
representative
of
as
the
the
The
the
6
of
are
no
and
on
band
groups:
While
sequences
the
cytophaga,
strain
results
growth
different
in
characterize
strains
smaller
patterns
no
Figure
can
color
of
show
or
no 9
.
to
were
corresponding
Figure
the
judged
numbers
8:
RFLP
to
to
have
pattern
in
these
the
distinct
phylogenetic
from
lanes
clone
band
was
sequenced.
patterns
library
trees.
DNA.
“L”
and
denotes
Numbers
PCR
Numbered
product
a
ladder.
correspond lanes
sulfur
sequences
Figure
bacteria,
9:
Phylogenetic
similar
Phylogenetic
a
0.1
to
ACMOI
tree
a
tree
from
appeared
clone
and
in library
the
a
psuedoalteromonad.
clone
(“PS”)
9
library,
and
ACMOI
as
did
PCR
purple
Psuedoalteromonads
products.
sulfur-
Purple
Vibrios
Chromatie,
Purple
Non-sulfur
arid
Note
Spirochaetes
Sulfur
Cytophage
purple
that
Becteria
acteria non
.
4
sequence
Figure
Phyogenetic
10:
from
Phylogenetic
ACMOI.
tree
positioning
of
the
Cytophaga-related
10
BzetIine3
Cytophaga
sequences,
Anaeroflexus
•
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fermentans
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odoratus
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the full
.
.
sterile-filtrated from normal
to
As degradation
was
in isolated.
the liquid
isolated
to
Figure
more
galactose-
mixed
be
Both
media
media
a
The
11:
likely
Chromatium.
0.
culture
the
of
culture.
1
Phylogenetic
was
two and
agar
containing
strain that
and
strains added
ACMOI added
that
glucose-containing
one
This
from
promoted
strain
to
did
to
positioning
galactose.
suggested
the formed
a
a
therefore
higher
7
central
provided
day
growth
large
of
old
density
—
that
In
white
Interaction
the
not
media
contrast,
shake-tube
a
colonies
L_PS31
90Th
ACMO1
of
Chromatium-related
substrate
11 have
______
colony
shake. PSB. I nus
______
inoculated
an
PSBI —
in
—
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and
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that
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test
SuuEndos
In
S78Roseu
formed
the
—Gamma.
—
101
promoted
Unknoli43
Gamma
overnight
Chromatium
tubes
a
this
1144,
a
of
purple
cBact6
nromatium
syntrophic
Thiocystis
Chromatium
sequences.
substrate
PSB.
1
hypothesis,
phototrophic
tiny
and
purpuratum
sulfur
growth
okenii
with
qelatinosa
colonies
As
Phototrophic
vinosum
grew
relationship.
from
PSBI a
ACMOI
tepidum
bacterium
control,
of
up
supernatant
compared
the
is
Bacteria
overnight
believed
was
other.
were It
.
Additionally,
the
PSB,
that
larger for
ACMOI
short-chain
PSB
produced
improved
confirming that
galactose
inspected
Figure
reduction
gas
produced
an
during
a
amounts
To
Another
12:
This
liquid
could
increase
is
test
and
microscopically
by Shake
during
hydrogen,
the
fatty
experiment
growth.
acetate
addition
of
PSB
glucose
discriminate
if
CO 2
result.
explanation
CO 2
CO 2 .
hydrogen
tubes
acid
in
degradation
culture
would
the
is
was
which
degraded
Hydrogen
by of of
Additionally,
generated
amount
was
PSB
short-chain
to
ACMOI,
added
dissolve
was
could
between
ensure
provides
could
repeated
with
of
by
incubated
of
be
has
(Figure
agar,
supematant
ACMOI
which
it
that
be
supernatant
and
a
could
Figure
the
fatty
not
that
PSB
possible
no
in
these
not
two
in
enhanced
been
13).
liquid
ACMOI
with
acids.
the
form
with
turn
12
3
produce
possibilities.
or
results
shows
gas
either
directly
metabolic
bubbles. reducing culture
enhances
media
is
bacteria
Combined
then
formed
the
that
bubbles.
could
a
added
growth
measured
N 2 /C0 2
(see
utilized,
was
equivalents
the
Only
linkage
during
the
point
superficially.
Figure
with
transferred
of
growth
growth
On
a
or
PSB.
possibly
toward
direct
the
degradation
between
but
H 2 /C0 2
the
3).
of
The
fact
that
of
one
other
PSB and
measurement
Here
PSB,
an
The
top-liquid
assimilated,
that
headspace.
could
might
ACMOI
proliferated.
excretion
hand,
is
supernatant
one
and
acid
of
generally
be
expect
agar
can
thereby
was
if
is
and
used
of
of
by
by
see
a of
from
Combined,
is organism
revealed
strong
this
possible from was
2001).
of
motility,
in
compound ACMOI
colonies
short-chain the
of supernatant
Figure
strictly
which the
H 2 ,
group
substrates
not
degradation
a
genus
There
Phylogenetic
it
evidence
sulfide
13:
Phylogenetic
Finally,
Both
This
tested.
are
in
is
fermentations
vary
phototrophic, a
at
belonging
Optical
and
reasonable
an
highly
these
or
low
fatty
able
acetate
is
is
significantly
Cytophaga.
in
attempt
relative
rich
it consistent
a
strong
are
Additionally,
densities
length
that
has
constant
to
density
acid.
of
results
motile
salt
possibly
cleave
agar
to
data
analysis
and
ACMOI
been
cell to
evidence
to
the
marsh
all
depending
products.
rod
of
indicate
regenerate
to
hydrogen
generated
say
of
enhanced
with
characteristic
Cytophagas
densities
number
group
PSBI
reported
3-glycosidic
PSBI.
ACMOI.
with
transferred
members
belongs
that
based
in
the
for
California.
grown
internal
Chromatium.
that
at
Based
observations
of
on
The
transfer
can
that
may
from
growth
on the
least
PSBI
This
Conclusions
to
growth
PSBI
with
from
are
a
be
bond,
presence
of between
the
satellite
hydrogen
influence
on
sulfur
clone
full-length
a
indicates
Chromatium
13 known
used
nitrogen
was
Agar
of
where
soluble
this
molecular
belong
Cytophaga.
conditions
a grow
Morphological
granules
library
of
metabolic
added
group
growth
formation.
ACMO1
degradations
of
to
ACMOI,
as
this
one
or
to aerobic
compound
that
168
the
be
well
hydrogen
the
and
and
have
phenomenon.
would
to
(Pfennig
small
by
fermentation
(pleomorphic)
a
and
rDNA
and
shake
Chromatiaceae.
short-chain
product
chemical
PSB
16S
phenotypic
even
as
PSBI
appeared
bacteriochlorophyll-a
expect
rods,
PSB.
examination
well
sequence and
is
is
sequencing
and
tubes
and
though
commonly
involved,
generated
only
with
anaerobic,
that
gradient
Since
either
a
Truper
product
fatty
with
in
data,
(Reichenbach
low
or
formed
possess
gliding
clone
placed
without
the
concentration one
of
acids
presumably
a
showed
observed
there
2001).
of dilution
by
PSBI
enhances
and
satellite
libraries
or
a
motility
ACMO1
gliding
ACMOI
are
acetate.
both
is
some
that
an
of
in
of a
.
and
Carrie
Diversity
analysis
bacterium.
both
cultures
H 2
Enrichments compounds
potentially
both) surrounding
either
points growth
or
Louie
cultures
CO 2
of
Harwood,
GC
Continued
towards
We
of
to
these
of
2001
Kerr.
PSBI.
is or
be
examine
would
PSBI
are
a
being
of
HPLC
on
identified
for
white
compounds
either
ACMOI
created
various
Bianca
would
their like
examination
It
produced,
was
are
the
colony
to
hydrogen
help
with
for
needed
products
definitively
Brahamsha,
acknowledge
substrates
by
not
and
gas
ACMOI
of
and
HPLC
possible
and
ACMOI
of
production/identification
is
or
for
support,
Acknowledgements
this
and
dependent
labeled
a
and
identify
a
would
and
short-chain
John
conclusive to
phenomenon
Future
their
all
is
GC
directly
exchanged
therefore
of
particularly
14
galactose
further
Waterbury,
utilization.
it
the
as
as
Work
of
well
faculty
identify
the
a
fatty
answer.
clarify
purple
generated
as
would
density
with
could
Jane
acid.
Also,
some
Dan
and
the
their
analysis
PSBI.
sulfur-
The
focus
Gibson,
be
students
compound
Buckley,
of
Direct
growth
wet
metabolism,
by
followed
both
formation
or
mainly
The
the
chemistry
would
measurements
purple
Alfred
strains.
rate
gradient
of
Jochen
compounds
through
but
Microbial
on
identify
experiments
of
Spoorman,
non-sulfur
and
what
evidence
analysis.
PSB
Mueller,
of
molecular
both
whether
one
colonies
could
with
(or for
0
van
Reichenbach
Pfennig
Krassilnikova
Bauld
Gemerden
Springer-Verlag. J,
Online.
526-528.
belonging 339.
vinosum
N
Favinger
and
H.
EN,
Springer-Verlag.
Truper
H.
from
to
2001.
JL,
Kondratyeva
1993.
the
Hamelin
Madigan
HG.
genus
http:Iink-springer-ny.com:6635
The
Microbial
2001.
Order
Pool,
Ectothiorhodospira
MT,
http:link-springer-ny.com:6635
EN.
The
Cytophagales.
mats:
Shark Gest
Literature
1984.
Family
H.
15
a
Bay,
joint
Growth
1987.
Chromatiaceae.
Cited
Australia.
venture.
in
In:
of
Obligately
the
The
different
dark.
Mar.
Curr.
Prokaryotes
Mikrobiologiya
halophilic
Geol.
purple
in:
Microbiol.
The
113:
bacterial
Prokaryotes
Online.
Chromatium
3-25.
14(6):
53(3):
species 335- Appendix A: PS Medium, shake instructions, fw and sw malate plates
.
16
.
After
-L—
1.
(partial) composition,
light Parameters obtain
In
Light Trace Final
Anoxygenic
Na 2 S9H 2 O
NaHCO 3
Enrichment
NHCI 12
KC1
KH 2 PO 4
CaCI 2 2H 2 O
NaC1 Media
“berries”, are
Subsamples
selectively well
higher
values
intensities.
up
Green
environmental This
sulfide,
Green
Green
the
autoclaving
vitamin inoculated
quality. to
pH
source as
element following
a
growth
5
(per
I wide
sulfur
light
above
Y”/L.’ and
different
mM,
6S
and
elemental
fluffy
changed
sol.
enrich
rRNA phototrophic
liter) purple
In After
the
intensities.
from
variety
purple
absorb mode
bacteria
sol.
7.1, and
contrast,
into
and
series
samples. formation
red/pink
pH
isolation
different gene
absorb either
cooling
--
sulfur
mineral
sulfur, include
will
isolation
of
values sulfur
light
of
grow purple
sequence. purple
Consequently,
thus
enrichments,
for
bacteria
bacteria
cell
light
(selects
>960nm >800nm
7.2-7.3
SLI2 of
and
polysulfides
I 1
under CR
in the
sources media
and
bacteria
ml
mM
at
green
wavelengths
be
pure
material
sulfur,
pH localization
sulfur
in
type
of
sulfide
used
-
(final)
an
the
are
grow
for
CR
values sulfur
culture,
anoxygenic
(laminated
of
N 2 /C0 2
infrared
bacteria purple
to
physiologically
BChlb)
different or
from
electron
different
or
concentrationsof
preferentially selectively
bacteria
CL:
between
17
20g
below
thiosulfate
0.25
0.50g
0.15
0.20
3
of
strains
I
puddles,
nonsulfur,
mM
(90/10)
elemental
wavelength
grow
mats
g g
g
donor
incubation
light
760
(final)
or
phototropliic
will
6.5
enrich
only
purple
from atmosphere,
pink nm,
as
sources
and
photolithoitotrophically added
and
be
and
sulfur
electron-donating
6.7-6.8
fluorescent
SLIO
2.5
1
at CL
the
and
characterized sand
phylogenetically
range
ml these
sand 7.0,
conditions
sulfur lower
green
mM
(organic/inorganic),
growth
(fluorescent
are
globules,
at
flats,
from
bacteria
(up
(final)
high
add sulfide
able
phototrophic
bacteria.
bacteria
light
to
black medium
-
sand
sterile
will
sulfide to
—
1030
based
cell from
exploit
concentrations,
bars
light, be
substrate
mats,
diverse.
nm),
solutions:
morphology
used
concentrations
are
on
a
in
bacteria.
variety
by
ER
pink low S
the
their employed
and tidal in
oxidizing light),
in
S
pH,
light
order __
require
j,igment
the
rivers)
of
at
and
and
as
light.
to
pH
to of
Filter Sodium
Na 2 WO 4 •2H 2 0
Na 2 SeO 3 5H 2 O ZnSO 4 •7H 2 0
Na 2 MoO 4 •2H 2 0
CuCI 2 •2H 2 0
CoCl 2 6H 2 O
NICI 2 •6H20 Add
MnCI 2 •4H 2 0
Adjust
H 3 B0 3
FeSO 4 7H 2 O Water
EDTA
Water
I
Store
NH 4 CI
lOOx
Keep
KH 2 P0 4
KCI CaCl 2 •2H 2 0 Water
MgCl 2 6H 2 O
NaCl
lx
Keep
KH 2 PO 4
KCI
CaCl 2 •2H 2 0 Water
MgCl 2 6H 2 O
NaCI
Composition
lOOx
000x
SW-Base
the
sterilize
Ammonium
in
in
FW-Base
on
pH EDTA-Chelated
Vanadate
following:
clean
Clean
shelf
to
6.0
for
bottle
in
Na/gene
of
with
for
Marine
clean
Chloride
solutions
Freshwater
NaOH
Trace
bottle
plastic
media
solution
used
8
25
6
36 2
24
30
2100
144
190
100
Elements
987
5200
25
I
4g
3
400
60 20
log
20
50g
40
10
bOg
I
bottle
mg
mg
mg
(S-.
liter
media
g
mg
mg
mg liter
mg
grams
g
liter
g
g
g
mg
mg
mg
ml
g
in
for
mg
mg
C-,
construction
freshwater
(S-.
and
Stock
C-.
N-free)
Solution
and
or
N-free)
of
marine
media
media
during
MBL 2000
Q
0.4
0.5
I
0.5
1
1
1
I I
I I
I
Sundry
I
Water
Cyanocobalamin
Titrate
Thiamine
I
NAD
Titrate 4-Aminobenzoic
Lipoic
Pyridoxine•HCI
Niacinamide
Nicotinic
Folic Thiamine•HCI
D-Ca-pantOtheflate
L-Ascorbic
Riboflavin
%
pH7.2
Phosphate
l000x
M
000x
M
M
M
M M
M
M
M
M
(wlv)
M
M
Sodium
Sodium
Sodium
Methanol,
Sodium
Sodium
MES
MES
MOPS
Fe(lll) Trimethoxybenzoic
4-Hydroxybenzoic
acid
Vitamin
acid
with
with
Solutions:
12-Vitamin
Resazurin
acid
Buffer,
Buffer,
pyrophosphate
Buffer,
HCI
acid
NaOH
Sulfide,
Acetate,
oxyhydroxide,
buffer,
Lactate,
Sulfate,
Bicarbonate,
filtered
B
until
pH
pH
acid
pH
until
Solution
Solution
10mM,
Dye,
stored
6.5,
6.8,
store
filtered
dissolved;
autoclaved
7.2,
dissolved;
acid,
filtered
stored filtered
acid,
autoclaved
in
filtered
prepared
under
clean
titrated
100
100
100
100mg
100
100
100
100
100
100
100
100
100
10
titrated
100
filter
on
N 2
mg
Filter
mg
ml
mg
mg
mg
mg
mg
mg
bottle mg
mg
mg
shelf mg
ml
sterilize
by
at
under
with
with
40
neutralizing
sterilize
in
on
C
NaOH
clean
NaOH
100%
slelf
(throw
and
and
bottle
freeze
until
until
CO 2
away
Fe(lll)Cl
freeze
crystals
crystals
in
in
when
serum
10
in
with
ml
10
dissolved,
dissolved,
no
bottles
aliquots
ml
NaOH,
longer
aliquots
filtered
washed
clear)
filtered
3x
with
H 2 0