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ABSTRACT

GLUCOSE AND MALTOSE TRANSPORT
IN BACILLUS POPILLIAE

 

By

Daniel Clifford Taylor

Glucose was found to be actively transported by Bacillus
popilliae, and the data indicate that the transport is via a
phosphoenolypyruvate (PEP) phosphotransferase system. Uptake of
[149 glucose and [14(2) methyl a-D-glucoside (aMG) was inhibited
by fluoride, and oxidation of glucose was neglibible in the pres-
ence of fluoride or azide. Considerable phosphorylation of glu—
cose by toluenized cells occurred with either PEP or ATP as the
phosphoryl group donor, but phosphorylation of a MC was largely
dependent on PEP. An ATP dependent hexokinase was demonstrated
in crude cell extracts, using glucose, fructose, or mannose as
substrates. Saturation studies indicated that maltose was diffu-
sing across the bacterial membrane. The uptake of 14C maltose
was not inhibited by energy poisons. A constitutive maltose

cleaving enzyme was active in crude extracts of E, popilliae.

GLUCOSE AND MALTOSE TRANSPORT

IN BACILLUS POPILLIAE

 

BY

Daniel Clifford Taylor

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

1976

DEDICATION

To my parents, Dr. and Mrs. Clifford B. Taylor, for all their help,
love and trust during the past three years.
To Vicki Chessin and Bob and Kay Zahorchak for their warmth and kind-

ness which never allowed me to despair when times were rough.

ii

ACKNOWLEDGMENTS

I wish to express my deepest appreciation and respect to Dr. R.N.
Costilow for his guidance and encouragement during this investigation
and his patience throughout 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 equip-

ment .

iii

TABLE OF CONTENTS

Page
DEDICATION . . . . . . . . . . . . . . . . . . . . . . . . . ii
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . iii
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . v

LIST OF FIGURES o o o o o o o o o o o o o o o o o o o o o o o 0 v1

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 3
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . 10
Organism and Growth Conditions . . . . . . . . . . . . . . . 10
Oxygen Uptake Experiments . . . . . . . . . . . . . . . . . . 10
Enzyme Assays . . . . . . . . . . . . . . . . . . . . . . . . 10
Uptake of [14C] Carbohydrates . . . . . . . . . . . . . . . . 12
Purification of Maltose and Maltase Assay . . . . . . . . . . 12

Other Procedures . . . . . . . . . . . . . . . . . . . . . . . 13

RESULTS 0 O O O O O O I O I O O O O O O O O O O O O O 0 O O O I O 15
Glucose Transport . . . . . . . . . . . . . . . . . . . . . . 15
Studies with Maltose . . . . . . . . . . . . . . . . . . . . 21

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 35

iv

LIST OF TABLES

Table Page
1. Phosphotransferase activities of_§. popilliae
cells . . . . . . . . . . . . . . . . . . . . . . . l7

2. Hexokinase activities in extracts of g. popilliae

cells . . . . . . . . . . . . . . . . . . . . . . . l9

Figure

10.

LIST OF FIGURES

Sodium fluoride inhibition of [14C] glucose uptake
by B. popilliae . . . . . . . . .

Sodium fluoride inhibition of [1443 methyl a-D-glucoside
uptake by B, popilliae . . . . . . . . . . . . .
Methyl a—D—glucoside inhibition of [@4Q] glucose up~
take by B. popilliae . . . . . . . . . . . . . .
Glucose inhibition of [14C] methyl a-D-glucoside up-
take bY.§° popilliae . . . . . . . . . . . . . .

Kinetics of [IACI methyl a-D—glucoside transport .
Lineweaver-Burk plot of kinetic data from Fig. 5 .
Effects of inhibitors on [14C] maltose uptake .
Inhibition of maltose oxidation . . . . . . .
Kinetics of [14C] maltose uptake by B. popilliae .

Constitutive oxidation of maltose . . . . . . . .

vi

Page

16

20

22

23
24
25
26
27
29

31

INTRODUCTION

The larvae of the Japanese beetle, Popillia japonica Newn., and the

 

European chafer, Amphimallon majalis (Razoum.) are susceptible to milky dis-
ease. A.massive accumulation of refractile endospores imparts a chalky color
to infected larvae. Dutky (11) was the first to demonstrate the causal rela—
tionship between the disease and two closely related, gram positive, spore-

forming bacilli. Bacillus popilliae, the causitive agent of Type A milky

 

disease, is characterized by the formation of a phase bright parasporal body
along with a heat resistant endospore. The parasporal body is a bipyrimidal
crystalline structure, and is composed mainly of protein, although some car-
bohydrate is present (6). Type B milky disease, which differs from the Type
A disease in that larvae turn chocolate brown due to extensive formation of

blood clots, is caused by Bacillus lentimorbus. Although both organisms are

 

similar in appearance during vegetative stages,_§. lentimorbus does not form

 

a parasporal crystal during sporulation.

Many attempts have been made to develop media and other appropriate
conditions which would support growth and eventual sporulation of either
species, with the hope of mass producing spores and using them as an alter-
native to chemical insecticides. llgflgiggg_sporulation of B. popilliae, a1-
beit in low frequency, was first reported by Steinkraus and Tashiro (41).
Later investigators isolated strains 0f.§3 popilliae which would sporulate
to a low but significant extent in artificial media (31, 36). However,
sporulation was inconsistent and in 31552 spores were not capable of pro-

ducing infection via ingestion in test larvae.

2

In an attempt to compare sporulation of B, popilliae to sporulation in
less fastidious Bacillus species, various physiological processes known to
be associated with endospore formation were investigated in B, popilliae.
Much of this work has been done by Costilow and co-workers (4, 7, 8, 9, 27,
28, 29, 30, 42). Recent studies by Bhumiratama §£_§l, (3) demonstrated that
trehalose, which is apparently the natural carbon and energy source for B.
popilliae, is transported via a phosphoenolpyruvate dependent phosphotrans-
ferase system. Strains of B, popilliae were found to contain phosphotrehalase,
and a mutant was isolated that could not grow on trehalose but accumulated
trehalose 6-phosphate intracellularly. This report prompted the present in-
vestigation into the mode of transport of two other sugars which support the

growth of B, popilliae, glucose and maltose.

LITERATURE REVIEW

Bacterial cells transport sugars by several methods. Members of the
same genus may transport a specific sugar by facilitated diffusion, active
tranSport, or group translocation, whereas one cell type may transport a var-

iety of sugars by all three processes (34). For example, Escherichia coli

 

and Salmonella typhimurium transport sugars by all three methods, while

 

Staphylococcus aureus appears to transport most sugars by group translocation

 

(34). In addition, E, ggli_transports galactose by four separate permease
systems (34). Many investigations have centered upon group translocation
since its discovery in 1964 (22).

Kundig, Ghosh, and Roseman (22) first detected the phosphotransferase
system when searching for a kinase in bacterial extracts that catalyzed the
phosphorylation of N-acyl-D-mannosamine, which is a step in the biosynthesis
of the sialic acids normally occurring in animal tissues. This transport
system is unique in that it requires several protein fractions and uses
phosphoenolpyruvate (PEP) as the phosphoryl group donor rather than a nucleo-
tide triphosphate. Extensive work with the PEP dependent phosphotransferase
system (PEP-PTS) of E. ggli and §, aureus has resulted in the characteriza-
tion of the components in each species (1, 18, 23, 24, 29, 37, 38).

Two proteins, Enzyme I and HPr, appear to be constitutively synthesized

 

members of the PEP-PTS of E, coli, §, aureus, and S, typhimurium (34). Both

 

are soluble proteins and lack sugar specificity. HPr has been purified to
homogenity from each of the above organisms. Enzyme I catalyzes the transfer

of the phosphoryl group from PEP to HPr. Studies with purified Enzyme I from

4

S, typhimurium indicate that the phosphoryl group is first attached to a

 

histidinyl residue in the protein; magnesium ion is required for this re-
action. In the second step, demonstrated with PEP-PTS components from_§.
coli, the phosphoryl group is transferred from Enzyme I to the N-l position
of the imidazole ring of a histidinyl residue in HPr. This reaction does
not require magnesium ion.

Although most sugar specific proteins are inducible, there are two con-
stitutive systems in E, ggli_catalyzing the transfer of the phosphoryl group
from P-HPr to carbohydrate (34). If E. ggli is grown on a salts medium with
glucose as the sole carbon source, the membrane fraction is capable of trans-
ferring the phosphoryl group from P-HPr to glucose, mannose, and fructose.
Extraction of the membrane with a combination of urea and n-butanol gives a
soluble protein fraction, designated II-A, and an insoluble fraction. II-A
can be further purified into three proteins, each specific for one of the sug-
ars. A protein labeled 11-8 is obtained from the pellet by fractionation in
the presence of deoxycholate, which also removes contaminating lipid. A mem-
brane lipid, phosphatidylglycerol, is an essential component of the Enzyme II
system. Using P-HPr, this system is able to phosphorylate a Specific sugar
(depending upon which II-A is used) if the components of the system are
mixed in the following sequence: II-B, Mg2+, phosphatidylglycerol, II-A (24).

The second system found to transfer the phosphoryl group from P-HPr also
involves two sugar-specific proteins (34). One is found in the soluble frac-
tion of crude extracts of E, coli, while the other is in the membrane frac-
tion. The soluble protein has been designated Factor III. Factor III is

tightly associated with a phosphatase that is inhibited by fluoride.

5
Fractionation experiments and studies with sulfhydryl reagents indicate that
the membrane protein is different from II-B. In contrast to the first sys-
tem, this one actively phosphorylates the galactoside analog methyl B—thio-
galactoside.

S, aureus contains a membrane bound Enzyme II and a soluble Factor III
which react with P-HPr to transfer the phosphoryl group to lactose. Factor
III is capable of accepting three phosphoryl groups from P-HPr, and chromat-
ographic studies of the phosphohistidine obtained from an alkaline hydrolysate
of the phospho—Factor III indicate that the phosphoryl groups are attached to
the N-3 position of the imidazole ring of histidine. Enzyme II-B is required
for the transfer of the phosphoryl group from P—III to lactose.

Saier 35 a1. (35) demonstrated a PEP-dependent system for the phosphory-
lation of fructose in two species of purple non-sulfur bacteria. The enzyme
system differs from that found in E, ggli or_§. aureus in that only two pro-

tein fractions are necessary for the reaction. In Rhodospirillium rubrum,

 

phosphotransferase activity was found in the particulate fraction of crude
extracts. Extracting the pellet with water removed a component which was
required in combination with the pellet to transfer the phosphoryl group from
PEP to fructose. Only two protein fractions were found essential for the

phosphorylation of fructose in crude extracts of Rhod0pseudomonas spheroides.

 

In this case, one fraction was associated with the membrane and the other
was found in the soluble fraction after centrifugation.

PEP-dependent phosphotransferase activity has been demonstrated in many
other bacterial species. Romano g£_al, (32) assayed a wide variety of bac-

teria for phosphotransferase activity. After studying representatives of

6
genera that were either strict aerobes or fermentative species, they conclu—
ded that PEP-dependent phosphotransferase activity was limited to those species
capable of anaerobic metabolism. Activity was found in species of Bacillus,

Cornynebacterium, Escherichia, Salmonella, and Staphylococcus, and was absent

 

 

 

in species of Arthobacter, Azotobacter, Micrococcus, Mycobacterium, and

 

 

 

Pseudomonas. It has been shown that Pseudomonas aeruginosa actively trans-

 

ports glucose intact, or alternatively, oxidizes it to gluconate by a mem-
brane bound glucose dehydrogenase and transports gluconate inside the cell
(17). Eisenberg g£_§l, (13) reported essentially the same mechanism for trans-

port of glucose by Pseudomonas fluorescens.

 

In addition to the examples mentioned above, PEP-PTS activity has been
found in a variety of mesophilic bacilli (21), a strain of a thermophilic
Bacillus (21), in all lactic streptococci transporting lactose (25), in Aero-

bacter aerogenes with respect to mannitol (43), and in Vibrio cholerae grow-

 

 

ing on galactose or fructose (2). Cirillo and Razin (5) demonstrated that

10 strains of Mycoplasma transported methyl u-D-glucoside by group transloca-

 

tion. This could not be demonstrated in non-fermentative strains.
Romano g£_gl, (32) noted that although phosphotransferase activity is

present in B, subtilis and Achromobacter parvulus, most strains are strict

 

aerobes. Both species, he stated, belong to genera that are predominantly
facultative anaerobes. However, there have been other reports which directly
contradict Romano's hypothesis. Cirillo and Razin (5) could find no phospho-

transferase activity for methyl u-D-glucoside in strains of Acholeplasma, a

 

species closely related to Mycoplasma, which metabolizes carbohydrates by

 

fermentation. And Sobel and Krulwich (40) reported that the strict aerobe,

7

Arthrobacter pyrindinolis, accumulates fructose via an inducible PEP-depen-

 

dent phosphotransferase system. They were able to separate the transport
system into membrane-associated and soluble components. Both fractions were
required for the phosphorylation of fructose with PEP. Some mutants of

A, pyrindonolis, having a slower growth rate with fructose as substrate than

 

the parent, were shown to have reduced levels of phosphotransferase activity.
It is possible that other strictly aerobic species will be found to
possess a phosphotransferase system. However, it is those species which
possess the capacity to anaerobically ferment sugars which benefit most from
such a system. As Roseman (34) points out, the presence of a phosphotrans-
ferase system in facultative organisms enables them to conserve metabolic
energy. It takes one molecule of ATP or its equivalent to transport a mole-
cule of sugar against a concentration gradient. In organisms where sugars
are oxidized to completion, the expenditure of one ATP to bring the sugar
into the cell is not particularly wasteful in view of the large amount of
ATP produced. However, under anaerobic conditions, the combined processes
of transport and glycolysis result in the production of only one ATP if the
result of transport is the non-phosphorylated sugar. If the sugar is phos-
phorylated during its transport, the subsequent fermentation yields two mole-
cules of ATP. The doubling of ATP production in organisms possessing a phos-
photransferase system gives them a tremendous selective advantage. The wide-
spread occurrence of group translocation testifies to such a selective pres-
sure and the logic of microbial economy.
E. ggli_actively transports the disaccharide maltose, although not by

group translocation. Accumulation of maltose was shown to be dependent on

8

the generation of energy, as sodium azide (0.01 M) inhibited the initial rate
of[;4qlmaltose uptake by 70% (47). Maltose permease activity, as well as the
enzymatic activities concerned with the metabolism of maltose, are inducible
(47). Receptors for the bacteriophage lambda are also induced by maltose
(19) .

Once inside cells of E. coli, maltose is cleaved by amylomaltase into
glucose and a polymer consisting of a-l,4-glucosidic units (46). In contrast

to E. coli, enzyme preparations of Neiserria menengitidis catalyze the direct

 

phosphorolysis of maltose to glucose and B-D-glucose l-phosphate (14). This
is analogous to the phosphorolytic cleavage of sucrose to fructose and glu-

cose-l-phosphate by enzyme preparations from Pseudomonas saccharophila (10).

 

The sucrose phosphorylase of P, saccharophila does not phosphorolyze maltose.

 

Wiesmeyer and Cohn (46) investigated the mechanism of purified amylomal-
teas, and demonstrated that glucose from the nonreducing end of maltose is
attached to the nonreducing end of the polymer. This reaction is reversible,
as shown by Doudoroff 33 El. (11). They added glucose to bacterial prepara-
tions which had synthesized polysaccharide, causing a rapid transformation
of the polysaccharide to a shortened form which no longer gave a blue-colored
complex with iodine.

If glucose is allowed to accumulate during the breakdown of maltose by
amylomaltase, the dextran formed consists of from 4 to 6 glucose units (11).
Since amylomaltase activity is reversible, longer polysaccharides can only
form in the absence of glucose. Once glucose becomes limiting, the dextran
is phosphorolyzed by maltodextrin phosphorylase, producing glucose l-phosphate.

Doudoroff g£_al, (11) demonstrated the reversibility of this activity by

9
adding glucose and glucose-l-phosphate to dry bacterial preparations. What
was observed was the production of maltose and reducing dextrins.
Doudoroff §£_§l, (11) has proposed that enzymes with catalytic properties
similar to amylomaltase be referred to as "transglycosidases". These would

include the sucrose phosphorylase of Pseudomonas saccharophila and the dextran-

 

and levan-forming enzymes of certain bacteria. Doudoroff suggested that this
type of enzyme plays an important part in the catabolism and synthesis of var-

ious carbohydrates.

MATERIALS AND METHODS

Organisms and growth conditions. B, pOpilliae NRRL B-2309MC was used

 

in all experiments. B, popilliae 2309MC is an asporogenic mutant derived
from strain NRRL B-2309M after cultivation in broth for several months (27).
It is characterized by a faster growth rate than 2309M. This strain is typi-
cal of B, popilliae since (1) it is an aerobic, non-motile rod, (2) vegeta-
tive cells are catalase negative, and (3) broth cultures demonstrate a char—
acteristic birefringence. The basal medium used contained 1.5% trypticase,
0.5% yeast extract, and 0.6% KZHPO4 (TY). Carbohydrates were filter steril-
ized separately and added to autoclaved basal media. Media were dispensed
in 50 or 200 ml amounts in 125- or 500-ml Erlenmeyer flasks, respectively.
Cultures for experiments were started with a 5% inoculum from starter cul-
tures which had reached late exponential or early stationary phase, and in-
cubated at 30 C on a rotary shaker. Stock cultures were maintained on 2%
agar slants of TY plus 0.2% glucose or 0.2% trehalose, and transferred every
two weeks.

Oxygen uptake experiments. B, popilliae was grown to mid- or late-

 

exponential phase in TY plus 0.2% glucose. Cells were harvested by centri-
fugation at 12,000 rpm for 15 min., washed twice with 0.05 M phosphate buf-
fer, pH 7.2, and resuspended in the same buffer. Oxygen uptake was measured
in a warburg apparatus, equilibrated at 30 C, by standard manometric tech-
niques (44).

Enzyme assays. All assays were performed at room temperature unless

 

otherwise specified. Hexokinase activity was assayed spectrophotometrically

10

11

by coupling the reaction to glucose 6-phosphate dehydrogenase and following
the reduction of nicotinamide adenine dinucleotide phosphate (NADP+). Each
reaction mixture (0.5 ml) contained 30 umoles glycylglycine buffer (pH 7.5),
3 umoles M3012, 0.3 umoles NADP+, 2 umoles ATP, 15 umoles carbohydrate, a
non-rate limiting amount of glucose 6-phosphate dehydrogenase, crude cell ex-
tract, and distilled water. Reduction of NADP+ was monitored by following
the increase in absorbancy at 340 nm with a Gilford model 2000 spectrophoto-
meter. One unit of enzyme was defined as the amount that reduced 1 umole of
NADP+ per min. The molar absorbance for reduced NADP+ is 6.2 x 103M'1cm'1.
Phosphoenolpyruvate (PEP) dependent phosphotransferase activities were
measured by a combination of the methods of Lee egflgi. (25) and Romano e£_§l,
(32). Cells were grown in TY plus 0.2% glucose to late exponential phase,
harvested (23,300 x g for 15 min.), washed twice with 0.05 M phosphate buf-
fer, pH 7.2, and either frozen overnight or used the same day. The cells
were suspended in 40 ml of the wash buffer; 2 ml of a 1:9 (vol/vol) mixture
of toluene-acetone was added, and the suspension was shaken vigorously for
5 min. The cells were centrifuged (27,000 x g for 20 min.) and resuspended
in a small volume of buffer. The reaction mixture (5 ml) consisted of 1 ml
toluene treated cell suspension, [l4élcarbohydrate, 3 mM MgC12, 0.03 M
KF, 0.015 M phosphate buffer (pH 7.2), distilled water, and either 1.5 mM
PEP or 1.5 mM ATP. A third reaction contained neither ATP or PEP and
served as a control. Reaction mixtures were incubated at 30-31 C in a shak-
ing water bath for 60 min. After centrifugation, (27,000 x g for 20 min.), 0.5
ml of the supernatant liquid was layered on a column (5.0 x 1.1 cm) of Dowex

AG l-x4 (100-200 mesh) in the formate form. The column was rinsed with 5 m1

12

distilled water, followed by 5 ml 0.5 M ammonium formate in 0.2 M formic
acid; 0.5 ml of each wash was counted in handifluor or PCS liquid scintilla-
tion counting fluor. Phosphotransferase activity was expressed as percent
carbohydrate phosphorylated.

Uptake of [léfilcarbohydrates. In this paper, all uptakes refer to the

 

amount of radioactivity accumulated in cells; this includes both the sugar
and any products produced by metabolism of the sugar. B, popilliae was grown
to mid- or late—exponential phase in TY plus 0.2% glucose, harvested by cen-
trifugation (23,300 x g for 15 min.), washed twice in 0.05 M phosphate buffer,
pH 7.2, and resuspended in the same buffer. Reaction mixtures (6.0 ml) con-
tained approximately 3.0 mg (dry weight) cells, [1403 carbohydrate, and buffer.
The reaction mixtures were incubated at 30-31 C in a shaking water bath; re-
actions were started by the addition of carbohydrate. At various intervals,
1 ml samples were filtered (0.45 um pore size, Millipore Corp.) and washed
with 5 ml ice cold buffer. Filters were baked in a hot air oven until com-
pletely dry, then counted in 10 m1 toluene based scintillation fluid. Where
indicated, variations of this procedure were used to determine transport kin-
etics, competition by analogs, and inhibition by energy poisons.

Purification p£_maltose and maltase assay. Our lot of maltose (Nutri-

 

 

tional Biochemicals Corporation) contained approximately 0.9% glucose contam—
ination as estimated by the spectrophotometric assay for hexokinase activity.
A small quantity of this maltose was boiled in 95% ethanol and filtered on
Whatmann #1 filter paper. The filter was washed several times with hot 95%
ethanol, the crystals were scraped off the filter, and the procedure was re-

peated three more times. Glucose contamination was reduced to 0.18%. Maltase

13
activity was assayed using the hexokinase assay. A non—rate limiting amount
of commercial hexokinase (yeast) was added to the reaction mixture to enzy-
matically remove the remaining glucose before cell extract was added.

Other procedures. The dry weight of cell suspensions were estimated by

 

measuring the OD at 620 nm. One OD unit equalled approximately 0.45 mg dry
cell weight per ml. Cells used to prepare cell extracts were grown to mid-
or late-exponential phase in TY plus 0.2% glucose, harvested by centrifuga-
tion (23,300 x g for 15 min.), and washed twice with 0.05 M phosphate buffer,
pH 7.2. The pellet was resuspended in an equal volume of 0.01 M tris (hydroxy-
methyl) aminomethane (Tris)-hydrochloride buffer (pH 7.2), containing 1 mM
dithiothreitol. Cells were disrupted by ultrasonic oscillation for four to
eight lS-sec. intervals at a peak to peak amplitude to approximately 7 pm;
the resulting crude extract was used to enzyme assays. Protein was deter-
mined by the method of Lowry e£.§l. (26), using bovine serum albumin, frac-
tion V, as standard.

Reducing sugars were measured using the glucostat reagent (Worthington
Biochemical Corp.), Method I-A, according to directions outlined by the manu-
facturers. The total volume of the assay was reduced to 2 ml. This assay
is sensitive to glucose concentrations as low as 5 pg per m1. Glucose was
used as a standard, and was measured between 5 and 40 pg per ml. The reaction
mixture (2.0 m1) contained 0.015 M phosphate buffer (pH 7.2), approximately
2000 pg maltose, and 0.2 m1 crude cell extract. Samples (0.1 ml) taken at
different time intervals were diluted in 1.9 ml H20 and immersed in boiling
water to stop the reaction, and 0.2 m1 of the dilution was used in the sugar

assay.

l4
Radioactivity was measured using a Packard Tri—Carb Liquid Scintilla-
tion Spectrophotometer (model 3320). Scintillation fluid consisted of 0.01
g l,4-bis-2-(5-phenyloxazole)-benzene and 6 g 2,5-diphenyloxazole in 1 liter
of toluene. Radioactive liquid samples were counted in handifluor (Mallin-

ckrodt Chemical Works) or PCS solubilizer (Amersham/Searle).

RESULTS

Glucose transport. Enolase, the enzyme which catalyzes the conversion

 

of 2-phosphog1ycerate to phosphoenolpyruvate, is inhibited by low concentra-
tions of fluoride (20). When cells of B. popilliae were incubated in the
presence of 0.01 M NaF,[?4§]glucose uptake was completely inhibited (Fig. 1).
Previous data indicated that 0.01 M fluoride inhibited glucose oxidation approx—
imately 50%, and that 0.1 M fluoride inhibited completely (29). The inhibition
of glucose oxidation by the cells was rechecked at different glucose concen-
trations. When the glucose level was 2.5 x 10'3 M or higher, 0.01 M NaF only
inhibited by about 50%, but complete inhibition of oxygen uptake was observed
when 5 x 10‘4 M glucose was used.

Since these findings indicate that glucose is transported by group trans-
location, assays for phosphotransferase activity were conducted. Trehalose
was the first substrate used in the assay, and served as a positive control,
since it has been shown that trehalose is transported via a phosphotransfer-
ase system (3). In addition to glucose, udmethyl D-glucoside (a MC), which
is not readily metabolized by glucose grown B. popilliae cells (4), was also
tested. It will be demonstrated later that uMG appears to function as a
glucose analog in uptake studies. The results clearly demonstrated the
dependence on PEP for the production of phosphorylated sugar from trehalose
(Table 1). In contrast, glucose was phosphorylated to about the same extent
when either PEP or ATP was present. However, two assays conducted with

aMG as substrate indicated that its phosphorylation was largely PEP-de-

pendent.

15

16

 

 

UPTAKE (NMOLES/MG DRY WT)

 

 

 

 

MINUTES

Fig. 1.--Sodium Fluoride (0.01 m) inhibition of‘ [Me]

glucose (10-5M, 1).:Ciylmole) uptake by _B_. popilliae.
Symbols: (9, without NaF; ., with NaF.

17

Table 1.-—Phosphotransfsrase activities of B, popilliae

 

 

cellsa.
Dry weight Substrate fiphosphorylation with:
(”9) ATP 959
149 Trehalossb 0.2 17.0
109 ciucosac 34.3 29.0
31 Methyl di-D-glucosided 3.3 12.5
153 Methyl cc-o-giucoaide" 0.1 4.9

 

8The extent of substrate phosphorylation was determined
by the chromatographic procedure of Romano et a1 (33).

Controls were run without ATP or PEP, and the appropriate

corrections made.

b

0.1 n [146] trehalose (0.02quiéumole)

c0.1 M [149] glucose (0.02/uCi/junole)

d

0.1 fl [14C] methyl arO-glucoside (0.1/utiéumols)

18

The generation of a phosphorylated glucose derivative by B, popilliae
utilizing ATP as the sole phosphoryl group donor may be due largely to a hexo-
kinase present in the cells since crude cell extracts demonstrated high kinase
activities (Table 2). No production of glucose 6—phosphate could be detected
when PEP was substituted for ATP in the spectrophotometric assay. Probably
the concentration of extract in the reaction was too low for PEP-PTS activity
to be detected (16). Fructose and mannose functioned as substrates for the
kinase assay, which is indicative of hexokinase, rather than glucokinase,
activity. Neither the mannose or fructose were checked for glucose contamina—
tion. However, glucose grown B, popilliae oxidizes mannose as rapidly as glu-
cose (4), suggesting that a relatively non-specific hexokinase is involved in
the metabolism of both sugars.

To further investigate the transport of glucose, we examined various non-
metabolizable analogs of glucose for one which could be accumulated as rapidly
as glucose. Romano e£_§l, (32) successfully used 2—deoxy-D-glucose in their
survey of phosphotransferase activity in various bacteria, but we discovered
that B, popilliae transported this analog very slowly. Uptake of N-acetyl-D-
mannosamine could be detected only at very high concentrations. Methyl a-D-
glucoside is frequently used as a glucose analog (33), and was found suitable
for our studies. This compound was taken up rapidly by cells, and its uptake
was inhibited by 0.01 M NaF (Fig. 2). Growth studies with B, popilliae in TY
media plus 0.2% aMG indicated that this culture would not metabolize the glu-
coside, and previous studies ( 4) demonstrated that cells of this strain could
only use it after a fairly long period of induction (4 to 5 hr. lag). The

uptake of[14§]glucose (10'5 M) was negligible in the presence of 5 x 10'4 M

19

Table 2.-stokinase activities in extracts of B.
popilliae cells‘.

 

Substrate TYG ggown cellsb TYM grown cellsc

 

ATP No ATP ATP No ATP
Glucose 0.0905 0 0.1037 0
Fructose 0.0851 0 0.0836 0
Hannose 0.0501 0 0.0309 0

 

9Assays were based on the reduction of NADP+ by glucose
6-phosphata dehydrogenase and were conducted as described
in Materials and Methods. Enzyme activities are expressed
as units of enzyme per mg protein.

bTY medium plus 0.2% glucose.

cTY medium plus 0.2% maltose plus 0.05% glucose.

20

 

UPTAKE(NMOLES/MG DRY WT)

 

 

 

 

 

 

 

l 2 3 4 ‘ 5 6

MINUTES

Fig. 2.--Sodium Fluoride (0.01 N) inhibition of [14C]
methyl OL-D-glucoside (10-5M, 1}JCl/}Jm018) uptake by B

popilliae. Symbols: 0, without NaF; O, with NaF.

21

glucoside (Fig. 3), and the uptake of [14C) glucoside (10'4M) was lowered in
the presence of 10'3 M glucose (Fig. 4). Unfortunately, neither the concen-
trations or ratios were the same in the two experiments and this may account
for the difference in the results obtained. However, the data do indicate
that the two sugars may share a single uptake system.

Since methyl u-D-glucoside was not immediately metabolized, we were able
to study the kinetics of its transport. Initial rates of transport over a
range of concentrations were estimated after 20 sec. of incubation with [14g
glucoside (Fig. 5). The transport system appeared to become saturated at
6 x 10‘5 M. The apparent Km value from a Lineweaver-Burk plot (Fig. 6) was
6.7 x 10‘6 M. A similar value was obtained with glucose, but a determination
of the Km was not attempted due to the rapid metabolism of glucose once inside
the cell.

Studies with maltose. Maltose is taken up bY.§° popilliae cells at a

 

slower rate than glucose. At 10‘5 M, an average of 142 picomoles of maltose
was taken up per min per mg dry cell weight, while 350 picomoles of glucose
was accumulated under identical conditions. Maltose uptake was not affected
by 0.01 M NaF or 0.01 M NaN3 (Fig. 7). However, oxidation of maltose was in-
hibited by 0.1 M concentrations of fluoride and azide (Fig. 8).

The kinetics of maltose uptake bY.§3 popilliae cells were completely dif-
ferent from those observed with glucose and aMG. Initial rates of transport
were calculated after allowing cells to incubate with [14C] maltose for 15 sec.
As shown in Fig. 9, intracellular accumulation of maltose increased as the
extracellular concentration increased and did not reach a limit even at 10'3 M.
These data indicate that maltose is diffusing, either mediatively or passively,

through the membrane.

22

 

UPTAKE (NMOLES/HG DRY WT)

 

 

 

 

 

MINUTES

Fig. 3.--Methyl Ot-D-glucoside (s x 10‘4m) inhibition
of‘ [140] glucose (1076M, 1pCi/jlmole) uptake by B.
popilliae. Symbols: ., without «MC; 0, with «MG.

23

 

 

 

 

 

 

5
I I I l I

4 3'— ._1
E:
Z
>4
m 3 -— __
Q
O
< #9
U)
m
._l
O
E e
V 2 in— —
[:3
M
E. .
b. I
D

l—- .—

1 I l l l l

l 2 3 4 5 6
MINUTES

Fig. 4.--Glucose (10‘3m) inhibition of‘ [MC] methyl
ct-O-glucoside (10-41“, 0.5}lCi/jlmole) uptake by B.
ponilliae. Symbols: ., without glucose; 0, with glucose.

24

 

UPTAKE (NMOLES/MIN/MG DRY WT)

 

 

 

31|||1||1||

20 40 60 80 100

 

METHYL a-D-GLUCOSIDE uM

Fig. 5. Kinetics of [14C] methyl ct-D-glucoside (0.5
‘pCiéumole) transport. Initial rates of transport were
estimated after incubation (30 C) for 20 sec. Each reac-
tion mixture contained 0.49 mg (dry weight) cells. Reac-
tions were terminated by the addition of 5 ml ice cold
phosphate buffer (0.05 M, pH 7.2).

25

 

l/[UPTAKE]

 

 

 

 

Fig. 6.-Lineweavsr-Burk plot of kinetic data From
Fig. 5.

26

 

800

600

400

200

UPTAKE (PICOMOLES/MG DRY WT/MIN)

 

Fig. 7.
(1}1Ci/jlmole)

of incubation

 

l L J L l

20 40 60 80 100

 

MALTOSE, uM

Effects of inhibitors on [14C] maltose

uptake. Uptake was determined after 1 min
at 30 C. Each reaction mixture contained

0.5 mg (dry weight) cells. Symbols: 0 , without inhibitor;

0, with 0.01 m NaF; A , with 0.01 m NaN

3.

27

 

 

 

 

30 I I I I I
E'4
3
>4
‘23
U 20'—
E
\
CJ
DJ
E
D __ —-4
U)
Z
O
U
ON
H 10,. ——
1
. C
0 *———- /' . --I
l l I I I
15 30 45 6O 75
MINUTES

Fig. 8. Inhibition of maltose (0.01 m) oxidation.
Oxygen uptake was measured in a Uarburg apparatus equili-
brated at 30 C. All values were corrected For endogenous
activity. Each Flask contained 5 mg (dry weight) cells.

Symbols: 0 , without inhibitors; A , with 0.1 M NaN 0
with 0.1 N KF.

33

28

Fig. 9. Kinetics of [146 maltose (2 ’uCi/pmole)
uptake by B. popilliae. Initial rates of transport were
estimated after incubation (30 C) for 15 sec. Each
reaction mixture contained 0.55 mg (dry weight) cells.
Reactions were stopped by addition of 5 m1 ice cold
phosphate buffer (0.05 M, pH 7.2).

29

 

 

 

 

 

 

 

 

 

 

 

lll

 

 

.10

00
O

\O
O
0

(EM 180 sw/le/saqonu) axvman

10

(x 104)

(x 105)

(x 105)

M

MALTOSE,

30

The cleavage of maltose by crude cell extracts was indicated by the
maltose-dependent reduction of NADP+ by glucose 6-phosphate dehydrogenase.
The specific activity of "maltase" in the crude extracts was 0.0256 umole
NADP+ reduced per min per mg protein. Cleavage of maltose resulted in the
production of either glucose, glucose 6-phosphate, or some derivative of glu—
cose which, in combination with the extract, formed glucose 6-phosphate. No
maltase activity was detected by the assay procedure when PEP was substituted
for ATP. In addition, no increase in the amount of reducing sugar occurred
when maltose was incubated with crude cell extracts. In this experiment, ATP
was omitted to prevent the further metabolism of any cleavage produce. Chlora-
mphenicol had no effect on the oxication of maltose by glucose grown cells
(Fig. 10), demonstrating that the maltase activity is constitutive. Further
support for this conclusion comes from our finding that extracts from glucose

grown cells contain spectrophotometrically detectable maltase activity.

31

 

 

 

 

50 I I I I
E‘
3
>4 .
a: I
0 I
E I
Q
ELI I
:3 I
D /
a ,I
53 30 r- ’ '—
N
O [I
I
I; /
I
I
I
20 r- ” “
I
’I
I ,’o
I ’I’ '
I ,0
I ’,v’ .
I
10 -- ‘ ’I’o -"3
I 1’
I
I ”’e
I ,’
15 30 45 60 75
MINUTES

Fig. 10. Constitutive oxidation of maltose. Cells
were grown in TY media plus 0.2% glucose. Respiration
was measured in a Uarburg apparatus equilibrated at 30 C.
All values were corrected for endogenous oxygen uptake.
Each Flask contained 5 mg (dry weight) cells. Symbols:
0 , with glucose (0.01 m); o , with maltose (0.01 m);
dashed lines, with chloramphenicol (0.1 mg/ml).

DISCUSSION

Saturation studies indicate that glucose and uMG are actively transported
bY.§- popilliae. aMG is an appropriate analog of glucose because it appears
to be transported as well as glucose. In addition, glucose uptake is inhibited
almost completely in the presence of a 50 fold excess of the glucoside. Pre-
sumably, uMG competitively saturates the glucose transport system.

Fluoride inhibits the uptake of both [I40] glucose and [14C] aMG by B.
popilliae. This inhibition of transport by an enzyme poison which affects the
production of phosphoenolpyruvate suggests that these sugars are accumulated by
a PEP-dependent phosphotransferase system. Freese e£_§l, (15) reported that that
PEP-dependent glucose phosphotransferase system of B. subtilis was able to phos-
phorylate aMG, whereas the glucokinase was not. Although we did not test
whether the hexokinase in 2309MC could phosphorylate uMG, ATP did not appear
to serve as a significant phosphoryl donor in the phosphotransferase assays
with the glucoside as substrate. Toluenized cell suspensions 0f.§- popilliae,
poisoned with fluoride, preferentially used PEP to phosphorylate aMG.

The nearly equal amount of glucose phosphorylation by toluenized cells
with either PEP or ATP is presumably a result of both phosphotransferase and
hexokinase activity. Since toluenized membranes allow a passive equilibration
of an intracellular small molecular weight compound with any given extracell-
ular concentration of the compound (16), glucose would not need to be actively
transported to cross the permeability barrier imposed by the cell membrane.

Unaltered glucose would be freely accessible for phosphorylation by the ATP-

32

33
dependent hexokinase. However, the large amount of phosphorylation obtained
with PEP suggests that it too is being utilized directly as a phosphoryl group
donor. Lee §£_§l, (25) similarly found that toluenized cells of lactic strep-
tococci would phosphorylate glucose with PEP or ATP and concluded that glucose
was transported by a phosphotransferase system.

While the data are not conclusive, it appears probable that B, popilliae
possesses a PEP-PTS for glucose transport. This system has been found in many
other members of this genus (21), and trehalose, the natural carbon and energy
source of B, popilliae, is phosphorylated by a PEP-dependent system during up-
take (3). In addition, possession of a phosphotransferase system allows organ-
isms to conserve metabolic energy (34).

Studies of maltose uptake over a range of concentrations indicated that this
sugar enters the cell by simple diffusion. There was no evidence of saturation
of the transport mechanism at substrate concentrations between 10"6 and 10"3 M.
Also, accumulation of maltose was not affected by the presence of energy poisons
at 0.01 M concentrations, although oxidation of maltose was inhibited by 0.1 M
fluoride and 0.1 M azide. At the higher concentrations, these poisons inhibit
electron transport, which would account for these observations (30).

Once inside the cell, maltose is cleaved into as yet unknown products, at
least one of which eventually becomes glucose 6-phosphate. Cleavage of mal-
tose may or may not occur without the simultaneous phosphorylation of one of
the products, since no activity could be detected when ATP was omitted from
the spectrophotometric assay. In £3.2211; maltose is cleaved into glucose and
a short dextran by amylomaltase, without the production of a phosphorylated
sugar (46). Maltose cleavage in B, popilliae may be dependent upon the pres-

ence of ATP, since we failed to find any increase in the amount of reducing

34
sugar when maltose was incubated with crude extracts without ATP. However,
the amount of reducing sugar produced may have been too small to be detected
by our assay, since the assay's limit of detection of 5 ug/ml (2.7 x 104 pico-
moles) would require a relatively large amount of cleavage to cause a change in
OD. No ATP was added to the assay for fear of metabolizing, and consequently
not detecting, any glucose produced.

Freese SE él- (15) found that glucose phorphotransferase activity of B.
subtilis declines toward the end of growth. The decrease in the ability to
transport glucose correlated with an inability of glucose to suppress sporula-
tion of cells. They noted that a mutant which grew slowly on glucose and could
sporulate even when glucose was present throughout growth lacked PEP-PTS activity.
It would be interesting to see if group translocation of trehalose ble. popilliae
similarly declines as cells enter stationary phase. Since trehalose is cleaved
to glucose and glucose 6-phosphate inside the cell, limiting trehalose accumu-
lation would decrease the intracellular glucose concentration. Possibly, this
is a method of limiting the accumulation of metabolites which could repress the
initiation of sporulation (15). Regulation of tranSport activity may be one
mechanism inherent in the lp yiyp situation which allows B, popilliae to con-

tinue its developmental cycle and sporulate efficiently.

LITERATURE CITED

35

10.

ll.

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A

 

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