Research Reports of Fukui National College of Technology Natural Science and Engineering No.46 (2012)
Research Reports of Fukui National College of Technology Natural Science and Engineering No.46 (2012)
Mechanisms regulating direction of spiral colony curvature in bacteria of genus
Mechanisms regulating direction of spiral colony curvature in bacteria of genus
Bacillus
Bacillus
Natsuyo UOMI 1, Maya ENOHATA1, Hajime HONDA2, Toshiyuki KAWAMURA1*
Natsuyo UOMI 1, Maya ENOHATA1, Hajime HONDA2, Toshiyuki KAWAMURA1*
1
Department of Chemistry and Biology Engineering , Fukui National College of Technology , Geshi-cho. Sabae-City . Fukui 916-8507 ,
Japan
1
Department of Chemistry and Biology Engineering , Fukui National College of Technology , Geshi-cho. Sabae-City . Fukui 916-8507 ,
2
Department of Bioengineering, Nagaoka University of Technology. Nagaoka, Niigata, 940-2188, JAPAN
Japan
2
Department of Bioengineering, Nagaoka University of Technology. Nagaoka, Niigata, 940-2188, JAPAN
Abstract: Among members of the genus Bacillus, B. mycoides are characterized by the formation of
radiating spider web-like colonies known as rhizoids. Di Franco et al. previously reported the existence of
Abstract: Among members of the genus Bacillus, B. mycoides are characterized by the formation of
two morphotypes of rhizoids in B. mycoides, whose filaments systematically curved in either a clockwise or
radiating spider web-like colonies known as rhizoids. Di Franco et al. previously reported the existence of
counter-clockwise direction. However, the mechanisms regulating the direction of curvature remain
two morphotypes of rhizoids in B. mycoides, whose filaments systematically curved in either a clockwise or
unknown. In this investigation, we isolated a Bacillus strain forming rhizoidal colonies from soil and
counter-clockwise direction. However, the mechanisms regulating the direction of curvature remain
demonstrated that its colonies change direction of curvature (either clockwise or counter-clockwise)
unknown. In this investigation, we isolated a Bacillus strain forming rhizoidal colonies from soil and
depending on the NaCl concentration of the culture media. In addition, we performed a molecular
demonstrated that its colonies change direction of curvature (either clockwise or counter-clockwise)
phylogenetic analysis based on the strain’s 16S rDNA gene sequence using the neighbor joining method and,
depending on the NaCl concentration of the culture media. In addition, we performed a molecular
utilizing a scanning electron microscope, examined its morphological characteristics.
phylogenetic analysis based on the strain’s 16S rDNA gene sequence using the neighbor joining method and,
utilizing a scanning electron microscope, examined its morphological characteristics.
Key words: Bacillus pseudomycoides, spiral colony, rhizoid
Key words: Bacillus pseudomycoides, spiral colony, rhizoid
Introduction
Introduction
Due to their ability to form endospores, members of the
genus
may to
still,
on endospores,
rare occasions,
causeoffood
Due toBacillus
their ability
form
members
the
poisoning,
even
in
food
that
has
been
heat
treated(1,2).
In
genus Bacillus may still, on rare occasions, cause food
the
majorityeven
of such
cases,
is believed to
poisoning,
in food
thatthe
hascausal
been agent
heat treated(1,2).
In
be
B.
cereus(3).
In
addition,
anthrax
(B.
anthracis)
is
a
the majority of such cases, the causal agent is believed to
highly
lethal
pathogen
and
is
feared
for
its
potential
use
as
be B. cereus(3). In addition, anthrax (B. anthracis) is a
ahighly
biological
Meanwhile,
var. natto
is
lethalweapon(4).
pathogen and
is feared B.
forsubtilis
its potential
use as
used
in the production
natto (fermented
soyvar.
bean)
and
a biological
weapon(4). of
Meanwhile,
B. subtilis
natto
is
polyglutamic
acid,
responsible
for
natto’s
slimy
texture,
for
used in the production of natto (fermented soy bean) and
use
as a moisturizer(5).
B. thuringiensis
produces
crystal
polyglutamic
acid, responsible
for natto’s slimy
texture,
for
proteins
(BT
toxins)
that
have
insecticidal
activity(6).
It
is
use as a moisturizer(5). B. thuringiensis produces crystal
therefore
expected
understanding
of the genus
proteins (BT
toxins)that
thatgreater
have insecticidal
activity(6).
It is
Bacillus
will
prove
useful,
not
only
from
the
standpoint
of
therefore expected that greater understanding of the genus
preventing
food
poisoning
and
preventing
or
treating
Bacillus will prove useful, not only from the standpoint of
infectious
but also and
in thepreventing
productionoroftreating
useful
preventing diseases,
food poisoning
materials
environmental
infectiousand
diseases,
but alsoremediation.
in the production of useful
It
is
important
to
characterize
each of these species;
materials and environmental
remediation.
however,
in observing
and analyzing
the these
influence
of
It is important
to characterize
each of
species;
various
on these bacteria,
given their
size (1
however,factors
in observing
and analyzing
the small
influence
of
to
3
µm)
and
simple
rod
shape
(long
and
narrow
capsules),
various factors on these bacteria, given their small size (1
the observation of phenotypic details of individual cells is
extremely
difficult
light microscopy.
Franco reported
the observation
of by
phenotypic
details ofDi
individual
cells is
the
existence
of
two
morphotypes
of
rhizoids
in B.
extremely difficult by light microscopy. Di Franco reported
mycoides,
whose
rhizoids
systematically
curved
either
a
the existence of two morphotypes of rhizoids ininB.
clockwise
or counter-clockwise
direction.
mycoides, whose
rhizoids systematically
curvedThe
eitherstrain
in a
isolated
in
this
investigation
also
characteristically
formed
clockwise or counter-clockwise direction. The strain
radiating
spider
web-like colonies
as rhizoid(7).
The
isolated in
this investigation
also known
characteristically
formed
above-mentioned
B. cereus,
B. anthracis,
B. rhizoid(7).
thuringienesis,
radiating spider web-like
colonies
known as
The
B.
mycoides,
and
B.
pseudomycoides
exhibit
a
high
degree
above-mentioned B. cereus, B. anthracis, B. thuringienesis,
of
genetic homology
and are similar
to the
point
that
B. mycoides,
and B. pseudomycoides
exhibit
a high
degree
identification
based on and
genetic
alone
difficult.
of genetic homology
are analysis
similar to
the ispoint
that
Accordingly,
B.
mycoides
and
B.
pseudomycoides(8),
identification based on genetic analysis alone is difficult.
whose
responseB.to environmental
can be read in the
Accordingly,
mycoides and signals
B. pseudomycoides(8),
form
changes
in rhizoid curvature
whose of
response
to environmental
signals candirection
be read inand
the
morphology,
may
be
considered
model
organisms
within
form of changes in rhizoid curvature direction and
this
genus. may be considered model organisms within
morphology,
In
investigation, colonies of a strain isolated from the
thisthis
genus.
soil
grew
in a clockwise
direction
2% LB
In this investigation,
colonies
of awhen
straincultured
isolatedonfrom
the
nutrient
agar.
We
discovered,
however,
that
the
direction
of
soil grew in a clockwise direction when cultured on 2% LB
curvature
andWe
morphology
rhizoids that
changed
when the
nutrient agar.
discovered,ofhowever,
the direction
of
concentration
of
nutrient
in
the
culture
media
was
varied.
curvature and morphology of rhizoids changed when
the
to 3 µm) and simple rod shape (long and narrow capsules),
concentration of nutrient in the culture media was varied.
Corresponding author. E-mail: [email protected]
䋪
Corresponding author. E-mail: [email protected]
䋪
10
Research Reports of Fukui
福井工業高等専門学校　研究紀要　自然科学・工学　第
National College of Technology Natural Science
46 and
号　2012
Engineering No.46 (2012)
Spiral colony curvature in Bacillus
We, therefore, conducted experiments focusing on the
influence of culture medium NaCl concentration on the
direction of rhizoid curvature.
sufficient PBS buffer was added to the petri dishes to cover
the culture media surface. Plates were left standing for five
minutes. After removal of PBS buffer, sufficient PBS
buffer was again added to plates to cover the culture media
surface, and these were left to stand for five minutes. PBS
buffer was then removed, and plates were flooded with
25% ethanol in PBS buffer. After removal of the 25%
ethanol in PBS buffer, plates were flooded with 50%
ethanol in PBS buffer. This process was repeated
sequentially using 75% ethanol in PBS buffer, then 99.5%
ethanol in PBS buffer. After removal of the 99.5% ethanol
in PBS buffer, plates were allowed to air dry in a laminar
flow hood. Samples, fixed and dried just prior to
observation under the scanning electron microscope, were
transferred to cover slips (by touching the culture media to
the cover slips) and coated with platinum to a thickness of
50 Å. Samples were observed using a scanning electron
microscope (JSM-6340F; JEOL Ltd.Tokyo, Japan) at an
accelerated voltage of 2.00 kV.
Materials and Methods
Culture media and reagents
LB Broth,Lennox(Nacalai Tesque, Kyoto, Japan,
code:20066-95) ‫ ޔ‬Agar
Powder(Nacalai
Tesque,
TM
code:01028-85)‫ޔ‬Bacto Peptone(BD, Franklin Lakes, NJ
USA, code:REF211677) ‫ ޔ‬Extract Yeast Dried(Nacalai
Tesque code:15838-45)‫ޔ‬Sodium Chloride(Nacalai Tesque
code:31320-05) ‫ ޔ‬Rose Bengale(WALDECK, Münster,
Germany, code:1A-182)
Isolation of bacteria
Soil collected from the grounds of Fukui National College
of Technology was diluted in sterile water and plated onto
culture media containing 2% LB (nutrient) and 1.5% agar,
and was incubated under aerobic conditions at 25°C.
Selective staining of rhizoids
As the rhizoids were difficult to observe in their natural
state, they were stained prior to optical microscopic
observation. Staining was accomplished using 0.1% rose
bengal aqueous solution.
Putative taxonomic identification based on 16S rDNA
base sequence and molecular phylogenetic analysis
Genomic DNA was extracted from a colony of the isolated
strain using InstaGene Matrix (Bio RAD, Hercules, CA).
16S rDNA was amplified by PCR using PrimeSTAR HS
DNA Polymerase (Takara Bio Inc., Shiga, Japan) and
prepared for sequencing using the BigDye Terminator v3.1
Cycle Sequencing Kit (Applied Biosystems, Foster city,
CA). Primers used were 9F and 1510R (9, 10) for PCR
amplification, and 9F, 785F, 802R and 1510R for the
sequencing reaction. Sequence reactions were performed
using the ABI PRISM 3130˜1 Genetic Analyzer System
(Applied Biosystems), and sequences were analyzed using
ChromasPro 1.4 software (Technelysium Pty Ltd.,
Tewantin, Australia). We searched for homologous
sequences in the Apollon DB-BA 6.0 database (Techno
Sugura Laboratory Co., Ltd., Shizuoka, Japan) and public
databases in the International Nucleotide Sequence
Database Collaboration (GenBank/DDBJ/EMBL) using
Apollon 2.0 software (Techno Suruga Laboratory Co., Ltd.).
The CLUSTAL W (11) multiple-alignment program and
MEGA ver 3.1 (12) were used for molecular phylogenetic
analysis.
Changes in direction of rhizoid curvature as a function
of nutrient (LB broth) concentration
Petri dishes with culture media containing 1.5% agar and
0.02%, 0.05%, 0.1%, 0.2% or 2% LB Broth were
inoculated using a bamboo skewer and incubated for three
days at 25°C. Colonies were observed after staining with an
aqueous solution of rose bengal. Twelve photographs were
taken of the underside of each of the 0.02%, 0.05%, 0.1%,
0.2%, and 2% LB petri dishes, and the curvature direction
(clockwise, counter-clockwise or straight) of 10 to 50
rhizoids on each plate were recorded.
Reversal of rhizoid curvature direction resulting from
addition of NaCl
When cultured on media containing 1.5% agar and 0.05%
LB Broth, colonies of the isolate grew in a
counter-clockwise direction. We conducted experiments to
determine whether the addition of 2% NaCl would induce a
change in colony morphology.
Scanning electron microscopy
Formaldehyde (4%) was added directly to nutrient agar
plates containing colonies of the isolate, and these were left
to stand for one day. After removing the 4% formaldehyde,
Direction of rhizoid curvature direction as a function of
NaCl concentration
Petri dishes with culture media containing 1.5% agar,
2
Mechanisms regulating direction of spiral colony curvature in bacteria of genus Bacillus
Research Reports of Fukui National College of Technology 46 (2012)
Research Reports of Fukui National College of Technology 46 (2012)
11
BSL2
BSL2
BSL3
BSL3
Figure
Molecular
phylogenetic
constructed
based
isolate
rDNA
sequence.
Line
at the
bottom
Figure
1.1.
Molecular
phylogenetic
treetree
constructed
based
on on
thethe
isolate
16S16S
rDNA
sequence.
Line
at the
bottom
leftleft
is ais a
scale
Numbers
at branch
nodes
represent
bootstrap
values.
A superscript
following
strain
designation
indicates
scale
bar.bar.
Numbers
at branch
nodes
represent
bootstrap
values.
A superscript
“T”“T”
following
the the
strain
designation
indicates
it isit is
a type
strain
a given
species.
BSL
indicates
a biosafety
level
or higher.
a type
strain
for for
a given
species.
BSL
indicates
a biosafety
level
of 2ofor2 higher.
isolate
belongs
to the
genus
Bacillus.
performed
isolate
belongs
to the
genus
Bacillus.
WeWe
thenthen
performed
a a
molecular
phylogenetic
analysis
after
acquiring
molecular
phylogenetic
analysis
after
acquiring
the the
16S16S
rDNA
sequences
from
strains
of thuringiensis
B. thuringiensis
rDNA
sequences
from
typetype
strains
of B.
andand
numerous
other
members
of
the
genus
Bacillus.
Molecular
numerous other members of the genus Bacillus. Molecular
phylogenetic
analysis
using
neighbor
joining
method
phylogenetic
analysis
using
the the
neighbor
joining
method
(14)
based
on
16S
rDNA
sequences
placed
the
isolate
(14) based on 16S rDNA sequences placed the isolate in ain a
cluster
comprising
members
of the
genus
Bacillus
(Fig.
cluster
comprising
members
of the
genus
Bacillus
(Fig.
1). 1).
Althoughthe theisolate
isolateformed
formeda acluster
clusterwithwithB. B.
Although
(77%),
pseudomycoides(8),
the
bootstrap
value
(15)
lowlow
(77%),
pseudomycoides(8), the bootstrap value (15) waswas
indicating
significant
distance
between
strains
(Fig.
Results
indicating
significant
distance
between
the the
twotwo
strains
(Fig.
Results
Based
above
results,
determined
it would
1). 1).
Based
on on
the the
above
results,
we we
determined
thatthat
it would
be
reasonable
to
identify
the
isolate
as
Bacillus
Isolate
identity
be reasonable to identify the isolate as Bacillus sp sp
Isolate identity
KOSEN(Fig.
1. AB703619:
Accession
number).
Sequence
homology
analysis
based
a Blast
search KOSEN(Fig.
1. AB703619:
Accession
number).
Sequence
homology
analysis
based
on on
a Blast
(13)(13)
search
of the
Apollon
DB-BA6.0
database
indicated
of the
Apollon
DB-BA6.0
database
indicated
thatthat
the the
16S16S
rDNA
sequence
of
the
isolate
was
highly
homologous
Scanning
electron
microscopy
rDNA sequence of the isolate was highly homologous to to Scanning
electron
microscopy
Based
on scanning
electron
microscope
observations,
it was
of members
of the
genus
Bacillus.
highest
degree Based
on scanning
electron
microscope
observations,
it was
thatthat
of members
of the
genus
Bacillus.
TheThe
highest
degree
determined
that
cells
of
the
isolate,
measuring
of
homology
(98.7%)
was
found
with
the
16S
rDNA
determined that cells of the isolate, measuring
of homology (98.7%) was found with the 16S rDNA
approximately
in length,
were
of the
bacillus
sequence
of thuringiensis
B. thuringiensis
strain
ATCC
10792.
Similarly, approximately
1.51.5
µmµm
in length,
were
of the
bacillus
typetype
sequence
of B.
strain
ATCC
10792.
Similarly,
characteristic
of the
genus
Bacillus
(Fig.
sequence
homology
analysis
using
GenBank/DDBJ/EMBL characteristic
of the
genus
Bacillus
(Fig.
2). 2).
sequence
homology
analysis
using
GenBank/DDBJ/EMBL
databases
also
indicated
high
homology
of
the
isolate
16S
databases also indicated high homology of the isolate 16S
rDNA
sequence
to that
of members
of the
genus
Bacillus.
Colony
staining
rDNA
sequence
to that
of members
of the
genus
Bacillus.
Colony
staining
Given
their
light
color,
detailed
observation
of rhizoids
Based
these
results,
there
a highly
likelihood
their
light
color,
detailed
observation
of rhizoids
in in
Based
on on
these
results,
there
is aishighly
likelihood
thatthat
the the Given
0.0125%
yeast
extract
0.025%
peptone
(comparable
0.0125%
yeast
extract
andand
0.025%
peptone
(comparable
to to
0.05%
media
used
elsewhere
in this
investigation),
0.05%
LBLB
media
used
elsewhere
in this
investigation),
andand
0.005%,
0.010,
0.020%,
0.025%,
0.050%,0.100%,
0%,0%,
0.005%,
0.010,
0.020%,
0.025%,
0.050%,0.100%,
1.000%
or
2.000%
NaCl
were
inoculated
and
incubated
1.000% or 2.000% NaCl were inoculated and incubated
for for
2 days.
undersides
each
dishes
were
2 days.
TheThe
undersides
of of
each
of of
the the
fivefive
dishes
were
photographed
at
8
locations,
and
the
curvature
direction
photographed at 8 locations, and the curvature direction
(clockwise,
counter-clockwise,
intermediate
(straight))
(clockwise,
counter-clockwise,
or or
intermediate
(straight))
of to
1040
to rhizoids
40 rhizoids
on each
location
recorded.
of 10
on each
location
waswas
recorded.
12
Research Reports of Fukui
福井工業高等専門学校　研究紀要　自然科学・工学　第
National College of Technology Natural Science
46 and
号　2012
Engineering No.46 (2012)
Spiral
colony
curvature
in Bacillus
Spiral
colony
curvature
in Bacillus
Figure
Scanning
electron
microscope
observation
Figure
2. 2.
Scanning
electron
microscope
observation
isolated
strain.
ofof
isolated
strain.
Figure4. 4.Difference
Differenceinindirection
directionofofrhizoid
rhizoidcurvature
curvature
Figure
dependingononnutrient
nutrient(LB
(LBBroth)
Broth)concentration
concentrationofofthethe
depending
culturemedia.
media.The
Theleftleftand
andright
rightpanels
panelsshow
showmedia
media
culture
containing0.2%
0.2%and
and0.02%
0.02%LBLBBroth,
Broth,respectively.
respectively.Scale
Scale
containing
represents
1 mm.
barbar
represents
1 mm.
Figure3. 3.Rhizoid
Rhizoidstained
stainedwith
withrose
rosebengal.
bengal.The
Theleftleftand
and
Figure
rightpanels
panelsshow
showthetherhizoid
rhizoidprior
priorto toand
andafter
afterstaining,
staining,
right
respectively.
Scale
bars
represent
1 mm.
respectively.
Scale
bars
represent
1 mm.
Figure
Direction
rhizoid
curvature
a function
Figure
5. 5.
Direction
ofof
rhizoid
curvature
asas
a function
ofof
culture
media
nutrient
(LB
Broth)
concentration.
culture
media
nutrient
(LB
Broth)
concentration.
their
natural
state
difficult.
such,
this
investigation,
their
natural
state
is is
difficult.
AsAs
such,
in in
this
investigation,
attemptedvarious
variousmethods
methodsforforstaining
stainingrhizoids
rhizoids
weweattempted
growing
culture
media.
We
found
that
colonies
could
growing
onon
culture
media.
We
found
that
colonies
could
bebe
sufficientlystained
stainedwith
withthetheaddition
additionofof0.1%
0.1%rose
rosebengal
bengal
sufficiently
aqueous solution
solution to to enable
enable detailed
detailed observation
observation ofof
aqueous
individual
filaments
on
culture
media
using a a
individual filaments on culture media using
stereomicroscope
(Fig.
stereomicroscope
(Fig.
3).3).
Change
rhizoid
morphology
a function
nutrient
Change
inin
rhizoid
morphology
asas
a function
ofof
nutrient
(LB
Broth)
concentration
(LB
Broth)
concentration
order
determine
influence
nutrient
levels
InIn
order
to to
determine
thethe
influence
ofof
nutrient
levels
onon
thethe
rhizoid
morphology,
we
prepared
culture
media
containing
rhizoid morphology, we prepared culture media containing
different
levels
Broth.
was
shownthat
that
tips
different
levels
ofof
LBLB
Broth.
It It
was
shown
tips
ofofthethe
rhizoids grown
grown onon media
media with
with high
high LBLB Broth
Broth
rhizoids
concentrations(0.2%)exhibited
exhibitedtight
tightclockwise
clockwisecurvature,
curvature,
concentrations(0.2%)
whilethose
thosegrown
grownononmedia
mediawith
withlow
lowLBLBBroth
Broth
while
concentrations(0.02%)
exhibited
more
gradual
curvature
concentrations(0.02%)
exhibited
more
gradual
curvature
oror
counter-clockwisecurvature
curvature(Fig.
(Fig.4).4).ToTofurther
furtherexplore
explore
counter-clockwise
thisrelationship,
relationship,weweobserved
observedthethecurvature
curvatureofofrhizoids
rhizoids
this
grown
on
culture
media
containing
0.02%,
0.05%,
0.10%,
grown on culture media containing 0.02%, 0.05%, 0.10%,
0.20%and
and2.00%
2.00%LBLBBroth
Broth(Fig.
(Fig.5).5).A Atotal
totalofof59.
59.
8㫧
0.20%
8㫧
3.1%(㫧:standard
standarderror
errorofofmean)
mean)and
and51.4㫧4.6%
51.4㫧4.6%ofof
3.1%(㫧:
㪣㪙㩷㪇㪅㪇㪌㩼䋫㪥㪸㪚㫃㩷㪉㩼
㪣㪙㩷㪇㪅㪇㪌㩼䋫㪥㪸㪚㫃㩷㪉㩼
Figure
Clockwise-curving
rhizoids
resulting
from
Figure
6. 6.
Clockwise-curving
rhizoids
resulting
from
supplementation
culture
media
with
NaCl.
Scale
supplementation
ofof
culture
media
with
NaCl.
Scale
bars
represent
2 mm.
bars
represent
2 mm.
rhizoids
low-LB
Broth
(0.02%
and
0.05%,
respectively)
rhizoids
onon
low-LB
Broth
(0.02%
and
0.05%,
respectively)
mediagrew
grewin ina acounter-clockwise
counter-clockwisedirection.
direction.InIncontrast,
contrast,
media
only8.3㫧2.2%
8.3㫧2.2%and
and6.4㫧0.5%
6.4㫧0.5%ofofrhizoids
rhizoidsononhigh-LB
high-LB
only
broth(0.20%
(0.20%and
and2.00%,
2.00%,respectively)
respectively)media
mediaexhibited
exhibited
broth
counter-clockwise
curvature,
whereas
majority
(69.6㫧
counter-clockwise
curvature,
whereas
thethe
majority
(69.6㫧
8.7%
and
70.2㫧4.9%,
respectively)
grew
in
a
clockwise
8.7% and 70.2㫧4.9%, respectively) grew in a clockwise
44
Mechanisms regulating direction of spiral colony curvature in bacteria of genus Bacillus
13
Research Reports of Fukui National College of Technology 46 (2012)
Research Reports of Fukui National College of Technology 46 (2012)
Figure 7. Direction of rhizoid curvature as a function of culture media NaCl concentration.
Figure 7. Direction of rhizoid curvature as a function of culture media NaCl concentration.
direction (Fig. 5).
direction (Fig. 5).
Identification of factors regulating direction of rhizoid
Identification
of factors regulating direction of rhizoid
curvature
curvature
Based
on the observations that LB Broth concentration
Based
on direction
the observations
LB Broth
influences
of colonythat
curvature
(Figs.concentration
4 and 5), we
influences
direction
of
colony
curvature
4 and 5),
we
conducted subsequent experiments to (Figs.
determine
which
conducted
to determine
which
component subsequent
of LB Brothexperiments
(peptone, yeast
extract or NaCl)
component
of LB We
Brothdid
(peptone,
yeast extract
or NaCl)
was responsible.
not observe
any change
in
was
responsible.
We
did
not
observe
any
change
in
direction of rhizoid curvature on media lacking either
direction
rhizoid
curvature
mediaIt was
lacking
peptone orofyeast
extract
(data not on
shown).
foundeither
that
peptone
or
yeast
extract
(data
not
shown).
It
was
found
while a large proportion of rhizoids grown on 0.050% that
LB
while
large proportion
rhizoids
grownmedia
on 0.050%
Broth a(containing
0.013%of NaCl)
culture
curvedLB
in
Broth
(containing
0.013%
NaCl)
culture
media
curved
in
the counter-clockwise direction (Fig. 5), rhizoids grown on
the
counter-clockwise
direction
(Fig.
5),
rhizoids
grown
on
the same media supplemented with 2.000% NaCl tended to
the
same
media
supplemented
NaCl tended to
curve
in the
clockwise
directionwith
(Fig.2.000%
6).
curve
in thetoclockwise
direction (Fig.
In order
further investigate
the 6).
relationship between
In
order
to
further
investigate
relationship
between
NaCl concentration and direction the
of rhizoid
curvature,
we
NaCl
concentration
and
direction
of
rhizoid
curvature,
we
generated culture media containing yeast extract and
generated
culture
media
containing
extract
and
peptone along
with 0%,
0.005%,
0.010%,yeast
0.020%,
0.025%,
peptone
along
with
0%,
0.005%,
0.010%,
0.020%,
0.025%,
0.050%, 1.000% or 2.000% NaCl. Petri dishes containing
0.050%,
1.000%
or were
2.000%
NaCl. Petri
dishes containing
each of these
media
inoculated
and observed
after two
each
of
these
media
were
inoculated
and
observed
after two
days. The majority of rhizoids on low-NaCl (0%
and
days.
The
majority
of
rhizoids
on
low-NaCl
(0%
0.005%) media grew in the counter-clockwise directionand
or
0.005%)
media
grew
in
the
counter-clockwise
direction
or
straight, with 54.1±10.3% and 50.4±2.9%, respectively,
straight,
with
54.1±10.3%
and
50.4±2.9%,
respectively,
curving in the counter-clockwise direction and only
curving
the 3.2±3.2%,
counter-clockwise
direction
andin only
5.7±1.1%inand
respectively,
curving
the
5.7±1.1%
and
3.2±3.2%,
respectively,
curving
in
clockwise direction. In contrast, the majority of rhizoids the
on
clockwise
contrast,1.000%
the majority
of rhizoids
on
high-NaCl direction.
(0.050%, In
0.100%,
and 2.000%)
media
high-NaCl
and with
2.000%)
media
grew in the (0.050%,
clockwise0.100%,
direction1.000%
or straight,
65.3±0.4%,
grew in the clockwise direction or straight, with 65.3±0.4%,
50.3±0.3%, 48.2±15.3% and 37.7%±1.9%, respectively,
50.3±0.3%,
48.2±15.3%
37.7%±1.9%,
curving in the
clockwiseand
direction
and onlyrespectively,
3.3±0.6%,
curving
in
the
clockwise
direction
and
only
3.3±0.6%,
15.8±2.0%, 13.5±11.5 and 11.8±0.1%, respectively,
15.8±2.0%,
13.5±11.5
and
11.8±0.1%,
respectively,
curving in the counter-clockwise direction (Fig. 7).
curving in the counter-clockwise direction (Fig. 7).
Discussion
Discussion
The direction of colony curvature changed with the LB
The
of colony
changed
with4 the
Brothdirection
concentration
in thecurvature
culture media
(Figs.
and LB
5).
Broth
concentration
in
the
culture
media
(Figs.
4
and 5).
We initially hypothesized that this was due to a tendency
of
We
initially
hypothesized
that
this
was
due
to
a
tendency
of
colonies to grow towards areas of higher nutrient
colonies
to grow
towardswhen
areasweof investigated
higher nutrient
concentration.
However,
the
concentration.
However,
when
we
investigated
the
influence of individual components of culture media
influence
of individual
components
of culture
media
(peptone, yeast
extract, and
NaCl), it was
found that
the
(peptone,
yeast
extract,
and
NaCl),
it
was
found
that
change in direction of curvature depended only on the
the
change
in
direction
of
curvature
depended
only
on
the
concentration of NaCl (Figs. 6 and 7). As organic matter
concentration
of NaCl
(Figs.also
6 and
7). As
organic
matter
from excreta and
dead cells
contain
salts,
it is possible
from
excreta
and
dead
cells
also
contain
salts,
it
is
possible
that Bacillus sp. in the soil environment tend to grow
that
Bacillus
in theNaCl
soil concentrations,
environment tend
to likely
grow
towards
areas sp.
of higher
which
towards
of higher
NaCl concentrations,
which Or,
likely
also offerareas
higher
concentrations
of other nutrients.
it
also
offer
higher
concentrations
of
other
nutrients.
Or,
it
could be that colonies grow towards higher osmotic
could
be that
towards
higher osmotic
pressures,
whichcolonies
depend grow
on NaCl
concentration
and
pressures,
which
depend
on
NaCl
concentration
and
represent favorable conditions for the acquisition of water
represent
favorable
conditions
for
the
acquisition
of
water
and nutrients.
and
nutrients.
Di Franco
et al. reported that the rhizoids of B. mycoides
Di
Franco
et al. reported
the rhizoids
B. identified
mycoides
curved systematically
in a that
specific
directionofand
curved
systematically
in a specific
two morphotypes
depending
on direction
whether and
the identified
rhizoids
two
morphotypes
depending
on
whether
the
rhizoids
curved in the clockwise direction or counter-clockwise
curved
in
the
clockwise
direction
or
counter-clockwise
direction(7). From the results of molecular phylogenetic
direction(7).
the rDNA
resultssequences,
of molecular
phylogenetic
analysis basedFrom
on 16S
we identified
the
analysis based on 16S rDNA sequences, we identified the
14
Research Reports of Fukui
福井工業高等専門学校　研究紀要　自然科学・工学　第
National College of Technology Natural Science
46 and
号　2012
Engineering No.46 (2012)
Spiral colony curvature in Bacillus
isolated strain as a Bacillus sp. (Fig. 1), more closely
related to B. pseudomycoides(10) than B. mycoides. Many
genes in B. mycoides, B. pseudomycoides, B. thuringiensis,
B. cereus and B. anthracis exhibit a high degree of genetic
homology, and 95% or more homology in their 16S rDNA
sequences(2, 16). Among these, B. cereus and anthrax (B.
anthracis) are known for their high toxicity and,
respectively, as a causal agent of food poisoning and a
potential biological weapon(4, 14). Unfortunately, Bacillus
species are also difficult to completely eliminate as they
can form endospores that are highly resistant to bactericides
and heat treatment (7, 8). While it is important to elucidate
the nature of these highly toxic species, because the
biosafety levels of B. cereus and B. anthracis are 2 and 3
(BSL in Fig. 1), respectively, their use in direct research is
challenging. In contrast, B. mycoides and the strain isolated
in this investigation are relatively safe to handle.
Furthermore, given that individual bacilli of members of
the genus Bacillus, including the strain isolated in this
investigation, are on the order of several microns in size
(Fig. 2), it is not possible to observe cellular-level changes
by optical microscopy. Thus, it is easier to elucidate the
nature of strains such as that isolated in this experiment,
whose colony morphology is readily observed and reflects
the influence of external factors.
Acid Produced by Bacillus subtilis (natto): Structural
Characteristics, Chemical Properties and Biological
Functionalities. J. Chin. Chem. Soc. 53:1363-1384.
6. Roh, J.-Y., J.-Y. Choi, M-.S. Li, B.-R. Jin, Y.-H.Je. 2007.
Bacillus thuringiensis as a specific, safe, and effective
tool for insect pest control. J. Microbiol.
Biotechnol.17:547-559.
7. Di Franco, C., E. Beccari, T. Santini, G. Pisaneschi, G.
Tecce. 2002. Colony shape as a genetic trait in the
pattern-forming Bacillus mycoides. BMC Microbiol.
2:33.
8. Nakamura, L.K. 1998. Bacillus pseudomycoides sp. nov.
Int. J. Syst. Bacteriol. 3:1031-1035.
9. Sato, T., M. Sato, J. Matsuyama, and E. Hoshino. 1997.
PCR-restriction fragment length polymorphism analysis
of genes coding for 16S rRNA in Veillonella spp. Int. J.
Syst. Bacteriol. 47:1268-1270.
10. Weisburg, W. G., S.M. Barns, D.A. Pelletier, and D.J.
Lane. 1991. 16S ribosomal DNA amplification for
phylogenetic study. J Bacteriol. 173:697-703.
11. Thomson, J.D., D.G. Higgins, and T.J. Gibson. 1994.
CLUSTAL W: Improving the sensitivity of progressive
multiple sequence alignment through sequence
weighting, positions-specific gap penalties and weight
matrix choice. Nucleic Acids Res. 22:4673-4680.
12. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3:
Integrated Software for Molecular Evolutionary Genetics
Analysis and Sequence Alignment. Brief. Bioinform.
5:150-163.
13. Altschul, S.F., T.F. Madden, A.A. Schäffer, J. Zhang,
Z.Zhang, W. Miller and D.J. Lipman,. 1997. Gapped
BLAST and PSI- BLAST: a new generation of protein
database search programs. Nucleic Acids Res.
25:3389-3402.
14. Saitou, N., and M. Nei. 1987. The neighbor joining
method㧦a new method for reconstructing phylogenetic
trees. Mol. Biol. Evol. 4:406-425.
15. Felsenstein, J. 1985. Confidence limits on phylogenies:
an approach using the bootstrap. Evolution. 39:783-791.
16. Bavykin, S.G., Y.P. Lysov, V. Zakhariev, J.J. Kelly, J.
Jackman, D.A. Stahl, A.Cherni, 2004. Use of 16S rRNA,
23S rRNA, and gyrB gene sequence analysis to
determine phylogenetic relationships of Bacillus cereus
group microorganisms. J. Clin. Microbiol. 42:3711-3730.
17. Wang, L., F. Lee, C. Tai, H. Kasai. 2007. Comparison
of gyrB gene sequences, 16S rRNA gene sequences and
DNA-DNA hybridization in the Bacillus subtilis group.
Int. J. Syst. Evol. Microbiol. 57:1846-1850.
Acknowledgements
We would like to thank Mr Jiro Nagatomo (Wakasa Wan
Energy Research Center) for technical assistance of
scanning electron microscope observations.
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6