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The effect of gluconate in promoting sporulation in Bacillus, cereus
Vol. 24, No. 5, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THEEFFE'3OFGLUCONATEIN PROMOTING SPORULATION IN BACILIUS CEREUSl H. L. Sadoff Microbiology and PublicHealth Michigan State University EastLansing,Michigan
ReceivedJuly 28, 1966 Sporulation in the Bacillaceae is a process of unicellular which results
in the fomation
is known of the control heat resistance.
of dmmnt,
resistance (Collier
acid (2,6-dicarboxypyridi,
and Nakata, 1958; Day and Costilow, 1964).
but acquired heat stability resistant
cells
1962).
While studying the
spores sooner than did the
in gluconate contained DPA, were heat labile,
at precisely
spores we= formed in the con-1
the sametime that refractile, cultures.
in this cmnuni cation show that the incorporation media results
Glucose dehy-
it was noted that cultures grown in media
containing sodium gluconate fmmed refxactile The spomlating
with heat
synthesized early in the
of Bacillus cereus (Bach and Sadoff,
role of this enzyme in sporulation,
controls.
Little
DPA)
Bacillus or Clostridium species concanitantly
dmgenaseis a spore-specificpmteinwhichis spomlation
cell forms.
of sporulation CIPof the mechanism(s) of dcmnamy or
However, dipicolinic
occurs in spomlating
heat resistant
mrphogenesis
The experiments described
of gluconate in sporulation
in a) reduction of the time required for the initiation
synthesis of morphologically
heat
of
recognizable spores; b) a bmad separation of
spore mrphogenesis fran heat resistance and c) a separation of the time of appemame of DPA from the occurrence of heat resistance.
'Journal Article University.
3691
Agriculture
Experiment Station,
691
Michigan State
Vol. 24, No. 5, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EXPERIMENTAL
These studies were conducted with -B. cereus grawn aerobically in 3 liter
volumes of modified G-mediumWashimto et - al -'
Brunswick fermntors. 5 liters transfers
The cultures were stirred
of air per min. of the culture
An active
at 30 C
1960) in New
at 450 rpm and aerated with
incculum was prepared by successive
in shake flasks of G-m&m
(Collier,
1957).
Identi-
cal inoculums were used for normal G-mediumand G-medim plus 0.2 per cent sodium gluconate.
The 3 litercultureswere
growth and sporulation
sampledatvaricustims
to ascertain the dry weight of cells/ml,
ml (Janssen -et al, 19581, refractile
spores, and heat resistant
should be pointed out that the ccqound ultimately in sane other functional
form in spores.
spores/ml were determined by direct a phase contrast microscope. crystal nutiient
violet.
Heat resistant
agar after
during
DPAcontent/ spores.
It
assayed as DPAmy exist
The refractile
CT phase bright
counts in a Petrwff-Hauser chamber using
Refractile
spores could not be stained with
spores/ml were determined by plating
on
heating suspensions at 80 C for 10 min.
2.5 2.0
i’
0.6
HEAT
.06
Figure 1.
STABLE
-o-
NG-
-.-
GO-MEDIUM
MEDIUM
The time course for the growth and sporulation of cultures of g. cereus in normal G-medium(NG) and G-mediumplus 0.2 per cent sodium gluconate (GG). Refractile spores are those which appear bright under the phase contrast microscope.
692
BIOCHEMICAL
Vol. 24, No. 5, 1966
Figurelshcws
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the growthme
forB.cereus inthetwomediaused. -did not affect the exponential growth of the
The presence of gluconate organism.
Refractile,
non-staining
scopically
in cultures
grown in gluconate
increased
exponentially
rnately 10g/ml.
for the increase development
became apparent micro-
medium at 9.5 hours, and they
in xxmber frun 107/ml to a final
Refnactile
13 hours, and the plot
spore structures
bodies in the wntrcl
of their
medium first
increasewasidenticalto
appeared at
the curve shawn
innumberofheatresistantspcres.
of heat resistant
ccunt of apprcxi-
The time cause
spares in gluwnate
and control
for the
media were
identical. Figure 2 presents the time course for the synthesis farrnation media.
of refractile
sp0res in cultures
The presence of gluwnate
for appearance of these by alxb
grown in gluconate
decreases the incubation fa
of DPA and for and control
time required
TWIXT.
DPA IN -o-o-
G G- Medium NG- Medium
SPORES
-z-X-
2
IO
Figure 2.
IN
GG- Medium NG-Medium
, 4 , , , , ( 14 “O”RIG 22
Correlation between the time of synthesis, of DPA and the appearrefractility and ance of refractile spores. Heat resistance, DPA synthesis occur sinultaneously in spores produced in normal G-medium (NG).
693
Vol. 24, No. 5, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCLESION The incorporation taining
of 0.2 per cent sodium gluconate
0.4 per cent glucose does not affect
--B. cereus nor the final that gluconate Gluconate
yield
of heat resistant
intermediate,
during
sporulation
spores.
have been demx&wated
cells
cultures
current
conditions,
to pyrzvate and
and pyruvate
These results
The differences particularly
of DPA,
occur within
differfromthose
and refractility
reported preceded thermal
might be attributed
the higher aeration
a very
to a variation
rate used in the
study.
Refrwtile
spores occurred four hours earlier
conate medium than control were the same. interval
medium but their
The principal
effect
The final
media were equal,
DPA content
grown in glu-
rates of synthesis
of gluconate.was
to shorten the time
growth and the appearance of spore
of spores frcm gluconate
and control
85 pg/mg dry spores.
The very close correspondence and DPA synthesis
to the conclusion
in cultures
respective
between the end of exponential
structures.
refractility
of acetate
spares, and heat resistance
by Hashimoto --et al (1960) where DPA synthesis
in cultural
as a key
and Nakata and Halvcxson (1960) and
of each other.
by 1.5 hours.
capacity,
grown in normal medium, the synthesis
the appearance of refractile short time interval
grcwth.
of g. cereus by Halvorson
Hanson --et al (1963) have shwn that the oxidation provides energy.for spore synthesis. In sporulating
vegetative
Enzymes for its oxidation
in spcsulating
Church (1957) and Doi --et al (1959);
growth rate of
These data suggest
in some control
or as an energy scurce.
G-mediwn con-
the exponential
is not used as an energy source during
may function
stability
into
between the percentage
in gluconate
that each refractile per
heat resistance
Furthermore,
70 per cent complete in the gluconate
spore
of
shown in Figure 2, leads
spore contains
of DPA. That is, DPA synthesis in that spore.
media,
completion
is canplete sporulation
its full
canplement
four hours preceding of the culture
is
medium before it begins in the control
694
Vol. 24, No. 5, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
medium. This very broad separation of the time of the Occurrence of DPAand heat stability species. resistance
has not been observed previously
Halvorson (1957) has shmn a separation of DPA synthesis and heat in an anaerobic spore former.
The over-all the initial Therefore,
in cultures of Bacillus
experimental results
stages of sporulation
indicate
but has little
that gluconate functions effect
later
in
in the process.
cultures grcwn in the gluconate rmzdim should be useful in study-
ing the sequence of events in sporulation, between the 0courrence of refractile
This investigation
in the time interval
spores and nature, heat stable spores.
was supported in part by research grant AI-01863
frcm the National Institute Health Service.
particularly
of Allergy
and Infectious
Diseases, U.S. Public
The author acknowledges the capable assistance of Ruth
McKay. REFERENCES
Bach, J.A. and H.L. Sadoff. J. Bacterial. 83, 699, (1962). Collier, R.E. In Spores, ed. H.O. Halvorson, Am. Inst. Biol. Sci., SymposiumSeries NO. 5, 1957, p. 10. Collier, R.E. and H.M. Nakata. Bacterial. Pmt., 1958, 42. Eay, L.E. and R.N. Costilow. J. Bacterial. z, 690, (1964). bi, R., H. Halvorson and B.D. Church. J. Bacterial. 77, 43, (1959). Halvorson, H. and B.D. Chum&. J. Appl. Bacterial. 20, 359, (1957). Halvorson, H.O. J. Appl. Bacterial. 20, 305, (1957). Hanson, R-S., V.R. Srinivasan and H.O. Halvorson. J. Bacterial. 86, 45, (1963). Hashinmto, T., S.H. Black and P. Gerhardt. Cm. J. Microbial. 6, 203, (1960). Janssen, F.W., A.J. Lund, and L.E. Anderson. Science 127, 26, T1958). Nakata, H.M. and H.O. Halvorson. J. Bacterial. &?$ 801, (1960).
695
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THEEFFE'3OFGLUCONATEIN PROMOTING SPORULATION IN BACILIUS CEREUSl H. L. Sadoff Microbiology and PublicHealth Michigan State University EastLansing,Michigan
ReceivedJuly 28, 1966 Sporulation in the Bacillaceae is a process of unicellular which results
in the fomation
is known of the control heat resistance.
of dmmnt,
resistance (Collier
acid (2,6-dicarboxypyridi,
and Nakata, 1958; Day and Costilow, 1964).
but acquired heat stability resistant
cells
1962).
While studying the
spores sooner than did the
in gluconate contained DPA, were heat labile,
at precisely
spores we= formed in the con-1
the sametime that refractile, cultures.
in this cmnuni cation show that the incorporation media results
Glucose dehy-
it was noted that cultures grown in media
containing sodium gluconate fmmed refxactile The spomlating
with heat
synthesized early in the
of Bacillus cereus (Bach and Sadoff,
role of this enzyme in sporulation,
controls.
Little
DPA)
Bacillus or Clostridium species concanitantly
dmgenaseis a spore-specificpmteinwhichis spomlation
cell forms.
of sporulation CIPof the mechanism(s) of dcmnamy or
However, dipicolinic
occurs in spomlating
heat resistant
mrphogenesis
The experiments described
of gluconate in sporulation
in a) reduction of the time required for the initiation
synthesis of morphologically
heat
of
recognizable spores; b) a bmad separation of
spore mrphogenesis fran heat resistance and c) a separation of the time of appemame of DPA from the occurrence of heat resistance.
'Journal Article University.
3691
Agriculture
Experiment Station,
691
Michigan State
Vol. 24, No. 5, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EXPERIMENTAL
These studies were conducted with -B. cereus grawn aerobically in 3 liter
volumes of modified G-mediumWashimto et - al -'
Brunswick fermntors. 5 liters transfers
The cultures were stirred
of air per min. of the culture
An active
at 30 C
1960) in New
at 450 rpm and aerated with
incculum was prepared by successive
in shake flasks of G-m&m
(Collier,
1957).
Identi-
cal inoculums were used for normal G-mediumand G-medim plus 0.2 per cent sodium gluconate.
The 3 litercultureswere
growth and sporulation
sampledatvaricustims
to ascertain the dry weight of cells/ml,
ml (Janssen -et al, 19581, refractile
spores, and heat resistant
should be pointed out that the ccqound ultimately in sane other functional
form in spores.
spores/ml were determined by direct a phase contrast microscope. crystal nutiient
violet.
Heat resistant
agar after
during
DPAcontent/ spores.
It
assayed as DPAmy exist
The refractile
CT phase bright
counts in a Petrwff-Hauser chamber using
Refractile
spores could not be stained with
spores/ml were determined by plating
on
heating suspensions at 80 C for 10 min.
2.5 2.0
i’
0.6
HEAT
.06
Figure 1.
STABLE
-o-
NG-
-.-
GO-MEDIUM
MEDIUM
The time course for the growth and sporulation of cultures of g. cereus in normal G-medium(NG) and G-mediumplus 0.2 per cent sodium gluconate (GG). Refractile spores are those which appear bright under the phase contrast microscope.
692
BIOCHEMICAL
Vol. 24, No. 5, 1966
Figurelshcws
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the growthme
forB.cereus inthetwomediaused. -did not affect the exponential growth of the
The presence of gluconate organism.
Refractile,
non-staining
scopically
in cultures
grown in gluconate
increased
exponentially
rnately 10g/ml.
for the increase development
became apparent micro-
medium at 9.5 hours, and they
in xxmber frun 107/ml to a final
Refnactile
13 hours, and the plot
spore structures
bodies in the wntrcl
of their
medium first
increasewasidenticalto
appeared at
the curve shawn
innumberofheatresistantspcres.
of heat resistant
ccunt of apprcxi-
The time cause
spares in gluwnate
and control
for the
media were
identical. Figure 2 presents the time course for the synthesis farrnation media.
of refractile
sp0res in cultures
The presence of gluwnate
for appearance of these by alxb
grown in gluconate
decreases the incubation fa
of DPA and for and control
time required
TWIXT.
DPA IN -o-o-
G G- Medium NG- Medium
SPORES
-z-X-
2
IO
Figure 2.
IN
GG- Medium NG-Medium
, 4 , , , , ( 14 “O”RIG 22
Correlation between the time of synthesis, of DPA and the appearrefractility and ance of refractile spores. Heat resistance, DPA synthesis occur sinultaneously in spores produced in normal G-medium (NG).
693
Vol. 24, No. 5, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCLESION The incorporation taining
of 0.2 per cent sodium gluconate
0.4 per cent glucose does not affect
--B. cereus nor the final that gluconate Gluconate
yield
of heat resistant
intermediate,
during
sporulation
spores.
have been demx&wated
cells
cultures
current
conditions,
to pyrzvate and
and pyruvate
These results
The differences particularly
of DPA,
occur within
differfromthose
and refractility
reported preceded thermal
might be attributed
the higher aeration
a very
to a variation
rate used in the
study.
Refrwtile
spores occurred four hours earlier
conate medium than control were the same. interval
medium but their
The principal
effect
The final
media were equal,
DPA content
grown in glu-
rates of synthesis
of gluconate.was
to shorten the time
growth and the appearance of spore
of spores frcm gluconate
and control
85 pg/mg dry spores.
The very close correspondence and DPA synthesis
to the conclusion
in cultures
respective
between the end of exponential
structures.
refractility
of acetate
spares, and heat resistance
by Hashimoto --et al (1960) where DPA synthesis
in cultural
as a key
and Nakata and Halvcxson (1960) and
of each other.
by 1.5 hours.
capacity,
grown in normal medium, the synthesis
the appearance of refractile short time interval
grcwth.
of g. cereus by Halvorson
Hanson --et al (1963) have shwn that the oxidation provides energy.for spore synthesis. In sporulating
vegetative
Enzymes for its oxidation
in spcsulating
Church (1957) and Doi --et al (1959);
growth rate of
These data suggest
in some control
or as an energy scurce.
G-mediwn con-
the exponential
is not used as an energy source during
may function
stability
into
between the percentage
in gluconate
that each refractile per
heat resistance
Furthermore,
70 per cent complete in the gluconate
spore
of
shown in Figure 2, leads
spore contains
of DPA. That is, DPA synthesis in that spore.
media,
completion
is canplete sporulation
its full
canplement
four hours preceding of the culture
is
medium before it begins in the control
694
Vol. 24, No. 5, 1966
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
medium. This very broad separation of the time of the Occurrence of DPAand heat stability species. resistance
has not been observed previously
Halvorson (1957) has shmn a separation of DPA synthesis and heat in an anaerobic spore former.
The over-all the initial Therefore,
in cultures of Bacillus
experimental results
stages of sporulation
indicate
but has little
that gluconate functions effect
later
in
in the process.
cultures grcwn in the gluconate rmzdim should be useful in study-
ing the sequence of events in sporulation, between the 0courrence of refractile
This investigation
in the time interval
spores and nature, heat stable spores.
was supported in part by research grant AI-01863
frcm the National Institute Health Service.
particularly
of Allergy
and Infectious
Diseases, U.S. Public
The author acknowledges the capable assistance of Ruth
McKay. REFERENCES
Bach, J.A. and H.L. Sadoff. J. Bacterial. 83, 699, (1962). Collier, R.E. In Spores, ed. H.O. Halvorson, Am. Inst. Biol. Sci., SymposiumSeries NO. 5, 1957, p. 10. Collier, R.E. and H.M. Nakata. Bacterial. Pmt., 1958, 42. Eay, L.E. and R.N. Costilow. J. Bacterial. z, 690, (1964). bi, R., H. Halvorson and B.D. Church. J. Bacterial. 77, 43, (1959). Halvorson, H. and B.D. Chum&. J. Appl. Bacterial. 20, 359, (1957). Halvorson, H.O. J. Appl. Bacterial. 20, 305, (1957). Hanson, R-S., V.R. Srinivasan and H.O. Halvorson. J. Bacterial. 86, 45, (1963). Hashinmto, T., S.H. Black and P. Gerhardt. Cm. J. Microbial. 6, 203, (1960). Janssen, F.W., A.J. Lund, and L.E. Anderson. Science 127, 26, T1958). Nakata, H.M. and H.O. Halvorson. J. Bacterial. &?$ 801, (1960).
695