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Journal of Bacteriology, May 1999, p. 3172-3177, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Sodium-Dependent Glutamate Uptake by an
Alkaliphilic, Thermophilic Bacillus Strain, TA2.A1
Catherine J.
Peddie,1
Gregory M.
Cook,2 and
Hugh W.
Morgan1,*
Thermophile and Microbial Biochemistry and
Biotechnology Unit, University of Waikato,
Hamilton,1 and Department of
Microbiology, School of Medical Sciences, University of Otago,
Dunedin,2 New Zealand
Received 17 November 1998/Accepted 2 March 1999
 |
ABSTRACT |
A strain of Bacillus designated TA2.A1, isolated from a
thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and
70°C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was
an obligate requirement for growth. Growth on glutamate was inhibited
by monensin and amiloride, both inhibitors that collapse the sodium
gradient (
pNa) across the cell membrane.
N,N-Dicyclohexylcarbodiimide inhibited
the growth of Bacillus strain TA2.A1, suggesting that an
F1F0-ATPase (H type) was being used to generate
cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of
V-type ATPases, did not affect the growth of Bacillus
strain TA2.A1. Glutamate transport by Bacillus strain
TA2.A1 could be driven by an artificial membrane potential (
),
but only when sodium was present. In the absence of sodium, the rate of
-driven glutamate uptake was fourfold lower. No glutamate
transport was observed in the presence of pNa alone (i.e., no
). Glutamate uptake was specifically inhibited by monensin, and
the Km for sodium was 5.6 mM. The Hill plot had
a slope of approximately 1, suggesting that sodium binding was
noncooperative and that the glutamate transporter had a single binding
site for sodium. Glutamate transport was not affected by the
protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for
glutamate transport. The rate of glutamate transport increased with
increasing glutamate concentration; the Km for
glutamate was 2.90 µM, and the Vmax was 0.7 nmol · min
1 mg of protein. Glutamate transport was
specifically inhibited by glutamate analogues.
| |
INTRODUCTION |
Bacteria display a remarkable
capacity to survive and grow in extremely hostile environments. Entire
groups of organisms (e.g., thermophiles, halophiles, and acidophiles)
have adapted their lifestyles to these extreme environments. Even
within a given group, a very wide range of environmental limits may be
tolerated. In general terms, microorganisms are able to grow over a
wide range of pH values, from 0 to 11.0 (1). For example,
alkaliphiles grow between pH 9.0 and 11.5 and maintain their
intracellular pH between 8.4 and 9.0 (2, 10, 13, 17). Most
aerobic alkaliphiles belong to the genus Bacillus (10,
13, 17). Bacillus species are gram-positive
sporeformers which have been isolated from a diverse range of
environments, including those of neutral, acidic, and alkaline pH. Few
bacteria growing at extremes of both pH and temperature have been
described (18, 19, 22). For example, a novel group of
anaerobic alkaliphilic bacteria capable of optimal growth at 55°C and
pH 9.3 (upper limit of pH 10.3) was described by Li et al. (18,
19). These bacteria extended the combined range of temperature
and pH for bacterial groups, and one of these organisms,
Clostridium paradoxum, was subsequently shown to maintain a
pH gradient of 1.3 pH units at an extracellular pH of 9.0 (2). Bacteria that grow aerobically at alkaline pH and
extreme temperature (optimum growth temperature above 65°C) have not
previously been described.
Bacteria that grow at alkaline pH are faced with bioenergetic problems
in terms of chemiosmotic energy generation and solute transport driven
by the proton motive force (p) (14). The alkaliphilic bacteria studied maintain an intracellular pH lower than the
extracellular pH (inverted pH gradient). It is generally accepted that
alkaliphiles use sodium/proton antiporters to acidify the cytosol and
generate an inwardly directed sodium motive force (pNa)
(16). The use of sodium as a coupling ion circumvents the
problem of a low p. Growth at extremes of temperature also poses the
additional problem of a leaky cytoplasmic membrane to protons
(28). Konings and coworkers (3, 4, 8, 25-27) and
Holtom et al. (9) have demonstrated high proton permeability
of membranes at high temperature, and some thermophilic bacteria
overcome this problem by using sodium as a coupling ion for solute transport.
In this study, we describe the growth and bioenergetic properties of an
aerobic, extremely thermophilic, alkaliphilic Bacillus strain, TA2.A1. This isolate can grow over a pH range from 7.7 to 10.5, with an optimum of pH 9.2 at 70°C. The growth of this bacterium and
glutamate transport were completely dependent on sodium, indicating
that this organism uses sodium rather than protons in bioenergetic processes.
| |
MATERIALS AND METHODS |
Chemicals.
L-[U-14C]glutamic acid
was from Amersham International (Little Chalfont, Buckinghamshire,
England). Amiloride-HCl, carbonyl cyanide
m-chlorophenylhydrazone (CCCP), and monensin were obtained from Sigma Chemical Co. (St. Louis, Mo.).
N,N-Dicyclohexylcarbodiimide (DCCD) and
sodium vanadate were from BDH Chemicals Ltd. (Poole, England).
Growth and maintenance.
Bacillus strain TA2.A1 was
isolated from a continuously enriched pool sample. The growth medium
contained (per liter) 0.5 g of Na2SO4,
0.1 g of (NH4)2SO4, 0.1 g
of MgSO4 · 7H2O, 0.2 g of
K2HPO4, 9.0 g of NaHCO3, 5.0 ml of dictyglomous trace elements (23), 0.1 g of
Trypticase (Oxoid), and 2 g of glutamate (BDH). The pH of the
medium was adjusted to 10 (20°C), which equates to a pH of 9.2 at
70°C after autoclaving, using 2 N NaOH. Then 100-ml aliquots of
the medium were dispensed into 500-ml flasks, which were stoppered
with nonabsorbent cotton wool bungs and autoclaved at 15 lb/in2 for 15 min. Cells were cultured in a 65°C orbital
shaking incubator and aerated by shaking at 100 rpm. In a typical
experiment, flasks were inoculated (1% inoculum) from an overnight
culture and cells were grown to mid-exponential phase (0.2 to 0.25 units of optical density at 450 nm).
Glutamate transport assays.
Cells were harvested by
centrifugation (12,000 × g, 5 min, 4°C) during
exponential growth (0.22 mg of protein/ml) and washed twice in Tris-HCl
buffer (50 mM, pH 9.2; 70°C). The cell pellet was resuspended in the
same buffer to achieve a concentration of 14 to 20 mg of protein per
ml. Aliquots (200 µl) of cell suspension were placed into tubes in a
shaking (70 rpm) water bath (Julabo; Labortechnik, GmbH) at 60°C, and
transport was initiated by the addition of 100 nCi of
L-[U-14C]glutamate (270 mCi/mmol). After
0 to 60 s, transport was terminated by the addition of ice-cold
LiCl (2 ml, 100 mM) and rapid filtration (0.45-µm-pore-size
cellulose-nitrate filter). Experiments were carried out where the
transport rate was first order with respect to protein, and initial
rates are reported here. The filters were washed once with 2.0 ml of
LiCl, dried for 30 min at 105°C, and counted by liquid scintillation.
Cells which were treated with monensin (10 µM) or incubated in the
absence of sodium showed essentially no
[14C]glutamate uptake, and this result indicated that
there was little nonspecific binding of
[14C]glutamate to cells.
Artificial membrane potentials.
To create an artificial
pNa and membrane potential (), washed cells (100 mM Tris-HCl
containing 100 mM KCl, pH 9.2) from exponentially growing cultures were
loaded with potassium by valinomycin treatment (10 µM, 0°C, 30 min)
and diluted 50-fold (4 µl) into 200 µl of Tris-HCl buffer
containing 100 mM NaCl (pH 9.0) plus 1 µM
[14C]glutamate. Potassium-loaded cells were diluted
into either 100 mM Tris-HCl buffer containing 100 mM NaCl and 100 mM
KCl to create a pNa in the absence of or 100 mM Tris-HCl
buffer alone to create a . Controls (no driving force) were
loaded with K+ or K+ and Na+ and
diluted into K+ or K+ and Na+,
respectively. Transport was initiated by a 50-fold dilution of
concentrated cells (4 µl) into buffer (200 µl) containing the radioactive glutamate (see above).
Competition and metabolic inhibitor experiments.
Competitive
substrates (amino acid) and metabolic inhibitors, tested as potential
inhibitors of glutamate uptake, were added to the transport assay
medium 5 min before [14C]glutamate. Unlabeled amino
acids were added at a final concentration of 5 mM. Metabolic inhibitors
were added at the final concentrations indicated in the text. All of
the water-insoluble inhibitors were dissolved in 95% ethanol and
compared with ethanol-treated controls. The results of competition
experiments were expressed as the mean of three determinations, and the
level of inhibition was expressed as the percent inhibition of the
initial rate of uptake compared to controls (nominally 100%) in the
absence of competitive substrate.
| |
RESULTS |
Growth of Bacillus strain TA2.A1.
Bacillus strain TA2.A1 grew rapidly on minimal medium
containing L-glutamate as the sole carbon and energy
source. The optimal conditions for growth were pH 9.2 and 70°C,
and the maximum specific growth rate was 0.35 h1 (data
not shown). Sodium was an absolute requirement for cell growth (Fig.
1a). Growth was barely evident below
a concentration of 5 mM NaCl, and the final optical density increased
as the sodium concentration in the growth medium was increased from 5 to 100 mM. Sodium concentrations greater than 100 mM were
inhibitory to cell growth. These results suggested that the growth of
Bacillus strain TA2.A1 may depend on sodium for energy
generation and bioenergetic processes (i.e., transport, motility,
etc.). To investigate this possibility in more detail, we tested the
effect of specific metabolic inhibitors on Bacillus strain
TA2.A1 to determine whether a p or pNa was required for growth.
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FIG. 1.
Effects of sodium ion concentration (a), monensin (0.1 µM) and CCCP (100 µM) (b), and vanadate (500 µM), DCCD (500 µM), and amiloride (500 µM) (c) on the growth of
Bacillus strain TA2.A1 in minimal medium
containing glutamate; 100 µl of a 100% ethanol solution was
added to controls where inhibitor was dissolved in ethanol. Arrows
indicate addition of inhibitor or ethanol.
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|
The p, and therefore the energized state of the membrane, can be
abolished by proton conductors or uncouplers such as CCCP (11). Monensin is a carboxylic ionophore that disrupts
sodium or potassium gradients or both across bacterial membranes
(21). When monensin was added to exponential-phase cells
growing on glutamate, there was an immediate cessation of growth, even
when as little as 0.1 µM was added (Fig. 1b). Growth was completely inhibited, and cells did not become resistant to monesin even after prolonged incubation. Cells challenged with CCCP (100 µM) in the exponential phase of growth were not initially inhibited, but after 2 h of further incubation there was a significant
decline in the growth rate (Fig. 1b). This effect was more dramatic if higher concentrations of CCCP (500 µM) were added. Because CCCP is a weak acid and causes a collapse of the p by cycling
between the protonated and unprotonated states, this process may be
somewhat reduced at high pH and hence the small effect seen on growth. When amiloride (500 µM), an inhibitor of
Na+/H+ antiporters (12), was
added to exponentially growing cells, there was an immediate cessation
of growth (Fig. 1c). Similarly, DCCD (25 µM), an inhibitor of
the F1F0-ATPase, also caused growth to
cease (Fig. 1c). Vanadate (500 µM), an inhibitor of V-type ATPases, had no effect on the growth of Bacillus strain
TA2.A1 (Fig. 1c).
[14C]glutamate transport.
Cells grown in
minimal medium containing glutamate and washed twice in Tris-HCl (100 mM, pH 9.0) containing 100 mM NaCl transported [14C]glutamate at an initial rate of 0.12 nmol
· min1 mg of protein (Fig.
2a). However, if cells were washed in
Tris-HCl (100 mM, pH 9.0) containing 100 mM KCl, little if any uptake
was observed (Fig. 2a). When sodium-washed cells were preincubated with
either monensin (10 µM) or amiloride (500 µM), the uptake of
[14C]glutamate was completely abolished (Fig. 2b).
Preincubation with CCCP (10 µM) had no effect on
[14C]glutamate transport. To better understand this
requirement for extracellular sodium, we performed experiments in
which the extracellular concentration of sodium was
varied. Cells resuspended in Tris-HCl (pH 9.0) containing 100 mM KCl
did not transport [14C]glutamate at a rate that could
be differentiated from that of nonspecific
[14C]glutamate binding to cells (Fig. 2c). When the
extracellular concentration of NaCl was increased to greater than 5 mM,
the rate of [14C]glutamate uptake increased and
reached a maximum at 25 mM extracellular NaCl (Fig. 2c). Further
increases in sodium concentration did not increase the rate
of [14C]glutamate transport. The
Km for sodium as calculated from an Eadie-Hofstee plot was 5.6 mM, and the slope of the Hill plot was
approximately 1.0, suggesting that sodium binding was noncooperative (Fig. 2c, inset).
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FIG. 2.
(a) [14C]glutamate transport by
washed cells of Bacillus strain TA2.A1 with either 100 mM NaCl or 100 mM KCl (without sodium). The final concentration of
[14C]glutamate was 1 µM. (b) Effects of monensin
(10 µM), CCCP (100 µM), and amiloride (100 µM) on
[14C]glutamate (1 µM) transport by washed cells of
Bacillus strain TA2.A1. The assay buffer contained 100 mM
NaCl. (c) Effect of extracellular sodium chloride concentration on
[14C]glutamate (1 µM) uptake by washed cells of
Bacillus strain TA2.A1. A Hill plot of the data is shown in
the inset.
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|
To demonstrate that sodium and therefore a
pNa was the driving force
for [
14C]glutamate transport, we used an artificially
generated
and
pNa to study [
14C]glutamate
transport. When K
+-loaded cells were diluted into Tris-HCl
containing 100 mM NaCl
to create a
pNa and
, the rate of
[
14C]glutamate uptake was 0.16 nmol · min
1 mg of protein (Fig.
3a). K
+-loaded cells diluted
in Tris-HCl containing both 100 mM NaCl
and 100 mM KCl to create a
pNa in the absence of
did not transport
[
14C]glutamate, suggesting that
was required
for glutamate transport.
An artificially generated
(K
+-loaded cells diluted in Tris-HCl) in the absence of a
pNa was
able to drive [
14C]glutamate transport,
but the level was fourfold lower, indicating
that sodium was required
in addition to a
. No [
14C]glutamate transport
was observed when K
+-loaded cells were diluted into
Tris-HCl containing KCl (no driving
force). Other ions were tested for
the ability to act as a coupling
ion for
[
14C]glutamate transport (Fig.
3b). Cells
(K
+ loaded) diluted into buffer containing 100 mM NaCl
transported
[
14C]glutamate at a rate of 0.19 nmol · min
1 mg of protein (Fig.
3b). The ions
Rb
+, Li
+, Cs
+, and
NH
4+ were ineffective in driving
[
14C]glutamate transport (Fig.
3b).
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FIG. 3.
(a) Transport of [14C]glutamate by
valinomycin-treated and potassium (100 mM KCl)-loaded TA2.A1 cells.
K+-loaded cells were diluted into 100 mM Tris-HCl buffer
(pH 9.2) to create a , Tris-HCl buffer containing either 100 mM
KCl (no driving force), 100 mM NaCl to create a and pNa, or
100 mM NaCl and 100 mM KCl to create a pNa in the absence of .
(b) Cation specificity of the glutamate uptake system of
Bacillus strain TA2.A1. Transport of
[14C]glutamate (1 µM) was measured in
valinomycin-treated, potassium-loaded cells, which were diluted into
Tris-HCl (pH 9.2) containing 100 mM KCl, NaCl, NH4Cl, RbCl,
LiCl, or CsCl.
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|
[14C]glutamate transport versus glutamate
concentration, temperature, and pH.
The rate of glutamate uptake
was studied over a range of glutamate concentrations, pH values, and
temperatures. The pH range for glutamate uptake correlated well with
the pH profile for growth, with the greatest uptake at pH 9.5 and low
levels of uptake at pH 7.5 and 11.0 (Fig.
4a). Maximum
[14C]glutamate uptake was observed at 60°C, and the
rate decreased rapidly above this temperature. Temperatures below
40°C decreased the rate by 50% (Fig. 4b). When the
extracellular glutamate concentration was increased from 1 to
50 µM, the rate of glutamate transport increased rapidly
and saturation kinetics were observed (Fig. 4c). The Eadie-Hofstee plot
was linear; the Km for glutamate was 2.90 µM,
and the Vmax was 0.7 nmol · min1 mg of protein (Fig. 4c, inset).
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FIG. 4.
Effects of extracellular pH (a), temperature (b), and
glutamate concentration (c) on the rate of
[14C]glutamate (1 µM) transport by washed cells of
Bacillus strain TA2.A1. An Eadie-Hofstee plot of glutamate
transport is shown in the inset in panel c.
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|
Competitive amino acids of [14C]glutamate
transport.
The specificity of the glutamate transport system was
determined by measuring the uptake of [14C]glutamate
in the presence of a 50-fold excess of other amino acids (Table
1). [14C]glutamate
uptake was specifically inhibited by L-glutamate, D-proline, and the glutamate analogues
DL-
-methylglutamate, L-cysteate, and
D- and L-aspartate (Table 1).
D-Glutamate had no effect on [14C]glutamate uptake, suggesting that the
transporter was specific for the L isomer.
| |
DISCUSSION |
Bacteria that grow at extremes of pH and temperature are
confronted by two bioenergetic problems. First, at high growth
temperatures, the cytoplasmic membrane becomes more permeable to
ions, including protons, and therefore the use of protons as coupling
ions for solute transport and ATP generation has limitations
(20, 27, 28). The use of sodium as a coupling ion for
bioenergetic processes can be an important energetic advantage because
phospholipid membranes are 6 to 10 orders of magnitude less permeable
for sodium compared to protons (5, 28). The second
bioenergetic problem is that of alkaline pH. The acidification of
cytoplasmic pH at alkaline extracellular pH creates special
bioenergetic problems. If the pH gradient is reversed (pHin < pHout), the electrical potential must increase to
prevent an overall decline in the total p. This chemiosmotically
adverse pH gradient is bypassed by the use of an electrochemical
gradient of Na+ rather than of protons to energize solute
uptake and motility (1, 16, 17, 22). In this study, we
describe the growth of an obligate aerobic, alkaliphilic, thermophilic
Bacillus isolate, strain TA2.A1, which has a pH and
temperature optimum of 9.2 and 70°C, respectively. Microbial growth
under these conditions has not been previously described. Growth of
Bacillus strain TA2.A1 was completely dependent on sodium
(>5 mM) and was inhibited by monensin and amiloride, inhibitors that
collapse the pNa and inhibit Na+/H+
antiporters, respectively (12, 21).
Because Bacillus strain TA2.A1 is an obligate aerobe and
has a growth requirement for sodium, it was of further
interest to determine what coupling ion this bacterium uses to
drive bioenergetic processes such as transport. Bacillus
strain TA2.A1 grew on glutamate as the sole carbon and energy
source in minimal medium. To study the precise nature of the driving
force for glutamate transport, glutamate uptake in response to an
artificially imposed ion gradient was used. Glutamate transport
could be driven by an artificially created pNa but only in the
presence of a . alone was a weak driving force for
glutamate transport. When both gradients, i.e., pNa and , were
applied, the highest rate of glutamate transport was observed. These
results demonstrate that glutamate uptake is driven by with
sodium as a coupling ion. Because CCCP had no effect on glutamate
transport, the transmembrane pH gradient (ZpH) does not seem to play
an important role in glutamate uptake. The use of different monovalent
cations to create an artificial membrane potential demonstrated that
only sodium was able to couple effective glutamate uptake by
Bacillus strain TA2.A1. Two glutamate transport mechanisms
have been described for thermophilic bacteria. In
Bacillus stearothermophilus, L-glutamate (or
L-aspartate) transport proceeds via a sodium/proton
symport mechanism with a 1:1:1 stoichiometry (3, 4,
8). Glutamate transport in other thermophilic bacteria has also
been shown to depend on an electrochemical gradient of sodium. For
example, Clostridium fervidus, a fermentative
bacterium, has been shown to transport glutamate electrogenically
( driven) in symport with two Na+ molecules and
not by ZpH (26). Glutamate transport by the aerobic
thermophilic bacterium Thermus thermophilus was also shown to be catalyzed by a sodium/glutamate symport mechanism (9).
Na+ is the predominant ion for solute transport in
obligately alkaliphilic bacteria, and often in these bacteria the
Na+-solute symporter has a low affinity for sodium
(16). The
Na+-H+-L-glutamate
transport system in the thermophilic organism B. stearothermophilus has a very high affinity for sodium
(Km < 5.5 µM) (8). In the present
study, the effect of sodium ion concentration on glutamate transport
followed Michaelis-Menten kinetics. The Hill plot suggested that
the glutamate transporter had only one binding site for sodium. The
Km for sodium was 5.6 mM, and glutamate transport was completely abolished by 0.1 µM monensin but not the
protonophore CCCP. The results of this study indicate that Bacillus strain TA2.A1 transports glutamate electrogenically
( driven) in symport with sodium. Like other sodium/glutamate
symporters (5), the affinity for sodium is low (>5 mM).
Initial rates of L-glutamate uptake in B. stearothermophilus have been shown to be strongly dependent on the
medium pH. Glutamate uptake was highest at low external pH (5.5 to 6.0)
and declined with increasing pH (4, 8). Glutamate transport
by Bacillus strain TA2.A1 was also affected by the
extracellular pH, and the maximum rate was observed at an extracellular
pH of 9.5. The Km for glutamate was 2.90 µM,
and glutamate uptake by Bacillus strain TA2.A1 was
specifically inhibited by the glutamate analogues
DL--methylglutamate, L-cysteate, and
D- and L-aspartate. The closely related amines L-glutamine and DL-asparagine did not inhibit
glutamate transport. The glutamate uptake system of C. fervidus is not competitively inhibited by -methylglutamate
(26), suggesting that the glutamate transporter from
Bacillus strain TA2.A1 is more like the glutamate transporter in Escherichia coli (6, 7) that is
specifically inhibited by this analogue (24). The glutamate
transporter in B. stearothermophilus is specific for acidic
amino acids and is also most similar to that of E. coli
(7, 24).
Despite the inverted pH gradient and the reduced p in alkaliphiles,
ATP synthesis occurs via completely proton-coupled oxidative phosphorylation, but the mechanism for this remains unknown (14, 15). The Na+-ATPase from the thermophile
C. fervidus functions as a Na+-extruding
ATPase, stimulated to the same extent by both LiCl and NaCl
(25). Speelmans et al. (25) found no evidence for an additional H+-pumping ATPase.
Bacillus strain TA2.A1 grows aerobically and presumably
generates the bulk of its p by respiration. CCCP had no effect on
glutamate transport, but growth did slow in the presence of this
protonophore, suggesting that the collapse of the p causes a
decrease in ATP synthesis and hence anabolic reactions such as
growth. DCCD, an inhibitor of the
F1F0-ATPase (H type), inhibited growth, but
vanadate, an inhibitor of V-type ATPases, had no effect on the
growth of Bacillus strain TA2.A1. Further studies are needed to determine the precise mode of ATP generation in
Bacillus strain TA2.A1.
| |
ACKNOWLEDGMENTS |
C. J. Peddie was supported by a University of Waikato
Post-Graduate Fees Scholarship. G. M. Cook was supported by a
travel grant from the School of Medical Sciences, University of Otago, Dunedin, New Zealand.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand. Phone: 64 7 8384705. Fax: 64 7 8384324. E-mail:
h.morgan{at}waikato.ac.nz.
| |
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Journal of Bacteriology, May 1999, p. 3172-3177, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
