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J. Biosci., Vol. 6, Number 2, June 1984, pp. 203–212.
© Printed in India.
Localization of 3-phosphoglyceric acid synthesis in the mother cell
compartments and forespores of Bacillus megaterium and the effects of
manganous ions on its metabolism
RAVENDRA PAL SINGH
Biochemistry Division, Regional Research Laboratory, Jorhat 785 006, India
MS received 13 December 1983; revised 26 March 1984
Abstract. Rapidly metabolizable compounds such as glucose or glycerol were not utilized by
Bacillus megaterium in the absence of manganese when grown in the supplemented nutrient
broth medium. Under these conditions, growth ceased at low cell titre, 3-phosphoglyceric acid
accumulated inside the cells and normal sporulation process was arrested. Addition of
manganese to the medium caused disappearance of 3-phosphoglyceric acid, growth resumed
and normal sporulation was observed. Synthesis of 3-phosphoglyceric acid occurred only in
the mother cell compartments and it was transported for accumulation inside the forespores of
Bacillus megaterium when grown in supplemented nutrient broth medium. Incubation of
forespores in the presence of glucose or glycerol had no effect on 3-phosphoglyceric acid
synthesis/accumulation, but it was completely utilized when forespores were incubated with
manganese plus ionophore (X 537A). No other metal(s) could substitute for manganese
suggesting that manganese plays crucial role in 3-phosphoglyceric acid metabolism.
Keywords. Bacillus megaterium; 3-phosphoglyceric acid; dipicolinic acid; germination; sporulation; ionophore.
Introduction
The processes of sporogenesis and spore germination in bacilli have been considered as
an unique model for studying the mechanism of differentiation because of the relative
simplicity and the ready application of biochemical and genetic analysis to this system
(Singh, 1983). During late stationary phase two distinct intracellular compartments,
namely mother cell and forespore are formed (Young and Fitz-James, 1959). The latter
compartment is metabolically active before becoming a dormant spore (Church and
Halvorson, 1957; Setlow and Kornberg, 1969). The most significant event during
sporulation is the accumulation of 3-phosphoglyceric acid (3-PGA) in the forespore/dormant spore which is utilized during the first minutes of spore germination
(Setlow and Kornberg, 1970; Singh et al., 1977; Singh and Setlow, 1979b). Among metal
ions, manganese has been shown to play an essential role during normal sporulation in
bacilli (Charney et al., 1951) and it is specifically required as a cofactor for
Abbreviations used: 3-PGA, 3-Phosphoglyceric acid; DPA, dipicolinic acid; SNB, supplemented nutrient
broth; buffer A, 0·6M sucrose, 0·1 M potassium phosphate buffer (pH7·0) and 16mM MgSO4; HSF, heat
stable forms.
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Singh
phosphoglycerate mutase (Oh and Freese, 1976; Singh and Setlow, 1979a) and fructose1, 6-diphosphatase (EC 3.13.11) (Opheim and Bernlohr, 1975).
In the present communication evidence is presented to show the significance of
manganese in the metabolism of 3-PGA and utilization of catabolizable substrates such
as glucose and glycerol during normal sporulation in B. megaterium. These results also
indicate the possible site in the mother cell compartment for 3-PGA synthesis which is
subsequently transported for accumulation in the developing forespore inside the
sporulating cell.
Materials and methods
Reagents and enzymes
[14C](U)-Glucose, [14C](U)-glycerol and 32Pi(H3PO4) were purchased from New
England Nuclear Corpn., Boston, Massachusetts, USA. Phosphoglycerate mutase (EC
2.7.5.3) and enolase (EC 4.2.1.11) were purified from actively growing vegetative cells of
B. megaterium (Singh and Setlow, 1978b; 1979a). ADP was freed from contaminating
ATP by incubating with hexokinase (EC 2.7.1.1) and glucose with subsequent boiling.
The ionophore (X 537A) (Pressman and de Guzman, 1975) was a gift from Dr. W. E.
Scott (Hoffman La Roche) and stock solution (1 mg/ml) was prepared in ethanol. All
other enzymes and chemicals were obtained from Sigma Chemical Co., St. Louis,
Missouri, USA.
Organism, cultural conditions, germination studies and isolation of forespores
B. megaterium QM B 1551 was obtained from Dr. Hillel S. Lovinson (US Army
Development Command Center, Natick, Massachusetts, USA). The organism was
grown at 30°C in supplemented nutrient broth (SNB) medium (Setlow, 1975) and
spores harvested.
The method for the isolation of forespores is a modification of Ellar and Postgate
(1974) procedure. Samples (50 ml) of sporulating cells were centrifuged (5 min,
10,000 g) and washed with 25 ml of warm (37°C) buffer A (0·6 M, sucrose; 0·1 M,
potassium phosphate buffer (pH 7·0) and 16 mM of MgSO4). The cell pellet was
suspended in 6 ml of buffer A. To this, lysozyme (10 mg) was added followed by
incubation for 10 min at 37°C. The lysozyme treated cells were washed twice with cold
buffer A. The cells were suspended in 6 ml of cold buffer A and sonicated (30 s to
1·5min, maximum output of sonifier cell Disrupter, Heat systems-Ultra Sonics, Inc.,
New York, USA) to release forespores. The sample was centrifuged twice for 3 min at
7,000 g and 3 min at 6,000 g respectively. The final forespore pellet had a very low level
of contamination with mother cells and cell debris as seen under phase contrast
microscope. Whole cell was used to study 3-PGA synthesis in mother cell compartment
which is whole cell minus forespore. Germination of dormant spores was carried in
KBr solution (Singh and Setlow, 1979b).
Labelling and identification of 3-PGA
The organism was grown in SNB medium containing 1 µCi of either glucose or
[
Metabolism of 3-PGA in B. megaterium
205
glycerol [14C] (U) or 32Pi per ml. To this, 20 mM glucose or glycerol was added when
the culture reached 3·5–5·0 absorbance at 600 nm. At various time intervals, 5ml
culture was filtered through a membrane filter (0·45 mµ) pore size (Milipore Corpn,
Bedford, Massachusetts, USA) and the filter was extracted with 2 ml ice cold formic
acid (0·5 M). The extracts were chromatographed on a column of AG1- × 8 (chloride
form; 100–200 mesh; 0·8 by 10 cm size) by elution with HC1 (20 mM) at a flow rate of
1 ml/min and fractions (3 ml) were collected. A 2 ml portion of each fraction was dried
in a scintillation vial and its radioactivity was determined using liquid scintillation
counter (Packard Co., Dowers Grove, Illinois, USA). A reference sample of 3-PGA
(0·1 µ mol) was also chromatographed under the same conditions and 0·1 ml of each
fraction was assayed colorimetrically for 3-PGA (Bartlett, 1959). The enzymatic
identification of 3-PGA was done using enzymatic coupling methods i.e. the formation
of glycerolphosphate or lactate from 3-PGA (Czok, 1974).
Extraction and determination of 3-PGA and DPA from sporulating cells, forespores and
dormant spores
Forespores were isolated at designated times (arrows in figure 1), sporulating cells
collected at about the time when DPA concentration was less than 5% and dormant
spores were prepared by growing-bacterium in SNB medium and cleaned (Singh,
1982b). 3-PGA and dipicolinic acid (DPA) were extracted from freeze dried samples of
forespores, sporulating cells and dormant spores (each from 50 ml culture) by boiling in
80% n-propanol (Setlow and Kornberg, 1970).
3-PGA was assayed using luciferase assay method (Singh and Setlow, 1979b) after
conversion of 3-PGA to ATP using ADP, enolase, phosphoglycerate mutase and
pyruvate kinase as reported earlier (Singh, 1982b). DPA was determined colorimetrically (Rotman and Fields, 1967). Heat stable forms (HSF) were evaluated by the
formula proposed earlier (Singh, 1982a).
Incubation of forespores
Forespores samples were incubated at 30°C with slow shaking (40 strokes/min) along
with either glycerol or glucose (20 mM) in buffer A to determine de novo synthesis of
3-PGA. Sporulating cells or dormant spores were also used under similar conditions.
At various intervals, samples were collected by centrifugation (5 min at 10,000 g), the
pellet lyophilized, used for 3-PGA extraction and determination. In some experiments,
ionophore dependent in vivo utilization of 3-PGA in the forespores was carried out in
buffer A containing ionophore (10 µg/ml) plus manganese (1 mM) and samples were
collected for 3-PGA estimation. The concentration of glucose in the growth medium
was enzymatically determined (Gutman and Wahlefield, 1974) after cells had been
removed from the culture by centrifugation.
Results
Effects of manganese on growth and sporulation
The organism grown in SNB medium accumulated 3-PGA about 1·5 h before DPA
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accumulation followed by the appearance of heat stable forms (figure 1) when the
concentration of manganese is about 20 µM. In the absence of manganese, the organism
failed to sporulate (1–3% spores when viewed under microscope), growth was
considerably low (figure 2) and DPA synthesis/accumulation was not noticed (figure 1).
The most interesting observation was the inability of the organism to utilize glucose in
the absence of manganese while glucose was completely metabolised when the medium
was supplemented with 20 µM manganese (figure 2) which suggested that the
supplementation of SNB medium with manganese and glucose greatly enhanced
Figure 1. Accumulation of 3-PGA, dipicolinic acid and development of heat stable forms
during sporulation of B.megaterium. The organisms was grown in SNB medium in the absence
or presence of manganese (20 µM). The forespores were isolated at designated times (shown by
arrows) as mentioned in the texts. 3-PGA, DPA and HSF were determined.
Figure 2. Growth and sporulation of B. megaterium in SNB medium plus glucose (20 mM)
in the presence or absence of manganese (20 µM). Utilization of glucose from culture medium
was done as mentioned in the texts.
Metabolism of 3-PGA in B. megaterium
207
growth and some of the spore specific events such as DPA synthesis and appearance of
heat stable forms.
Dependence of 3-PGA metabolism on manganese levels in the medium
More than 95% of glucose or glycerol from the medium was metabolized when the
organism was grown in SNB medium containing manganese (20 µM) but these
carbohydrates were not metabolized in the absence of manganese (figure 2) (data on
glycerol utilization not given). To identify the metabolic block in the absence of
manganese, the organism was grown in SNB medium containing 32Pi (1 µCi), when the
optical density reached 3·5, a sample was taken for 3-PGA extraction and 20 mM
glycerol was added to the rest of the culture. Thirty min later, another sample was taken
and 50 µM of MnCl2 was added to the remaining medium. After another 30 min, a third
sample was taken. Thin layer chromatography of cell extract in all the samples showed
the accumulation of radioactive 3-PGA after glycerol addition which disappeared after
manganese addition (data not shown). Also 3-PGA was further identified by isolating
extracts from cells grown either in the presence of [14C] glycerol or [14C] glucose
(1 µCi/ml) in the medium without manganese followed by Dowex-1-column chromatography of the extracts. A major peak appeared at the same position as the peak of
authentic 3-PGA sample (figure 3).
The amount of 3-PGA that accumulated inside the cells and in the culture medium
were also estimated (table 1). The intracellular concentrations of 3-PGA increased
significantly when glucose (data with glucose not given) or glycerol was added in SNB
medium in absence of manganese (table 2). Some 3-PGA also leaked out into the
medium and its concentration did not change significantly even after manganese
addition, whereas intracellular 3-PGA was no longer detectable in the cells.
Table 1.
5
Accumulation of 3-PGA under various cultural conditions.
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Figure 3. Column chromatographic separation of [14C] or [32P] labelled compounds in
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cells. For labelling, 20 mM (U- C)-glycerol or glucose or [ Pi] (1 µCi) was added to the
culture in SNB medium and after 30 min incubation cells were extracted with formic acid.
A column (0·8 × 12 cm) anion exchanger AG1- × 8 chloride (100–200 mesh) was charged with
the extract and eluted with HC 1 (20 mM) at a flow rate of 1 ml/min as mentioned in the texts.
A. Elution pattern of the [32P]-labelled extract. B. Elution pattern of the [14C] glycerol or
glucose extract. C. Mixture of the [14C] or [32P]-labelled extracts and reference 3-PGA, all
eluted from the same column.
Table 2. Effects of glycerol on 3-PGA accumulation in forespores and
sporulating cells
a
Sporulating cells were harvested at (A6OO nm = 5·5) washed twice and suspended in
warm buffer A + glycerol (20 mM). 3-PGA was extracted before and after incubation
(30 min) and assayed as described in the text.
b
Forespores samples were prepared from the culture isolated at designated by arrows
1 to 3 in figure 1. Incubation etc. was done as stated above.
c
Dormant spores were isolated from culture when > 95% free when viewed under
phase contrast microscope.
[
Metabolism of 3-PGA in B. megaterium
209
Accumulation of 3-PGA in forespores and spores
It has been well documented that 3-PGA is accumulated late during stages of
sporulation i.e. about 1·5 h before the accumulation of DPA (figure 1). Previous work
has shown that more than 90% of 3-PGA was accumulated within the forespores which
are destined to form dormant spores but the site of 3-PGA synthesis whether mother
cell compartment or forespore was not determined (Singh et al., 1977). Results
presented in table 2 indicated that forespores (containing about 5–10% DPA) when
incubated with either glycerol or glucose failed to synthesize 3-PGA while sporulating
cells isolated at the corresponding times were able to synthesize significant amount of
3-PGA and transported for accumulation inside the developing forespores (table 2).
Under these experimental conditions, mother cell compartment represents whole cell
(sporulating cell minus forespore). These data clearly demonstrated mother cell
compartment as the site of 3-PGA synthesis. Interestingly, the intracellular concentrations of 3-PGA in the sporulating cells or forespore could not be increased once an
initial 3-PGA (21 nmol) level was attained (table 2), though synthesis of 3-PGA was
observed which was excreted out in the medium.
In vivo utilization of 3-PGA in the forespores and dormant spores
Forespores prepared at the designated times (arrows in figure 1) have stable 3-PGA
pool (table 3) which suggested that one of the enzymes needed for its catabolism may
not be fully active. Earlier studies (Singh and Setlow, 1978a; 1979b have shown that
the phosphoglycerate mutase requires manganese as a cofactor and was inactive in
forespores and dormant spores. Results presented in figure 4, showed that the
intracellular level of 3-PGA was constant when forespores were incubated with or
without manganese or ionophore alone (data not shown) but 3-PGA level declined
rapidly when forespores were incubated with manganese plus ionophore (figure 4). As
expected manganese alone cannot cross the permeability barrier of forespore
membrane and is facilitated by the ionophore.
Earlier studies have demonstrated that the 3-PGA was utilized to produce ATP and
NADH needed during early phases of spore germination (Setlow and Setlow, 1977;
Singh et al., 1977). Incubation of dormant spores with manganese plus ionophore has
Figure 4. Effect of ionophore plus manganese on 3-PGA levels in isolated forespores. Forespores isolated when containing
about 60% of the maximum amount of 3PGA were incubated at 30°C in buffer A. The
control represents incubation with either
ionophore or manganese alone. 3-PGA extracted and analysed as described in the texts.
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no effect on 3-PGA utilization (data not shown) probably because the dormant spore
has two distinct membranes namely, cortex and plasma membrane which this
ionophore probably failed to penetrate. During the germination of dormant spores in
salt solution (KBr), 3-PGA was completely metabolised within 30 min (table 3).
Addition of NaF (10 mM) arrested 3-PGA utilization under these conditions.
Table 3. Levels of 3-PGA during various stages of
growth.
Cells or spores were extracted and the extracts were passed through
Norit column to remove nucleotides before 3-PGA assaying.
a
Harvested at the point noted by arrow No. 2 in figure 1.
Discussion
A large amount of 3-PGA was accumulated during late stages of sporulation, about
1·5h before DPA synthesis (figure 1). This 3-PGA pool was stable in both isolated
forespores and dormant spores inspite of the presence of all the necessary enzymes
needed for its catabolism namely, phosphoglycerate mutase, enolase and pyruvate
kinase (Singh et al., 1977; Singh and Setlow, 1978a, 1979b). Since 3-PGA accumulation
occurred in developing forespores even though the enzymes for 3-PGA catabolism
were detected in extracts (forespore or spore) at levels similar to those in growing cells
or germinated spores (Singh et al., 1977), at least one of these enzymes must have had
very little activity in vivo and was activated upon spore germination. 3-PGA was
completely metabolised during first minutes of spore germination to generate much of
the ATP and NADH needed for out growth (Setlow and Setlow, 1977; Setlow and
Kornberg, 1970). As expected, 3-PGA utilization was completely inhibited when
germination was carried out in KBr medium containing NaF suggesting that the
3-PGA catabolism occurred in the forward direction of glycolytic pathway i.e. 3-PGA
to pyruvate as NaF is a known inhibitor of enolase (Singh and Setlow, 1978b). It cannot
be ruled out that the slight decrease in 3-PGA levels in germinated spores in the
presence of NaF may be due to the operation of other pathways not involving enolase.
Earlier studies have shown that phosphoglycerate mutase was inactive in the
forespores and dormant spores and was activated upon spore germination (Oh and
Freese, 1976; Singh and Setlow, 1978b, 1979a). This enzyme specifically requires
manganese (Watabe and Freese, 1979) as a cofactor and not 2,3-diphosphoglycerate
as reported in the literature (Grisolia and Carreras, 1975). Recently it was
proposed that free manganous ions are chelated under in vivo conditions by a
small molecular weight protein which was synthesized at about the same time that
3-PGA starts accumulating in the developing forespores (Singh, 1982b). These
|
Metabolism of 3-PGA in B. megaterium
211
observations suggest that the intracellular levels of free manganous ions play a
significant role in 3-PGA metabolism when the organism was grown under various
culture conditions (table 2). Similar observations were reported by Oh and Freese
(1976) using various mutants of B. subtilis. Our earlier data on manganese uptake by the
bacterium suggested that the sporulating cells were able to accumulate more than 95%
manganese of the medium. About 70% of the total manganese present in dormant
spores was released along with DPA and other smaller spore integuments during
germination and the remaining 30% manganese could be bound loosely to spore
components as evident from the manganese exchange data (Singh and Setlow, 1979b).
Most interesting observations were the in vivo utilization of 3-PGA when the
forespores were incubated with ionophore plus manganese (figure 4). These data
support the earlier suggestions that the phosphoglycerate mutase was inactivated due
to nonavailability of free manganous ions and could be activated in vivo upon
incubation of forespores with ionophore plus manganese, suggesting that the free
manganese was not available to the enzyme in sufficient amount.
Experiments were also done to determine the site of 3-PGA synthesis i.e. mother cell
compartment and/or forespore. Results indicated that the incubation of forespore
samples either with glycerol or glucose (data not shown) has no effect on 3-PGA
synthesis (table 2). On the other hand, in the incubation of sporulating cells which
represents mother cell compartment plus forespore under these conditions, there was a
significant increase in intracellular 3-PGA pools suggesting that the 3-PGA was
synthesized in the mother ceil compartment and transported for accumulation in the
forespores.
Acknowledgement
The author is grateful to the Director, Regional Research Laboratory, Jorhat, for a
permission to publish.
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