From the Neurobiotechnology Center and Departments of Biochemistry
and Medical Biochemistry, The Ohio State University,
Columbus, Ohio 43210
An open reading frame, ORF3, first
identified adjacent to the mycocerosic acid synthase gene in
Mycobacterium bovis BCG encodes a protein with acyl-CoA
synthase (ACoAS) activity. Genes homologous to acoas are
found adjacent to other multifunctional polyketide synthase genes in
the mycobacterial genome. To test whether these gene products are
necessary to esterify the fatty acids generated by the adjacent
polyketide synthase gene products, the acoas gene was
disrupted in M. bovis BCG using a suicide vector containing the acoas gene with an internal deletion and the
hygromycin-resistant gene as selection marker. Allelic exchange at the
acoas locus was confirmed by Southern hybridization and
polymerase chain reaction amplification of both flanking regions
expected from homologous recombination. Immunoblot analysis indicated
that the 65-kDa ACoAS protein product was absent in the mutant.
Chromatographic analysis of lipids derived from
[1-14C]propionate showed that the mutant did not produce
mycocerosyl lipids, although it produced normal levels of mycocerosic
acid synthase. These results suggest that ACoAS is involved in the synthesis of mycocerosyl lipids of the mycobacterial cell wall.
 |
INTRODUCTION |
Tuberculosis is the leading cause of death from a single
infectious agent, accounting for approximately 26% of all preventable adult deaths in the world. It is estimated that approximately 0.6 billion people are infected with the causative agent,
Mycobacterium tuberculosis, with 8-10 million new cases and
3 million deaths occurring annually (1). Mycobacterial cell walls have
a very high lipid content (50-60%), which constitutes an effective
permeability barrier to antimycobacterial therapies and contributes to
the survival of this pathogen within the host (2). In addition, conventional antimycobacterial treatments largely directed against cell
wall lipids unique to pathogenic mycobacteria have been rendered less
effective because of the increasing incidence of multidrug resistance.
The increased incidence of tuberculosis associated with the AIDS
pandemic has provided an added impetus to identify alternative targets
that would allow for the development of novel therapeutic drugs (3,
4).
Other classes of cell wall lipids found solely in pathogenic
mycobacteria include the phenolphthiocerols (mycosides) and the phthiocerols (5, 6) both of which have been reported to play a key role
in the host-pathogen interaction (7, 8). In each of these lipid
classes, multimethyl-branched long chain fatty acids, known as
mycocerosic acids, are esterified to two long chain diols, the
phenolphthiocerols and the phthiocerols, respectively (5, 8). This
laboratory has previously cloned and characterized the mycocerosic acid
synthase gene (mas) (9) and has identified a gene cluster
involved in phthiocerol and phenolphthiocerol synthesis
(pps) in Mycobacterium bovis BCG (10). In
addition, we recently reported that a small open reading frame, ORF3,
is located at the 5' end of the mas gene; amino acid
sequence homology and enzyme assays using purified ORF3 protein
indicated that this open reading frame encoded an
ACoAS1 (11).
Genes homologous to acoas have been identified adjacent to
the mas gene in M. tuberculosis
(GenBankTM accession number Z83858) and Mycobacterium
leprae (GenBankTM accession number U00010), as well as
5' to a polyketide synthase gene and 3' to a mas-like gene
in M. tuberculosis (GenBankTM accession numbers
Z74697, U00024, and Z77826). Given the high degree of homology among
these genes (54-99% identity; 69-99% similarity), it seems likely
that all these genes play a role in acyl transfer. The multiplicity and
relative location of these genes adjacent to large polyketide
synthase-like enzymes lead us to speculate that their gene products may
be involved in the selective transfer of the acyl products of the
neighboring synthase genes either directly or indirectly to the
ultimate biological acceptors (11). Purified MAS fails to release
mycocerosic acids, and mycocerosic acids are not found free in the
cytosol but instead are esterified to the diols, which suggests the
involvement of a separate transferase enzyme (12). A possible
interaction of the mas and acoas gene products
may be facilitated by their co-localization on the cell wall;
immunogold labeling experiments indicate that MAS is associated with
the cell membrane (13) and recent results also suggest that the ACoAS
protein may be loosely bound to the membrane (11). However, in
vitro studies indicated that purified ACoAS protein catalyzed the
activation of mycocerosic acids to their corresponding thioesters only
at extremely low rates. Furthermore, ACoAS failed to stimulate MAS
activity and did not cause measurable release of mycocerosic acids
either in the free form or as phthiocerol derivatives when
phenolphthiocerol or glycosylated phenolphthiocerol was provided as
acceptor (11). Although such in vitro reconstitution experiments failed to reveal the function of ACoAS, additional factors
may be necessary for this protein to function in the transfer of
mycocerosic acids to the diol acceptors.
The recent development and application of targeted gene disruption
technology to slow growing mycobacterial species (10, 14-16) allowed
us to directly test the in vivo function of ACoAS in
M. bovis BCG. In this paper, we report the targeted
disruption of the acoas gene in M. bovis BCG and
show that this mutant is incapable of producing mycocerosyl lipids,
although it has normal levels of mycocerosic acid synthase activity.
These results suggest that acoas-like genes function in the
transfer of acyl groups, generated by the products of adjacent
polyketide synthase genes, to their ultimate biological acceptors.
 |
EXPERIMENTAL PROCEDURES |
Bacterial Strains and Growth Conditions--
Strain descriptions
and cultivation conditions for all the mycobacterial and
Escherichia coli strains used in this study have been
presented previously (11).
Generation of an acoas Gene Disruption Construct--
A
pUC19-based plasmid, D2, which had previously been subcloned from a
MAS-containing M. tuberculosis BCG cosmid (10), contained the entire 1.749-kb coding region of the acoas gene, 0.8-kb
5'-flanking region and 0.4-kb 3'-flanking region. A 181-base pair
BglII fragment internal to the acoas gene was
deleted and replaced with the hygromycin gene (hyg) from
Streptomyces hygroscopicus. The hyg gene was
excised as a 1.8-kb BglII fragment from plasmid pIJ963
(donated by Dr. John Hopwood, John Innes Center, Norwich, United
Kingdom) and ligated with BglII digested D2, creating
plasmid pORF3C35. The structure of this construct was verified by
restriction enzyme digestion, Southern hybridization analysis, and
sequencing of the ligation junctions.
Electroporation in M. bovis BCG--
Mid-log phase BCG cultures
were subjected to glycine treatment and electroporated with 2 µg of
XbaI digested pORF3C35 using previously outlined
transformation parameters (17). Hygromycin-resistant transformants were
selected on Middlebrook 7H11 plates (Difco) supplemented with oleic
acid-albumin-dextrose complex (Difco) and hygromycin B (50 µg/ml)
(Calbiochem) following incubation at 37 °C for 3-4 weeks.
Genomic DNA Isolation and Southern Blot Analysis--
High
molecular weight chromosomal DNA was isolated according to the method
of Jacobs et al. (17). DNA samples were digested with
appropriate restriction enzymes, transferred to nylon membranes (Nytran
Plus, Schleicher and Schuell), and hybridized with
[
-32P]dCTP-labeled probes according to standard
protocols (18).
PCR Analysis--
To screen hygromycin resistant M. bovis BCG transformants for homologous recombination, PCR
amplification was performed directly on crude lysate of boiled cells.
PCR amplification using standard protocols (Perkin Elmer) and
Taq polymerase (Boehringer Mannheim) was performed using the
following primer pairs: 5'-CGCGTTTAATAGCGCCCAGTCTAG-3' (A)
and 5'-TTGAACTAGCGGCACGAAGAACAA-3' (B);
5'-CGGCGACGTCGTCAAGGAACCCAC-3' (C) and
5'GACACCGCCCCCGGCGCCTGACGC-3' (H1); 5'
CGGCCAGCGTCCAAGAAAATACCG-3' (D) and
5'-TGGACCTCGACGACCTGCAGGCAT-3' (H2);
5'-TGATCCGACCGATGATGAAC-3' (E) and
5'-ACGGCGACAACGTCGGTAAT-3' (F).
Biochemical Analysis of Cell Wall Lipids in the Wild Type and
ACoAS-disrupted M. bovis BCG
Strains--
Na[1-14C]propionate (25 µCi, specific
activity 55 Ci/mole) (American Radiolabeled Chemicals, MO) was added to
100 ml of 12-day-old cultures and incubation was continued at 37 °C
in roller bottles for a further 18 h. Cell pellets, collected
following centrifugation at 15,000 × g for 10 min,
were extracted with an excess of chloroform:methanol (2:1) at room
temperature. Total lipids were extracted by the Folch method (19) and
assayed for 14C in Scintiverse BD using a Beckman LS3801
liquid scintillation spectrometer. Total lipids were separated on
silica gel G plates (20 × 20-cm, 0.5-mm depth) using either
chloroform:methanol (95:5) or n-hexane:ethyl ether (90:10)
as solvent systems. Lipids were visualized by spraying chromatograms
with 5% K2Cr2O7 in 50% sulfuric acid and heating at 180 °C for 10 min. 14C-Labeled
lipids were detected by scanning chromatograms in a Berthold
Tracemaster 20 TLC linear analyzer and by autoradiography. Silica gel
containing labeled phenolphthiocerol and phthiocerol compounds was
removed and lipid material eluted with ethyl ether. These lipids were
refluxed with 5% KOH in 2-methoxyethanol containing 12%
H2O for 3 h, the mixture was acidified with HCl,
extracted with chloroform and the organic solvent was evaporated to
dryness under reduced pressure. The residue was refluxed with 14% BF3 in methanol for 2 h and the products, recovered by chloroform extraction after quenching the reaction mixture with the addition of
water, were subjected to thin-layer chromatography on silica gel G
plates with n-hexane:diethyl ether (90:10) as the solvent. Methyl esters were extracted and analyzed by radio-gas chromatography using a Varian model 3300 gas chromatograph and a Lablogic GC-RAM radioactivity monitor (INUS Systems) operated under previously described parameters (16).
Extraction and Analysis of Total Proteins--
Cells from 500 ml
of 12-day-old cultures of the wild type and acoas mutant
strains were harvested by centrifugation, disrupted in a French press,
and centrifuged at 105,000 × g for 90 min at 4 °C.
Proteins present in the high speed supernatant were separated by
SDS-PAGE on either a 3.5% stacking and 10% separating gel (ACoAS analysis) or a 3% stacking and 5% separating gel (MAS analysis) and
protein bands were visualized by Coomassie Blue staining (20). For
immunoblot analysis, electrophoresed proteins were transblotted onto an
Immobilon polyvinylidene difluoride membrane (Millipore Corp., Bedford,
MA) as described previously (11). MAS and ACoAS antibodies were diluted
1:500 and 1:1000, respectively, and 125I-labeled protein A
was used as the secondary detection reagent.
Determination of MAS and ACoAS Activities--
The cell-free
extract was fractionated on a 1.8 × 12.5-cm DEAE-Sephadex (Sigma
A25) column, and the column was washed with 2 bed volumes of 0.1 M potassium phosphate buffer, pH 6.8, containing 10%
glycerol (v/v), 1 mM dithioerythritol, and 1 mM
EDTA. Proteins were eluted with a 0.1-0.7 M potassium
phosphate buffer gradient (5 bed volumes at a flow rate of 0.8 ml/min)
and the MAS-containing fractions were identified by slot-blot analysis
using the MAS specific antibody and the Supersignal ULTRA
chemiluminescent detection system according to the manufacturer's
recommendations (Pierce). MAS-containing fractions were pooled and
concentrated by ultrafiltration using a PM30 membrane (Amicon, MA). MAS
activity was quantified using labeled methylmalonyl-CoA as described
previously (12), and activity is expressed as picomoles of
methylmalonyl-CoA incorporated into hexane-extractable material per
minute per milligram of protein. ACoAS activity was determined by
quantifying the amount of fatty acyl-CoA in the aqueous phase following
extraction of the reaction mixtures with organic solvents as described
(21). ACoAS activity is expressed in units of fatty acyl-CoA formed per
milligram of protein. One unit is defined as the amount of enzyme which
forms 1 nmol of fatty acyl-CoA per minute. Protein concentrations were determined by the Bradford method (22) with bovine serum albumin as
standard.
 |
RESULTS |
Disruption of the acoas Gene by Allelic Exchange--
To examine
the role of ACoAS in cell wall lipid metabolism, disruption of the
acoas gene in M. bovis BCG was undertaken by allelic exchange. A pUC-based suicide plasmid, pORF3C35, was
constructed containing the entire 1.749-kb acoas-coding
region, 0.8-kb 5'-flanking region and 0.4-kb 3'-flanking region. An
internal 181-base pair BglII fragment of acoas
was replaced with a 1.8-kb hygromycin gene (hyg) as the
selectable marker. Hygromycin-resistant transformants, generated with
the linearized plasmid, were screened by PCR using a set of primers
flanking the deleted internal segment of acoas (Fig.
1, primers A and
B). This primer pair generates a single 0.7-kb PCR product
from the wild type strain, 0.7-kb and 2.4-kb products in a mutant
generated from a single crossover homologous recombination event, and a
single 2.4-kb product in a mutant generated from double crossover
homologous recombination. Analysis of approximately 500 hygromycin-resistant transformant colonies using this PCR screening
strategy identified four mutants with single crossover events and two
mutants with double crossover events. PCR amplification of
representative single and double crossover mutants (RF3147 and RF320,
respectively) using primers A and B is depicted in Fig.
2.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic organization of the construct used
to disrupt the M. bovis BCG acoas gene by
allelic exchange. Hatched, stippled,
checkered, and unshaded boxes represent
acoas coding sequences, acoas-flanking sequences,
acoas internal deleted region, and regions on the
mycobacterial genome outside those used to make the disruption
construct, respectively. Black boxes represent the
hyg gene, which was used as both a disruption element and
selection marker. Relevant sites are indicated as follows:
Bg-BglII; B-BamHI, and S-SphI. Primer
pairs A/B, C/H1, D/H2 and E/F were used for PCR analysis of homologous
recombination as described in the text. P1-P5 denote acoas
segments used as probes in hybridization experiments with PCR products
(Figs. 2-4) and genomic DNA (Fig. 5).
|
|

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 2.
Screening of M. bovis BCG
hygromycin-resistant transformants for disruption of the
acoas gene by homologous recombination. A, PCR
amplification of genomic DNA from M. bovis BCG wild type and
mutant strains with primers A and B; B, Southern
hybridization analysis of PCR products generated in A with
probe P1. C, Southern hybridization analysis of PCR products
generated in A with probe P2. Lane 1, mutant
RF3147; lane 2, wild type M. bovis BCG; and
lane 3, mutant RF320. The 1-kb ladder was used as size
marker.
|
|
Disruption of acoas by allelic exchange was confirmed by
further PCR analysis using two other sets of primers, each containing a
hyg primer and a primer in the mycobacterial genome directly outside the acoas-flanking segments used to make the
disruption construct (Fig. 1, primer pairs C and
H1, and D and H2). In mutant RF320,
these primer pairs generated 2.3- and 1.2-kb PCR products, respectively, sizes consistent with homologous recombination by double
crossover, whereas in mutant RF3147 only the larger PCR product was
obtained, indicating that integration of the disruption construct
occurred at the 5' end of the acoas gene (Fig.
3A). To confirm that the PCR
products generated from either side of the hyg gene
represent genuine acoas-flanking segments, regions representing the 5' and 3' ends of acoas were used to probe
these PCR products. As anticipated, the 5' probe hybridized only with the 2.3-kb PCR product and the 3' probe hybridized only with the 1.2-kb
product (Fig. 3, B and C). Furthermore, primers
specific for the internal deleted segment of acoas (Fig. 1,
primers E and F) failed to amplify this region in
mutant RF320, whereas a 167-base pair fragment was present in both the
wild type and mutant RF3147 (Fig.
4A). The identities of these
PCR products were confirmed by hybridization with the internal
BglII fragment deleted from acoas (Fig.
4B).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 3.
Confirmation of acoas gene
disruption by homologous recombination. A, PCR amplification
of genomic DNA from M. bovis BCG wild type and mutant
strains with primers C and H1 (lanes 1-3) and primers D and
H2 (lanes 4-6). B, Southern hybridization
analysis of PCR products generated in A with probe P3.
C, Southern hybridization analysis of PCR products generated
in A with probe P4. Lanes 1 and 4,
mutant RF3147; lanes 2 and 5, wild type M. bovis BCG; and lanes 3 and 6, mutant RF320.
The 1-kb ladder was used as size marker.
|
|

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 4.
A, PCR amplification of genomic DNA from
M. bovis BCG wild type and mutant strains with primers E and
F. B, Southern hybridization analysis of PCR products
generated in A with probe P2. Lane 1, mutant
RF3147; lane 2, wild type M. bovis BCG; and
lane 3, mutant RF320. The 123-base pair ladder was used as
marker.
|
|
Southern hybridization analysis confirmed that replacement of the
acoas gene had occurred by allelic exchange in mutant RF320. When genomic DNA from the wild type and mutant strains was digested with BamHI and SphI, which results in excision of
the hygromycin element from RF320, the wild type produced a single
hybridization band at approximately 3 kb; RF3147 yielded three
hybridization bands at 3, 2.2, and 0.8 kb; and RF320 produced two bands
at 2.2 and 0.8 kb. This result is consistent with integration of the hyg-disrupted acoas gene and replacement of the
wild type allele in mutant RF320 (Fig.
5).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 5.
Southern analysis of BamHI- and
SphI-digested genomic DNA from M. bovis BCG
wild type and mutant strains hybridized with probe P5. Lane
1, mutant RF3147; lane 2, wild type M. bovis
BCG; and lane 3, mutant RF320.
|
|
Immunological and Biochemical Characterization of the acoas
Gene-disrupted Mutant--
When total proteins from the wild type and
mutant RF320 were separated by SDS-PAGE and subjected to immunoblot
analysis using an ACoAS specific antibody, the wild type showed a
specific cross-reacting band of approximately 65 kDa, whereas this
hybridizing band was not visible in the acoas-disrupted
mutant (Fig. 6). This mutant expressed
ACoAS activity at approximately 20% of wild type levels (0.17 units/mg
protein versus 0.85 units/mg protein in the wild type) when
14C-labeled palmitic acid was used as the substrate.
Therefore disruption of the acoas gene in mutant RF320
results in a failure to express a functional 65-kDa protein that
encodes acyl-CoA synthase activity.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Immunoblot analysis of ACoAS expression in
M. bovis BCG wild type (lane 1) and mutant
RF320 (lane 2). Rabbit anti-ACoAS IgG was used as the
primary antibody, and 125I was used for detection.
|
|
To examine the effect of acoas gene disruption on the
synthesis of mycocerosyl lipids in M. bovis BCG,
incorporation of [1-14C]propionate into total lipids was
examined in both the wild type and acoas-disrupted mutant.
After 18 h of incubation, approximately 18 and 6% of the total
administered 14C was incorporated into total lipids by the
wild type and acoas-disrupted mutant, respectively.
Thin-layer chromatography identified two major labeled lipid fractions
in the wild type that corresponded to phenolphthiocerol esters
(mycosides) and phthiocerol esters. However, in the
acoas-disrupted mutant only the phthiocerol ester fraction
was present (Fig. 7, left).
Charring of chromatograms showed that only phthiocerol esters but not
mycosides were present in the acoas-disrupted mutant,
whereas the wild type contained both (Fig. 7, right).
Approximately equal amounts of label were present in the mycosides and
phthiocerol esters in the wild type (approximately 46 and 48%,
respectively), whereas in the mutant the bulk of the label
(approximately 75%) was in the phthiocerol esters. Following base
hydrolysis, the majority of the label in both strains (in excess of
80%) was found in the acyl portion of the lipids. Radio-gas
chromatographic analysis of the methyl esters from the mycosides in the
wild type indicated that 14C-labeled propionate was
predominantly incorporated into C29 and C32
mycocerosic acids, whereas this fraction was missing from the
acoas-disrupted mutant. The phthiocerol ester fraction of the wild type also contained significant quantities of the
C29 and C32 mycocerosic acids, whereas only
shorter chain fatty acids were present in the corresponding fraction
from the acoas disruptant (Fig.
8). Thus, mycocerosic acids generated by
MAS were not found in the lipids of the acoas-disrupted
mutant.

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 7.
Radio thin-layer chromatographic analysis of
total lipids derived from [1-14C]propionate in M. bovis BCG and the ACoAS-disrupted mutant RF320. Left,
autoradiograms; right, chromatograms charred with
K2Cr2O7/H2SO4.
Lane 1, M. bovis BCG wild type; lane
2, mutant RF320. The arrow indicates mycosides (absent
in the mutant).
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8.
Radio gas-liquid chromatograms of methyl
esters from the phthiocerol esters derived from
[1-14C]propionate in M. bovis BCG wild type
and the ACoAS disruptant RF320. A, fatty acids from the
phthiocerol esters in the wild type M. bovis BCG;
B, fatty acids from the phthiocerol esters in the ACoAS
disruptant RF320. Retention times of methyl esters of
n-fatty acids with carbon atoms indicated are shown by
arrows. MA and MB are C29
and C32 mycocerosic acids, identified as before (12).
|
|
To investigate whether the inability of the ACoAS disruptant to produce
mycosides is a consequence of some type of interference in the
expression of the mas gene, production of the MAS protein was monitored in both the wild type and the ACoAS-disrupted mutant strains. SDS-PAGE and immunoblot analysis indicated that a similar level of MAS protein was produced in both the wild type and the ACoAS-disrupted mutant (Fig. 9). In
addition, MAS protein, partially purified by DEAE-Sepharose
fractionation from the ACoAS disruptant, incorporated radiolabeled
methylmalonyl-CoA into mycocerosic acids at a level similar to that
observed with the wild type strain (approximately 34 pmol of
methylmalonyl-CoA incorporated per min per mg protein) and radio-gas
chromatographic analysis of the fatty acid methyl esters generated by
both the wild type and mutant strains were identical (Fig.
10).

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 9.
SDS-PAGE (A) and immunoblot
(B) analysis of MAS protein expression in M. bovis BCG wild type (lane 2) and ACoAS disruptant RF320
(lane 3). Purified goose fatty acid synthase
(FAS) was used as marker (lane 1). Rabbit
anti-ACoAS IgG was used as the primary antibody and 125I
was used for detection.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 10.
Radio gas-liquid chromatograms of methyl
esters synthesized from [methyl-14C]
methylmalonyl-CoA by MAS protein partially purified by DEAE-Sepharose
fractionation from M. bovis BCG wild type (A)
and ACoAS disruptant RF320 (B). Enzyme assays were
performed as described previously (12) using
n-C20-CoA primer.
|
|
 |
DISCUSSION |
The process by which pathogenic mycobacteria transfer
multimethyl-branched mycocerosic acids onto the diol acceptors on the cell wall is poorly understood. Previous results indicate that the
M. bovis BCG mas gene lacks a chain-terminating
thioesterase domain (9) and that mycocerosic acids remain attached to
the synthase (12). Mycocerosic acids are found exclusively as esters of
phenolphthiocerol and phthiocerol (7, 8), suggesting the involvement of
a separate transferase system that mediates direct transfer of the
newly synthesized mycocerosic acids from the synthase to the diol
acceptors. Recently, we identified a small open reading frame at the 5'
end of the mas gene, ORF3, which displays distinct homology
to a number of ACoAS-like enzymes and purified ORF3 protein has ACoAS
activity (11). However, we could not demonstrate the involvement of
ACoAS in the in vitro activation of mycocerosic acids,
mycocerosic acid release from MAS, or transfer of mycocerosyl groups
from MAS to the diols (11). Therefore, we resorted to the in
vivo disruption of acoas to determine the possible role
played by this gene in mycocerosyl lipid synthesis. The recent
successful application of allelic exchange technology to slow-growing
mycobacterial species made such an approach feasible. To date,
reciprocal recombination has been achieved using the mas
gene (16), the pps gene cluster (10), and the
ureC gene (14) in M. bovis BCG, and the
leuD gene in M. tuberculosis (15). In the present
study, the following evidence clearly showed that the M. bovis BCG acoas gene was disrupted by homologous
recombination. Mutant RF320 lacks the internal segment deleted from the
acoas gene, contains the hyg gene, and PCR
amplification using a combination of hyg and flanking
primers show that the disrupted allele has integrated into the correct
region on the mycobacterial genome. Furthermore, the mutant fails to
produce the 65-kDa protein encoded by acoas and has a
drastically decreased ACoAS activity.
Disruption of the acoas gene in M. bovis BCG
results in the production of a mutant that lacks the ability to produce
mycosides and mycocerosyl phthiocerol esters. Radiolabeling studies
with [1-14C]propionate, used to specifically label
mycocerosic acids, show that even though mycocerosylated
phenolphthiocerol esters are abundant in the wild type, this fraction
is absent in the acoas disruptant. In addition, an
examination of charred chromatograms confirms the total absence of
mycoside production in the mutant. Radio-gas chromatography indicates
that mycocerosic acids are significant components of the phthiocerol
wax esters in the wild type strain. However, in the ACoAS disruptant
mycocerosyl phthiocerols were not present; only shorter chain fatty
acids were esterified to the phthiocerols.
Studies using the acoas mutant clearly show that this strain
produces mycocerosic acid synthase. Partial purification of MAS by
anion-exchange chromatography from the wild type and mutant strains
yielded similar elution profiles. The purified proteins from both
strains were discernible at similar levels following SDS-PAGE and
immunoblot analysis, displayed comparable specific activities, and
generated similar relative proportions of radiolabeled products from
methylmalonyl-CoA. Therefore, it appears that while mycocerosic acid
synthase is produced at wild type levels in the mutant, mycocerosic
acids are not esterified onto the diol acceptors on the cell wall. This
finding directly implicates ACoAS in the process by which mycocerosic
acids are incorporated onto the diol acceptors in the cell wall of
M. bovis BCG. With mandatory coupling of the synthesis and
transfer, MAS would be unable to produce mycocerosic acids in the
absence of a functional transfer mechanism. Alternatively, it is also
possible that disruption of acoas affected the supply of the
acyl-CoA primers required for the elongation of methylmalonyl-CoA by
MAS and thus prevented mycocerosyl lipid synthesis. We consider this an
unlikely possibility because highly homologous acoas genes
are present near other polyketide synthase-like genes in the
mycobacterial genome and these acoas genes are likely to
encode acyl-CoA synthases that would have substituted for the disrupted
one unless there is strict substrate specificity. Considering the
composition of the long chain methyl-branched fatty acids present in
the mycobacteria, C16, C18, and C20
primers would be adequate for the synthesis of all of these acids. If
the acyl-CoA synthases encoded by the multiple acoas genes
produce any of these primers, mycocerosic acids or their homologues
would have been produced by the mutant as MAS is known to be able to
use any of these primers (12). On the other hand it would appear more
likely that the acoas products are specific for the transfer
of the elongated acids generated by the synthases encoded by the gene
adjacent to each acoas in the genome. Such a specificity may
not only depend on the nature of the acyl chain but also on the
interaction of this enzyme with a membrane bound partner that is
probably involved in the acyl transfer to the final acceptor.
The observed lack of mycocerosyl lipids in the acoas
disruptant is consistent with the participation of ACoAS in mycocerosyl transfer to the diols. Previous studies have shown that only
mycocerosic acids are esterified to phenolphthiocerol and that shorter
chain fatty acids cannot substitute in the acylation steps; a
mas mutant that lacks the ability to synthesize mycocerosic
acids also fails to generate any mycosides or mycocerosic
acid-containing phthiocerols (16). Recent studies revealed that unlike
MAS, a purified synthase that produces short chain branched acids
releases its products directly as free fatty acids (23), which can then
be esterified directly to the acceptors without requiring a separate
ACoAS transfer system. Therefore, in the absence of a coupling of
shorter chain branched fatty acid synthase and ACoAS transferase
activities, disruption of ACoAS still permits synthesis of shorter
chain fatty acids and their subsequent incorporation into the
phthiocerol esters.
A possible explanation for the inability of ACoAS to cause mycocerosic
acid transfer from MAS to the diols in vitro may be that
this enzyme alone is not sufficient to effect transfer. In E. coli, ACoAS forms a transient membrane complex with a separate acyltransferase to promote the direct transfer of acyl moieties from
enzyme bound fatty acyl-CoA to the transferase (24). At the C terminus
of the mas gene, a short open reading frame has been
identified which shares homology with polyketide synthase-type condensation enzymes that are involved in ester and amide bond synthesis (9, 25). This enzyme may function with ACoAS in transferring
mycocerosic acids from MAS to the diols without involving release of
free acids and activation of such released acids as discrete separate
steps.
Genes displaying homology to the M. bovis BCG
acoas gene have been identified adjacent to mas
(GenBankTM accession number Z83858), mas-like
genes (GenBankTM accession numbers Z77826 and Z97188) and
polyketide synthase-like genes (GenBankTM accession numbers
U00024, Z74697, and Z84725) in the M. tuberculosis genome.
An acoas homologue is also present on the M. leprae genome adjacent to mas (GenBankTM
accession number U00010). Overall, acoas-like genes located adjacent to polyketide synthases display 45-99% identity. Despite this high degree of conservation, acoas-like genes do not
appear to be functionally interchangeable. We succeeded in creating a stable M. bovis BCG mutant with an altered pattern of lipid
metabolism even though the other acoas-like genes were
presumably intact in this mutant. The presence of additional
acoas-like genes in the vicinity of large multifunctional
polyketide synthases further supports our suggestion that the product
of each acoas-like gene functions specifically with the
products of the neighboring synthase gene to directly transfer their
products to the appropriate acceptors on the mycobacterial cell
wall.
Termination of a biosynthetic process, catalyzed by the product of a
multifunctional synthase gene, by a small protein encoded by an
adjacent open reading frame appears to be a commonly used mechanism not
only in the synthesis and assembly of complex polyketides but also in
the synthesis of other natural products. A coupling of multifunctional
synthase and transferase activities is believed to occur during
gramicidin biosynthesis; a thioesterase-like domain, located at the 5'
end of the gramicidin synthase gene and displaying homology to fatty
acid thioesterases, has been suggested to function as a transacylase in
the chain termination step (26, 27). Similar mechanisms also appear to
be involved in the synthesis of nonribosomal peptide synthases such as
-(L-
-aminoadipyl)-L-cysteinyl-D-valine synthase, which is involved in penicillin and cephalosporin
biosynthesis (28, 29) and the bialaphos antibiotic synthesizing gene
cluster (30). In mycobacteria each acoas-like gene, located
adjacent to each of the many multifunctional polyketide synthases,
probably functions at the terminal step to channel the product of the
synthase to the appropriate acceptor in the cell wall as demonstrated
in this paper for mycocerosyl lipid synthesis.
We thank Linda Rogers and Norvin Fernandes
for assistance with radio-gas chromatographic analysis.