.........

.......
------
--------
-.-..

...........

THE EFFECT or GLUCOSE AND swam oN-me 2 "  .._ __ _
smmmm or A FASTIDIOUS BAchws SPEClES ._ ‘ .5 .1 ;. . '

Thesis tor the Degree- of M; S.

MICHEGAN STME unwassm

mm PHEUP POPGVEC ’
1972‘

 

WWI!!!”WWWf

31 293(01733443BEM , LIBR/‘VY

       

 

 

   

 

Michigan 3 to
University

 

This is to certify that the
thesis entitled
The Effect of Glucose and Salicin
on the Sporulation of a Fastidious Bacillus species

presented by

John Philip Popovec

has been accepted towards fulfillment
of the requirements for

M.S . Microbiology and

Public Health

Major professor

degree in

Date September 6, 1972

 

0-169

 

 

 

328QEE-0 92002

 

 

 

 

ABSTRACT

THE EFFECT OF GLUCOSE AND SALICIN ON THE
SPORULATION OF A FASTIDIOUS
BACILLUS SPECIES

BY

John Philip POpovec

An unidentified Bacillus species was found to rapidly
sporulate when growth on Salicin as the major carbon and
energy source. The growth rate was restricted below that in
a glucose supplemented medium in which sporulation was
strongly repressed. Very low levels of glucose completely
repressed sporulation whereas salicin levels remained in
great excess when maximum spore yields were achieved.
Salicin was utilized by sporulating cells and was required
as an exogenous energy source for maximum sporulation. An
examination of growth characteristics in both glucose and
salicin media showed that volatile acid was produced in
both instances but only in the salicin medium was the acid
utilized and the pH observed to increase. Furthermore,
only sporulating cells exhibited a maximally induced citric
acid cycle capable of oxidizing acetate at a maximum rate.

Preliminary data indicate that acetate was accumulated in

John Philip Popovec

glucose medium and that glucose grown cells were unable to
significantly oxidize this acid. Respiration studies per-
formed on resting cells showed that salicin was catabolized
much slower than glucose but was more completely oxidized.
The oxidation of salicin was found to be inducible and
energy dependent in resting glucose grown cells. Additional
sporulation studies revealed that the uptake of calcium
preceded the synthesis of dipicolinic acid and the acquisi-

tion of heat resistance by about 4 hr.

THE EFFECT OF GLUCOSE AND SALICIN ON THE
SPORULATION OF A FASTIDIOUS

BACILLUS SPECIES

BY

John Philip Popovec

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1972

DEDICATION

I dedicate this thesis to my wife, Alberta, for
her love, understanding, and patience throughout the course
of my graduate education and to my daughters, Pamela and
Jennifer, for providing the spark of laughter at many

discouraging times.

ii

‘E

 

ACKNOWLEDGMENTS

I wish to express my sincere gratitude to
Dr. R. N. Costilow for his guidance and encouragement
throughout this investigation and during the preparation
of this thesis.

I also wish to express my sincere thanks to
Dr. H. L. Sadoff and Dr. R. R. Brubaker for the use of

their laboratory facilities and equipment.

iii

TABLE OF CONTENTS

Page
DEDICATION . . . . . . . . . . .
ACKNOWLEDGMENTS . . . . . . . . . . . . . iii
LIST OF TABLES . . . . . . . . . . . . . V

LIST OF FIGURES . . . . . . . . . . . . . Vi

INTRODUCTION 0 O O O O I O O I I O O O O 1
LITERATURE REVIEW . . . . . . . . . . . . 3
MATERIALS AND METHODS . . . . . . . . . . . 9
Culture . . . . . . . . . . 9
Media and Cultural Methods . . . . . . . . . 9
Growth and Sporulation Studies . . . . . . . lO
Oxidation Experiments . . . . . . . . . . ll

45Ca++ Uptake . . . . . . . . . . . . . 12
Analytical Methods . . . . . . . . . . . 13
Glucosidase . . . . . . . . . . . . . . 13

RESULTS . . . . . . . . . . . . . . .' . 14

Growth and Sporulation in SPY . . . . . . . . 14
Growth in GPY . . . . . . . . . . . . . 17

Growth in PY . . . . . . . . . . . . l7
Volatile Acid Production . . . . . . . . 20
Calcium Uptake and Dipicolinic Acid Synthesis . . 20
Oxidation of Substrates . . . . . . . . . . 25

Growth Rates . . . . . . . . . . . . . 36

DISCUSSION . . . . . . . . . . . . . . . 39

LITERATURE CITED . . . . . . . . . . . . . 43

iv

LIST OF TABLES

Table Page

1. Comparison of 1-14C-acetate oxidation by cells

harvested at various times during growth in
SPY medium . . . . . . . . 28

2. Differences in glucose and salicin catabolism by

salicin grown cells . 3O

LIST OF FIGURES

Page

Growth, sporulation, and salicin utilization
in SPY medium . . . . . . . . . . . 15
Growth and sporulation in GPY medium . . . . 18

Comparison of volatile acid production and pH
changes in GPY and SPY media . . . . . . 21

Relationship between 45Ca++ uptake, dipicolinic
acid (DPA) synthesis, and heat resistance . 23

Salicin and acetate oxidation rates by cells
harvested at various times during growth in
SPY medium . . . . . . . . . . . . 26

Oxidation of salicin from cells harvested 0,
15, and 30 minutes after inoculation of SPY
medium 0 O O O O O O O O O O O O 31

Effect of glucose on the induction of salicin
oxidation . . . . . . . . . . . . 34

Effect of various sugars on the growth of UB‘
in batch culture . . . . . . . . . . 37

Vi

INTRODUCTION

The_physiologica1 requirements controlling sporula-
tion in the bacilli have been examined in considerable
detail. However, the organisms most studied have been non-
fastidious with respect to nutritional requirements, and
as a consequence have permitted their cultivation in simple
chemically defined media. It has been possible, therefore,
to study various individual nutrients and their influence
upon sporulation. Similar investigations with the

fastidious bacilli, notably Bacillus popilliae and E.

 

lentimorbus, have been hampered because of the complex

 

media required for growth and the inability of these
organisms to sporulate significantly in vitro. Therefore,
it is the purpose of this investigation to study a
fastidious Bacillus species capable of Sporulating to a
high degree under certain defined conditions.

Elucidating the factors repressing sporulation or

inducing good sporulation of B. pOpilliae and B. lentimorbus

 

 

in vitro would be of great economic importance since both
organisms are pathogenic for Japanese beetle larvae via
the ingestion of spores. Sporulation of the insect

pathogens in vitro would offer a practical and inexpensive

means of producing a biological insecticide. It is
ultimately hoped that understanding the mechanism control-
ling the sporulation of the organism under study may be
helpful in devising media and cultural conditions for the
sporulation of the insect pathogens in vitro.

The concept supporting the present investigation
is based on the idea that a common sporulation control
mechanism may exist between the test organism and the
insect pathogens. This assumption is based on preliminary
investigations by Dr. R. N. Costilow: first, sporulation
of the test organism is strongly repressed by low concen-

trations of glucose as is the case with B. pgpilliae;

 

second, the test organism has weak catalase activity and
is in close agreement with the catalase negative character-
istics of the insect pathogens; third, the test organism

and selected strains of B. popilliae sporulate when the

 

carbon source is a sugar which is used slowly by the
organism; and fourth, an exogenously added sugar is

required for appreciable growth in broth media.

LITERATURE REVIEW

In the past few decades considerable emphasis has

been given to B. pppilliae and B, lentimorbus as possible

 

 

 

control agents in Japanese beetle (Popillia japonica)

 

infestations. Dutky (5) first described these two organ-
isms as the causative agents of the fatal milky disease of
the insect larvae. ‘The milky appearance of the larvae is
due to high concentrations of spores which are formed
during the course of infection. The spores are resistant
to dessication and remain viable for many years in the soil.
Subsequent ingestion of the spores by healthy larvae
repeats the infectious cycle. Therefore, the large scale
production of spores presents a logical approach to the
development of a biological insecticide with both economic
and ecological significance.

The production of large masses of spores of B.

popilliae has not been achieved. Up to the present time,

 

the only means available for obtaining significant quanti-
ties of infective spores has been by harvesting diseased
larvae after artificial infection (6). However, this is
not a practical method of production for the large
quantities needed. In vitro methods of sporulation would

circumvent these problems.

In vivo, B. popilliae and B. lentimorbus multiply

 

 

in the haemolympth of E. japonica larvae and sporulate to
concentrations of about 2 to 3 x lolo/ml (20). The normal
course of infection requires from 14 to 21 days during
which time spores appear to be formed concommitantly with
vegetative cell growth (20). The entire process of growth
and sporulation in the larval haemolymph occurs in a very
rich nutritive environment (25). In vitro, both organisms
are nutritionally fastidious requiring complex media for
cultivation. However, Sylvester and Costilow (26)

delineated the growth requirements for B, popilliae and

 

have devised both synthetic and semisynthetic media which
support growth.
In contrast to in vivo Spore yields, the sporula-

tion of B. p0pilliae on laboratory media has been generally

 

poor. Rhodes et a1. (22) isolated strain NRRL B-23098
which sporulated on acetate supplemented media but only
under certain defined conditions. The frequency of spores
was only 0.1 to 0.3% after lengthy incubation. Sharpe

et a1. (24) achieved 20% sporulation of strain NRRL B—2309M
on a solid medium supplemented with trehalose. However,
Costilow and Coulter (4) could only obtain 2-6% sporulation
with this strain. Also, tests indicate that the in vitro
produced spores of this strain were not infective by

feeding to larvae (24). Spores have been formed in broth

 

 

media containing activated carbon (9, 10) although counts
have never risen above 106/ml (10).

In the host, B. pgpilliae and B. lentimorbus

 

 

sporulate in the presence of high concentrations of
trehalose (21).” Trehalose levels remain high throughout
the course of infection although diseased larvae do show

a reduced level (21). However, the incorporation of

 

trehalose:hnbroth media does not support spore formation

in E. pgpilliae (23). Also, the addition of trehalose to

 

sporulation media exerts no significant effect on spore
yields (4, 22). Rhodes (20) points out that trehalose may
be bound in the larval haemolymph and, thus, not readily
available to the proliferating organisms.

Glucose and other readily utilized sugars repress
Sporulation in the nonfastidious bacilli (1, 16). In B.

popilliae, low concentrations are sufficient to repress

 

sporulation (4, 22). However, some species of bacilli
sporulate when the carbon and energy source is present in
amounts above that required for growth (14). Hsu and Ordal
(12) reported that carbohydrates restricting growth below

that of glucose permitted sporulation in Clostridium

 

thermosaccharolyticum even though they were supplied at

 

high concentrations. Feeding glucose in limiting amounts

exerted a similar effect (11). In B. popilliae, Costilow

 

and Coulter (4) showed that sporulation was stimulated by

high concentration of a-methylmannoside (a-MM) and various

other carbon and energy sources. Increasing the concen-
trations of o-MM in a stepwise manner increased the
frequency of sporulation. A concentration of 0.5% was

necessary to achieve an equivalent sporulation frequency

 

obtained in 0.05% glucose medium. However, concentratiOns
of a-MM above 0.5% reduced the spore yields. Growth in
salicin containing medium resulted in swollen cells
characteristic of sporangia, although no spores were pro-
duced.

Bhumiratana and Costilow (submitted for publication)
showed that the oxidation of a-MM was inducible and its
utilization was rate limiting. They concluded that the
induced glucosidase had a low affinity for a-MM and that
the slow hydrolysis restricted the rate of utilization.
Hsu and Ordal (12) proposed that membrane permeation or
some other mechanism was responsible for limiting the

growth rates in E. thermosaccharolyticum on glucosides

 

since glucosidase activity appeared not to be rate limiting.
The oxidation of a-MM occurred in all strains of

B. popilliae capable of utilizing acetate (Bhumiratana and

 

Costilow, submitted for publication), and McKay et a1. (15)
showed that acetate oxidation occurred with all strains
demonstrating some degree of spore formation in vitro.
However, all but one asporogenous strain also oxidized
acetate. The wild type strain (2309) which has not been

sporulated in vitro oxidized the acid but only after

several subcultures in broth media. Strain 2309 was pre-
viously reported not to oxidize acetate (2). The ability
to oxidize acetate plays a critical role in the sporulation
of some bacilli (l, 8). Mutants devoid of this activity
are asporogenous (7). However, a recent investigation of

tricarboxylic acid cycle negative mutants of B. subtilis

 

indicates that this is not a prerequisite for normal
sporulation in this species (3).

B. popilliae catabolizes glucose to acetate,

 

lactate, and CO2 (18) and is, therefore, not unique among

the bacilli (16). In B. pgpilliae considerably more lactic

 

acid is produced at lower oxygen tensions. However, glucose
is not catabolized under strictly anaerobic conditions.
Hydrogen peroxide is generated in older broth cultures of

E. popilliae although such cultures are devoid of catalase

 

or peroxidase activity (19).
Physiological events commonly associated with

sporulation in B. popilliae were studied by Costilow and

 

Coulter (4). The pH profile of the medium during growth
. and sporulation of an oligosporogenous strain was similar
to that of other sporulating bacilli. However, the only
sporulation-specific marker observed was a heat stable
catalase. Neither antibiotics nor proteolytic enzymes
could be detected.

Two theories have recently been advanced in

attempting to understand the physiological control

mechanisms operating in the sporulation of B. pppilliae

 

in vitro. First, on the basis of their work with a-MM,
Costilow and Coulter (4) proposed that a growth rate
limitation is essential for Sporulation to occur. Such
sugars could be supplied in a growth rate limiting form

or at concentrations imposing a similar restriction on
growth kinetics. Second, McKay et a1. (15) suggested that
the citric acid cycle is strongly repressed in the wild
type strains cultured under the normal laboratory condi-
tions. However, they emphasized that this is not the only
block to sporulation in vitro since several asporogenous
strains oxidized acetate at levels greater than that

exhibited by the oligosporogenous varients.

MATERIALS AND METHODS

Culture
The organism used in this study is an unidentified
Bacillus species (hereafter referred to as UB) isolated
as a contaminant by Dr. R. N. Costilow while conducting

research with B. popilliae. The organism is nutritionally

 

fastidious and requires a sugar for good growth.

Media and Cultural Methods

 

The basal medium (PY) employed contained 0.5%
yeast extract, 0.5% peptone, 0.01% MnSO4-H20, and 0.01%
CaC12-2H20. PY plus glucose (0.2%) will be referred to
as GPY, and PY plus salicin (0.2%) as SPY. Sporulation
medium is synonomous with SPY. Agar (2.0%) was added when
a solid medium was required. All ingredients, including
the carbon sources, were sterilized by autoclaving for 15
min at 121 C. The media were adjusted to pH 7.4 prior to
sterilization.

UB was maintained on agar slants of SPY. Slants
were incubated at 30 C until spores had formed, and then

were stored at 4 C. Broth cultures were incubated at 30 C

on a rotary water bath shaker.

 

1“
=4“.

10

A spore suspension was routinely used as an inoculum.
Spores were harvested from slants of SPY by washing with 5
m1 of sterile distilled water. This suSpension was then
heat shocked for 20 min in a 60 C water bath and then
stored under refrigeration until needed. In all instances
a small volume of the spore suspension was inoculated into
GPY broth medium and incubated overnight (”10 hr). A 10%

inoculum was used in the appropriate medium.

Growth and Sporulation Studies

 

Because UB clumps severely during sporulation in
SPY, growth and sporulation experiments were conducted in
20 x 150 mm test tubes. The entire fluid content of a
test tube was examined at selected times. Each test tube
contained 4.5 ml of the apprOpriate medium to which 0.5 ml
of an inoculum grown in GPY was added to initiate the
experiment. It was observed by Dr. Costilow that the cell
clumps in SPY could be dispersed by resuspending the
centrifuged pellet in a protein fraction from spent sporu-
1ation medium. In the present investigation, cell-free
sporulation medium was fractionated to 80% saturation with
ammonium sulphate. The precipitate was then resuspended
in a small volume of 0.05M potassium phosphate buffer,
pH 7.4 and dialyzed in the cold against the same buffer.

This was then filtered sterilized using a Nalgene Filter

11

Unit (Nalge Sybron Corp.) suspended to a volume equivalent
to 10% of the original unfractionated medium, and frozen
in 10 ml aliquots until needed. During the growth and
sporulation studies the clumped cells were centrifuged, the
pellet resuspended in an equivalent volume of the above
fractionated medium, and incubated at 30 C under constant
agitation until the clumps had dispersed (approximately
30 min).

Growth was monitored by following optical density
(O.D.) with a Bausch and Lomb Spectronic 20 colorimeter at
a wavelength of 620 nm. Vegetative and heat resistant
counts were determined by the spread plate technique. Cell
dilutions were prepared in 0.85% sterile saline. Dilutions
were plated in triplicate on trypticase soy agar. To
obtain heat resistant (spore) counts, the cell suspensions
were heated in a 60 C water bath for 20 min. Measurements

of pH were performed with a Beckman, model G, pH meter.

Oxidation Experiments

 

The oxidation of various substrates by resting
cells were conducted by manometric techniques described by
Umbreit et a1. (27). Cells were harvested, washed one
time, and suspended in 0.05M potassium phosphate buffer
(pH 7.4 for sugar oxidations and pH 6.8 for acetate
oxidations). Dry weights were determined by drying the

suspensions at 110 C and correcting for the phosphate

 

12

present. All experiments were carried out at 30 C. Each

Warburg vessel contained 1.0 ml of the cell su3pension and
0.5 ml 0.05M potassium phosphate buffer at the appropriate
pH in a total volume of 3.0 ml. Substrates were added at

concentrations indicated in the results. Two-tenths ml

of 20% KOH was added to the center well of the Warburg

 

flasks when measuring oxygen uptake. For those experiments
in which the fluid was analyzed after incubation, 0.2 ml 4N
sulfuric acid was added from a side arm at the end of the
incubation period to terminate the reaction. The acidified
fluids were neutralized prior to assaying. In experiments
using labeled acetate, 14CO was trapped and counted accord-

2
ing to the method described by McKay et a1. (15).

45Ca++ Uptake

 

The incorporation of calcium in sporulating cells
was followed by the use of radioactive calcium. The
45CaCl2 was added to SPY at a concentration of 10.4 poi/ml.
At selected times, duplicate test tubes of cell suspensions
were each filtered through a 25mm millipore membrane filter
and washed with 4 x 5 ml aliquots of chilled 0.001N HCl
(28). The filters were dried in a 110 C oven and then

dissolved in a toluene based scintillation fluid for

counting (15).

13

Analytical Methods

 

Volatile acid was determined using the method
described by Neish (l7). Dipicolinic acid was measured by
the method of Janssen et a1. (13). Salicin was assayed by
measuring total reducing sugars after acid hydrolysis.
Hydrolysis was achieved by adding 0.1 ml of concentrated
HCl to 1.0 ml of sample fluid and steaming for one hr. The
mixture was then neutralized with base and the total
reducing sugars determined by the method of Neish (17).
Corrections were made for reducing sugars detected prior

to hydrolysis.

Glucosidase

 

Uninduced and induced cells were analyzed for
glucosidase activity by measuring total reducing sugars
released during incubation with salicin. Cells to be

tested were suspended to an O.D. of about 10 and 2 drops

620
of toluene were added to 0.9 ml of the cell suspension.
The mixtures were then shaken vigorously for 30 min or
until the odor of toluene could no longer be detected.
After incubating the toluene treated cells with salicin
(0.1 m1 of 0.1M solution), the reactions were stopped at

selected times by immersing the test tubes in boiling

water for 10 min.

RESULTS

The sporulation of UB was found to be maximally
induced by salicin. On the other hand, free glucose
strongly repressed sporulation even at very low concentra-
tions. Therefore, a number of comparisons were made of the

response of UB to these two sugars.

Growth and Sporulation in SPY

 

The relationship between growth and sporulation of
UB in SPY medium was quite similar to what has been observed
with many spore forming bacteria (Figure 1). Thus, spore
formation was initiated at the end of exponential growth
and proceeded at about the same rate as was observed for
‘exponential growth of the cells. Sporulation was essentially
completed within 24 hr, and the population of heat stable
spores was approximately equal to the total viable popula-
tion.

Salicin utilization was evident throughout the
experiment, and the rate of disappearance of the sugar was
essentially as rapid during sporulation as during
exponential growth. It is evident that salicin was in
excess since almost two-thirds of the amount added was

still present after 48 hr incubation.

14

 

15

Figure l.--Growth, sporulation, and salicin utilization
in SPY medium. Optical density (O.D.) ,0;
viable cell count,A; heat resistant count
(spores), O; salicin, E] .

l6

 

 

 

 

 

0.0| l l 1 1

 

HOURS

 

 

 

2.4

2.2

2.0

l.8

t;

17

A clumping of the cells prior to sporulation was
consistantly observed in SPY medium. The reason for this
is not known. Clumping appeared after 8 hr growth and was
maximum at about 24 hr, as observed visually. A very
small amount of clumping was present at 48 hr when the
experiment was terminated. Only swollen cells and
sporangia containing spores appeared to clump. The
massive clumping precluded average population samplings
in Erlenmyer flasks and, therefore, necessitated conducting
the experiments in test tubes. Resuspending the clumped
cells in an 80% ammonium sulfate fraction from spent

sporulation medium dispersed the clumps.

Growth in GPY

 

Figure 2 shows that glucose strongly represses
sporulation. The onset of exponential death after 8 hr may
be due to the high acid conditions of the medium, since as
evident below, considerable acid accumulated. However,
the failure of GPY to support sporulation is not due to
lethal acid conditions since no spores were formed when
the medium was buffered to neutrality (Costilow, unpublished

data). No clumping of the cells was detected in GPY.

Growth in PY

 

Growth in the basal medium without added sugars
resulted in a maximum spore yield of 1.8 x 107/ml after

36 hr, which is less than 10% that observed in SPY. After

 

18

Figure 2.--Growth and sporulation in GPY medium.
Optical density (O.D.) ,.; viable cell
count, A; heat resistant count (spores) ,O .

0.0.,520

|.0

O.l

0.0 l

19

 

 

 

 

 

 

l T r r
410°
C
«no7
6
-IO
~105
1 1 ‘ 1 1 . '04
IO 20 30 4o 50

HOURS

No./ml

20

36 hr incubation, cultures contained some large fluffy
clumps. Microsc0pic examination of the clumps revealed
some free spores suspended in a matrix of an unknown
material. Continued incubation for two weeks beyond the
initial stage of clumping showed no change in the nature
of the clumps. Resuspending the pelleted clumps in
fractionated Spent sporulation medium did not disrupt

the clumps.

Volatile Acid Production

 

Figure 3 shows that volatile acids were produced
in both salicin and glucose supplemented media. However,
only in the SPY medium was the acid utilized and the pH
observed to increase. The volatile acid produced from
glucose catabolism.was accumulated and the pH remained at
a very low level. Assay of the spent glucose medium by
gas chromatography revealed acetate as the volatile acid
produced.

Calcium Uptake and Dipicolinic
AcidCSynthesis

 

 

Several characteristics commonly associated with
sporulation were investigated in order to define more
clearly the sporulation of UB. Figure 4 shows the cor-
relation between bound 45Ca++, dipicolinic acid (DPA)

formation, and heat resistance. All the parameters

appeared to increase at approximately the same rate. The

21

Figure 3.--Comparison of volatile acid production and
pH changes in GPY and SPY media. Volatile
acid, —; pH, --; GPY,.; SPY,O.

23

Figure 4.--Relationship between 45Ca++ uptake,

dipicolinic acid (DPA) synthesis, and heat
resistance in Sporulating cells in SPY medium.

'++ .
45Ca , Q; DPA, A; heat re51stance, O .

24

 

 

 

40

IO

 

- _ - —

 

 

IOO-

0 O 0 0
8 6 4. 2

. 23282;. no hzmommm

50

3O

20

HOURS

25

temporal sequence of events shows that the uptake of

+ .
bound 45Ca + preceded both DPA synthes1s and heat
resistance by about 4 hr. The increase in heat resistance

during Sporulation appeared concomittantly with increased

levels of DPA.

Oxidation of Substrates

 

The oxidation of acetate and salicin was examined
in cells harvested at various times during the growth of
U3 in sporulation medium (Figure 5). Acetate was oxidized
at all stages of cultural growth but was maximal at the
time sporulation was initiated. The ability of glucose
grown cells to oxidize acetate was not directly tested
but it appears probable that it is strongly repressed as
indicated by the low 002 values for acetate of cells at
zero time.

The oxidation of salicin was at a maximum in cells
4 hr prior to maximum acetate oxidation. No salicin was
oxidized at zero time. It is evident that salicin was
oxidized in sporulating cells which also have the ability
to oxidize acetate. Evidently sporulating cells use both
carbon sources as energy since volatile acid and salicin
are utilized during sporulation in batch culture.

The respiratory quotient (R.Q.) for the oxidation
of salicin was consistently about 1.0. In contrast, Table 1

shows that the R.Q.'s for the oxidation of acetate were

26

Figure 5.--Salicin and acetate oxidation rates by cells
harvested at various times during growth in
SPY medium. The Warburg flasks contained 5 to
9 mg (dry weight) of cells for acetate
oxidations and 7 to 14 mg (dry weight) of cells
for salicin oxidations. 30 umoles of salicin
or 27 umoles of labeled acetate were tipped in
from a side arm to initiate the reactions.
1-14C-acetate was supplied at a concentration
of 4.8 x 10"3 uci/umole acetate for 0, 4, 8, 12,
and 24 hr samples and 4.0 x 10'2 uci/umole acetate
for the 16 and 20 hr samples. Two-tenths ml of
4N sulfuric acid was tipped in to terminate the
reactions. Appropriate contfiols were added for
endogenous respiration and 1 C-acetate distilla-
tion. 00 values represent the uliters oxygen
taken ul/Hr/mg dry weight of cells. Salicin,();
acetate, Q; heat resistant counts (spores) , A;
optical density (O.D.) , A .

no.

:5 .02 A

 

]._Od

1 to

T O;

 

 

rfifido

¢N

ON

manor
m.

N.

 

 

 

 

 

CV

00

28

TABLE l.--Comparison of 1-14C—acetate oxidation by cells
harvested at various times during growth in SPY

 

 

 

medium.*
umoles
Hours
R.Q.**
14co2 02
produced utilized
4 0.24 0.85 0.57
8 4.19 17.23 0.49
12 1.04 5.09 0.41
16 3.88 8.35 0.93

 

*
Data obtained from the oxidation experiments
shown in Figure 5.

**
Represents R.Q. values using corrected values

for CO2 production (see text).

variable. The R.Q. for the oxidation of acetate was
calculated using a corrected value for CO2 production.
This was necessary since the fate of the unlabeled carbon
atom is not known. Therefore, I assumed that acetate is
oxidized to completion and the unlabeled carbon atom is
evolved as C02. As a result, the data were corrected by
doubling the amount of 14CO2 produced. It is realized
that this manipulation may not be completely justified,
but it does offer a means for comparison of R.Q. values.

Utilizing the corrected values for CO2 production, the data

indicate that a larger amount of oxygen was taken up than

29

CO2 evolved in the 4, 8 and 12 hr old cells and was
reflected in R.Q. values being considerably less than 1.0.
However, in the 16 hr cell suspension the R.Q. approached
1.0.

Because glucose and salicin dramatically affect
the sporulation of UB it was necessary to look at the
metabolism of both sugars by resting cell suspensions. The
rates of oxygen uptake by salicin grown cells were
essentially the same with either glucose or salicin as
substrate. However, much more glucose was utilized during
the reaction than salicin (Table 2). On the basis of
volatile acid production, it is obvious that glucose is
less completely oxidized than salicin. Only a small amount
of volatile acid accumulates during salicin oxidation.
Because of the significant amount of volatile acid pro-
duced by endogenous respiration, it is not known for sure
if the acid detected in the presence of salicin is truly
a result of its oxidation or of oxidation of endogenous
substrates. It is evident, however, that salicin does
exert some effect on endogenous metabolism since twice as
much acid is produced in its absence.

There was no oxidation of salicin by resting cells
harvested from glucose containing medium although the
reaction mixture was incubated for an extended period of
time. Glucose, however, was immediately oxidized. Because

salicin was utilized in batch culture (SPY) when inoculated

30

TABLE 2.--Differences in glucose and salicin catabolism
by salicin grown cells.*

 

 

 

umoles
. . Volatile
Utilized 02 CO2 Acid
Glucose ' 21.7 37.2 36.1 15.6
Salicin 7.0 30.7 31.4 3.2
Endogenous - 1.1 3.0 6.1

 

*Cells were harvested after 4 hr growth in SPY
medium. The Warburg flasks contained 7 mg (dry weight) of
cells and 30 umoles of glucose or salicin. The reactions
were terminated by tipping in 0.2 ml of 4N sulfuric acid.

with glucose grown cells, but not in a resting cell sus-
pension by glucose grown cells, it was decided to look at
its induction in both dividing and nondividing cells.

The next series of experiments were designed to study
these phenomena.

Salicin oxidation was obviously induced rapidly
after inoculation of cells into SPY (Figure 7). Cells
harvested immediately after inoculation did not oxidize
salicin. However, cells harvested after 15 min growth
showed appreciable oxidation and those harvested after 30
min were even more active. In addition, the extent of
salicin oxidation by cells harvested after 30 min incuba-

tion in SPY containing'lmaug/ml chloramphenicol was

31

Figure 6.--Oxidation of salicin from cells harvested 0,
15, and 30 min after inoculation of SPY medium.
The Warburg flasks contained 8 to 10 mg (dry
weight) of cells and 30 umoles of salicin.
Values have been corrected for endogenous
respiration. Zero time, Q; 15 min,O;
30 min, A .

32

 

 

 

 

5 T l l l
4.. ..
as- .
'3.
N
22- -
Ir '1
O . 4 H—J
l 2 3 4

HOURS

33

approximately 80% less during the first 60 min incubation
than the control. These data strongly suggest that the
oxidation is inducible.

Resting cells from a glucose medium failed to show
any oxygen uptake with salicin as substrate even after
prolonged incubation even when 0.4% yeast extract and/or
0.4% peptone was added to the resting cell suspension.
Apparently the cells need a readily available energy source
for the induction, since on the addition of 5 umoles of
glucose along with 30 umoles salicin to the Warburg vessel
there was an apparent induction of salicin oxidation after
about two hr (Figure 7). This increased oxygen uptake rate
was inhibited by chloramphenicol. Apparently glucose
serves as an energy source needed for induction. The
failure to demonstrate induction in resting cells suspended
in peptone-yeast extract is reasonable since the small
amount of glucose present in the yeast extract would be
used rapidly by the large number of cells present thereby
imposing an energy shortage.

In order to confirm that salicin oxidation is truly
inducible, the ability of toluenized cells to hydrolyze
salicin was investigated. It was observed that glucose
grown cells were unable to hydrolyze the molecule. On the
other hand, cells grown in SPY did so readily. Therefore,

the hydrolysis of salicin is inducible.

34

Figure 7.--Effect of glucose on the induction of salicin
oxidation. Cells were harvested from GPY
medium. The Warburg flasks contained 6 mg
(dry weight) of cells. Chloramphenicol was
added at a final concentration of 214 ug/ml
where indicated. The vessels contained the
following sugars: glucose (30 umoles),();
salicin (30 umoles) plus glucose (5 umoles)
with «3) and without (A) chloramphenicol, and
salicin (3O umoles) , O .

 

IO

35

 

 

60

 

l20 ISO 240 300 360
MINUTES

36

Growth Rates

 

Examination of Figures 1 and 2 shows that the rate
of exponential growth of UB was higher in glucose than in
salicin. However, the rates of oxygen uptake by salicin
grown cells were approximately the same with either sugar
as substrate (Table 2). Therefore, a direct comparison of
growth response to the two sugars was made. It is evident
in Figure 8 that the growth rate is slower with salicin as
the primary energy source than with glucose. The
respective doubling times were approximately 2 hr and 1.2
hr. Of particular interest is the fact that the growth
rate in salicin was initially lower than that in the basal
medium without sugar, but the addition of 0.2% salicin
along with glucose (0.2 or 0.05%) had no effect.
Apparently salicin inhibits growth only when the concentra-

tion of glucose is very low.

37

Figure 8.--Effect of various sugars on the growth of
UB in batch culture. The sugars and their
concentrations in the basal medium were:
glucose (0.2%),.; salicin (0.2%),0; saliéin
(0.2%) plus glucose (0.05%),A; and no
sugars, A .

 

 

38

 

 

 

 

 

 

0N0.Q.O

0.0l

HOURS

DISCUSSION

Many of the characteristics of UB during growth and
sporulation are very similar to those observed with other
sporeforming bacilli. The most obvious sporulation
associated characteristics observed were: the pH profile
of the medium; the close correlation between dipicolinic
acid (DPA) and heat resistance; the uptake of bound radio-
active calcium prior to synthesis of detectable levels of
DPA; and the induction of acetate oxidation. UB differs
from many other bacilli in that it apparently requires an
oxidizable carbohydrate during spore formation.

The oxidation of acetate by sporulating cells was
associated with oxygen consumption above theoretical expecta-
tions. In theory, two moles of oxygen are required for the
complete oxidation of one mole of acetate. The increased
oxygen demand is apparently due to the oxidation of

endogenous substrates. Bhumiratana and Costilow (submitted

 

for publication) have shown that in E. popilliae the co-
presence of acetate and a-methylmannoside (a-MM) stimulated
oxidation of both compounds. Perhaps the added acetate
stimulated the oxidation of salicin not removed from the

cells during washing.

39

40

The data presented demonstrate that salicin is
essential for massive sporulation in UB and can be supplied
in a great excess. It is especially noted that salicin
restricts the growth rate below that of glucose which
strongly represses sporulation. The growth rate restriction
imposed by salicin in UB is not unique since salicin and
other carbohydrates supplied in high concentrations
restrict growth rates below that of glucose in g. thermo-

saccharolyticum and also derepress sporulation (12).

 

Feeding glucose in growth rate limiting amounts also
induced sporulation in this fastidious anaerobe and served
as a source of energy for sporulation (ll). Sporulating
cells of UB also require an exogenous energy source since
salicin is utilized throughout sporulation. Apparently,
the slow utilization of salicin restricts the flow of
glucose allowing a derepression of sporulation. Therefore,
it is possible that one might feed glucose at slow enough
a rate to permit spore formation.

The incomplete metabolism of glucose in resting
cells is consistent with the large accumulation of volatile
acid in dividing cells in glucose medium. This is of
particular interest since UB does have the potential to
oxidize volatile acid and does so during sporulation. It
was initially thought that the extreme acid conditions

were responsible for the asporogenic nature of glucose

41

medium. Murrell (16) has reviewed similar observations
reported by other investigators and has suggested that low
pH values interfere with induced enzyme formation and the
activity of the citric acid cycle enzymes. However,
buffering glucose medium does not derepress sporulation in
UB (Costilow, unpublished data). In fact, only 1 x 106
spores/ml were formed when 0.05% glucose was added to the
basal medium. Approximately 1 x 107 spores/m1 were formed
in the basal medium alone. Calculated estimates show the
basal medium to contain about 0.006% glucose. In the non-
fastidious bacilli, glucose repression is transient and
depletion of the sugar from the medium derepresses the
citric acid cycle enzymes permitting oxidation of the
accumulated acid and sporulation (1). Since UB apparently
requires an oxidizable sugar during sporulation, sporula-

tion does not occur in a glucose medium. This is of

particular interest since B. popilliae apparently requires

 

an oxidizable sugar during spore formation (4), and sporu-
lation is also repressed by low concentrations of glucose.
The oligosporogenous varients and the asporogenous wild

type strain of B. popilliae oxidize acetate but only at

 

severely repressed rates (15). McKay et a1. (15) have

proven that B. pOpilliae has the genetic potential to

 

oxidize acetate but that it is strongly repressed in the

wild type strains under normal cultural conditions.

42

Salicin oxidation is inducible in UB. Induction
is rapid in cells inoculated in a growth medium. The
fact that salicin reduces the growth rate of UB below that
observed in the basal medium probably accounts for the
failure to detect a diauxie response. Hsu and Ordal (12)
have postulated that various glucosides (including

salicin) inducing sporulation in E; thermosaccharolyticum

 

might restrict growth rates by their slow permeation of
the cell membrane. Bhumiratana and Costilow (submitted

for publication) showed that in B. popilliae the rate

 

limiting utilization of o-MM was due to a slow enzymatic
hydrolysis of the alpha linkage. It is of interest,

however, that in B. p0pilliae a prolonged period of

 

induction was associated with a-MM oxidation along with

a diauxie growth response.

LITERATURE CITED

43

LITERATURE CITED

Bernlohr, R. W., and C. Leitzman. 1969. Control of
sporulation, p. 183-213. In G. W. Gould and A.
Hurst (ed.), The bacterial spore. Academic Press
Inc., New York.

Bulla, L. A., G. St. Julian, R. A. Rhodes, and C. W.
Hesseltine. 1970. Physiology of spore forming
bacteria associated with insects. I. Glucose
catabolism in vegetative cells. Can. J. Microbiol.
'1§:243-248.

Carls, R. A., and R. S. Hanson. 1971. Isolation and
characterization of tricarboxylic acid cycle mutants
of Bacillus subtilis. J. Bacteriol. 106:848-855.

 

Costilow, R. N., and W. H. Coulter. 1971. Physio-
logical studies of an oligosporogenous strain of
Bacillus popilliae. Appl. Microbiol. 22:1076-1084.

 

 

Dutky, S. R. 1940. Two new spore-forming bacteria
causing milky diseases of Japanese beetle larvae.
J. Agr. Research. 61:57-68.

Dutky, S. R. 1963. The milky diseases, p. 75-115.
In E. A. Steinhaus (ed.), Insect pathology, vol. 2.
Academic Press, New York.

Fortnagel, P., and E. Freese. 1968. Analysis of
sporulation mutants. II. Mutants blocked in the
citric acid cycle. J. Bacteriol. 95:1431-1438.

Hanson, R. S., V. R. Srinivasan, and H. O. Halvorson.
1963. Biochemistry of sporulation. I. Metabolism
of acetate by vegetative and sporulating cells.

J. Bacteriol.‘ 85:451-460.

Haynes, W. G., and Lenora J. Rhodes. 1966. Spore
formation by Bacillus popilliae in liquid medium
containing activated carbon. J. Bacteriol. 91;
2270-2274.

 

44

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

45

Haynes, W. C., Lenora J. Weih, and C. D. Crowell.
1971. Sporulation of Bacillus popilliae in liquid
medium as affected by method of steriIizing
activated carbon. Bacteriol. Proc., p. 57.

 

Hsu, E. J., and Z. J. Ordal. 1969a. Sporulation of
Clostridium thermosaccharolyticum under conditions
ofirestrIcted growth. J. Bacteriol. 2151511-1512.

 

Hsu, E. J., and Z. J. Ordal. 1969b. Sporulation of
Clostridium thermosaccharolyticum. Appl. Microbiol.

Ig3958-ggoo

 

Janssen, F. W., A. J. Lund, and L. B. Anderson. 1958.
Colorimetric assay for dipicolinic acid in bacterial
spores. Science. 127:26-27.

Majumder, S. K., and M. C. Padma. 1957. Screening
of carbohydrates for sporulation of bacilli in fluid
medium. Can. J. Microbiol. 3:639—642.

McKay, L. L., A. Bhumiratana, and R. N. Costilow.
1971. Oxidation of acetate by various strains of
Bacillus popilliae. Appl. Microbiol. 22:1070-1075.

 

Murrell, W. G. 1967. The biochemistry of the bacterial
spore, p. 133-151. {p.A. H. Rose and J. F. Wilkinson
(ed.), Adv. microbiol. physiol., vol. I. Academic
Press, Inc., New York.

Neish, A. C. 1952. Analytical methods for bacterial
fermentations. Report no. 46-8-3, 2nd Ed. Prairie
Regional Laboratory, Saskatoon, Canada.

Pepper, R. E., and R. N. Costilow. 1964. Glucose
catabolism by Bacillus popilliae and Bacillus
lentimorbus. J. BacterioI. §ZT303-3I0.

 

 

 

Pepper, R. E., and R. N. Costilow. 1965. Electron
transport in Bacillus popilliae. J. Bacteriol.
89:271-276.

 

Rhodes, R. A. 1965. Symposium on microbiol. insecti-
cides. II. Milky disease of the Japanese beetle.
Bact. Reviews. 29:373-381.

Rhodes, R. A. 1968. Milky disease of the Japanese
beetle, p. 85-91. In Proc. Joint U.S.-Japan Sem.
Microbial Control of_Insect Pests. Shukisha
Printing Co., Ltd., Fukuoka, Japan.

22.

23.

24.

25.

26.

27.

28.

46

Rhodes, R. A., M. S. Roth, and G. R. Hrubant. 1965.
Sporulation of Bacillus pOpilliae on solid media.
Can. J. Microbiol. 21:779-783.

 

Rhodes, R. A., E. S. Sharpe, H. H. Hall, and R. W.
Jackson. 1966. Characteristics of the vegetative
growth in Bacillus popilliae. Appl. Microbiol.
14:189-195.

 

Sharpe, E. S., G. St. Julian, and C. Crowell. 1970.
Characteristics of a new strain of Bacillus popilliae

 

sporogenic in vitro. Appl. Microbioll 19:681-688.

Shotwell, O. L., G. A. Bennett, H. H. Hall, C. H.
Van Etten, and R. W. Jackson. 1963. Amino Acids
in the haemolymph of Popillia japonica (Newman)
larvae. J. Ins. Physiol. 9:35-42.

 

Sylvester, C. J., and R. N. Costilow. 1964. Nutri-
tional requirements of Bacillus popilliae. J.
Bacteriol. 81:114-119.

 

Umbreit, W. W., R. H. Burris, and F. G. Stauffer.
1957. Manometric techniques, 3rd Ed. Burgess
Publishing Co., Minneapolis.

Vinter, V. 1962. Spores of microorganisms. X.
Interference of tetracycline antibiotics with
sporogenesis of bacilli. Fol. Microbiol. 1:275-287.

HICHIGQN STATE UNIV. LIBRARIES

 

llll |||1| | mm |l|| ill)

312930 7334438