Journal of General Microbiology (1989, 131, 1061-1067.
Printed in Great Britain
1061
Glutamine Synthetase from Pseudomonas syringae pv. tabaci: Properties
and Inhibition by Tabtoxinine-B-lactam
By M I C H A E L D . T H O M A S * ? A N D R I C H A R D D . D U R B I N
Department of Plant Pathology and ARS-USDA, University of Wisconsin, Madison,
WI53706, U S A
( Received 10
October I984 ;revised 19 December 1984)
Glutamine synthetase from Pseudomonas syringae pv. tabaci was purified 500-fold. Maximum
activity was observed with 10 mwglutamate, 20 mM-ATP and 4 mM-NH,Cl. The enzyme
exhibited substrate inhibition; higher levels of glutamate, Mg .A T P or NH,Cl decreased its
activity. The y-glutamyltransferase activity was inhibited by Mg2+(75% at 1OmM-Mg”). The
enzyme was heat stable and there appeared to be only one form present. Tabtoxinine-P-lactam, a
hydrolytic product of tabtoxin produced by pv. tabaci, inactivated the enzyme. This inhibition
was linear with respect to the concentration of the inhibitor, and enzyme activity could not be
recovered by dialysis, acetone precipitation or incubation with crude cell lysate. Mg . A T P and
ammonium ions were required for binding of the inhibitor: incubation of tabtoxinine-P-lactam
with the enzyme in the presence of both Mg . ATP and ammonium ions resulted in a greater
decrease in synthetase activity than incubation with either one or neither component.
Tabtoxinine-/)-lactam did not inhibit the y-glutamyltransferase activity of the enzyme if A D P
was used in the assay, but did when ATP was used.
INTRODUCTION
A number of plant pathogens produce toxins whose targets are shared by the host and
pathogen. In these cases, to paraphrase Demain (1974), what keeps the potential suicide from
succeeding? Although this question has been extensively considered with regard to antibioticproducing micro-organisms, there is very little information available about the situation in plant
pathogens. To a large extent this is because the mechanism of action of most phytopathogenproduced toxins is unknown.
Essentially all the information we currently have comes from studies on phaseolotoxin,
produced by Pseudomonus sjsrirzgae pv. phaseolicola, which inhibits ornithine carbamoyltransferase (Patil rt ul., 1972). The evidence strongly suggests that pv. phaseolicola possesses at
least two mechanisms for self protection. First, it produces a form of ornithine
carbamoyltransferase which is relatively unaffected by phaseolotoxin (Ferguson et al., 1980).
Interestingly, this form occurs only at temperatures allowing phaseolotoxin production; a t high
temperatures when no phaseolotoxin is produced, a sensitive form of the enzyme occurs
(Staskawicz et ul., 1980). Second, induced mutants of Salrrzonella typhimurium and Escherichia
coli resistant to the toxin lose their transport or permease system for phaseolotoxin uptake
(Staskawicz & Panopoulos, 1980). Possibly then, phaseolotoxin may be selectively excluded
from the oligopeptide transport systems in pv. phaseolicola. Other mechanisms of self protection
may be involved too (e.g. internal compartmentalization separating phaseolotoxin from its
target), but as yet there is no experimental evidence for them.
A closely related pathovar, pv. rubaci, produces tabtoxin (Fig. 1 ; Stewart, 1971 ; Taylor et al.,
1972) which, when hydrolysed by the host and/or bacteria, releases tabtoxinine-&lactam
t Present address: ARS-USDA, National Cotton Pathology Research Laboratory, PO Drawer JF, College
Station, TX 77840, USA.
Ahhrrziutiuns : IM buffer, imidazole/MgCl, buffer; MSO, L-methionine sulphoximine.
0001-2253 $3 1985 SGM
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1062
M . D . THOMAS A N D R . D . D U R B IN
H,N-CH-CO-NH-CH-CO,H
I
I
CHOH
I
(7HJ2
OC-C-OH
R
I I
HN-CH,
(4
H ,N-C H-C 02H
I
(7HJ2
OC-C-OH
I I
HN-CH2
I
HN-CH-CO2H
I
(y32
OC-C-OH
I
H2N-CH2
(4
(b)
Fig. 1. Structure of tabtoxin from P . syringae pv. tabaci and its products. (a) Tabtoxins, R = H or CH3
for [Ser2]-tabtoxinor [Thr'l-tabtoxin, respectively ; (b) tabtoxinine-&lactam ; (c) tabtoxinine-b-lactam
(biologically inactive).
(Uchytil & Durbin, 1980). We have shown that tabtoxinine-Q-lactam inhibits glutamine
synthetase from pea seeds (Thomas et al., 1983). There are similarities between this
host/pathogen interaction and that of pv. phaseoficola.For instance, the chlorotic symptoms can
be reversed by addition of the product of the target enzyme [i.e. arginine (Patil et a f . , 1972) or
glutamine (Sinden & Durbin, 1968)l. If the self-protection mechanisms in the two bacteria are
similar, one would expect that either glutamine synthetase from pv. tabaci would be unaffected
by tabtoxinine-P-lactam, or there are two forms of the enzyme differing in sensitivity. Indeed,
multiple forms of glutamine synthetase have been found in other bacteria such as Rhizobium
japonicum (Darrow & Knotts, 1977), Agrubacterium tumefaciens (Fuchs & Keister, 1980) and
Bacillus caldolyticus (Wedler et a f . , 1980). However, only one form of glutamine synthetase was
found in three species of Pseudomonas (Meyer & Stadtman, 1981).
Here we report the purification and characteristics of glutamine synthetase from pv tabaci,
and its inhibition by tabtoxinine-P-lactam. The characteristics of the enzyme are distinctly
different from other reported bacterial glutamine synthetases, and it is markedly inhibited by
tabtoxinine-P-lactam. Possible mechanisms of self protection including cellular compartmentalization and detoxification are discussed.
METHODS
Cellgrowth. Pseudomonas syringae pv. tabaci TDI, a virulent, tabtoxin-producing clone of strain ATCC 11528,
was grown in shake culture overnight in a medium containing 30 mM-K2HP0,, 7 mM-KH,HPO,, 0.4 mMMgSO,, 55 mM-glucose and 10 mM-histidine. The culture was centrifuged, and the bacteria were resuspended at
one-tenth of the original culture volume in saline (0.85% NaCI) and used to inoculate fresh medium, at an initial
density of lo8 cells ml-', which was then incubated for an additional 24 h.
Enzyme purlfjcation. The following procedures were done at 4 "C,except for DEAE-cellulose and Sepharose 6B
chromatography (20 "C). Cells (up to 100 g wet wt) were collected by centrifugation (14500 g, 5 min), washed once
in 20 mM-imidazole (pH 7.5) and 10 mM-MgC1, (IM buffer), and resuspended in IM buffer containing 1 mM-8mercaptoethanol at the ratio of 1 g wet wt of cells to 2 ml buffer. The cells were disrupted by three passages through
a French press (1 10 MPa) and the lysate was clarified by centrifugation (40000g, 30 min). A protein fraction
containing glutarnine synthetase activity was precipitated by adding (NH.&SO, to give 50% saturation. The
precipitate was collected by centrifugation (40000 g, 10 rnin), resuspended at half the original lysate volume in IM
buffer and dialysed against column buffer (IM buffer plus 50 mM-KCI adjusted to pH 8.3). This crude preparation
was centrifuged (40000g, 10 min) and the supernate applied to a column of DEAE-cellulose (1 x 20 cm)
equilibrated with column buffer. Non-adsorbed protein was washed from the column with column buffer until the
effluent had no absorbance at 280nm, and the enzyme eluted by applying a linear gradient (400 ml) of 50 to
700 mM-KCI in IM buffer (pH 8.3). Fractions having glutamine synthetase activity were pooled (usually 30 ml
total), precipitated by the addition of (NH,)?SO, to 50% saturation, centrifuged (40000g, 10 min) and
resuspended in 1 ml IM buffer. After dialysis against 20 mM-imidazole (pH 7.5) and 1 mM-MgCI,, the preparation
was heat-treated for 30 min at 60 "C, centrifuged for 2 min (Eppendorf microfuge, 12000g) and subjected to gel
filtration on a Sepharose 6B column (1-8 x 118 cm) eluted with buffer containing 20 mM-imidazole (pH 7.5) and
1 mM-MgCl?. Fractions (1 ml) were monitored with respect to ,4180 and transferase activity; the active fractions
were pooled (5 to 10ml total volume).
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P . syringae pv. tabaci glutamine synthetase
1063
Table 1. Pur$cation procedure and yields of glutamine synthetase from P . syringae pv. tabaci
Step
Clarified crude lysate
DEAE effluent
Heat treatment
Sepharose 6B effluent
Specific
activity*
0.077
5-0
6.6
41.6
* Units are pmol y-glutamylhydroxamate formed (mg protein)-'
Percentage
yield
100
41
31
25
min-I at 24 "C.
Analytical procedures. The y-glutamyltransferase assay of Stumpf et al. (1951) was modified to allow direct
comparisons with the assay of Shapiro 8t Stadtman (1970). The assay mixture contained 0.5 ml40 mM-imidazole
(pH 7.2), 60 mM-NH,OH. HCI, 3 m~-MnCl,,20 ~ M - K H ~ A s O20
, , mM-glutamine and either 0-4 mM-ATP or
0.4 mM-ADP. The transferase assay using ADP was used to monitor enzyme purification, Mg2+ sensitivity and
enzyme identification on non-denaturing polyacrylamide gels. The transferase assay using either ADP or ATP
was used to study toxin inhibition. In this assay, the assay components and the enzyme were incubated for 30 min
before the addition of the inhibitor and glutamine. Synthetase activity was measured by phosphate liberation
(Shapiro & Stadtman, 1970). The assay was modified by stopping a 0.2 ml reaction mixture with 1 ml 52 mMFeSO, in 0.015 M-H,SO,, and colour was developed with 1 m18 ~M-(NH,),Mo,O,, in 0.55 M-H2SO4.All assays
were run for 15 min at 23 f 2 "C.
Non-denaturing and SDS discontinuous polyacrylamide gel electrophoresis (PAGE) were done with a GE-2/4
LS system (Pharmacia) by the methods provided by Pharmacia. Up to 1Opg protein was applied to 5 % nondenaturing polyacrylamide gels (80 mM-H, B03, 90 mM-Tris, pH 8.3, running and sample buffer). SDS-PAGE
used a 5% stacking gel (0.1% SDS, 60 mM-Tris, pH 6-8)and a 10% separating gel (0.1 % SDS, 375 mw-Tris, pH 8.8)
with 0.1% SDS, 25 mM-Tris, 192 mwglycine running buffer. Gels were stained with Coomassie brilliant blue and
A m 0 1 -X8 mixed-bed resin (Bio-Rad) was added to the destaining solvent. Protein concentration was determined
by the method of Bradford (1 976). Tabtoxinine-/3-lactam and tabtoxin preparation has been described elsewhere
(Thomas et al., 1983). Other chemicals and enzymes were obtained from Sigma.
Tabtoxin and alkali-boiled tabtoxinine-/3-lactam (this treatment converts the /3-lactam to the biologically
inactive d-lactam form) were used as controls for synthetase inhibition studies. L-Methionine (SR)-sulphoximine
(MSO) and glutamine synthetase from pea seed and E. coli were used for comparison in transferase inhibition
studies. Glutamine synthetase from E. coli, isolated under conditions favouring adenylylation (Streicher & Tyler,
1980), was used as a control for experiments to investigate the Mg2+ sensitivity of the pv. tabaci enzyme.
RESULTS
Typical purification and yield of glutamine synthetase from pv. tabaci, based on transferase
activity, are presented in Table 1. Routinely, a 500-fold increase in specific activity was obtained
with yields ranging between 25 and 40%. The enzyme was heat stable for I h at 60 "C; this aided
in the purification procedure. Glutamine synthetase eluted as a single peak from DEAEcellulose at approximately 300 mM-KC1. The enzyme preparation exhibited a single band on
native PAGE. Based on SDS-PAGE the subunits have a molecular weight of approximately
60 000.
Hexadecyltrimethylammonium bromide, often reported to aid in glutamine synthetase
purification, destroyed enzyme activity in crude extracts. Very low yields, due to weak binding,
were obtained with crude and semi-purified glutamine synthetase when either Affi-gel blue
(Bio-Rad) or ADP-agarose (Sigma) affinity chromatography was attempted. Purified glutamine
synthetase from pv. tabaci did not focus and less than 5 % of the original activity was recovered
on a 20 ml PBE chromatofocusing column (Pharmacia) previously equilibrated with 25 mMimidazole (pH 7.4), and eluted with Polybuffer 74 (pH 4.0), presumably due to sequestration of
the Mg2+cofactor by Polybuffer 74. Glutamine synthetase from pv. tabaci was also isolated by
the methods of Shapiro & Stadtman (1970) and Streicher & Tyler (1980); however, yield and/or
purity was not as good as with the method described here.
Glutamate concentrations between 50 and 1 0 0 m ~a, range commonly used in glutamine
synthetase assays, inhibited glutamine synthetase from pv. tabaci. A Lineweaver-Burk plot over
a 200-fold glutamate concentration range illustrates this (Fig. 2). The inhibition was
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M . D . T H O M A S A N D R. D. D U R B I N
9
8
7
6
a 5
- 4
3
2
\
'I0.5
1.0
1.5
1 /(Glutamate concn, mM)
2.0
0.1
0.2
0.3
1/(Mg .ATP concn, mM)
Fig. 3
0.4
Fig. 2
Fig. 2. Response of glutamine synthetase to various glutamate concentrations. Assay components :
MgCI,, ATP, NH,Cl (10 mM each) and 50 mM-imidazole (pH 7.5).
Fig. 3. Activity of glutamine synthetase at various concentrations of Mg. ATP. Assay components:
NH,Cl and glutamate (10 mM each) and 50 mM mM-imidazole (pH 7.5). Separate blanks lacking
enzyme were used at each Mg . ATP concentration to correct for extraneous phosphate.
1.0
1.5
2.0
2.5
10
20 30 40
50
60
MgCI, concn (mM)
l/(NH,CI concn. mM)
Fig. 4
Fig. 5
Fig. 4. Activity of glutamine synthetase at various concentrations of NH4Cl. Assay components :
MgC12, ATP, glutamate (1OmM each) and 50 mM-imidazole (pH 7.5).
0-5
Fig. 5. Response of glutamine synthetase to Mg2+in the transferase assay. Assay components: 40 mMimidazole (pH 7.2). 60 mM-NH20H, 3 mM-MnCI,, 20 mM-KN,AsO,, 0.4 mM-ADP and 20 mMglutamine.
independent of ammonium concentration in the range 5-50 mM-NH,Cl. The enzyme showed a
linear response from 0.5 mM- to 2.5 mM-glutamate (correlation coefficient 0.99). High
concentrations of ATP inhibited the enzyme to a lesser extent (Fig. 3). The response of the
enzyme to ammonium was linear over the range 0.4 mM to 1 mM (correlation coefficient 0.99;
Fig. 4). Ammonium concentrations greater than 10 mM slightly decreased the velocity.
Maximum velocity was observed at 10 mM-glutamate, 20 mM-Mg .ATP and 4 mM-NH,Cl.
After dialysis to replace Mg2+with Mn2+,the y-glutamyltransferase activity of glutamine
synthetase from pv. tabaci cells was assayed with various concentrations of MgClz (Fig. 5).
Velocity was maximum in the absence of Mg2+,and at 10 mM it had decreased by 75%. The
adenylylated form of the enzyme in other bacterial systems is the only form sensitive to high
levels of Mg2+(Shapiro & Stadtman, 1970; Stadtman et al., 1970; Streicher & Tyler, 1980).
Adenylylated glutamine synthetase from E . coli had lowered activity without Mg2+, optimal
activity at 3 mM-Mg2+,and significantly lowered activity at Mg2+concentrations greater than
30 mM. In contrast, the presence of any Mg2+decreased the transferase activity of glutamine
synthetase from pv. tabaci.
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P. syringae pt'. tabaci glutamine synthetase
1065
20
I
I
1
I
I
I
0.3
0.4
0.5
Tabtoxinine-p-lactam concn (mM)
0.1
0.2
Fig. 6. Response of glutamine synthetase to various concentrations of tabtoxinine-P-lactam. Assay
components: MgCl,, ATP, NH4CI, glutamate (10 mM each) and 50 mM-imidazole (pH 7.5).
Table 2. Eject of incubation of enzyme, tabtoxinine-P-lactam and components of the assay
mixture on inhibition of pv. tabaci glutamine synthetase activity
Preincubation was for 30 min; all treatments contained 50 mM-imidazole (pH 7.9,O.l mM-tabtoxininep-lactam and glutamine synthetase. The concentrations of glutamate, Mg .ATP, Mg .ADP and NH4Cl
were each 10 mM. The incubation treatment containing ADP was corrected for phosphate
contamination of the ADP and inhibition of synthetase activity by ADP.
Preincubation
mixture
Mg.ATP
NH,CI
Mg . ATP
N H,Cl
+
-
Mg.ADP
+ NH,CI
Assay
incubation
Glutamate
NH,Cl
glutamate
Mg.ATP
glutamate
Mg.ATP
NH,CI
gl u tama te
Mg.ATP +glutamate
+
+
+
+
Percentage
of control*
11
42
100
96
91
* Mean of four experiments. The control consisted of 0.1 mM-tabtoxinine-P-lactam added at assay initiation:
100% = 0.14 pmol Pi released. A control without tabtoxinine-&lactam was 220% of the control with it.
Tabtoxinine-P-lactam at 0.25 mM inhibited the synthetase activity of pv. tabaci enzyme by
90%; tabtoxin and tabtoxinine-6-lactam did not inhibit this activity. The inhibition of
glutamine synthetase by tabtoxinine-P-lactam was linear over the range 0.01 mM to 0.5 mM
(correlation coefficient 0.99; Fig. 6). Various components of the synthetase assay were first
incubated with 0.1 mM-tabtoxinine-P-lactam and the enzyme for 30 min, at which time the
remaining assay components were added along with the glutamate to initiate the assay. The
preincubation mixture containing both Mg.ATP and NH4C1 showed a marked decrease in
activity over the control, the mixture containing Mg.ATP was intermediate and the other
treatments showed negligible decrease (Table 2).
Recovery of enzyme activity was attempted from glutamine synthetase inhibited by
incubation with 2 mM-tabtoxinine-P-lactam by: (i) 24 h dialysis against IM buffer, (ii) 24 h
dialysis against synthetase assay mixture containing glutamate, (iii) incubation for 30 min with
crude cell lysate at 24 "C, and (iv) precipitation by the addition of 0.5 vol. acetone (- 20 "C)
followed, after centrifugation, by resuspension of the pellet in IM buffer. No glutamine
synthetase activity was recovered by any of the above treatments.
Tabtoxinine-P-lactam at 0.5 mM inhibited the y-glutamyltransferase activity of glutamine
synthetase from pv. tabaci when ATP, but not ADP, was present. The transferase assay using
ATP incorporates a 30 min incubation of assay components and enzyme before the addition of
glutamine to start the reaction. Tabtoxinine-P-lactam was added at 30min along with the
glutamine; the assay using ADP was done in the same manner. Separate experiments using the
transferase assay with ADP but without this 30 min incubation period showed that tabtoxininep-lactam did not inhibit the enzyme when ADP was used in the assay. Using ATP, the
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1066
M . D. THOMAS A N D R . D. D U R B I N
transferase activity in the presence of tabtoxinine-&lactam was 10% of the control activity,
whereas the analogous reaction using ADP was 102% of its control. In parallel experiments,
tabtoxinine-P-lactam inhibited the transferase activity of glutamine synthetase from both pea
seed and E . coli when either ADP or ATP was used in the assay.
DISCUSSION
Glutamine synthetase from pv. tabaci has two unusual characteristics when compared with
other bacterial glutamine synthetases. First, moderate to high levels of each substrate used
decreased its activity. The inhibition observed with each substrate was not affected by either
saturating (inhibiting) or optimal concentrations of the other two substrates. Second, the
enzyme was sensitive to low concentrations of Mg2+in the transferase assay. It was much more
sensitive than adenylylated glutamine synthetase from E. coli. This sensitivity to Mg2+ may
indicate that the enzyme from pv. tabaci was isolated in an adenylylated state. However, if it
responds like other adenylylated enzymes, one would have expected to see negligible synthetase
activity in the presence of Mg2+:this was not observed.
Incubation of the synthetase assay components indicated that both NH4Cl and Mg.ATP
were required for inhibition by tabtoxinine-/I-lactam. Incubation of the enzyme with the toxin in
the presence of these substrates decreased the remaining activity over the non-incubated
treatment. The assay chemicals were not totally free of ammonium; separate experiments with
shorter preincubation periods showed that the decrease in activity of the NH4C1-free incubation
treatment was the result of this residual ammonium. Inhibition by tabtoxinine-P-lactam of
transferase activity of the pv. tabaci enzyme was different from that of the enzyme from pea
(Thomas et al., 1983) or E. cofi. Also, this inhibition was different from that caused by MSO.
Tabtoxinine-/I-lactam inhibits transferase activity of glutamine synthetase from pea and E. cofi
when either ADP or ATP is present in the assay mixture : it inhibited the pv. tabaci enzyme only
if ATP was present. All three enzymes are inhibited by MSO when either ADP or ATP is
present in the mixture. This may indicate that ATP is an absolute requirement for the binding of
tabtoxinine-Q-lactam to pv. tabaci glutamine synthetase and that ADP will suffice for its binding
to the pea and E. coli enzymes along with the binding of MSO to all three enzymes.
It seems unlikely that the self protection of pv. tabacifrom tabtoxinine-/I-lactam is the result of
multiple forms of the enzyme. Purification by three different methods yielded an enzyme which
was not separable by electrophoresis, heat stability or Mg2+sensitivity, characteristics often
used for distinguishing different forms. Further, heat treatment of crude lysate resulted in no loss
of total glutamine synthetase activity. It is possible that another form of the enzyme may be
produced under other growth conditions, but we found no evidence to support this.
We are currently investigating the possibility that this self protection is the result of either
strict intracellular compartmentalization or excretion of tabtoxinine-P-lactam upon formation.
Tabtoxin and tabtoxinine-&lactam have not been found within cells of pv. tabaci (R. D. Durbin,
unpublished results). The unique substrate interactions observed with pv. tabaci glutamine
synthetase may also play a role in the tolerance of the organism. Altered substrate affinity has
been reported to confer resistance to MSO in mutants of Salmonella typhimurium (Miller &
Brenchley, 1981). The glutamine synthetases from these mutants are still irreversibly inactivated
by MSO, but they also have significantly higher K , values for both glutamate and ammonium.
These bacteria are capable of growing at a slightly lower rate in a concentration of MSO which is
completely inhibitory to wild-type S. typhimurium.
Tabtoxin does not inhibit glutamine synthetase from pea (Thomas et al., 1983) or pv. tabaci,
and tabtoxin at concentrations of at least 5 0 0 does
~ ~ not inhibit the growth of pv. tabaci,
whereas 250 pM-tabtoxinine-/I-lactam will inhibit the growth of pv. tabaci for a period of 6 to
30 h, depending on the strain used. It is conceivable that the formation of tabtoxin is a
detoxification mechanism ; however, the proportions of tabtoxin and tabtoxinine-P-lactam
formed in culture can be altered by manipulating the medium (Durbin & Uchytil, 1984).
Elucidation of the mechanism of glutamine synthetase inhibition by tabtoxinine-&lactam
and a complete understanding of the self protection exhibited by pv. tabacimay be important for
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P.syringae p v . tabaci glutamine synthetase
1067
understanding the host-parasite interactions of tabtoxin-producing P . syringae pathovars. In
addition, the unusual characteristics of pv. tabaci glutamine synthetase have the potential of
adding information to our understanding of this complex enzyme in relationship to its regulation
and occurrence in other organisms. It is apparent that glutamine synthetases from
pseudomonads are more diverse than was previously thought.
Supported in part by U S D A Competitive Research Grant Program, grant no. 83-CRCR-1-1201
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