Marine
Introduction
capability through section this variety
environments
published Isolation Alicyclobacillus, physiological
Pasteuria,
and and
can red, in
(Gibson,
seen
trait.
be
genera.
Thermoactinomyces. endos
yellow
The
At 13
of
found. University
different
In
in
pore-forming
of
1935), to metabolic
marine
Sporohalobacter,
The
the Sporosarcina. genera production
and
the
survive
In
of
and
and
Aerobic
the end physiological
Bergey’s
sea pink Am
habitats. endospore-forming
environments,
have
of
which,
last
genetic
phi
water process
of adverse
Puerto
pigments.
endospore-forniing years,
clinical
of
1992,
Manual
Sporosarcina Controversy
(Wood,
Sporolactobacillus,
Most
taken
diversity
Rico,
and
environmental
José
new
diversity
and
the
Bacillus,
Evidence organisms
of
of
products
Department
together,
1946),
R.
industrial Systematic
endospore-formers
endospore-forming
ureae
(Berkeley Pérez-Jiménez
exists
by
among
and
have
for
Clostridium,
bacteria
several
which
remarkably
can
conditions,
1
bacteria
form
today
importance.
Sporosarcina, the
Bacteriology
soil
of
been
endospore-forming
be
Biology,
and
differences
contribute
from
bacterial
in isolated
a
about
added
the
Ali,
group
distinct
genera
were
the
and
Desulfotomaculum,
from
Mayaguez
marsh
(Sneath
the
Sulfobacillus,
to
base
1994).
from
ensures
to
between genus
some
allocated
of
have
validity
the
from
marine
and
of
garden et
some
survival bacteria
provides
of the
been
al.,
PR a
the
These open
both
this Perez-Jimenez,
wide
of trees
1986)
00681
Syntrophospora,
to morphological,
terrestrial
placed
and
genera.
some
sources
ocean
environments
in
Oscillospira,
13 includes dispersion
genera
where
them
based
field
different
validly
species
in
exhibit
1996
ones
the dogs
soils the
are
on
a Marine
have
from
pigmented
population the
aerobic
isolation
salt
urinated
endospore-forming
Work
and
marsh
aerobic
were
anaerobic
required
done
(Pregerson,
soil
sporeformers.
spore-forming
in by
bacteria
media
population
Germany
Singer
1973).
containing
and
The
(Claus
bacteria of
Sporosarcina
endospore-forming
Leadbetter
objective
sea
et
from
al.,
water
2
1983).
of
halophila
Sippewisset
(year?)
and
this
that
study
bacteria
has
resulted
less
salt
is
been
than
to
from
marsh.
isolate
in
isolated
1%
marine
the
Perez-Jimenez,
They
of
and
the
isolation
exclusively
sources.
show
bacterial
compare
1996
that
of
YEG-SW)
cultivated
aerobic pasteurized
from hr.,
Culture-
Table
source
Massachusefts.
marsh,
Sample-Samples
Materials
Marine
Integrated
and
each
SM-V
SM-TV
SM-rn
SM-il
SM-I
Ri
MBL-3
MBL-2
MBL-1
1.
GB1
and EP1
anaerobic
(Table
endospore-forming
Except
MBL
Origin
and
aerobically
sample
(heat
anaerobically
Sample
anaerobically.
and
Beach,
1).
where
and
10
was
cultures
These
were
Methods
mm
composition
on
diluted
Garbage Aquarium
specify MBL
Sippewisset
Sippewisset MBL
MBL Sippewisset
Garbage Sippewisset
Eel
Sippewisset
bacteria
SWC
collected
were
at
on
at
Pound
80°C
Beach
Beach
Beach 25°C
After
different
and
processed
102
other
Beach,
Beach
and Ramp
of
Source
to
from
for
NA-SW.
Salt
Salt
Salt
Salt Salt
incubation, integrated
way,
iO
cool
7
Eel media
water days.
Marsh
Marsh
Marsh
Marsh
Marsh
in
as
aerobic
3
rapidly
sterile
Pound,
integrated
All
One
and
(Table
samples.
colonies
plates
cultures
sea
on
gram
marine
and
mats
water
sludge
soil
dry
sea
sea
sea
sediment
sea
sea
ice).
water.
2).
samples
were
water
and water water
sand
aquarium
water
water
of
were
were with
sediments
and
Dilutions sediment
Dilutions
water
incubated
Each
Components
under
and
and
and
and
enumerated
and
colloidal
grass
incubated
according
near
ramp
sediments
sediments
sediments
sediments
sediments
diluted
water
soil
(or
of
102
iO
to
aerobic
sulfate
1
Sippewisset
at
were
Pérez-Jiménez,
bridge
at
mL
to
to
sample
from
Woods
30°C
i0
the
of
cultured
(except
water)
SWC,
for
original
were
was
Hole,
48
salt 1996
the
Biochemical
production
Colony
for
microscopy
Microscopy
a
Yeast
Nutrient
Medium
Glucose-Yeast
made
Sea
Table
conditions.
microscopic
generate
NA-SW
0.05%;
Marine
[glucose,
[peptone,
sterilization.]
glucose,
[tryptic
agar,
[glucose, catalase
[peptone,
Gram
Water
in
Extract-Glucose
2.
15
1
agar,
endos
morphology-Colonies
staining. liter
Agar
Media
for
soy
g]
5 and
10
pure
and
5 10 and
5 g;
Complete
to
Tests-The
g;
pore-forming
g; S.
of
g;
broth, 15
g;
examination-The Cultures agar,
examination,
YEG-SW
beef
determine
(NA-SW)
yeast
sea
their
ureae
glycerol
Extract-Peptone
yeast
oxidase
g]
culture
used
water:destillated
15 extract
27.5 Mediaa
extract, form,
(Sur)
extract,
g;
for
(SWC)
physiological
(YEG-SW) were
3
g;
pH bacteria
plates
test,
and
mL;
isolation
motility
8
yeast
elevation
g;
8.1.
5
5
stored
agar,
colony
and
identify
g;
agar,
g;
to extract,
Urea
cysteine,
peptone,
were
water
(GYE)
microscopic
estimate
hydrolysis
15
and
15
of
at
and g]
characterization 1%
morphology,
g]
different
(3:1).
their
4°C
described
5
natural
after
5
g;
margin
g;
in population.
4 genus.
of
NA-SW
endospore-forming
shape
examination
endospore-forming Isolation
Sporosarcina forming
Isolation
starch
Isolation
Isolation
endospore-forming
Isolation
(Smibert
on
several
Identification
plates.
of
of
bases
and
Different
bacteria
the the
and
of
and
of
and
and
biochemical
casein.
Sporosarcina
Sporolactobacillus
halophila)
isolates
cells;
enumeration
was
enumeration
Krieg,
of
enumeration
(with
Propose colonies
of
intracellular
bacteria.
and
bacteria
For
bacteria
done
1994).
genus
was
special
the
light
tests
ureae
made
were
of
Perez-Jimenez, catalase
of using
was
of
microscopy
endospore
interest
and
anaerobic
isolated
pigment
through
based
phase
growth
test,
on
1996
on to
Bauer
cocci
G
In
as nutritional
media),
incubation
42
Anaerobic
30,
study.
Growth
around
agar hydrolysis
to
was
(1.5% while
isolates.
done peroxide
a
Marine
+
situ
qualitative
and
small
detect
C
37,
isolated
assayed
15
(1996).
and
hybridization-Several
no
w/v)
55
adding
Isolates endospore-forming
42
the
NA-SW
g;
conditions-Oxygen,
portion
change
°C).
The
preferences
incubation
in
sea
to
dearing
universal
and of
colony
were on
measurement.
the
observe
on
casein
production
water,
Light
the
sea
were
55°C
fresh
dark
in
(complex
of
used
was
zones
oxidase
color
water:destiled
fresh
was
probes
effect
750
was
plating
the
cultures
and
to
on
bacteria
considered
as
determined
determine mL;
was
bases
evolution
of
around
culture
done
control.
was
exposed
media)
purple
reagent
to
samples
on
temperature,
distillated
considered
growing
tested
of
detect
in
NA-SW
for
growth.
the
anaerobic
positive water
The
color
of
and
to
the
(Difco
for
every
were
by
bacteria
bubbles
endospore-forming
light.
procedure
on
oxygen
water;
cultures
NA
the
in
and
(3:1).
5
as
plates
subjected
In
light
isolate
reaction.
the
Co.;
inoculation
chamber
negative
Isolates
all
(non
incubate
as
250
as
colony
Gram
cases,
and and
containing
on
Detroit,
positive
was
positive
was
mL).
sea
casein
to
nutritional
temperature
were
reaction.
(25°C)
done
iodine was
the
aerobic
in
added
water)
of
After
situ
bacteria.
MI)
result.
amount
cultured
isolates
sea
interpreted
reaction
starch
as
and
was
hybridization
describe
incubation,
and
on
water
to
plates
The
were
Gas
one
added
required
The
fresh
of
(0.2%
in
anaerobically
in
hydrolysis
One
for
growth
NA-SW
agar
evaluated
Pak
SWC
drop
to
as oxidase
by
colonies
Pérez-Jiménez,
this
w/v)
to
rod
compare
positive
clearing
Nierzwicki (casein,
jars
for
using
(a
the
of
was
test.
and
and
minimal
and
of
(30, hydrogen
growth.
test
colonies
in
at
of
Low used
starch
their
result
one 50
zone
this
the
agar
37,
25,
The
was
1996
the g; Marine endospore-forming bacteria Pérez-Jimenez, 1996
Results and Discussion
Endospore-forming bacteria can be found in all environments. They were obtained from marine sources in every sample of this study. This organisms produce
endospore that ensure their survival in adverse environments. The production of
endospore by several bacterial genus make us to suppose that they could be found in
high quantity and equally distributed in nature. In this study, the population of
endosporers was variable among places evaluated and incubation conditions (Table 3).
Table 3. Population of endospore-forming bacteria from marine sources. Sample Aerobic Isolationa Anaerobic Isolationa
SWC NA-SW YEG-SW swca NA-SW’
Eel’Pound 3 x 108 1 x 108 1 x io > 1O > iO Garbage Beach 3 x 2 x io 100 6 x 3i0 157 MBL Beach 1 > 10 5 x 10 6 x iO 5 x iO 126
MEL Beach 2 3 x 108 5 x 1W 0 0 0
MBL Beach 3 > 108 > 108 0 0 0
Ramp Aquarium 1 x 108 2 x 106 0 5 x iO > 1W SaltMarshl lx 108 lx iO 0 34x10 148 Salt Marsh II 3 x 10 3 x 10 0 4 x 1W > 1W
SaitMarshifi 3 x 1W 2 x 108 0 5 x > 1W
Salt Marsh IV 2 x 108 1 x 108 0 4 x 1W > iW
Salt Marsh V 2 x 108 2 x 108 0 4 x iO > 1W acell/mL org bpjflpOjflt colorless colonies
Aerobic population was obtained in range in iO to more than 108 cells/g or mL; while the anaerobic was obtained mainly in range less than 1600 cell/g or mL. This data suggests that endosp ores grown aerobically predominate in the environments studied.
6
forming
accidentally
the
3).
extract
Table
was
intense
influence
The
Marine
Nutrient
isolation
Salt
Salt
Salt
Salt
Salt
Ramp
MBL
MBL
MBL
Garbage
Eel
more
endospores
4.
Nutrient
(Table
bacteria
Marsh Marsh
Marsh
Marsh
Pound
Marsh
endospore-forming
The
intracellular
Pigments
Beach
Beach Sample
Beach
Aquarium
in
diverse
observed
Agar
of
Beach
marine
their
2).
ifi
IV
II
V
Sporosarcina
I
3
2
well
Agar-Sea
1
In
(containing
survival
produced
seem
on
both
endosporer
pigmented
pigments.
seven
NA-SW
bacteria
media,
to
water
halophila and
days
be
from
MgC1 2
white,
white
than
white,
white
white
white
white
white
distribution.
white
white
white
exposed
bacteria
grow
The
differs,
media
after
aerobic
SWC
(Claus
and
SWC
isolation
yellow
yellow
with
inoculation
is
were
NaC1)
at to
(Table
7
isolates.
et
lower
richer
least,
different
al.,
isolated
of
and
Pigments
4).
1983).
level
pigmented
than
from
when
Marine
stress
in
orange,
pigmentation orange,
orange,
pink,
yellow
pink
white
pink,
orange,
orange,
orange,
SWC
Small
soil
clean
order
produced
Agar
white,
yellow
ones
or
endospore-forn-iing
pigmented
because
pink,
pink,
pink,
the
pink,
pink,
1O
pink,
selective
are
NA-SW
lab cells/g
on
yellow
white,
yellow
white,
on
white,
white,
white,
recommended
up.
the
it
SWC.
Perez-Jiménez,
colonies
contains
or
pressures
yellow
yellow
production
yellow
yellow
Endospore
yellow
mL
Also,
bacteria
(Table
were
beef
for
that
a
1996 of
Table
incubated
isolates
unpigmented
a
Oxygen
TableS.
produced
the
under
endospore-forming
unpigmented
pigmented
salt
higher
Marine
Anaerobic
Aerobic
relative
6.
marsh,
Pigmented
Some
aerobic
endospore-forming
demonstrated
The
pigment
Condition
Production
only
under
abundance
by
ones
biochemical
isolates
oxygen
Eel
ones
old incubation.
atmosphere
diversity
colonies
for
colonies
pound
for
to
of
bacteria.
availability
each
Isolation
growth
pigment
seem
bacteria
to
of
(more
and
each
of
be
and
sample were
This
contrary
colonies
to
morphological
than
fast
facultatively
Table
pigmented
under
Garbage
table
have
more
7
and
seems
days Pigment
in
to
was
5
TOTAL
White
OrangeBrowna
different
TOTAL
Yellow
White
Pink represent
Orange
every
some
shows
the
incubation)
abundant
more
Beach
8 obtained
group
to
initial
anaerobic
Produced
natural
dilution
traits
have
that
complex
oxygen
the
in
(Table
isolation
of
from
the
and
more
environments
number
effect
isolates.
(Data
sample.
condition
when
sources
morphologically
4).
nutritional
pigment
Number
(Table
on
of
not
This
they
bacterial
pigment
rich
shown).
of
6).
can
evidence
variety
13
11
71
of
isolation.
44
was
21
2
9
4
on
Isolates
requirements
provides
organic
isolates
cultured
Pérez-Jimenez,
production
similar
was
Most
suggest
it.
matter
obtained
of
but
than
and
the
than
that
not
1996
by as
Table
bacteria
dark
when
pigmentation
support
produce
under
Marine
Starch-
Starch
Oxidase
Oxidase
Casein-
Casein+
Anaerobic
Facultatively
Motility
Catalase
Catalase
Shape-rod
Shape-cocci
(Brock
7.
cells
anaerobic
The
b
endospore-forming
only
Trait
our
Qualitative
percentage
not
pigment
+
are
aerobic
idea
Growth et
-
+
determined produce
-
+
of
al.,
cultivated
Halobacterium,
of
conditions
of
when
1994).
pigmented
endospore-forming
pigmentation
isolates
pigment,
Orange
bacteria
14
71 40
60
52
48
they
81
70
15
85
85
15
anaerobically
(Table
are
isolates
bacterioruberins,
bacteriochiorophyll,
grown
level
Pink
25
100
75
25
100
75
50 100
50
75
7).
0
0
exhibited
under
bacteria
aerobically
(Brock
Aerobic
The
9
different
White
anaerobic
require
30
70
41
59
65
35
36
34
low
decreases
93
82
18
et
7
Pigment
Isolatesa
aL,
(Table
aerobically
pigmentation
growth
oxygen
1994).
Levela
pigmented
Yellow
from
7).
56
100
44
22
100
78 56
44
56
86
14
0
conditions.
to
The
in
purple
The
produce
either when
following
non-sulfur
isolates
Total
28
68
37
63
60 40
to
42
48
13
87
84
16
were
the
Perez-Jiménez,
pigment.
red
light
two
cultivated
or
failed
Anaerobic
Isolatesa
purple
orange
or
NDb
ND
cases
100
100
40
60
100
100
62
0
The
0
0
the
1996 to
carotenoids
are
energy
viability
chlorophyll
absorb
chlorophyll
causes
desiccation
al., proposed
Mycobacterium
activated
pigment
a
Marine
comparison
exposed
Temperature>
1994).
Temperature
Tight
Dark
Non
SWC
Anaerobic
Aerobic
source
various
much
The
endospore-forming
of
Sea
intensity
as
The
or
culture
is
(Brock
and
to (Brock
made
light
Incubation
photoprotective
water
Condition
of
(Brock
of
induced
the
Incubation
pigment
(Brock
photooxidation
obvious
the
this
with
25-37°C
effect
when
light,
et
et
of
37°C
media
photosynthetic
et
original
al.,
harmful
aL,
Halobacterium
et
by
al.,
bacteria
was
as
protects
they
advantage.
1994).
1994).
(NA)
al.,
light
well
1994).
isolation
also
are
agent
light
1994).
as
reaction
as
By
the
grown The
considerated
photosynthetic
conditions
Since
in
apparatus
and
been
other
incubated
cell
bright
photosynthetic
Aerobic
Previously,
in
against
thus
that
endospore-forming
10
reported
the
side,
light
Low
High
(cultivation
Low
High
Low
Low
Low
High
can
dark
provide
itself
in
anaerobically
Isolates
light
harmful
can
this
bacteria,
actually
the
for (Table
(Brock
be
bacteria
has
on
study.
a
photochromogenous
carotenoid
often
NA-SW
light,
shield
been
7).
the
et
lead
bacteria
Anaerobic
in
harmful
and
Our
al.,
The
at
photoprotective
shown
the
for
30°C
to
1994).
Low
High
Deinococcus
Low
Low
Low
Low
bacteria
High
Low
pigment
absence
pigments
the
the
in
for
to
to
Isolates
marine
48
destruction
light
Perez-Jimenez,
cells
radiation
The
maintain
hr.)
decrease
seems
of
species
has
in
sensitive (Brock
carotene
sources
organic
role
that
to
been
and
the
of
of
1996
be
on
et
of it Marine endospore-forming bacteria Pérez-Jimenez, 1996
In bases of the colors exhibited by endospore-forming bacteria colonies (orange, pink and yellow) probably they have carotenoid pigments. They are the most
widespread accessory pigments. They are present in Staphylococcus aureus, Mycobacterium,Deinococcus,mycoplasmas and photosynthetic, halophilic, green and non-
sulfur purple bacteria. They absorb light in the blue range of the spectrum to exhibit yellowish, reddish, brownish and greenish colors. They protect photosynthetic bacteria
from harmful light, some mesophilic bacteria from high temperature, S. aureus from
phagocytic cells, Deinococcusfrom UV radiation, mycoplasma from osmotic lysis and
phototrophic and airborne bacteria from toxic form of oxygen (Brock et aL, 1994). Also
they are involved in the metabolism of halophilic Archea and photosynthetic bacteria.
The application of pigment production as classification trait was evaluated for endospore-forming bacteria from marine environments. Microscopic examination and
several biochemical tests were done to the isolates. Isolates were grouped in bases of
their pigmentation in order to compare several morphological and biochemical
characteristics (Table 6). This analysis show that most of the isolate share the main
traits for endospore-forming bacteria: catalase and motility. But the strains into each
pigmented group have not reaction in common for shape, oxidase, starch nor casein.
With the currently schemes, the pigment production by endospore-forming bacteria is not useful for their identification and taxonomical classification. Similar idea has been
adopted for purple and green bacteria that also have carotenoid pigments. Pigment production has been correlated to taxonomic group in Mycobacterium and by now it seems to be useful for speciation.
Our data suggest the presence of at less two genus of endosporers in the studied
places: Sporosarcina and Bacillus (Figure 1). Sporosarcina is the unique cocci-shaped
endospore former bacteria. Bacillus is a rod-shaped endosporer bacteria and
11 Marine endos pore-forming bacteria Pérez-Jimenez, 1996
facultatively anaerobic. Although, more examination is needed to identify definitely each isolate and lock for the possible new taxons.
In comparison to previous work in this specific topic, this research confirm the
isolation of high population of aerobic endosporer from marine environments but is
contrary to the requirement of sea water for growth and to the use a minimal media for
isolation purpose (Singer and Leadbetter, 19??).
By other way, fluorescence in situ hybridization was useful applied for the
detection of endosporer from marine samples (Table 8). Samples pausterized from salt
marsh, MBL Beach, Garbage Beach, Eel Pound and Aquarium Ramp showed
fluorescence rod-shaped bacteria Low G + C either universal probes. Rod and cocci
endosporing isolate from marine environment were used as control with Low G + C
probe. Only the rod was detected. It suggests that Sporosarcina,the endosporing cocci,
is refractile to the with Low G + C probe.
Table 8. In situ Hybridization assay to marine samples. Sample Low G + C Probe Universal Probe Rod isolate + NDa Cocci isolate - ND MixEP,GBandR + ND MBLBeachl + ND MBLBeach2and3 - ND Salt Marsh I + + Salt Marsh II + + Salt Marsh Ill + + Salt Marsh W + - Salt Marsh V - + a Not Determined
Study the pigment production by endospore-forniing bacteria from marine environments would allow us to understand their role and explore possible applications.
12
physiological
Molecular
Marine
endos
pore-forming
biology
and
genetics
and
bacteria
spectrophotometrical
aspects
of
the
endospore-forming
13
techniques
would
bacteria.
be
used
Pérez-Jimenez,
to
research 1996
the
and
their
discussions
Acknowledgments
Marine
course
Daniel
special
endospore-forming
William
Tom
Madeline
Abigail
Microbial
S.
about
Pit
collaboration
Grosh
Salyers
and
Townsend
Vargas,
this
Jim
Diversity,
Foundation
bacteria
project.
and
Comely
Caroline
and
Porter
Ed
Marine
their
for
R.
for
Foundation,
Plugge,
your
Leadbetter
extraordinary
their
Biological
collaboration
14
contribution
Ana
for
American
Scavino-Fernández
Laboratory.
support.
their
with
to
guidance
my
Society
the
financial
microscopy
for
and
and
Cell
very
aid
Jorge
Pérez-Jimenez,
equipment.
Microbiology
support
productive
Olmos
1996
for for
Wood,
Snaeth,
Smibert
Singer,
Nierzwicki-Bauer,
Pregerson,
Gibson, Claus, Brock,
Berkeley
Balows,
Marine
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S F
•
o ir
S , ‘S 14 p 4 •• C I. p 4.s 3.. S g•. , I 5’. - ,
+3 V
cit
I ..• • • 0 •1
S ., S ‘ .,
U ‘I SI
Figure 1-Phase contrast (100X)photomycrograph of aerobic isolates from marine environments in wet mount. Cocci tetrads of Sporosarcina(left) and rod chains of Bacillus(right).
16