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Journal of General Microbiology (1984), 130, 309-31 8. Printed in Great Britain
309
Isoleucine Synthesis by Clostridium sporogenes from Propionate or
a-Methylbutyrate
By D . J . M O N T I C E L L 0 , t R . S. H A D I O E T O M 0 , S A N D R . N . C O S T I L O W *
Department of Microbiology and Public Health, Michigan State University, East Lansing,
MI 48824-1101, USA
(Received I0 June I983 ;revised I4 September 1983)
Preliminary studies demonstrated that Clostridium sporogenes synthesized isoleucine by a
pathway not involving threonine or threonine dehydratase. Radiotracer experiments with cells
grown in a defined carbohydrate-free medium showed that radioactivity from [U-l Tlserine, [314C]pyruvate, [ 14C]NaHC03 and [ 1-1, [2-] and [3-14C]propionate was incorporated into
isoleucine. Conversely, there was no detectable incorporation of 4C into isoleucine during
growth with [U-14C]glutamate, [U-14C]threonine, [U-14C]valine, [U-14C]leucine or [U14C]methionine.Crude extracts of the bacteria grown in a minimal medium contained levels of
a-acetohydroxyacid synthase activities comparable to those in Escherichia coli K 12 grown in
minimal medium. Stepwise degradation of isoleucine obtained from C . sporogenes grown in the
presence of specifically-labelledprecursors indicated that C. sporogenes can make isoleucine via
the reductive carboxylation of propionate to yield a-oxobutyrate, which is metabolized to
isoleucine in the classical fashion. Isoleucine was also formed by C. sporogenes via the reductive
carboxylat ion of a-methyl butyrate to a-0x0-P-methylvalerate.
INTRODUCTION
The amino acid requirements for growth of Clostridium sporogenes and Clostridium botulinum
type A were examined several years ago (Campbell & Frank, 1956; Kindler & Mager, 1956;
Mager et al., 1954). These bacteria were found to be indistinguishable in nutritional
requirements, and have since been shown to share 100% homology of DNA (Lee & Riemann,
1970; Wu et al., 1972). The mechanisms by which the clostridia use amino acids as both electron
donors and acceptors (the Stickland reaction) have been studied extensively (Barker, 1981). Less
consideration has been given to the synthesis of amino acids by strictly anaerobic bacteria. The
development in our laboratory of a simple defined medium for C . sporogenes (Hadioetomo, 1980)
has allowed us to initiate studies of branched-chain amino acid metabolism in these bacteria.
The mechanisms and regulation of isoleucine, leucine and valine synthesis in bacteria have
been studied in some detail (Umbarger, 1978; Iaccarino et al., 1978). The principal aerobic
pathway of isoleucine synthesis involves the dehydration and deamination of threonine to aoxobutyrate, followed by a condensation with pyruvate to yield a-aceto-a-hydroxybutyrate,
which is then converted through a series of reactions to isoleucine. A similar condensation of two
pyruvate molecules is used to generate a-acetolactate, the precursor of leucine and valine. The
mechanisms for the conversion of a-acetohydroxyacids to their corresponding branched-chain
amino acids appears highly conserved among the bacteria (Umbarger, 1978), but several
alternate sources of a-0x0-butyrate have been reported. L-glutamate (Phillips et al., 1972), Lmethionine (Barker, 1981) and phosphoserine (Schildkraut & Greer, 1973) are converted to a-
t Present address: Miles Laboratories Inc., PO Box 40, Elkhart, I N 46515, USA.
$ Present address : Fak. Pertanian Bogor, JL, Pajajaran Bogor-Indonesia.
Abhreriation : AH AS, acetohydroxyacid synthase.
0022-1287/84/0o01-1308 $02.00 0 1984 SGM
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3 10
D . J . MONTICELLO, R . S . HADIOETOMO A N D R . N . COSTILOW
oxobutyrate by some bacteria. The spirochaete Leptospira appears to generate a-oxobutyrate
from pyruvate via a novel pathway (Charon et al., 1974). Anaerobic rumen bacteria are able to
make isoleucine from a-methylbutyrate via reductive carboxylation and subsequent transamination (Robinson & Allison, 1969) and we have shown previously that C. sporogenes can utilize
presumably similar reactions to make valine from isobutyrate (Monticello & Costilow, 1982).
The objectives of this study were (a) to determine the source(s) of carbon atoms for isoleucine
synthesis in C . sporogenes growing in a carbohydrate-free medium, and (b) to identify the
pathway of biosynthesis.
METHODS
Culture and growth medium. Clostridium sporogenes ATCC 7955 was used in all experiments. Procedures for
maintaining the culture and for preparation of inocula were as previously described (Costilow, 1977). Except
where noted, the minimal medium was that described in Results. The bacteria were grown in an anaerobic
chamber (Coy Manufacturing, Ann Arbor, Mich, USA) at 37 "C in an atmosphere of 5 % (v/v) C 0 2 ,and 10%(v/v)
H2 in N2. Growth of C. sporogenes was estimated by measuring the OD600 value using a Mini Spec 20
spectrophotometer (Bausch & Lomb), and a 13 mm O D cuvette.
L-ubelling and fractionation of cells. 14C-Labelled compounds were added to exponentially growing cells (10 ml
cultures, ODbo0= 0-3),incubated for 4 h (about one generation) and harvested by centrifugation. Cell pellets were
extracted twice at 0 "C with 5 ml 10% (w/v) TCA for 15 min, then for 30 min at 90 "C with 2.5 ml 10% TCA.
Pellets were washed with 2.5 ml hot (90 "C) TCA, resuspended in 1.5 m16 M-HCIand hydrolysed at 1 10 "C for 24 h
in an argon-flushed, sealed ampoule. The hydrolysates were dried under a stream of nitrogen at 60 "C, dissolved in
500 pl water and absorbed on a cation exchange resin (AG 50W-X4, hydrogen form; Bio-Rad) packed in a
disposable Pasteur pipette (1.6 ml resin bed). The resin was washed with 15 ml water and the amino acids eluted
with 1 M-ammonium hydroxide. The eluate was evaporated and then dissolved in 200pl water and stored at
- 20 "C.
Preparation of fermentation products of ~-[U-~~Clisuleucine.
Cells were grown in 100 ml of minimal medium,
harvested during exponential growth (anaerobically, at 40000g, 20 min, 4 "C) and washed twice with 0.1 Mpotassium phosphate buffer, pH 7.4. The cells (0.27 g wet wt) were suspended in 2.0 ml of the buffer with 10 mM-Lisoleucine, 20 p c i L-[U-'4C]isoleucine (specific activity 360 pCi prilol-l ; 13.32 MBq pmol- l), and 20 mM-Lproline (an electron acceptor). The mixture was incubated anaerobically at 37 "C for 4 h. Following centrifugation
(20 rnin at 40000g, 4 "C), the supernatant fluid was made acid with 6 M-HCIand adsorbed to a cation exchange
resin (AG 50W-X4, hydrogen form; Bio-Rad). The fluid which passed through the column was collected and the
total radioactivity determined. Although this fraction may have contained traces of other radioactive volatile fatty
acids, a-methylbutyric acid was the major product since it is the major volatile fatty acid formed from isoleucine
(Barker, 1981). Analysis of an identical, non-radioactive sample by GLC showed the preparation to be greater
than 98% a-methylbutyrate. A yield of 28% was obtained based on radioactivity counting, corresponding to a
2.8 mwsolution with a specific activity of 0.97 pCi pmol- (35.89 kBq pmol- I ) .
Enzyme assays. Acetohydroxyacid synthase (AHAS; EC 4.1.3.18)activity was estimated in crude extracts of
C. sporogenes and Escherichia coli K12 (ATCC 14948) by measuring the rate of a-acetolactate formation from
pyruvate (Stsrmer & Umbarger, 1964). Escherichia coli K 12 was grown aerobically at 37 "C on a rotary shaker in
the minimal medium (without citrate) of Davis & Mingoli (1950). Crude extracts of both organisms were made
from cells harvested (20 rnin at 40000 g, 4 "C) from 500 ml exponentially-growing cultures. Centrifuged, washed
cells were suspended in 3 ml of the buffer used for washing [50 mM-potassium phosphate, 10% (v/v) glycerol and
0.5 mhi-dithiothreitol, pH 7.51, and sonicated for 3 x 30 s, with 1 min cooling intervals. Cellular debris was
removed by centrifugation (20 min at 48000g, 4 "C) and the extract was used immediately. Specific activity was
determined as pmol a-acetolactate formed h - l (mg protein)-'. The assays were performed at 37 "C in an
anaerobic chamber.
Threonine dehydratase [L-threonine hydro-lyase (deaminating), EC 4.2.1.161was assayed as follows: assay
mixtures (1-0ml final volume) contained 5 mwdithiothreitol, 5 mM-ADP, 0.15 mM-NADH, 15 pg rabbit muscle
lactic dehydrogenase type I1 (L-lactate : NAD' oxidoreductase; EC 1. I . 1.27) (Sigma), 75 mwpotassium
phosphate buffer (pH 8.0), 20 mM-L-threonine and cell extract (about 0.2 mg protein). The assay mixture was
preincubated at 37 "C for 5 min and the reaction initiated with the addition of L-threonine. The rate of NADH
oxidation at 340 nm was monitored with a Gilford 2000 recording spectrophotometer (Gilford Instrument Co.)
with a cuvette chamber maintained at 37 "C by use of a Haake temperature regulator (PolyScience Corp.). In all
assays, protein concentrations were determined by the Lowry method.
Amino acid determinations. Most amino acids were separated and quantitated by use of a Beckman Model 121
programmable amino acid analyser (Beckman). Two programs were used for separation of samples. Program I
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Isoleucine synthesis in Clostridium
311
with 0.02 M-sodium citrate buffer, pH 2.99, containing 3% (v/v) n-propanol was used to separate aspartate,
threonine, serine, glutamate, glycine, and alanine. Program I1 with 0.2 M-sodium citrate buffer, pH 3.39,
containing 3 % (v/v) methanol was used to separate valine, methionine, isoleucine, and leucine. All separations
were performed at 52 “C on a 0.9 x 65 cm column of spherical sulphonated styrene copolymer resin (Type AA-I 5,
8% crosslinking; Beckman). The flow rates for programs I and II were 28 ml h-l and 70 ml h - * , respectively.
Before each run, the column was flushed with 0-2M-NaOH (70 ml h - l ) for 8 min, and then equilibrated for 35 min
with the appropriate buffer at 70 ml h - l . After injection of the sample (600 pl), the flow rate was adjusted to that
required for each program.
For analysis of radioactive samples, the elution stream from the analyser was interrupted before reaction with
ninhydrin and diverted to a Gilson Microfractionator Type FC-80K fraction collector (Gilson Medical
Electronics). The point at which the eluant stream was diverted was determined by reference to the elution profile
of a calibration mixture, which was used as a guide. Fractions were collected in the time mode at 1 min intervals.
Every second or third fraction was quantitatively transferred to a glass vial, mixed with aqueous scintillation fluid
(Formula 963; New England Nuclear) and the radioactivity was counted. In all cases, plots of radioactivity versus
elution time gave discrete peaks which coincided with those obtained with calibration standards. Fractions of
individual peaks were pooled and the total label was quantitated by counting duplicate samples. All values were
corrected for quenching; the counting efficiency was 80-84% as measured against an internal standard. The
recovery of label by this method (based on analysis of a mixture of radioactive amino acid standards) was 98100%.
Lysine was routinely separated from other radioactive amino acids by electrophoresis on Whatman no. 1 paper
using a 0.25 M-sodium acetate buffer at pH 4.3. Arginine was not synthesized by this organism so it did not
interfere.
In later experiments, amino acids were separated by TLC, preparative descending paper chromatography or
high-voltage paper electrophoresis. Whatman LK-5-D silica gel linear K preadsorbant plates, developed with a
methylethylketone/pyridine/water/glacial
acetic acid solvent system (73 :5 :20 : 2 by vol.) (McBride et al., 1979)
were used to separate isoleucine from other amino acids. Aspartate and glutamate were separated from other
amino acids and each other by paper electrophoresis on Whatman no. 1 paper in 0.25 M-sodium acetate buffer at
pH 4.3. Samples of protein hydrolysates (10 pl) were spotted on the paper and electrophoresed at 17-35 V cm- I
for 90 min. Radioactive isoleucine was obtained for degradation by preparative descending paper chromatography with a butanol/acetic acid/water solvent system (1 20 : 30 : 50 by vol.). Hydrolysates were spotted on sheets
of Whatman no. 1 paper (46 x 57 cm) and following development, the isoleucine/leucine region of the
chromatogram was identified and removed, and the amino acid eluted with water. The coelution of isoleucine and
leucine in this solvent system was not a problem, since leucine was never labelled (as confirmed by TLC
separations). Amino acids were located by comparison with adjacent lanes sprayed with ninhydrin (0.1 %, w/v, in
acetone).
Decarboxylarion of radioacrioe amino acids. Glutamic acid and all monocarboxylic amino acids except isoleucine
were decarboxylated with chloramine-T according to the procedure, of Kemble & McPherson (1954). The
radioactive sample was placed in a Warburg flask with 0.5 ml 12%(w/v) chloramine-T solution in the side arm
and 0.4 ml hyamine hydroxide (Packard Instruments) in the centre well to trap the “TO2. The reaction was
initiated by tipping the chloramine-T solution into the sample. The reaction was allowed to proceed for 2 h at
30 “C.At the end of incubation, the contents of the centre well were transferred to a glass vial; the well was rinsed
with 1 ml methanol and the washings added to the same vial. Toluene-based scintillation fluid (10 ml) was added
to the vial and the radioactivity was counted. A vial containing 0.4 ml hyamine hydroxide and 1 ml methanol in
10 ml of the same scintillation fluid served as a reagent blank.
Aspartic acid and isoleucine were decarboxylated with ninhydrin by a modification of the procedure of
Greenberg & Rothstein (1957). The “TO, produced in the reaction vessel was driven by a stream of nitrogen into
two receiving vessels containing barium hydroxide solution. The precipitated barium carbonate was collected and
transferred to a Warburg flask containing 0.5 ml 2 M-HCI in the side arm and 0-4ml hyamine hydroxide in the
centre well. The flask was sealed, and the acid tipped into the sample. The flask was held at room temperature for
2 h to allow for complete absorption of the liberated lsCOz by the alkali. The rest of the procedure was as
described above.
tsoleucine degradation. The location of IJC label incorporated into isoleucine from labelled precursors was
determined by a stepwise degradation (Strassman er al., 1956) of [ lJC]isoleucine purified from protein
hydrolysates. [ “C]Isoleucine was combined with 250 mg L-isoleucine in 35 ml water and decarboxylated with the
addition of 0.15 ml 80% (v/v) phosphoric acid and 1 g ninhydrin and incubation at 70 “C for 1 h. Released CO,
was trapped as barium carbonate, and the a-methylbutyraldehyde formed was distilled into 20 ml 5 M-C~O,in
25% (v/v) H2S0, and directly oxidized to a-methylbutyrate (Robinson & Allison, 1969). The a-methylbutyrate
was recovered by steam distillation and degraded to 2-butylamine via the Schmidt reaction (Phares, 1951).This
product was oxidized to methylethylketone with KMnO, and subsequently degraded to propionate and COz.
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3 12
D. J .
MONTICELLO, R. S.
HADIOETOMO A N D
R.
N . COSTILOW
Radioactivity in I4C trapped as barium carbonate was determined by releasing the COz with concentrated
H2S04and trapping it in hyamine hydroxide in a closed vessel. After 12 h, the hyamine hydroxide solution was
dissolved in toluene-based scintillation fluid and the radioactivity was determined.
Materials. Amino acids of the highest purity available were purchased from Sigma. I4C-Labelled compounds
and aqueous scintillation cocktail were obtained from New England Nuclear. Toluene-based cocktail was
prepared by mixing 6.0 g PPO and 0.1 g POPOP in 1 litre of toluene; both chemicals were obtained from Research
Products International Corp.
RESULTS
Evidence for unusual isoleucine biosynthesis
A glucose-free minimal medium for C. sporogenes was developed which contained salts and
vitamins (Perkins & Tsuji, 1962), 0.05 % sodium thioglycollate and eight amino acids: L-serine,
25 mM; L-arginine, 20 mM; L-phenylalanine, 10 mM; L-methionine, 10 mM; L-valine, 10 mM; L-
leucine, 10 mM; L-tyrosine, 2 mM and L-tryptophan, 2 mM. All of these amino acids were
required for growth except that (a)serine could be replaced with alanine, and (b)methionine was
only stimulatory. The apparent ability of this organism to synthesize isoleucine but not leucine
or valine prompted our further investigation.
Studies of the incorporation of 14C-labelledsubstrates into cellular amino acids indicated that
C. sporogenes probably synthesizes most of the amino acids not added to the medium by
conventional biochemical pathways (Table 1). Thus, the distribution of 14C in all of the amino
acids assayed except for isoleucine was consistent with these pathways. However, the relative
amount of radioactivity in isoleucine produced from either [U-14C]serineor [3-14C]pyruvatewas
only about one-half the level expected if isoleucine was synthesized via threonine through the
classical pathway. Also, an unusually high percentage of the 14C in isoleucine from L-[U14C]serinewas present in C-1. Glycine was the only cellular amino acid other than threonine
found to be labelled during growth of C. sporogenes in the presence of L-[U-14C]threonine(Table
1). Other similar incorporation experiments demonstrated that there was no significant
incorporation of 4C into isoleucine from [U-14C]glutamic acid, [U-14C]leucine, [U-l 4C]valine
or [ U-l 4C]methionine.
Threonine dehydratase activity
Since threonine usually enters the isoleucine biosynthetic pathway via dehydration to aoxobutyrate, we examined threonine dehydratase activity in C. sporogenes. The addition of
threonine (42 mM), isoleucine (30 mM), or glucose (30 mM) to the growth medium had no effect
on the specific activity of the dehydratase in crude cell extracts. Neither was there any inhibition
of the activity after addition of isoleucine (5 mM) to assay mixtures. The addition of 5 mM-ADP
to the mixture caused a two-fold increase in specific activity [from 0.43 to 0.81 pmol threonine
deaminated min- (mg protein)- ] at low L-threonine concentrations (4 mM). No activation of
threonine dehydration by ADP was detected at a higher L-threonine concentration (20 mM), and
enzyme activity was unaffected by AMP (5 mM) at either threonine concentration. Kinetic
analysis showed that the apparent K, for L-threonine was 2.5 mM in the presence of ADP and
33.3 mM in its absence. These results suggest that the threonine dehydratase in C.sporogenes
grown in the minimal medium performs a catabolic function, as has been reported for this
activity in C. tetanomorphum (Whitely & Tahara, 1966) and anaerobically-grown E. coli
(Umbarger, 1973).
Incorporation of a-amino-[P'4C]butyrate into isoleucine
Since none of the previously reported (Umbarger, 1978) amino acid precursors of isoleucine
were incorporated into isoleucine by C. sporogenes, a-aminobutyrate was used to examine the
role of a-oxobutyrate in isoleucine synthesis. a-Oxobutyrate is a key intermediate in all of the
previously reported pathways for isoleucine synthesis. After growth for one generation (4 h)
with ~-amino-[3-~~C]butyrate,
over 90% of the radioactivity in protein was recovered in
isoleucine (Fig. 1). Similar results were obtained with a-amino-[ l-14C]butyrate.
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Isoleucine synthesis in Clostridium
Table 1. Distribution of radioactivity in amino acids from protein hydrolysates of C . sporogenes
grown with T-labelled substrates
The following labelled substrates were added to cultures (10 ml) at OD,,, = 0.3 and the cultures
incubated for 4 h (about one generation): 25 pCi ~-[U-'*C]serine(162 pCi pmol-I), 25 pCi [3-14C]pyruvate (200 pCi pmol-I), 50 pCi [1JC]NaHC03 (6.2 pCi pmol- I ) , or 25 pCi ~-[U-I*C]threonine
(210 pCi pmol- I ) (1 pCi = 37 kBq). Specific activities of the amino acids from cell proteins were based
on the average amino acid composition as determined by amino acid analysis. Numbers in parentheses
indicate the percentage of radioactivity released by decarboxylation.
Radioactivity (d.p.m. nmol- ') from:
Amino acid
L-[U-'TISerine
Alanine
Serine
Glycine
Aspartate
Threonine
Lysine
Glutamate
Isoleucine
Valine
Leucine
Total d.p.m.
recovered
142 (35)
237 (25)
103 (57)
184 (50)
131 (25)
231 (13)
245 (1 8)
120 (44)
8
277 (0)
438 (1)
37 (0)
328 (0)
297 (2)
554 (0)
622 (0)
220 (0)
ND
ND
ND
57 (101)
39 (-)
28 (-)
235 (104)
222 (20)
188 (21)
213 (0)
165 (18)
12 (107)
14 (-1
76340 (87%)
1 17740 (94%)
7 1 340 (97%)
ND,
1
[ "C] N a H CO,
[3-1*C]Pyruvate
L-[U- I 4C]Threonine
ND
ND
283
14
10 125
9
11
8
ND
7
I27 360 (1 05%)
Not detected; -, not determined.
reucine
900 -
Glutamate
700
+
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Valine Isoleucine
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.-c
;6 0 0 3 500-
Aspartate Proline
g 600
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800
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Aspartate Proline e . . . . " ;
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Section no.
Fig. 1
Fig. 1. Incorporation of a-amino-[3-'*C]butyrate into amino acids by C. sporogenes. Cells were grown
for one generation with 4C-labelled substrate and the cellular protein was extracted and hydrolysed.
Samples (10 pl) of the protein hydrolysate were applied to TLC plates and developed in a
methylethylketone/pyridine/water/glacialacetic acid solvent system (73 :5 :20 :2 by vol.). After
development, 5 mm fractions were scraped from the plates and radioactivity determined. The location
of amino acid standards chromatographed on an adjacent lane are shown at the top of the Figure.
Fig. 2. Incorporation of [2-'*C]propionate into amino acids by C. sporogenes. The procedure was as for
Fig. 1.
a-Acetohydroxyacid synthase activity in crude extracts
Since a-oxobutyrate appeared to be a precursor of isoleucine, we assayed crude extracts for
AHAS, a critical enzyme in the classical pathway for branched-chain amino acid synthesis. The
AHAS of E. coli catalyses two reactions: the condensation of two pyruvate molecules to form
a-acetolactate and C 0 2 (for valine and leucine synthesis) and the condensation of pyruvate and
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D. J.
MONTICELLO, R. S. HADIOETOMO
A N D R. N. C V S I I L O W
a-oxobutyrate to form a-aceto-a-hydroxybutyrateand C 0 2 . In crude extracts of C.sporogenes
and in E. coli K 12 cells grown in minimal media, the specific activities of a-acetolactate synthase
were 2-3 pmol h- (mg protein)- and 2.1 pmol h- (mg protein)- l , respectively.
Incorporation of [ 4Clpropionate into isoleucine
Reductive carboxylation reactions are relatively common among anaerobic bacteria
(Buchanan, 1972). Consequently, propionate was evaluated as a potential source of aoxobutyrate. Approximately 68 % of the radioactivity in protein from cells grown with
[2- 4C]propionate was recovered in isoleucine (Fig. 2). Similar results were obtained with
[ l-14C]propionate and [3-14C]propionate.
Analysis of the culture supernatant fluids following growth for 4 h with [1-14C],[2-14C]or [314C]propionateshowed that 65 % of the 14Cpresent was in a-aminobutyrate. (The 14C-labelled
substance was identified as a-aminobutyrate on the basis of its co-migration with authentic aaminobutyrate during ascending paper chromatography with three different solvent systems.)
These results suggest that propionate is rapidly converted to a-aminobutyrate, presumably via aoxobutyrate, and thus assimilated into isoleucine. During growth in the minimal medium,
propionate must be produced from serine, alanine, or pyruvate. Addition of 0.1 mg sodium
propionate ml-l to a 10 ml culture with 10 pCi (370 kBq) [U-14C]serine decreased the
incorporation of 14Clabel into isoleucine by about 50%, while no effect on the incorporation of
label into aspartate or glutamate was observed.
Degradation of isoleucine from C . sporogenes grown with [3-14Clpyruvateor [I-l4C1propionate
Labelled isoleucine from C. sporogenes was degraded in a stepwise fashion to locate the
position of the label in the isoleucine molecule. Decarboxylation did not release significant 14C
label from [3-l 4C]pyruvate- or [ 1-14C]propionate-labelled isoleucine (5600 c.p.m. and
1300 c.p.m. initial total, respectively). Decarboxylation of [U-14C]isoleucine (55 000 c.p.m.)
yielded 4900 c.p.m. (53% of theoretical recovery). Cleavage of the remaining five-carbon
fragment (a-methylbutyrate) yielded no detectable radioactivity in the C-2 of the [3-14C]pyruvate-labelled molecule, while over 22% of the initial label was released as C 0 2 from the [ 114C]propionate-labelledmolecule. No radioactivity was detected in the subsequently isolated
four-carbon fragment (2-aminobutyrate) from the [ 1- l 4C]propionate-labelled preparation, while
16% of the original label (900/5600 c.p.m.) remained in the [3-14C]pyruvate-labelledsample,
and 6.5% of the 14C label remained in this fragment from a uniformly labelled sample.
Cleavage of the [3-l 4C]pyruvate-labelledmolecule into propionate (C-3, C-4, C-5) and iodoform
(C-6) released label into both fractions (7% and 2.5% of the original 14C label, respectively).
Cleavage of the uniformly labelled sample yielded 1'4% of the original label in propionate and
0.25% of the original label in iodoform. These results show that most (if not all) of the 14Clabel
in the [ 1- l 4C]propionate-labelled isoleucine resided in C-2, while label from [3-l4C]pyruvatelabelled isoleucine resided in both C-6 and the C-3, C-4, C-5 fragment.
Incorporation of a-[ U- 4C]methylbutyrate into isoleucine
a-Methylbutyrate is a precursor of isoleucine in some anaerobic rumen bacteria (Robinson &
Allison, 1969). We sought to determine its role in isoleucine synthesis in C. sporogenes. This
organism normally required L-valine and L-leucine for growth, but leucine could be replaced by
high concentrations (10 mM) of a-methylbutyrate (Hadioetomo, 1980). Growth at these
concentrations was probably the result of contamination of the commercial a-mkthylbutyrate
with isovalerate, a potential percursor (via reductive carboxylation) of leucine. Analysis of the amethylbutyrate by GLC revealed it was about 9.0% isovalerate. Similarly, isobutyrate can
replace valine in the growth medium (Monticello & Costilow, 1982). These observations
suggested that C. sporogenes might reductively carboxylate a-methylbutyrate to isoleucine in the
same way. This proved to be true. Following growth with a-[U-14C]methylbutyrateprepared by
fermentation of [U-14C]isoleucine(see Methods), 95 % of the total radioactivity (87 000 d.p.m.)
in the protein hydrolysate was found in isoleucine and no label was released upon
decarboxylation of the purified [ 4C]isoleucine with ninhydrin.
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Isoleucine synthesis in Clostridium
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I
5
I
10
3 15
I
15
a-Methylbutyric acid concn (mM)
Fig. 3. Effect of a-methylbutyrate on the incorporation of [U-14C]pyruvate ( 0 )and 14C02 ( 0 )into
isoleucine. Closrridium sporogenes was grown for one generation in a minimal medium supplemented
with a-methylbutyrate and the appropriate 14C-labelled compound. Since the specific activity of
aspartate was not affected by a-methylbutyrate, the ratio of d.p.m. in isoleucine to d.p.m. in aspartate is
shown on the ordinate axis.
The effect of increasing the concentration of a-methylbutyrate in the medium on the
incorporation of 4C02and [U-l 4C]pyruvate into isoleucine was determined. [U-14C]Pyruvate
(10 pCi; 370 kBq) or [ 14C]sodiumbicarbonate (10 FCi; 370 kBq) and varying concentrations of
a-methylbutyrate were added to 10 ml cultures and the cells were allowed to grow for one
generation. Increased concentrations of a-methylbutyrate led to a significant decrease in the
incorporation of label from [U-14C]pyruvate into isoleucine relative to aspartate (Fig. 3). No
such change was seen with the 14C02label, indicating that COz is incorporated into isoleucine
regardless of the concentration of a-methylbutyrate in the medium.
DISCUSSION
Unusual L-[ U- 4C]serine-, [3- 4C]pyruvate- and 4C0,-labelling patterns, and the observation that isoleucine was not made via threonine by C. sporogenes prompted us to investigate
branched-chain amino acid synthesis in this organism. The evidence presented here indicates
that the synthesis of isoleucine in the minimal medium used proceeds via the classical pathway
(Umbarger, 1978) except that a-oxobutyrate is derived primarily from the carboxylation of
propionic acid (Fig. 4). The propionate is presumably produced from serine, which is the
primary carbon and energy source in the medium. These conclusions are supported by the fact
that : (a) radioactivity from a-[3-14C]aminobutyrate was incorporated almost exclusively into
isoleucine, (b) extracts of cells of C. sporogenes contained the first enzyme (AHAS) in the
classical biosynthetic pathway with activities comparable to that found in E. coli K12, (c) 14C
label from [ 1-14C],[2-14C]and [3-14C]propionatewas incorporated into cellular isoleucine and
into a-aminobutyrate which accumulated in the culture medium, (d) essentially all of the 14C
label in isoleucine from cells grown in the presence of [ l-14C]propionate was found in C-2, which
is the ultimate location of C-2 of a-oxobutyric acid when isoleucine is synthesized by the classical
pathway, (e) the 14C label from [3-14C]pyruvateincorporated into isoleucine was found in at
least two of the last four carbon atoms as would be expected if a-oxobutyrate is produced from
serine added to the medium, and (f)addition of low levels of propionate decreased the amount
of radioactivity from [U-14C]serinewhich cells incorporated into isoleucine but had no effect on
the amounts found in aspartate or glutamate.
A number of sources of a-oxobutyrate in bacteria are known. Both threonine and methionine
can be converted to a-oxobutyrate by C. sporogenes (Barker, 1981), but neither of these amino
acids contributed a significant amount of carbon to isoleucine synthesis in these experiments.
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3 16
D . J . MONTICELLO, R . S . HADIOETOMO AND R . N . COSTILOW
a-Methylbutyrate
9
-+c~~h;mT,cIIfiDI~MM
1 9
O
Propionate
a-Oxobutyrate
a-Acetoa-hydroxybutyrate
M3
a-0x0p-rnethylvalerate
b
lsoleucine
Fig. 4. Proposed pathway for isoleucine synthesis in C. sporogenes.
Leptospira synthesizes isoleucine via a-oxobutyrate produced from acetate and pyruvic acid
(Charon et al., 1974). Glutamic acid (Phillips et al., 1972) and phosphohomoserine (Schildkraut
& Greer, 1973) are other known sources of this 0x0-acid. Data herein indicate that the reductive
carboxylation of propionic acid as described by Buchanan (1969) and by Bush & Sauer (1976) is
the primary source of a-oxobutyrate used in isoleucine synthesis by C. sporogenes. Allison et a f .
(1979) reported that a number of rumen bacteria synthesized glutamic acid primarily from aoxoglutarate produced from the reductive carboxylation of succinic acid. However, this is not
the case with C. sporogenes since essentially no I4C was found in C-1 of glutamate from cells
grown in the presence of sodium [14C]bicarbonate.
a-Methylbutyrate can also serve as a precursor of isoleucine for C. sporogenes. We have
previously shown that isobutyrate can replace valine in the mineral medium (Monticello &
Costilow, 1982). These conversions and the production of a-oxobutyrate from propionate must
depend on reductive carboxylation reactions (Buchanan, 1972). Such reactions have been well
documented as occurring in heterotrophic bacteria from the rumen and other anaerobic habitats
(Allison & Bryant, 1963; Allison & Robinson, 1970; Allison et al., 1979; Buchanan, 1969;
Buchanan & Arnon, 1969; Buchanan & Evans, 1965; Bush & Sauer, 1976; Robinson & Allison,
1969; Sauer et al., 1975). Reductive carboxylation reactions are only possible in hiihly reduced
environments, because of the requirement for a very electronegative electron donor such as
reduced ferredoxin. The significance of such reactions in natural anaerobic environments has
not been established, but the apparent ubiquity of oxo-acid synthase activity among the
anaerobes suggests a possible role in the assimilation of branched-chain acids from the
environment.
If isoleucine is synthesized by C. sporogenes from a-oxobutyrate and pyruvate via the classical
pathway, one might expect valine and leucine to be synthesized from pyruvate, but we found no
incorporation of [3-14C]pyruvate into either amino acid (Table 1). The accumulation of aaminobutyric acid in the medium as observed in this study as well as in a complex medium
(Mead, 1971) may explain this phenomenon. In E. coli, a-oxobutyrate is usually produced from
threonine, and the levels of a-oxobutyrate in the cell are strictly controlled by feedback
regulation of threonine dehydratase by isoleucine and to a lesser extent valine and leucine
(Iaccarino et al., 1978). We have found that threonine is not the primary source of a-oxobutyrate
in C. sporogenes, nor is threonine dehydratase activity regulated by isoleucine. a-Aminobutyrate
is known to be an antimetabolite of valine in Bacillus anthracis (Gladstone, 1939)and E. coli K 12
(Iaccarino et al., 1978). Gladstone (1939) observed that growth of B. anthracis in a minimal
medium was inhibited by a-aminobutyrate in concentrations as low as 0-04mM, and the
inhibition could be overcome by the addition of L-valine to the medium. The inhibitory effects of
a-aminobutyrate may therefore be responsible for the inability of C. sporogenes to grow in the
minimal medium without valine and leucine. Inhibition of valine synthesis by a-aminobutyrate
may be the result of increased a-aceto-a-hydroxybutyratesynthesis at the expense of acetolactate
production, since increased concentrations of a-oxobutyrate (from a-aminobutyrate) would
competitively inhibit the formation of acetolactate. In E. coli the condensation of two pyruvate
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Isoleucine synthesis in Clostridiurn
317
molecules to form acetolactate (for valine and leucine) and the condensation of a-oxobutyrate
and pyruvate to form a-aceto-a-hydrokybutyrate(for isoleucine) are catalysed by the same
AHAS enzymes. In experiments with purified AHAS I from E. coli, Grimminger & Umbarger
(1979) showed that as the pyruvate :a-oxobutyrate ratio in their assay was changed from 10 :1 to
1 :1, the acetolactate :a-aceto-a-hydroxybutyrate formed ratio changed from 5.3 :1 to 1 : 1-5.
Since the inhibition of acetolactate formation is competitive, at higher a-oxobutyrate
concentrations acetolactate synthesis would be even more diminished, with a concomitant
increase in a-aceto-a-hydroxybutyrateformation. When a-aminobutyrate-resistant mutants
were selected from E. coli K12, they had derepressed levels of AHAS (Iaccarino et al., 1978),
which would overcome competitive inhibition. Such inhibition of acetolactate formation by the
AHAS enzyme(s) in C. sporogenes by a-aminobutyrate may be responsible for the cell’s inability
to make leucine or valine, despite the presence of the appropriate enzymes.
Journal article no. 10502 of the Michigan Agricultural Experiment Station.
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