Radiorespirometry was used to compare the primary pathways of glucose catabolism in 18 strains of Bacillus thuringiensis representing the 12 established serotypes. Every strain utilizes the Embden-Meyerhof-Pamas pathway almost exclusively; pentose-phosphate pathway participation is minor. The Embden- Meyerhof-Parnas pathway predominates regardless of whether the cells were grown in a minimal medium or one containing yeast extract. The results indicate that the absolute requirement for citrate and related compounds is not a result of defective citrate or glucose transport and metabolism

Bulla, Lee A., Jr.

Nickerson, Kenneth W.

St. Julian, Grant

English

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University of Nebraska - Lincoln 
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Papers in Microbiology Papers in the Biological Sciences 
July 1974 
Physiology of Sporeforming Bacteria Associated with Insects: 
Radiorespirometric Survey of Carbohydrate Metabolism In the 12 
Serotypes of Bacillus thuringiensis 
Kenneth W. Nickerson 
University of Nebraska-Lincoln, knickerson1@unl.edu 
Grant St. Julian 
Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 
Peoria, Illinois 
Lee A. Bulla Jr. 
Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 
Peoria, Illinois 
Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro 
 Part of the Microbiology Commons 
Nickerson, Kenneth W.; St. Julian, Grant ; and Bulla, Lee A. Jr., "Physiology of Sporeforming Bacteria 
Associated with Insects: Radiorespirometric Survey of Carbohydrate Metabolism In the 12 Serotypes of 
Bacillus thuringiensis" (1974). Papers in Microbiology. 48. 
https://digitalcommons.unl.edu/bioscimicro/48 
This Article is brought to you for free and open access by the Papers in the Biological Sciences at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an 
authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
APPLIED MICROBIOLOGY, JUly 1974, p. 129-132
Copyright 0 1974 American Society for Microbiology
Vol. 28, No. 1
Printed in USA.
Physiology of Sporeforming Bacteria Associated with Insects:
Radiorespirometric Survey of Carbohydrate Metabolism In the
12 Serotypes of Bacillus thuringiensis
KENNETH W. NICKERSON,I GRANT ST. JULIAN, AND LEE A. BULLA, JR.'
Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
Peoria, Illinois 61604
Received for publication 4 March 1974
Radiorespirometry was used to compare the primary pathways of glucose
catabolism in 18 strains of Bacillus thuringiensis representing the 12 established
serotypes. Every strain utilizes the Embden-Meyerhof-Pamas pathway almost
exclusively; pentose-phosphate pathway participation is minor. The Embden-
Meyerhof-Parnas pathway predominates regardless of whether the cells were
grown in a minimal medium or one containing yeast extract. The results indicate
that the absolute requirement for citrate and related compounds is not a result of
defective citrate or glucose transport and metabolism.
In an attempt to define the metabolic charac-
teristics of bacteria with insecticidal properties,
we previously compared Bacillus thuringiensis,
B. alvei, B. lentimorbus, and B. popilliae with
regard to their catabolism of glucose (3) and
oxidation of certain tricarboxylic acid cycle
intermediates (2). These studies were necessar-
ily restricted to single isolates of each species. In
the present investigation, we examined the
metabolic homogeneity of B. thuringiensis as a
group by using radiorespirometry to compare
the primary pathways of carbohydrate catabo-
lism in 18 natural isolates. The percent partici-
pation of the Embden-Meyerhof-Parnas (EMP)
and pentose-phosphate (PP) pathways was cal-
culated for each strain.
Extensive taxonomic studies have been con-
ducted on the numerous isolates of B. thuringi-
ensis (5, 13). A classification system dependent
on the flagellar H antigen has been devised, and
12 serotypes are now recognized (6). The extent
of the differences and similarities among these
serotypes is of interest. We have already ob-
served that the serotypes do not differ signifi-
cantly with regard to their nutritional require-
ments (10). Results presented in this communi-
cation demonstrate that the 18 strains of B.
thuringiensis also form a cohesive unit when
compared metabolically.
Apparently, B. thuringiensis is closely related
' Present address: Department of Plant Pathology, Univer-
sity of Nebraska, Lincoln, Neb. 68503.
2Present address: U.S. Grain Marketing Research Center,
1515 College Ave., Manhattan, Kan. 66502.
to Bacillus cereus (13). Whether this related-
ness derives from a single evolutionary diver-
gence to crystal-forming ability is not known.
Norris (12) showed that B. thuringiensis and B.
cereus possess phage in common, whereas Som-
erville and Jones (15) used nucleic acid hybridi-
zbtion to compare these two groups of bacilli.
Our metabolic data also reflect a close relation-
ship between B. thuringiensis and B. cereus.
In a separate series of experiments, we em-
ployed B. thuringiensis var. entomocidus as a
test organism to study glucose metabolism in
the defined minimal media described in the
previous paper (10). Particularly, we found that
when grown in a glucose-salts medium B. thu-
ringiensis exhibits an absolute requirement for
either citrate, aspartate, or glutamate; in con-
trast, a citrate-salts medium (4) was reported to
support growth in the absence of glucose. Be-
cause of this apparent paradox, we considered
the possibility that either glucose uptake or
metabolism was defective in our defined media
(10). For example, Romano and Kornberg (14)
found that glucose uptake was selectively inhib-
ited when Aspergillus nidulans was grown on an
acetate-containing medium. Alternatively, glu-
cose catabolism could be defective if our defined
media were deficient in thiamine, lipoic acid, or
another cofactor required for glycolysis. How-
ever, when glucose radiorespirometry was per-
formed on B. thuringiensis cells grown in the
defined media (10), the results differed only
slightly from those obtained when the cells were
grown in a medium containing yeast extract. B.
129
NICKERSON, ST. JULIAN, AND BULLA
thuringiensis does not require citrate because of
any defect in glucose uptake or metabolism.
(Presented in part at the 72nd Annual Meet-
ing of the American Society for Microbiology,
Philadelphia, 1972.)
MATERIALS AND METHODS
Organisms and cultural conditions. The strains
of B. thuringiensis listed in Table 1 were obtained
from the Agricultural Research Service Culture Col-
lection, Peoria, Ill. Vegetative cells suitable for radio-
respirometry were grown in GYS medium, which
consisted of 0.2% (NH4),SO4, 0.2% yeast extract, and
0.05% KHPO4 (pH 7.3). Aseptic addition of 0.1%
glucose, 0.02% MgSO4, 0.008% CaCl2 2H20, and
0.005% MnSO4 *H2O followed autoclaving. A uniform
spore inoculum (10) was put into 500 ml of GYS
medium contained in a Fernbach flask; the culture
was aerated by rotary agitation at 200 rpm. Cells were
harvested after 4 h of growth at 28 C.
In addition, vegetative cells of B. thuringiensis var.
entomocidus were cultured in the glucose-glutamate-
salts and glucose-aspartate-salts media described by
Nickerson and Bulla (10). Such cells were washed
twice in 0.003 M phosphate-salts medium after har-
vesting.
Radiorespirometry. The procedures used were
modified from those of Bulla et al. (2, 3). Respirome-
ter flasks containing about 60 mg of a vegetative cell
suspension were incubated at 28 C in a modified
Gilson differential respirometer. The cells were ac-
climated 30 min in 30 ml of glucose-free growth
medium to reduce endogenous metabolism. Carrier
glucose (1 mg) was added to each flask, and after an
additional 20 min of incubation 0.25 uCi of radioac-
tive glucose was added. D-Glucose specifically labeled
at C1, C3,4, and C6 was purchased from New
England Nuclear Corp., Boston, Mass. The flasks
were shaken while air was passed through at a rate of
60 cm3/min. Respired "4CO2 was trapped in 10 ml of a
mixture of absolute ethanol and monoethanolamine
(2:1, vol/vol) that was removed and replenished at
half-hour intervals. The trapping solutions then were
adjusted to 15 ml with absolute ethanol, and a 5-ml
portion was mixed with 10 ml of toluene containing 6
mg of 2,5-diphenyloxazole per ml and 0.1 mg of
1, 4-bis- [2]-(5-phenyloxazolyl)benzene per ml. The
mixtures were placed in scintillation vials, and a
liquid scintillation spectrometer was used to measure
radioactivity.
At the end of each experiment, the cells were
separated by centrifugation at 4 C, homogenized in
NCS solubilizer (Amersham/Searle Corp., Des
Plaines, Ill.) for 36 h at 45 C, and counted in a
scintillation fluid containing: toluene/2, 5-diphenyl-
oxazole/1, 4-bis- [2]-(5-phenyloxazoly) benzene: Triton
X-100: ethanol (8:4:3; vol/vol/vol). This scintillation
mixture was also used to count 1-ml samples of the
cell-free supernatant following centrifugation.
Estimation of pathway participation. A simplifi-
cation of the procedure of Wang (16) was used to
estimate participation of the EMP and PP pathways.
The following equation shows how the fraction of
glucose catabolized via the PP pathway (Ga) was
estimated from the yields of "4CO, recovered from
D-[1-"C]glucose (GD) and D-[6-"4C]glucose (G.):
TABLE 1. Glucose catabolism in B. thuringiensis
ARS culture Recovery as "4CO, (%)b Pathway
collection H antigen participation (%)nollectlo ariety name serotype
no.a C-1 C-3,4 C-6 EMP PP
B-4039 berliner 1 5.2 60.9 1.7 96.5 3.5
B-4040 finitimus 2 3.6 75.5 4.3 100 0
B-4041 alesti 3a 2.6 57.7 3.1 100 0
B-4055 kurstaki 3a3b 5.9 32.8 2.3 96.4 3.6
B-4042 sotto 4a4b 3.2 55.4 0.6 97.6 2.4
B-4043 dendrolimus 4a4b 9.5 58.4 2.5 93 7
B-4044 kenyae 4a4c 3.4 45.2 2.9 99.5 0.5
B-4045 galleriae 5a5b 3.1 42.2 2.5 99.4 0.6
B-4056 canadensis 5a5c 1.9 43.4 2.4 100 0
B-4046 entomocidus 6 1.2 43.4 3.1 100 0
B-4047 entomocidus-limassol 6 3.4 70.2 2.2 98.8 1.2
B-4057 subtoxicus 6 2.5 82.1 3.2 100 0
B-4048 aizawai 7 6.3 44.9 5.2 98.9 1.1
B-4049 morrisoni 8 1.8 65.5 2.4 100 0
B-4050 tolworthi 9 3.6 37.0 2.5 98.9 1.1
B-4058 darmstadiensis 10 1.1 83.8 4.4 100 0
B-4059 toumanoffi 11 1.9 52.8 2.8 100 0
B-4060 thompsoni 12 2.5 64.1 2.4 100 0
U.S. Department ofa ARS, Agricultural Research Service, Northern Regional Research Laboratory,
Agriculture, Peoria, Ill.
Averages of triplicate experiments.
130 APPL. MICROBIOL.
CARBOHYDRATE METABOLISM IN B. THURINGIENSIS
G -G-Gp=_
1- G6 50 - 60
The fraction of glucose catabolized via the EMP4
pathway (GE) was calculated by difference: GE = 1 - /
Gp. The "CO2 yields from specifically labeled D- O 40 / E 20
glucose were calculated as follows: (i) interval "4CO2 a.?
recoveries as counts per minute were summed (most
experiments entailed three half-hour intervals); (ii)
interval recoveries were multiplied by three because @ 30
only 5 ml of trapping solution was counted; (iii) total /
counts per minute were converted to disintegrations l
per minute by dividing by the overall counting
efficiency (approximately 85%); (iv) the percent yield X 20
of "4CO2 then was derived by dividing the total\
disintegrations per minute by 555,000 (the number of = - -_
disintegrations per minute in 0.25 yCi of radioactive 010
glucose used in these experiments) and multiplying
by 100. .*
This simplified procedure is valid because of the
tremendously high yield of "CO2 from D- [3,4- 0
"4C]glucose (Table 1) and because tricarboxylic acid 1 2
cycle activity is minimal during vegetative growth of Time (hr.)
B. thuringiensis in GYS medium (2, 3). A similar
phenomenon of diminished tricarboxylic acid cycle FIG. 1. Aerobic radiorespirom
activity is true for several other bacilli (1, 7, 8, 9). If cose utilization by vegetative cei
the tricarboxylic acid cycle were active, increased var. entomocidus grown in a gIl
14CO2 yields from D-[1-"4C]glucose and D-[6- medium (10). C1, CS.,4, and C. C
'4C]glucose would hinder estimation of G, by this labeled carbon atoms. One-half
method (1). cally labeled substrate (0.25 AC
reaction flask. Final volume per
RESULTS AND DISCUSSION
Eighteen strains of B. thuringiensis, including the
12 recognized serotypes (6), have been surveyed with X 30
regard to their pathways of glucose catabolism. The _ Q
"CO2 recovery and pathway participation percent- x 20
ages are listed in Table 1. The high yields of"=CO2 1
from D- [3,4- ]4Cglucose indicate that the EMP path- Q 1
way is predominant in every strain tested. With one
exception (B. thuringiensis var. dendrolimus), 96 to
100% of the glucose is catabolized via the EMP ° 30
pathway. As such, B. thuringiensis represents a cohe- *
sive group by the criterion of metabolic homogeneity. C
The observed small differences in PP pathway partici- 20/%
pation are as great within a single serotype as between
serotypes. , -
In addition to substantiating the unity of B. thurin- / \
giensis as a group, the uniform dominance of the EMP W 10 _/ -_
pathway points out the relatedness of B. thuringiensis X / ,.......
to B. cereus. Previous radiorespirometric studies dem- '
onstrated that, whereas 98% of glucose catabolism is .D I
via the EMP pathway in B. cereus (8), only 65% is 2
routed that way in B. subtilis (7, 17). Time (hr.)
Figures 1 and 2 show the aerobic radiorespirometric FIG. 2. Aerobic radiorespiron
patterns of glucose utilization by B. thuringiensis var. cose utilization by vegetative ce
entomocidus cells grown in glucose-glutamate-salts var. entomocidus grown in a gi
and glucose-aspartate-salts media, respectively. The medium (10). C1, C3.4, and C6
rapid appearance of 4C02 from D- [3,4-14C ]glucose is labeled carbon atoms. One-half
indicative of a functional EMP pathway and, more cally labeled substrate (0.25 uC
specifically, demonstrates pyruvate dehydrogenase reaction flask. Final volume per
3 4
Metric patterns of glu-
!lls of B. thuringiensis
ucose-glutamate-salts
designate specifically
milligram of specifi-
,i) was added to each
flask was 30 ml.
3 4
netric patterns of glu-
Ills of B. thuringiensis
lucose-aspartate-salts
designate specifically
f milligram of specifi-
"i) was added to each
r flask was 30 ml.
131VOL. 28, 1974
NICKERSON, ST. JULIAN, AND BULLA
(pyruvate: lipoate oxidoreductase, EC 1.2.4.1) activ-
ity. The high yield of "CO2 from D-[6- 4C]glucose
(Fig. 2) indicates that B. thuringiensis var. en-
tomocidus possesses an intact tricarboxylic acid cycle
(1) when grown in the glucose-aspartate-salts me-
dium. This observation confirms previous radiorespi-
rometric work with radioactive glutamate (2, 3) indi-
cating the intactness of the tricarboxylic acid cycle.
Taken together, these results demonstrate that the
absolute requirement shown by B. thuringiensis for
citrate or a related compound (10) is not caused by a
defect in either citrate or glucose metabolism or in
glucose uptake. We have shown in the accompanying
paper (10) that the requirement for citrate cannot be
replaced by biotin, thiamine, lipoic acid, or any other
vitamin tested.
It is known that in other bacilli glucose and
glutamate are required together to repress enzymes of
the tricarboxylic acid cycle (9). Our results are
consistent with this view because tricarboxylic acid
cycle activity (based on 4CO2 evolution from [6-
"4C glucose during the first 2 h of incubation) is
absent in vegetative cells grown in either glucose-
glutamate-salts (Fig. 1) or GYS media, but is present
in those cells grown in the glucose-aspartate-salts
medium (Fig. 2). We have no explanation for the
higher 14CO2 yield from D-[6- 4C]glucose than from
D-[1-_4C]glucose in Fig. 2, except that D-glucose may
be rapidly converted to uridine 5'-diphosphate glu-
cose and subsequently to uridine 5'-diphosphate-
galacturonic acid, which in turn undergoes oxidative
decarboxylation to give the observed order of 14CO2
evolution. However, because the tricarboxylic acid
cycle is inactive in the glucose-glutamate-salts-grown
cells we can calculate from the data in Fig. 1 that the
percent contribution of the PP pathway is 5%. This
slight PP activity probably supports biosynthetic
needs. Table 1 shows that glucose catabolism was
100% via the EMP pathway when the cells were grown
in GYS medium, wherein biosynthetic needs are
minimal.
Though enzyme assays demonstrate the simultane-
ous presence in cell-free extracts of the EMP and PP
pathways (L. A. Bulla et al., unpublished data), we
have used radiorespirometry to ascertain that the
EMP pathway is the dominant energy-generating
system in all strains of B. thuringiensis tested.
ACKNOWLEDGMENT
K.W.N. is the recipient of a postdoctoral resident research
associateship established by the Agricultural Research Serv-
ice, U.S. Department of Agriculture, in association with the
National Academy of Sciences-National Research Council,
1971-1973.
LITERATURE CITED
1. Blumenthal, H. J. 1965. Changes in carbohydrate metabo-
lism of Bacillus cereus spores during the return to
vegetative growth, p. 222-236. In L. L. Campbell and
H. 0. Halvorson (ed.), Spores III. American Society for
Microbiology, Ann Arbor, Mich.
2. Bulla, L. A., G. St. Julian, and R. A. Rhodes. 1971.
Physiology of sporeforming bacteria associated with
insects. III. Radiorespirometry of pyruvate, acetate,
succinate, and glutamate oxidation. Can. J. Microbiol.
17:1073-1079.
3. Bulla, L. A., G. St. Julian, R. A. Rhodes, and C. W.
Hesseltine. 1970. Physiology of sporeforming bacteria
associated with insects. I. Glucose catabolism in vege-
tative cells. Can. J. Microbiol. 16:243-248.
4. Conner, R. M., and P. A. Hansen. 1967. Effects of valine,
leucine, and isoleucine on the growth of Bacillus
thuringiensis and related bacteria. J. Invertebr. Pathol.
9: 12-18.
5. de Barjac, H., and A. Bonnefoi. 1968. A classification of
strains of Bacillus thuringiensis berliner with a key to
their differentiation. J. Invertebr. Pathol. 11:335-347.
6. de Barjac, H., and A. Bonnefoi. 1973. Mise au point sur la
classification des Bacillus thuringiensis. Entomophaga
18:5-17.
7. Goldman, M., and H. J. Blumenthal. 1963. Pathways of
glucose catabolism in Bacillus subtilis. J. Bacteriol.
86:303-311.
8. Goldman, M., and H. J. Blumenthal. 1964. Pathways of
glucose catabolism in Bacillus cereus. J. Bacteriol.
87:377-386.
9. Hanson, R. S., J. Blicharska, and J. Szulmajster. 1964.
Relationship between the tricarboxylic acid cycle en-
zymes and sporulation of B. subtilis. Biochem. Bio-
phys. Res. Commun. 17:1-7.
10. Nickerson, K. W., and L. A. Bulla. 1974. Physiology of
sporeforming bacteria associated with insects: minimal
nutritional requirements for growth, sporulation, and
parasporal crystal formation in Bacillus thuringiensis.
Appl. Microbiol. 28:124-128.
11. Nickerson, K. W., J. DePinto, and L. A. Bulla. 1974.
Sporulation of Bacillus thuringiensis without concur-
rent derepression of the tricarboxylic acid cycle. J.
Bacteriol. 117:321-323.
12. Norris, J. R. 1963. Bacterial insecticides. Sci. Progr.
(London) 51:188-197.
13. Rogoff, M. H., and A. A. Yousten. 1969. Bacillus thurin-
giensis: microbiological considerations. Annu. Rev.
Microbiol. 23:357-386.
14. Romano, A. H., and H. L. Kornberg. 1968. Regulation of
sugar utilization by Aspergillus nidulans. Biochim.
Biophys. Acta 158:491-493.
15. Somerville, H. J., and M. L. Jones. 1972. DNA competi-
tion studies within the Bacillus cereus group of bacilli.
J. Gen. Microbiol. 73:257-265.
16. Wang, C. H. 1972. Radiorespirometric methods, p.
185-230. In J. R. Norris and D. W. Ribbons (ed.),
Methods in microbiology, vol. 6B. Academic Press Inc.,
London.
17. Wang, C. H., and J. K. Krackov. 1962. The catabolic fate
of glucose in Bacillus subtilis. J. Biol. Chem.
237:3614-3622.
132 APPL. MICROBIOL.


Physiology of Sporeforming Bacteria Associated with Insects: Radiorespirometric Survey of Carbohydrate Metabolism In the 12 Serotypes of \u3ci\u3eBacillus thuringiensis\u3c/i\u3e

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Microbiology Papers in the Biological Sciences July 1974 Physiology of Sporeforming Bacteria Associated with Insects: Radiorespirometric Survey of Carbohydrate Metabolism In the 12 Serotypes of Bacillus thuringiensis Kenneth W. Nickerson University of Nebraska-Lincoln, knickerson1@unl.edu Grant St. Julian Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois Lee A. Bulla Jr. Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro  Part of the Microbiology Commons Nickerson, Kenneth W.; St. Julian, Grant ; and Bulla, Lee A. Jr., "Physiology of Sporeforming Bacteria Associated with Insects: Radiorespirometric Survey of Carbohydrate Metabolism In the 12 Serotypes of Bacillus thuringiensis" (1974). Papers in Microbiology. 48. https://digitalcommons.unl.edu/bioscimicro/48 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. APPLIED MICROBIOLOGY, JUly 1974, p. 129-132Copyright 0 1974 American Society for MicrobiologyVol. 28, No. 1Printed in USA.Physiology of Sporeforming Bacteria Associated with Insects:Radiorespirometric Survey of Carbohydrate Metabolism In the12 Serotypes of Bacillus thuringiensisKENNETH W. NICKERSON,I GRANT ST. JULIAN, AND LEE A. BULLA, JR.'Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,Peoria, Illinois 61604Received for publication 4 March 1974Radiorespirometry was used to compare the primary pathways of glucosecatabolism in 18 strains of Bacillus thuringiensis representing the 12 establishedserotypes. Every strain utilizes the Embden-Meyerhof-Pamas pathway almostexclusively; pentose-phosphate pathway participation is minor. The Embden-Meyerhof-Parnas pathway predominates regardless of whether the cells weregrown in a minimal medium or one containing yeast extract. The results indicatethat the absolute requirement for citrate and related compounds is not a result ofdefective citrate or glucose transport and metabolism.In an attempt to define the metabolic charac-teristics of bacteria with insecticidal properties,we previously compared Bacillus thuringiensis,B. alvei, B. lentimorbus, and B. popilliae withregard to their catabolism of glucose (3) andoxidation of certain tricarboxylic acid cycleintermediates (2). These studies were necessar-ily restricted to single isolates of each species. Inthe present investigation, we examined themetabolic homogeneity of B. thuringiensis as agroup by using radiorespirometry to comparethe primary pathways of carbohydrate catabo-lism in 18 natural isolates. The percent partici-pation of the Embden-Meyerhof-Parnas (EMP)and pentose-phosphate (PP) pathways was cal-culated for each strain.Extensive taxonomic studies have been con-ducted on the numerous isolates of B. thuringi-ensis (5, 13). A classification system dependenton the flagellar H antigen has been devised, and12 serotypes are now recognized (6). The extentof the differences and similarities among theseserotypes is of interest. We have already ob-served that the serotypes do not differ signifi-cantly with regard to their nutritional require-ments (10). Results presented in this communi-cation demonstrate that the 18 strains of B.thuringiensis also form a cohesive unit whencompared metabolically.Apparently, B. thuringiensis is closely related' Present address: Department of Plant Pathology, Univer-sity of Nebraska, Lincoln, Neb. 68503.2Present address: U.S. Grain Marketing Research Center,1515 College Ave., Manhattan, Kan. 66502.to Bacillus cereus (13). Whether this related-ness derives from a single evolutionary diver-gence to crystal-forming ability is not known.Norris (12) showed that B. thuringiensis and B.cereus possess phage in common, whereas Som-erville and Jones (15) used nucleic acid hybridi-zbtion to compare these two groups of bacilli.Our metabolic data also reflect a close relation-ship between B. thuringiensis and B. cereus.In a separate series of experiments, we em-ployed B. thuringiensis var. entomocidus as atest organism to study glucose metabolism inthe defined minimal media described in theprevious paper (10). Particularly, we found thatwhen grown in a glucose-salts medium B. thu-ringiensis exhibits an absolute requirement foreither citrate, aspartate, or glutamate; in con-trast, a citrate-salts medium (4) was reported tosupport growth in the absence of glucose. Be-cause of this apparent paradox, we consideredthe possibility that either glucose uptake ormetabolism was defective in our defined media(10). For example, Romano and Kornberg (14)found that glucose uptake was selectively inhib-ited when Aspergillus nidulans was grown on anacetate-containing medium. Alternatively, glu-cose catabolism could be defective if our definedmedia were deficient in thiamine, lipoic acid, oranother cofactor required for glycolysis. How-ever, when glucose radiorespirometry was per-formed on B. thuringiensis cells grown in thedefined media (10), the results differed onlyslightly from those obtained when the cells weregrown in a medium containing yeast extract. B.129NICKERSON, ST. JULIAN, AND BULLAthuringiensis does not require citrate because ofany defect in glucose uptake or metabolism.(Presented in part at the 72nd Annual Meet-ing of the American Society for Microbiology,Philadelphia, 1972.)MATERIALS AND METHODSOrganisms and cultural conditions. The strainsof B. thuringiensis listed in Table 1 were obtainedfrom the Agricultural Research Service Culture Col-lection, Peoria, Ill. Vegetative cells suitable for radio-respirometry were grown in GYS medium, whichconsisted of 0.2% (NH4),SO4, 0.2% yeast extract, and0.05% KHPO4 (pH 7.3). Aseptic addition of 0.1%glucose, 0.02% MgSO4, 0.008% CaCl2 2H20, and0.005% MnSO4 *H2O followed autoclaving. A uniformspore inoculum (10) was put into 500 ml of GYSmedium contained in a Fernbach flask; the culturewas aerated by rotary agitation at 200 rpm. Cells wereharvested after 4 h of growth at 28 C.In addition, vegetative cells of B. thuringiensis var.entomocidus were cultured in the glucose-glutamate-salts and glucose-aspartate-salts media described byNickerson and Bulla (10). Such cells were washedtwice in 0.003 M phosphate-salts medium after har-vesting.Radiorespirometry. The procedures used weremodified from those of Bulla et al. (2, 3). Respirome-ter flasks containing about 60 mg of a vegetative cellsuspension were incubated at 28 C in a modifiedGilson differential respirometer. The cells were ac-climated 30 min in 30 ml of glucose-free growthmedium to reduce endogenous metabolism. Carrierglucose (1 mg) was added to each flask, and after anadditional 20 min of incubation 0.25 uCi of radioac-tive glucose was added. D-Glucose specifically labeledat C1, C3,4, and C6 was purchased from NewEngland Nuclear Corp., Boston, Mass. The flaskswere shaken while air was passed through at a rate of60 cm3/min. Respired "4CO2 was trapped in 10 ml of amixture of absolute ethanol and monoethanolamine(2:1, vol/vol) that was removed and replenished athalf-hour intervals. The trapping solutions then wereadjusted to 15 ml with absolute ethanol, and a 5-mlportion was mixed with 10 ml of toluene containing 6mg of 2,5-diphenyloxazole per ml and 0.1 mg of1, 4-bis- [2]-(5-phenyloxazolyl)benzene per ml. Themixtures were placed in scintillation vials, and aliquid scintillation spectrometer was used to measureradioactivity.At the end of each experiment, the cells wereseparated by centrifugation at 4 C, homogenized inNCS solubilizer (Amersham/Searle Corp., DesPlaines, Ill.) for 36 h at 45 C, and counted in ascintillation fluid containing: toluene/2, 5-diphenyl-oxazole/1, 4-bis- [2]-(5-phenyloxazoly) benzene: TritonX-100: ethanol (8:4:3; vol/vol/vol). This scintillationmixture was also used to count 1-ml samples of thecell-free supernatant following centrifugation.Estimation of pathway participation. A simplifi-cation of the procedure of Wang (16) was used toestimate participation of the EMP and PP pathways.The following equation shows how the fraction ofglucose catabolized via the PP pathway (Ga) wasestimated from the yields of "4CO, recovered fromD-[1-"C]glucose (GD) and D-[6-"4C]glucose (G.):TABLE 1. Glucose catabolism in B. thuringiensisARS culture Recovery as "4CO, (%)b Pathwaycollection H antigen participation (%)nollectlo ariety name serotypeno.a C-1 C-3,4 C-6 EMP PPB-4039 berliner 1 5.2 60.9 1.7 96.5 3.5B-4040 finitimus 2 3.6 75.5 4.3 100 0B-4041 alesti 3a 2.6 57.7 3.1 100 0B-4055 kurstaki 3a3b 5.9 32.8 2.3 96.4 3.6B-4042 sotto 4a4b 3.2 55.4 0.6 97.6 2.4B-4043 dendrolimus 4a4b 9.5 58.4 2.5 93 7B-4044 kenyae 4a4c 3.4 45.2 2.9 99.5 0.5B-4045 galleriae 5a5b 3.1 42.2 2.5 99.4 0.6B-4056 canadensis 5a5c 1.9 43.4 2.4 100 0B-4046 entomocidus 6 1.2 43.4 3.1 100 0B-4047 entomocidus-limassol 6 3.4 70.2 2.2 98.8 1.2B-4057 subtoxicus 6 2.5 82.1 3.2 100 0B-4048 aizawai 7 6.3 44.9 5.2 98.9 1.1B-4049 morrisoni 8 1.8 65.5 2.4 100 0B-4050 tolworthi 9 3.6 37.0 2.5 98.9 1.1B-4058 darmstadiensis 10 1.1 83.8 4.4 100 0B-4059 toumanoffi 11 1.9 52.8 2.8 100 0B-4060 thompsoni 12 2.5 64.1 2.4 100 0U.S. Department ofa ARS, Agricultural Research Service, Northern Regional Research Laboratory,Agriculture, Peoria, Ill.Averages of triplicate experiments.130 APPL. MICROBIOL.CARBOHYDRATE METABOLISM IN B. THURINGIENSISG -G-Gp=_1- G6 50 - 60The fraction of glucose catabolized via the EMP4pathway (GE) was calculated by difference: GE = 1 - /Gp. The "CO2 yields from specifically labeled D- O 40 / E 20glucose were calculated as follows: (i) interval "4CO2 a.?recoveries as counts per minute were summed (mostexperiments entailed three half-hour intervals); (ii)interval recoveries were multiplied by three because @ 30only 5 ml of trapping solution was counted; (iii) total /counts per minute were converted to disintegrations lper minute by dividing by the overall countingefficiency (approximately 85%); (iv) the percent yield X 20of "4CO2 then was derived by dividing the total\disintegrations per minute by 555,000 (the number of = - -_disintegrations per minute in 0.25 yCi of radioactive 010glucose used in these experiments) and multiplyingby 100. .*This simplified procedure is valid because of thetremendously high yield of "CO2 from D- [3,4- 0"4C]glucose (Table 1) and because tricarboxylic acid 1 2cycle activity is minimal during vegetative growth of Time (hr.)B. thuringiensis in GYS medium (2, 3). A similarphenomenon of diminished tricarboxylic acid cycle FIG. 1. Aerobic radiorespiromactivity is true for several other bacilli (1, 7, 8, 9). If cose utilization by vegetative ceithe tricarboxylic acid cycle were active, increased var. entomocidus grown in a gIl14CO2 yields from D-[1-"4C]glucose and D-[6- medium (10). C1, CS.,4, and C. C'4C]glucose would hinder estimation of G, by this labeled carbon atoms. One-halfmethod (1). cally labeled substrate (0.25 ACreaction flask. Final volume perRESULTS AND DISCUSSIONEighteen strains of B. thuringiensis, including the12 recognized serotypes (6), have been surveyed with X 30regard to their pathways of glucose catabolism. The _ Q"CO2 recovery and pathway participation percent- x 20ages are listed in Table 1. The high yields of"=CO2 1from D- [3,4- ]4Cglucose indicate that the EMP path- Q 1way is predominant in every strain tested. With oneexception (B. thuringiensis var. dendrolimus), 96 to100% of the glucose is catabolized via the EMP ° 30pathway. As such, B. thuringiensis represents a cohe- *sive group by the criterion of metabolic homogeneity. CThe observed small differences in PP pathway partici- 20/%pation are as great within a single serotype as betweenserotypes. , -In addition to substantiating the unity of B. thurin- / \giensis as a group, the uniform dominance of the EMP W 10 _/ -_pathway points out the relatedness of B. thuringiensis X / ,.......to B. cereus. Previous radiorespirometric studies dem- 'onstrated that, whereas 98% of glucose catabolism is .D Ivia the EMP pathway in B. cereus (8), only 65% is 2routed that way in B. subtilis (7, 17). Time (hr.)Figures 1 and 2 show the aerobic radiorespirometric FIG. 2. Aerobic radiorespironpatterns of glucose utilization by B. thuringiensis var. cose utilization by vegetative ceentomocidus cells grown in glucose-glutamate-salts var. entomocidus grown in a giand glucose-aspartate-salts media, respectively. The medium (10). C1, C3.4, and C6rapid appearance of 4C02 from D- [3,4-14C ]glucose is labeled carbon atoms. One-halfindicative of a functional EMP pathway and, more cally labeled substrate (0.25 uCspecifically, demonstrates pyruvate dehydrogenase reaction flask. Final volume per3 4Metric patterns of glu-!lls of B. thuringiensisucose-glutamate-saltsdesignate specificallymilligram of specifi-,i) was added to eachflask was 30 ml.3 4netric patterns of glu-Ills of B. thuringiensislucose-aspartate-saltsdesignate specificallyf milligram of specifi-"i) was added to eachr flask was 30 ml.131VOL. 28, 1974NICKERSON, ST. JULIAN, AND BULLA(pyruvate: lipoate oxidoreductase, EC 1.2.4.1) activ-ity. The high yield of "CO2 from D-[6- 4C]glucose(Fig. 2) indicates that B. thuringiensis var. en-tomocidus possesses an intact tricarboxylic acid cycle(1) when grown in the glucose-aspartate-salts me-dium. This observation confirms previous radiorespi-rometric work with radioactive glutamate (2, 3) indi-cating the intactness of the tricarboxylic acid cycle.Taken together, these results demonstrate that theabsolute requirement shown by B. thuringiensis forcitrate or a related compound (10) is not caused by adefect in either citrate or glucose metabolism or inglucose uptake. We have shown in the accompanyingpaper (10) that the requirement for citrate cannot bereplaced by biotin, thiamine, lipoic acid, or any othervitamin tested.It is known that in other bacilli glucose andglutamate are required together to repress enzymes ofthe tricarboxylic acid cycle (9). Our results areconsistent with this view because tricarboxylic acidcycle activity (based on 4CO2 evolution from [6-"4C glucose during the first 2 h of incubation) isabsent in vegetative cells grown in either glucose-glutamate-salts (Fig. 1) or GYS media, but is presentin those cells grown in the glucose-aspartate-saltsmedium (Fig. 2). We have no explanation for thehigher 14CO2 yield from D-[6- 4C]glucose than fromD-[1-_4C]glucose in Fig. 2, except that D-glucose maybe rapidly converted to uridine 5'-diphosphate glu-cose and subsequently to uridine 5'-diphosphate-galacturonic acid, which in turn undergoes oxidativedecarboxylation to give the observed order of 14CO2evolution. However, because the tricarboxylic acidcycle is inactive in the glucose-glutamate-salts-growncells we can calculate from the data in Fig. 1 that thepercent contribution of the PP pathway is 5%. Thisslight PP activity probably supports biosyntheticneeds. Table 1 shows that glucose catabolism was100% via the EMP pathway when the cells were grownin GYS medium, wherein biosynthetic needs areminimal.Though enzyme assays demonstrate the simultane-ous presence in cell-free extracts of the EMP and PPpathways (L. A. Bulla et al., unpublished data), wehave used radiorespirometry to ascertain that theEMP pathway is the dominant energy-generatingsystem in all strains of B. thuringiensis tested.ACKNOWLEDGMENTK.W.N. is the recipient of a postdoctoral resident researchassociateship established by the Agricultural Research Serv-ice, U.S. Department of Agriculture, in association with theNational Academy of Sciences-National Research Council,1971-1973.LITERATURE CITED1. Blumenthal, H. J. 1965. Changes in carbohydrate metabo-lism of Bacillus cereus spores during the return tovegetative growth, p. 222-236. In L. L. Campbell andH. 0. Halvorson (ed.), Spores III. American Society forMicrobiology, Ann Arbor, Mich.2. Bulla, L. A., G. St. Julian, and R. A. Rhodes. 1971.Physiology of sporeforming bacteria associated withinsects. III. Radiorespirometry of pyruvate, acetate,succinate, and glutamate oxidation. Can. J. Microbiol.17:1073-1079.3. Bulla, L. A., G. St. Julian, R. A. Rhodes, and C. W.Hesseltine. 1970. Physiology of sporeforming bacteriaassociated with insects. I. Glucose catabolism in vege-tative cells. Can. J. Microbiol. 16:243-248.4. Conner, R. M., and P. A. Hansen. 1967. Effects of valine,leucine, and isoleucine on the growth of Bacillusthuringiensis and related bacteria. J. Invertebr. Pathol.9: 12-18.5. de Barjac, H., and A. Bonnefoi. 1968. A classification ofstrains of Bacillus thuringiensis berliner with a key totheir differentiation. J. Invertebr. Pathol. 11:335-347.6. de Barjac, H., and A. Bonnefoi. 1973. Mise au point sur laclassification des Bacillus thuringiensis. Entomophaga18:5-17.7. Goldman, M., and H. J. Blumenthal. 1963. Pathways ofglucose catabolism in Bacillus subtilis. J. Bacteriol.86:303-311.8. Goldman, M., and H. J. Blumenthal. 1964. Pathways ofglucose catabolism in Bacillus cereus. J. Bacteriol.87:377-386.9. Hanson, R. S., J. Blicharska, and J. Szulmajster. 1964.Relationship between the tricarboxylic acid cycle en-zymes and sporulation of B. subtilis. Biochem. Bio-phys. Res. Commun. 17:1-7.10. Nickerson, K. W., and L. A. Bulla. 1974. Physiology ofsporeforming bacteria associated with insects: minimalnutritional requirements for growth, sporulation, andparasporal crystal formation in Bacillus thuringiensis.Appl. Microbiol. 28:124-128.11. Nickerson, K. W., J. DePinto, and L. A. Bulla. 1974.Sporulation of Bacillus thuringiensis without concur-rent derepression of the tricarboxylic acid cycle. J.Bacteriol. 117:321-323.12. Norris, J. R. 1963. Bacterial insecticides. Sci. Progr.(London) 51:188-197.13. Rogoff, M. H., and A. A. Yousten. 1969. Bacillus thurin-giensis: microbiological considerations. Annu. Rev.Microbiol. 23:357-386.14. Romano, A. H., and H. L. Kornberg. 1968. Regulation ofsugar utilization by Aspergillus nidulans. Biochim.Biophys. Acta 158:491-493.15. Somerville, H. J., and M. L. Jones. 1972. DNA competi-tion studies within the Bacillus cereus group of bacilli.J. Gen. Microbiol. 73:257-265.16. Wang, C. H. 1972. Radiorespirometric methods, p.185-230. In J. R. Norris and D. W. Ribbons (ed.),Methods in microbiology, vol. 6B. Academic Press Inc.,London.17. Wang, C. H., and J. K. Krackov. 1962. The catabolic fateof glucose in Bacillus subtilis. J. Biol. Chem.237:3614-3622.132 APPL. MICROBIOL.

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Physiology of Sporeforming Bacteria Associated with Insects: Radiorespirometric Survey of Carbohydrate Metabolism In the 12 Serotypes of \u3ci\u3eBacillus thuringiensis\u3c/i\u3e

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