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Journal of Bacteriology, October 2000, p. 5771-5778, Vol. 182, No. 20
Department of Microbiology, Colorado State
University, Fort Collins, Colorado 80523-16771;
Università di Pisa, Dipartimento di Scienze
Farmaceutiche, 56126 Pisa, Italy2; and
School of Microbiological, Immunological, and Virological
Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne,
United Kingdom3
Received 22 May 2000/Accepted 25 July 2000
Mycobacterium smegmatis has been shown to contain two
forms of polyprenyl phosphate (Pol-P), while Mycobacterium
tuberculosis contains only one. Utilizing subcellular fractions
from M. smegmatis and M. tuberculosis, we show
that Pol-P synthesis is different in these species. The specific
activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and
heptaprenyl diphosphate were the main products synthesized in vitro,
whereas in M. tuberculosis only decaprenyl diphosphate was
synthesized. The data from both organisms suggest that geranyl
diphosphate is the allylic substrate for two distinct prenyl
diphosphate synthases, one located in the cell membrane that
synthesizes Polyprenyl phosphates (Pol-P) are
involved in the biosynthesis of bacterial cell walls (14),
and their availability is rate limiting for several aspects of cell
wall synthesis in Staphylococcus aureus (15) and
Bacillus spp. (2). It has also been suggested that the rate of synthesis of lipid I (in peptidoglycan synthesis) of
Escherichia coli may be dependent on the pool level of Pol-P (26), and Baddiley (4) reported that Pol-P levels
could regulate the rate of bacterial cell wall synthesis in vivo.
Mycobacterium smegmatis is known to contain two forms of
Pol-P that are covalently attached to mannose (25). These
are structurally unusual in that the decaprenyl phosphate contains one
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Polyprenyl Phosphate Biosynthesis in
Mycobacterium tuberculosis and Mycobacterium
smegmatis
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
,E,Z-farnesyl diphosphate and
the other present in the cytosol that synthesizes ,E,E,E-geranylgeranyl
diphosphate. In M. smegmatis, the
,E,Z-farnesyl diphosphate is utilized by a
membrane-associated prenyl diphosphate synthase activity to generate
decaprenyl diphosphate, and the ,E,E,E-geranylgeranyl
diphosphate is utilized by a membrane-associated activity for the
synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however,
,E,E,E-geranylgeranyl
diphosphate is not utilized for the synthesis of heptaprenyl
diphosphate. Thus, the difference in the compositions of the Pol-P of
M. smegmatis and M. tuberculosis can be
attributed to distinct enzymatic differences between these two organisms.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-isoprene unit, one E-isoprene unit, and eight
Z-isoprene units (mono-E, poly-Z)
(28) and the heptaprenyl phosphate consists of either four
saturated isoprene units on the omega end of the molecule, two
E-isoprene units, and one Z-isoprene unit
(6) or four saturated and three Z-isoprene units
(27) (Fig. 1A) (the
stereochemical configuration of the isoprene units is always listed
starting at the omega end of the molecule). It appears that
Mycobacterium tuberculosis may be more typical than M. smegmatis, as a single predominant Pol-P (decaprenyl phosphate)
was identified in this species, however; the stereochemistries of the
individual isoprene units were not determined (24).
View larger version (21K):
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FIG. 1.
Structures of heptaprenyl diphosphate and decaprenyl
diphosphate (A) and prenyl diphosphate synthesis scheme (B). The
structure of the heptaprenyl diphosphate is drawn as described for
heptaprenyl phosphoryl mannose by Wolucka and de Hoffmann
(27). The stereochemistry of the two isoprene units
indicated by the arrow is ambiguous, as they have been reported to be
Z (27) (shown) or E (6).
The structure of the decaprenyl diphosphate is drawn as described by
Wolucka et al. (28). Panel B shows the chain elongation of
E-prenyl diphosphates by head-to-tail condensation of
various allylic diphosphates (used as reaction primers in this study)
with IPP.
The fact that both forms of Pol-P in M. smegmatis are glycosylated suggested that both could be involved in the synthesis of cell wall polysaccharides. Our laboratory has shown that M. smegmatis utilizes its unusual Pol-P molecules in many stages of cell wall biosynthesis. Mature mycolic acids appear to be formed from precursors while attached to a heptaprenyl phosphate molecule (6). Decaprenyl-P-arabinose is a precursor of the arabinan portions of arabinogalactan, arabinomannan, and lipoarabinomannan (28). A polyprenyl diphosphate carrier lipid has been implicated in the synthesis of the linker unit galactan of M. smegmatis (17) and in the synthesis of linear forms of lipoarabinomannan (5).
Despite the crucial role of Pol-P in bacterial cell wall biogenesis, little is known about its biosynthesis, especially in Mycobacterium spp. Pol-P is typically synthesized by enzymes that catalyze the 1'-4 condensations of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates (reaction primers) in order to generate longer, physiologically appropriate, allylic prenyl diphosphates (Fig. 1B). The diphosphates are subsequently dephosphorylated to form the appropriate Pol-P. The importance of this biosynthetic pathway in mycobacterial biology is demonstrated by the fact that all species of mycobacteria tested (reference 19 and our unpublished data) are susceptible to the antibiotic bacitracin, which specifically binds prenyl diphosphate intermediates in Pol-P synthesis (22). Prenyl diphosphate synthases are very widespread in nature, but of the hundreds in existence, only a few have been studied (20).
Thus, Pol-P synthesis is clearly important in the rate of bacterial growth and the synthesis of cell wall components essential for the viability of mycobacteria. The intriguing structural differences in Pol-P from M. smegmatis and M. tuberculosis prompted us to initiate an investigation of the biosynthesis of Pol-P in these two species to explain the enzymatic basis of these observations.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials.
[14C]IPP (55 mCi/mmol) was
purchased from Amersham Life Science Inc. (Arlington Heights, Ill.),
potato acid phosphatase was purchased from Boehringer Mannheim
(Indianapolis, Ind.), and dimethylallyl diphosphate (DMAPP),
,E,E,E-geranylgeranyl diphosphate
(,E,E,E-GGPP), farnesol (mixed
stereoisomers), ,E,E-farnesol, and
,E-geraniol were purchased from Sigma (St. Louis, Mo.).
,E,E-Farnesyl diphosphate (,E,E-FPP) and ,E-geranyl
diphosphate (GPP) were synthesized as described by Davisson et al.
(8). Authentic prenols and prenyl phosphates of various
chain lengths were obtained from the Institute of Biochemistry and
Biophysics, Polish Academy of Sciences (Warsaw, Poland), and
,E,E,Z-geranylgeraniol was a gift from C. J. Waechter (University of Kentucky). Kieselgel 60 F254 thin-layer chromatography (TLC) plates were from EM
Science (Gibbstown, N.J.), and LKC18F reverse-phase TLC
plates were from Whatman (Maidstone, England).
Subcellular fractionation. M. tuberculosis (H37Rv) was grown to mid-log phase in glycerol-alanine-salts medium, washed with saline, and harvested by centrifugation. The resulting pellet was irradiated for 18 h at 2,315 rads/min using a JL Shepard instrument with a 137Cs source. This exposure was calculated to kill 100% of the bacteria but retain 90% of enzyme activity. M. smegmatis was grown to mid-log phase in nutrient broth (Difco, Detroit, Mich.). Cells were harvested by centrifugation, washed with a 0.9% saline solution, and centrifuged again. Some M. smegmatis cultures were harvested and subjected to the same irradiation protocol in order to confirm the effect of irradiation on prenyl diphosphate synthases. The prenyl diphosphate synthase activities in the irradiated preparations were 76% (average of 10 experiments) of those seen in nonirradiated controls.
The washed cell pellets from both species were resuspended in homogenization buffer containing 50 mM MOPS (morpholinepropanesulfonic acid) (pH 7.9), 0.25 M sucrose, 10 mM MgCl2, and 5 mM 2-mercaptoethanol and disrupted by probe sonication on ice with a Sanyo Soniprep 150 (10 cycles of 60 s on and 90 s off). The resulting suspension was centrifuged at 15,000 × g for 15 min. The pellet was discarded, and the supernatant was centrifuged at 200,000 × g for 1 h in a Beckman Ti70.1 rotor. The resulting supernatant (cytosol) was divided into 1-ml aliquots and frozen at
70°C until used. The 200,000 × g pellet
(membranes) was resuspended in homogenization buffer, divided into
aliquots, and frozen at 70°C. The protein concentrations of the
fractions were estimated using a bicinchonchinic acid protein assay kit
(Pierce, Rockford, Ill.).
Prenyl diphosphate synthase assays. Prenyl diphosphate synthase activity was assayed in mixtures containing 50 mM MOPS (pH 7.9), 10 mM sodium orthovanadate, 5 mM MgCl2, 2.5 mM dithiothreitol, 0.3% Triton X-100, 100 µM allylic diphosphate, 30 µM [14C]IPP, and 200 to 400 µg protein in a final volume of 200 µl. After incubation at 37°C for 10 to 60 min (M. smegmatis) or 1 to 12 h (M. tuberculosis), the reaction was stopped by the addition of 1 ml of water saturated with NaCl. Reactions were assayed under conditions linear for both time and protein concentration. Radiolabeled products were extracted with butanol saturated with water, and an aliquot was taken for liquid scintillation spectrometry. The radiolabeled products were characterized by TLC before and after treatment with potato acid phosphatase.
Enzymatic dephosphorylation of reaction products. Dephosphorylation of prenyl diphosphates to determine the length of the prenyl chain and the stereochemistry of the isoprene units was accomplished essentially as described by Fujii et al. (11). Samples were dried under a stream of nitrogen and dissolved in 5 ml of buffer containing 100 mM sodium acetate (pH 4.8), 0.1% Triton X-100, and 60% methanol. After a brief bath sonication, 1.5 U of potato acid phosphatase were added, and the mixture was incubated at room temperature overnight. Dephosphorylated products were extracted three times with 1 ml of n-hexane, the pooled extracts were washed with water, and the solvent was evaporated under nitrogen. Samples were redissolved in chloroform-methanol (2:1, vol/vol), and aliquots were taken for liquid scintillation spectrometry and analysis by TLC.
Product analysis. Analysis of the chain length of the dephosphorylated products was accomplished by TLC on LKC18F plates developed in methanol-acetone (8:2, vol/vol). Radioactive spots were located with a System 200 Imaging Scanner (Bioscan Inc., Washington, D.C.) or by autoradiography. Standard polyprenols were located with an anisaldehyde spray reagent (9).
The radioactive spots derived from the dephosphorylated, enzymatically labeled products that were identified as either farnesol or geranylgeraniol were scraped from the reverse-phase TLC plates and extracted from the gel with chloroform-methanol (2:1, vol/vol). The extracts were pooled, dried under nitrogen, and dissolved in chloroform-methanol (2:1, vol/vol) containing authentic, nonradioactive,E,E- and
,E,Z-farnesol or
,E,E,Z- and
,E,E,E-geranylgeraniol. The
stereochemistry of the radiolabeled product when one isoprene unit was
added to an allylic primer of known stereochemistry was determined by
TLC on Silica Gel G60 plates developed with toluene-ethyl acetate (7:3,
vol/vol).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Enzymatic transfer of radiolabeled prenyl groups from
[14C]IPP into allylic prenyl diphosphates.
The
cytosolic and membrane fractions prepared from M. smegmatis
and M. tuberculosis contain enzymatic activities that
incorporate radioactivity from [14C]IPP into longer
allylic diphosphate products using DMAPP, GPP, ,E,E-FPP, and
,E,E,E-GGPP as reaction primers
(Table 1). The cytosolic fractions from
both organisms were most active in the presence of GPP. The membrane
activities were also most active in the presence of GPP but could
utilize the longer-chain primers ,E,E-FPP and
,E,E,E-GGPP more effectively,
especially in M. smegmatis. When no allylic primer was added
to the reaction mixtures containing either cytosol or membranes, no
prenyl diphosphates were formed, even though a small amount of
radioactivity was incorporated into butanol-extractable compounds in
the case of M. smegmatis.
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Characterization of chain lengths of enzymatically labeled products synthesized by mycobacterial cytosol. In order to determine the chain lengths of the radiolabeled products synthesized by the enzymatic reactions, they were extracted, dephosphorylated, and analyzed by reverse-phase TLC. Although the rates of the reactions were low in M. tuberculosis fractions, the incorporation of IPP into product was linear for up to 12 h. Analysis of the products showed that the ratios of prenyl diphosphates produced in each assay remained the same at all time points up to and including 12 h (data not shown). These properties allowed the generation of sufficient product for subsequent chain length and stereochemical analysis.
Figure 2 shows TLC analysis of 14C-labeled prenols generated by dephosphorylation of prenyl diphosphates synthesized by M. tuberculosis and M. smegmatis cytosol incubated in the presence of [14C]IPP and GPP. The major product was identified as geranylgeraniol (GGPP prior to dephosphorylation), with significantly smaller amounts of FPP being produced. When M. smegmatis cytosol was the enzyme source, relatively small amounts of heptaprenyl diphosphate and decaprenyl diphosphate were also detected (Fig. 2B).
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,E,E-FPP was used as the reaction primer (data not shown).
Characterization of chain lengths of enzymatically labeled products
synthesized by mycobacterial membranes.
The low rate of synthesis
of prenyl diphosphates by M. tuberculosis membranes from
[14C]IPP and DMAPP resulted in very small amounts of
product for analysis, thus preventing unequivocal identification (Fig.
3A). However, M. tuberculosis
membranes incubated with GPP as the reaction primer resulted in FPP and
decaprenyl diphosphate being the major products, but products that
correspond to prenyl diphosphates having eight and nine isoprene units
were also formed (Fig. 3B). Only geranylgeranyl diphosphate and octa-,
nona-, and decaprenyl diphosphate were synthesized when
,E,E-FPP was used as the reaction primer, and
when ,E,E,E-GGPP was used as the
reaction primer, octaprenyl diphosphate was formed at a very low rate
(~1 pmol/mg/min).
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,E,E-FPP
as the reaction primer, decaprenyl diphosphate was the major product,
but products that correspond to prenyl diphosphates of intermediate
chain length were seen. When
,E,E,E-GGPP was used as the
reaction primer, heptaprenyl diphosphate was the major product. The
specific activity of the membrane-associated heptaprenyl diphosphate
synthesis from M. smegmatis membranes was 85 pmol/mg/min.
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Characterization of stereochemistries of enzymatically labeled
products.
When the relatively small amount of FPP produced by
M. tuberculosis cytosol incubated with either DMAPP or GPP
as the primer (Fig. 2) was dephosphorylated and analyzed for
stereochemistry by TLC, it was apparent that the
,E,Z configuration was dominant (Fig.
5B). Small, but reproducible, amounts of
,E,E-FPP were seen in these reactions.
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,E,E-FPP was identified as ,E,E,E-geranylgeraniol after it had
been dephosphorylated (Fig. 5E). The farnesol that was produced by
dephosphorylation of the radiolabeled products generated by incubation
of M. tuberculosis membranes with [14C]IPP and
GPP is virtually all ,E,Z-farnesol (Fig. 5C).
When the geranylgeraniol that was produced by the dephosphorylation of
the radiolabeled products generated when M. tuberculosis
membranes were incubated with ,E,E-FPP as the
primer was analyzed for stereoconformation, the material comigrated
with authentic
,E,E,E-geranylgeraniol (Fig. 5F).
The stereochemistries of short-chain prenyl diphosphates synthesized by
subcellular fractions derived from M. smegmatis were essentially the same as those described for the products synthesized by
the fractions derived from M. tuberculosis. There was,
however, one notable exception. When the geranylgeraniol that was
produced by the dephosphorylation of the radiolabeled products
generated when M. smegmatis membranes were incubated with
,E,E-FPP as the reaction primer was
analyzed, ,E,E,Z-geranylgeraniol
was dominant, although a significant amount of
,E,E,E-GGPP was also produced (Fig.
6). Synthesis of
,E,E,Z-GGPP was not observed in
incubations in which M. tuberculosis membranes were used as
the enzyme source.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results from these experiments suggest that there are several prenyl diphosphate synthase activities found in association with the membrane and cytosol fractions derived from both M. smegmatis and M. tuberculosis. The data shown in Table 1 suggest that the cytosolic prenyl diphosphate synthases prefer short-chain allylic primers as substrates, whereas the membrane enzymes prefer longer primers. However, the specific activity of the prenyl diphosphate synthases in M. smegmatis was much higher than that in M. tuberculosis.
It is presently unclear whether the differences in the rates of synthesis of prenyl diphosphates in M. tuberculosis and M. smegmatis are due to relative differences in the abundances of the enzymes or in the catalytic efficiencies of the enzymes. It will be interesting to determine if the activities of these enzymes are limiting for the formation of cell wall in M. tuberculosis and M. smegmatis (slow growers and fast growers, respectively), as speculated earlier (2, 4, 15, 26). It is clear, however, that M. tuberculosis and M. smegmatis have Pol-P synthetic pathways that are significantly different both quantitatively and qualitatively.
Short-chain cytosolic prenyl diphosphate synthases in
mycobacteria.
In both species the major product seen when cytosol
was incubated with [14C]IPP and DMAPP, GPP, or
,E,E-FPP was
,E,E,E-GGPP. Interestingly, no
radioactive GPP was synthesized from DMAPP, even though GPP is an
obligate intermediate in the synthesis of all of the larger prenyl
diphosphates. Similarly, only small amounts of
,E,E-FPP were found, although it is also an
obligate intermediate in the conversion of DMAPP and/or GPP to
,E,E,E-GGPP. The majority of the
FPP synthesized by both the cytosol and membranes was
,E,Z-FPP, which is unlikely to be an
intermediate in the synthesis of
,E,E,E-GGPP. Since
,E,E,E-GGPP was the only isomer of
GGPP isolated from the cytosolic incubations and both isomers of FPP
are water soluble (and hence available in the reaction mixtures), these
results suggest that there may be a single multifunctional enzyme
synthesizing ,E,E,E-GGPP from DMAPP
in mycobacterial cytosol. If this is the case, the enzyme would be
analogous to several eukaryotic ,E,E-FPP synthases that are also multifunctional. For example, purified and
crystallized avian ,E,E-FPP synthase
synthesizes primarily ,E,E-FPP when incubated
with DMAPP as the allylic primer and releases only trace amounts of the
GPP intermediate (18).
Membrane-associated prenyl diphosphate synthases in
mycobacteria.
The prenyl diphosphate synthase activities derived
from the membrane fractions of M. tuberculosis and M. smegmatis differed in several aspects. The membrane activities
from M. smegmatis were capable of utilizing DMAPP as a
reaction primer, whereas the membrane activities from M. tuberculosis could not. The predominant products of reactions
using M. tuberculosis membranes and GPP as the reaction
primer were ,E,Z-FPP and decaprenyl
diphosphate. However, when ,E,E-FPP was used
in the reaction, the primary products were decaprenyl diphosphate and
,E,E,E-geranylgeranyl diphosphate;
M. tuberculosis did not synthesize heptaprenyl diphosphate. In contrast, M. smegmatis membranes incubated with
[14C]IPP and DMAPP, GPP, or
,E,E-FPP, yielded a series of products ranging
in size from GPP to decaprenyl diphosphate, with heptaprenyl diphosphate as a major product.
,E,E,E-GGPP produced a single major
product. M. tuberculosis membranes synthesized octaprenyl
diphosphate (at a very low rate), while M. smegmatis membranes synthesized heptaprenyl diphosphate. It is unlikely that
the octaprenyl diphosphate synthesized by M. tuberculosis is
a precursor of decaprenyl diphosphate since very little decaprenyl diphosphate was synthesized in the presence of
,E,E,E-GGPP but decaprenyl
diphosphate was the primary product when GPP was the reaction primer.
Since it is not possible to assign stereoconformations to molecules to
which more than one isoprene unit is added (using the techniques
reported here), it was not clear whether the octaprenyl diphosphate
synthesized by M. tuberculosis membranes when GPP was used
as a primer was identical to the octaprenyl diphosphate synthesized
when ,E,E,E-GGPP was used. It is
also unclear what the function of the octaprenyl diphosphate is; the
molecule is synthesized in much lower quantities than the heptaprenyl
diphosphate of M. smegmatis and has not been reported as a
mannose carrier (24).
Heptaprenyl diphosphate synthesis is particularly interesting due to
its presence in M. smegmatis and absence in M. tuberculosis. Only a small amount of one intermediate (probably
hexaprenyl diphosphate) can be seen in reaction mixtures in which
M. smegmatis membranes are incubated with
[14C]IPP and
,E,E,E-GGPP (Fig. 4D). The
synthetic activity appears to be due to the presence of a peripheral
membrane enzyme(s) that can be released with 1 M KCl. Subsequent
precipitation by ammonium sulfate and anion-exchange chromatography did
not separate the activity into components (data not shown). Therefore,
this activity appears to be due to a single multifunctional enzyme.
This observation is more consistent with the structure of the
heptaprenyl phosphoryl mannose reported by Wolucka and de Hoffmann
(27) than with the structure reported by Besra et al.
(6).
When ,E,E-FPP was used in reactions with
M. smegmatis membranes, both decaprenyl diphosphate and
heptaprenyl diphosphate were synthesized, which is probably due to the
fact that the membrane activities synthesize both
,E,E,E-GGPP and
,E,E,Z-GGPP (Fig. 6).
However, when ,E,E-FPP was incubated with the
M. tuberculosis membranes, decaprenyl diphosphate was
synthesized, even though we believe that
,E,Z-FPP should be the precursor for
decaprenyl diphosphate (assuming that the decaprenyl phosphate from
this organism has the same stereochemistry as decaprenyl phosphate from
M. smegmatis [28]), and there was no
observable formation of ,E,E,Z-GGPP
(Fig. 5). This observation suggests that the enzyme that initiates the
additions of IPP to FPP in order to form decaprenyl diphosphate is not
strictly specific for one stereoisomer, as reported for other prenyl
diphosphate synthases that are capable of utilizing allylic primers of
various chain lengths and stereochemistries as substrates (7, 10,
16).
Isoprenoid chain elongation in mycobacteria.
Isoprenoid chain
elongation in Mycobacterium spp. appears to be more complex
than previously reported for eubacteria and for mammals. It has been
reported that there are four prenyl diphosphate synthases that
synthesize ,E,E-FPP,
,E,E,E-GGPP,
all-E-octaprenyl diphosphate, and a mixed
Z,E-polyprenyl diphosphate in E. coli (12) and Bacillus subtilis (23).
Mammalian cells typically contain an ,E,E-FPP
synthase, an ,E,E,E-GGPP synthase,
an all-E-prenyl diphosphate synthase (for ubiquinone
synthesis), and a Z-prenyl diphosphate synthase (for
dolichyl phosphate synthesis) (13). In each of these
examples, one of the products was a ubiquinone or menaquinone
precursor. M. smegmatis could have as many as 10 prenyl
diphosphate synthases. This is probably an overestimate, as it has been
shown that some prenyl diphosphate synthase enzymes catalyze the
addition of IPP to FPP to yield products of more than one chain length
(3). However, it is also possible that the estimate of 10 enzymes is accurate, as some prenyl diphosphate synthases generate
products with only one chain length (1, 23). The actual
number of prenyl diphosphate synthases in M. smegmatis
probably lies between 4 and 10.
,E,Z-FPP and a
decaprenyl diphosphate synthase, respectively (21).
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ACKNOWLEDGMENT |
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This work was supported by grant AI-18357 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677. Phone: (970) 491-3308. Fax: (970) 491-1815. E-mail: dcrick{at}cvmbs.colostate.edu.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, October 2000, p. 5771-5778, Vol. 182, No. 20
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