From the Department of Chemistry, University of
Washington, Seattle, Washington 98195-1700 and the
§§ Lehrstuhl für Pharmazeutische Biologie,
Friedrich-Wilhelms-Universität,
D-53115 Bonn, Germany
Received for publication, October 23, 2000, and in revised form, January 3, 2001
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ABSTRACT |
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To investigate a novel branch of the shikimate
biosynthesis pathway operating in the formation of
3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor
of rifamycin and related ansamycins, a series of target-directed
mutations and heterologous gene expressions were investigated in
Amycolatopsis mediterranei and Streptomyces
coelicolor. The genes involved in AHBA formation were inactivated
individually, and the resulting mutants were further examined by
incubating the cell-free extracts with known intermediates of the
pathway and analyzing for AHBA formation. The rifL,
-M, and -N genes were shown to be involved in
the step(s) from either phosphoenolpyruvate/D-erythrose
4-phosphate or other precursors to
3,4-dideoxy-4-amino-D-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G,
and -J genes resemble enzymes involved in the shikimate
biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J.,
Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim,
C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998)
Chem. Biol. 5, 69-79). Mutants of the rifH and
-J genes produced rifamycin B at 1% and 10%,
respectively, of the yields of the wild type; inactivation of the
rifG gene did not affect rifamycin production
significantly. Finally, coexpressing the rifG-N and
-J genes in S. coelicolor YU105 under the
control of the act promoter led to significant production
of AHBA in the fermented cultures, confirming that seven of these genes
are indeed necessary and sufficient for AHBA formation. The effects
of deletion of individual genes from the heterologous expression
cassette on AHBA formation duplicated the effects of the genomic
rifG-N and -J mutations on rifamycin
production, indicating that all these genes encode proteins with
catalytic rather than regulatory functions in AHBA formation for
rifamycin biosynthesis by A. mediterranei.
3-Amino-5-hydroxybenzoic acid
(AHBA)1 has been identified
as the common starter unit (mC7N unit) for the biosynthesis
of ansamycins (1-3) and mitomycins (4). Ansamycins are a class of
natural compounds produced by a variety of microorganisms and plants
and are characterized by a macrocyclic structure consisting of an aromatic ring system connected to an aliphatic chain that forms an
amide linkage to the amino group of the aromatic moiety (5). Based on
the structure of the AHBA-derived aromatic moiety, this family of
compounds can be further subdivided into a benzenic and a naphthalenic
subgroup. The benzenic ansamycins, such as geldanamycin, ansatrienin A,
and ansamitocin (Fig. 1), have been isolated from actinomycetes or
higher plants and are mainly cytotoxic agents against eukaryotes
(6-13). Naphthomycin, streptovaricin, rifamycin B, and
tolypomycin Y (Fig. 1) represent the naphthalenic ansamycins and
have antibacterial activity, particularly against Gram-positive
bacteria and Mycobacterium tuberculosis (14-19).
The biosynthesis of AHBA has been studied in organisms producing
various ansamycins and mitomycin C through the incorporation of
13C- and 14C-enriched glucose, glycerate, and
other precursors. This work led to the hypothesis that the seven-carbon
mC7N unit is derived from the shikimate biosynthesis
pathway (20-23). Genetic investigations on aromatic amino
acid-deficient mutants of Amycolatopsis mediterranei N813
further revealed that the mC7N unit of the rifamycin
chromophore must be derived from early intermediates of the shikimate
biosynthesis pathway (24-27). However, there has never been success in
any attempts to obtain incorporation of labeled shikimic acid, quinic
acid, or 3-dehydroquinic acid (DHQ) into the mC7N unit (20,
23, 28-30).
Based on our previous studies (31-33), we have proposed a novel
pathway for the formation of AHBA, which parallels the early stages of
the shikimate biosynthesis pathway (Fig.
1). Nitrogen is introduced at the
earliest biosynthetic step to form an amino analog of
3-deoxy-D-arabino-heptulosonate 7-phosphate
(DAHP). Proposed intermediates such as
3,4-dideoxy-4-amino-D-arabino-heptulosonate 7-phosphate (aDAHP), 5-deoxy-5-amino-3-dehydroquinate (aDHQ), and
5-deoxy-5-amino-3-dehydroshikimate (aDHS) were synthesized and shown to
be efficiently converted into AHBA in crude cell-free extracts of the
rifamycin B producer, A. mediterranei S699, and the
ansatrienin A producer, Streptomyces collinus Tü1892.
However, the normal shikimate biosynthesis pathway intermediate, DAHP, did not seem to give rise to AHBA under the same conditions, although phosphoenolpyruvate plus erythrose 4-phosphate were indeed converted into aDAHP and AHBA, albeit in very low yield.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparison of early biosynthetic steps of the
shikimate biosynthesis pathway with the proposed pathway for the
formation of AHBA (32, 33) (A) and AHBA-derived
antibiotics (B).
The enzyme which aromatizes aDHS to form AHBA has been purified from
A. mediterranei S699 (33). The encoding gene,
rifK, has been cloned, sequenced, and verified through gene
inactivation to be essential for the biosynthesis of the
mC7N unit of rifamycin (33). Along with the rifK
gene, a region of 95 kb of DNA (Fig. 2)
has been isolated and sequenced (34). This revealed five large open
reading frames (ORFs) coding for a modular type I polyketide synthase,
various putative modifying and regulatory genes, and a subcluster of
ORFs, the rifG--N genes, some of which are
homologous to genes involved in the shikimate biosynthetic and quinate
utilization pathways of plants, bacteria and fungi (34). The
rifG, -H, and -I genes, encoding
homologues of a DHQ synthase, a plant-type DAHP synthase, and a
shikimate or quinate dehydrogenase, respectively, are located upstream
of the rifK gene. Surprisingly, the rifJ gene,
which appears to encode a type II DHQ dehydratase homologue, is located
outside this subcluster about 26 kb downstream from the rifK
gene. Presumably, the rifH product has a similar enzymatic activity as DAHP synthase to condense phosphoenolpyruvate and erythrose
4-phosphate in the formation of aDAHP. The formation of aDHQ and aDHS
would be expected to involve the rifG and -J products to catalyze the cyclization and dehydration, respectively. The
rifK product, AHBA synthase, then aromatizes aDHS to AHBA. The presence of the rifI gene is curious, as it suggests
that the interconversion of aDHQ and 3-deoxy-3-aminoquinic acid or aDHS
and 5-deoxy-5-aminoshikimic acid might play a role in AHBA formation.
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The nitrogen of AHBA was originally proposed to originate from the amide nitrogen of glutamine by action of an amidohydrolase (31). However, a subsequent 15N experiment did not support this notion (32), and no homology to known amidohydrolases was seen in the entire rif cluster. The only gene obviously related to nitrogen metabolism is orf9, located 11 kb downstream of the rifK gene, which encodes an aminotransferase predicted to catalyze amination of a keto group to an aminosugar (34). Curiously, there are four genes, rifL, -M, -N, and -O, located immediately downstream of rifK, which are not obviously shikimate pathway-related. RifO is homologous to certain regulatory genes but its inactivation did not decrease rifamycin B formation.2 The gene product of rifL is similar to a class of oxidoreductases that have been implicated in interconversions between hydroxyl and carbonyl groups, such as glucose-fructose oxidoreductase which oxidizes glucose to gluconolactone and reduces fructose to sorbitol in Zymomonas mobilis (37). The deduced peptide sequence of the rifM gene has considerable similarity with the CBBY family of phosphoglycolate phosphatases involved in glycolate oxidation (38). The rifN gene product shows a significant similarity with the glucose kinase from Streptomyces coelicolor and Bacillus megaterium involved in glucose repression (39, 40). In the context of the current model of rifamycin biosynthesis, the observed homologies of the gene products of rifL, -M, and -N do not clearly reveal their functions. However, the juxtaposition of the rifK and rifL-N genes suggests that there is an organized subcluster or a potential operon that may be responsible for AHBA biosynthesis.
In the present study we have examined the involvement of the
rifG-N, orf9, and rifJ genes
in AHBA formation. We describe inactivation experiments for each of
these genes and the biochemical characterization of the mutants
generated. The rifG-N and -J genes were further subcloned and coexpressed in S. coelicolor YU105.
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MATERIALS AND METHODS |
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Strains and Culture Conditions
All strains and plasmids used in this work are listed in Table I. A. mediterranei S699 was a gift from Dr. G. C. Lancini (Lepetit S.A., Varese, Italy) and was grown as described previously (33). SM medium was used for sporulation. The E. coli strain XL-1 Blue (Stratagene) was routinely used as the host strain for DNA manipulations and for constructing the gene-inactivated suicide vectors. The strain was grown in LB medium supplemented with carbenicillin (100 µg/ml) or hygromycin (100 µg/ml). S. coelicolor YU105 served as the host for the coexpression of the rifG-N and rifJ genes and was cultured in modified R5 medium (without sucrose) (35).
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DNA Manipulation and Analysis
The total genomic DNA of A. mediterranei was isolated as described (33). Southern blotting analysis was carried out as described (33) or using the DIG luminescent labeling and detection kit (Roche Molecular Biochemicals). Oligonucleotides were obtained from Life Technologies, Inc. PCR was carried out in a TEMPTRONIC thermal cycler (Thermolyne). General cloning procedures and manipulation of DNA were performed according to Sambrook et al. (36). The gene sequence of the rifamycin biosynthetic gene cluster, including rifG-O, has been deposited at GenBankTM under accession numbers AF040570 and AF040571.
Mutagenesis in A. mediterranei
A 1.7-kb DNA fragment carrying the hyg gene for
hygromycin resistance either from pIJ693 (41) or
pIJ56073 was routinely used
as the selection marker in the generation of the gene-inactivated
constructs. The DNA fragments containing the targeted genes were
subcloned into pUC119 (43) or pBluescript SK() (Stratagene). The DNA
fragments containing the region to be mutated were as given below:
RifG Inactivation--
A 3.8-kb XhoI fragment
containing the rifG gene was cloned into pBluescript II
SK(), cut at the unique AscI site on the N terminus of the
rifG gene, blunt-ended, and religated to create pHGF7101. A
new BssHII site was generated and led to a frameshift mutation in the N terminus of the rifG gene. The replacement
suicide vector pHGF7102 was created by insertion of a 1.7-kb
KpnI fragment carrying the hygromycin resistance gene from
pIJ5607 into KpnI-treated pHGF7101.
RifG-I Inactivation--
A 5.6-kb
XhoI-SacI fragment containing the C terminus of
the rifE gene, the entire rifF-I
genes and the N terminus of the rifK gene, was cloned into
pBluescript SK(), partially digested with NotI, and
religated to create pHGF7206. A 2496-bp NotI fragment carrying the C terminus of the rifG gene, the entire
rifH gene, and the N terminus of the rifI gene
was deleted. The replacement suicide vector pHGF7207 was created by the
insertion of a 1.7-kb KpnI fragment carrying the hygromycin
resistance gene from pIJ5607 into KpnI-treated pHGF7206.
RifI Inactivation-- A 3.4-kb BsiWI-EcoRI fragment containing the rifI gene was cloned into LITMUS 29 (New England Biolabs, New England), cut at the unique XhoI site on the N terminus of the rifI gene, blunt-ended, and religated to create pHGF7202. A new PvuI site was generated and led to a frameshift mutation in the rifI gene. The replacement suicide vector pHGF7203 was created by the insertion of a 1.7-kb KpnI fragment carrying the hygromycin resistance gene from pIJ5607 into KpnI-treated pHGF7202.
RifL Inactivation-- The 1.6-kb EcoRI-XhoI and 1.65-kb XhoI-BamHI fragments containing the N terminus and C terminus of the rifL gene, respectively, were ligated and cloned into pHGF008 to create pRM04. A 624-bp XhoI fragment was deleted in the rifL gene. The replacement suicide vector pRM05 was created by the insertion of a 1.7-kb KpnI fragment carrying the hygromycin resistance gene from pIJ5607 into KpnI-treated pRM04.
RifM Inactivation-- An 8.6-kb EcoRI-KpnI fragment containing the rifM gene was cloned into pUC119, cut at the unique MluI site on the N terminus of the rifM gene, blunt-ended, and religated to create pRM031. A new BssHII site was generated and led to a frameshift mutation in the rifM gene. The replacement suicide vector pRM032 was created by the insertion of a 1.7-kb KpnI fragment carrying the hygromycin resistance gene from pIJ5607 into KpnI-treated pRM031.
RifN Inactivation-- An 8.6-kb EcoRI-KpnI fragment containing the rifN gene was cloned into pUC119, cut at the unique BsrGI site on the N terminus of the rifN gene, blunt-ended, and religated to create pRM036. A new BsaAI site was generated and led to a frameshift mutation in the rifN gene. The replacement suicide vector pRM035 was created by insertion of a 1.7-kb KpnI fragment carrying the hygromycin resistance gene from pIJ5607 into KpnI-treated pRM036.
RifJ Inactivation-- A 2.5-kb BamHI-EcoRI fragment containing the rifJ gene was cloned into pUC119, cut at the unique BglII site on the N terminus of the rifJ gene, blunt-ended, and religated to create pRM046. A new ClaI site was generated and led to a frameshift mutation in the rifJ gene. The replacement suicide vector pRM051 was created by the insertion of a 1.7-kb BglII fragment carrying the hygromycin resistance gene from pIJ963 into BglII-treated pRM046.
Orf9 Inactivation-- A 3.8-kb BamHI-EcoRI fragment containing the orf9 gene, was cloned into pUC119, digested with PstI, and religated to create pHGF102. An 897-bp PstI fragment was deleted in the orf9 gene. The replacement suicide vector pHGFAT108 was created by the insertion of a 1.7-kb BglII fragment carrying the hygromycin resistance gene from pIJ963 into BglII-treated pHGF102.
The mycelia of A. mediterranei S699 were cultured to an
early log-phase in a 500-ml Erlenmeyer flask with springs containing 100 ml of YMG medium, harvested by centrifugation, and washed twice
with ice-cold 10% glycerol. The prepared mycelia could be stored at
80 °C for at least 6 months. The DNA replacement vectors (~2
µg) were heat-denatured, immediately cooled on ice, and then used for
transformation via electroporation. The transformed mycelia were
transferred directly into 60 ml of YMG medium and grown at 28 °C for
16-36 h to increase the chance for homologous recombination between
the introduced vectors and the chromosomal DNA in growing mycelia.
Regenerated mycelia were then plated onto YMG agar plates containing
hygromycin (100 µg/ml) and continually grown for 1-2 weeks.
Integrated transformants start to appear as visible colonies after 3 days. The colonies resulting from the single crossover recombination
were plated on nonselective SM medium and screened for
hygromycin-sensitive recombinants derived from a second crossover event, through which the vector with the resistance marker is excised.
A dilution series of the harvested spores was prepared and plated again
on nonselective YMG agarose plates. After a sufficient growth, these
colonies were replicated to YMG agarose plates containing hygromycin.
Colonies growing on the nonselective, but not the selective, agar
plates were isolated, and their total genomic DNA was prepared and
analyzed by Southern blotting to determine whether the rif
genes had been replaced with their corresponding inactivated versions
or the colony was a revertant to the wild type. Typically, 10-20
transformants were picked in the initial gene disruption step. On
average, 0.5-5% of gene replacement clones are obtained with this
procedure depending on the length of DNA provided for homologous recombination.
AHBA Feeding and Rifamycin B Analysis
Cultures of the mutants were grown until they reached the early
stationary stage, split equally into two portions, and then transferred
into a new flask. AHBA (1-10 mg) was added to one of the cultures. The
cultures were then grown for 4 more days. The culture broths were
acidified with 1 N HCl to pH 2-3 and extracted twice with
equal volumes of ethyl acetate. After drying and removal of the organic
solvent in a vacuum, the residue was dissolved in 1 ml of methanol and
analyzed by HPLC (System GOLD, Beckman). HPLC was performed on an RP-18
column (250 × 4.6 mm, gradient MeOH:0.05% HOAc at
t0 min = 30:70, at
t20 min = 100:0; 1.0 ml/min flow rate) with
detection at = 256 and 425 nm. Rifamycin B
tret = 12.5 min. The eluted rifamycin B samples were collected, and their identity was confirmed by electrospray-mass spectrometry analysis.
The Vector for Expression of the rif Genes in S. coelicolor YU105
The actII-orf4/pactIII-actI Promoter Cassette-- A 3.1-kb HindIII-PacI fragment containing the divergent promoters (pactIII-actI) and the regulatory gene (actII-orf4) from pRM5 (44) was cloned into pNEB193 (New England Biolabs). The unique NdeI site on the lacZ gene in pNEB193 was then cut, blunt-ended, and eliminated to generate pHGF7502. To introduce unique DNA cloning sites to increase the cloning capacity and to eliminate nonessential sequences, two further steps were carried out. First, a pair of oligomers, TIN004 and TIN005 (Table II), were annealed and cloned into HindIII-SacI-treated pHGF7502 to replace the redundant HindIII-SacI fragment and incorporate BglII and PmeI sites upstream of actII-orf4 to generate pHGF7503. Further, using pHGF7503 as the DNA template, two separate PCR reactions were performed. The PCRI mixture contained oligomers TIN006 and M13-40 (Table II) as primers to obtain a 260-bp DNA fragment containing the pactIII-actI promoter region. The PCRII mixture used oligomers TIN001 and TIN007 (Table II), resulting in a product with the 1.1-kb fragment carrying the whole actII-orf4 gene. After treatment with XbaI, the products of PCRI and PCRII were assembled, digested with HindIII and EcoRI, and cloned into pHGF7503 to introduce the modified actII-orf4/pactIII-actI DNA fragment, leading to pHGF7505. The sequence of the 1.4-kb HindIII-EcoRI assembled product (Fig. 3) was confirmed, using an Applied Biosystems model 377 sequencer.
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Coexpressing rif Genes--
An 8.6-kb
EcoRI-HindIII fragment containing the C terminus
of the rifK gene and the whole rifL-N genes from
pRM028 (Table I) was cloned into pHGF7406 and replaced the 1.6-kb
EcoRI-HindIII fragment (45) to create pHGF7409.
Using pHGF018 (Table I) as the DNA template and primers TIN010 and
TIN011, a 0.5-kb PCR product containing the whole rifJ gene
was amplified and cloned into pHGF7409 to replace the 4.6-kb
NcoI-HindIII DNA fragment located downstream of
the rifN gene. The 4.5-kb NdeI-XbaI
fragment carrying the whole rifK-N and -J genes
from the resulting construct, pHGF7414, was further relocated to
pHGF7505 to generate pHGF7504. To introduce the PacI cloning
site in front of the rifG gene, a 1.1-kb PCR product
containing the N-terminal part of rifG was generated using the DNA template, pHGF7100 (Table I), and primers RM021 and RM024 (Table II). The resulting PCR product was subcloned into pCRScript digested with SrfI, and then the 0.5-kb
AscI-HindIII fragment was further replaced with
the 11.4-kb AscI-HindIII fragment from pRM028
(Table I) to create pRM043. The 3.4-kb PacI-EcoRI
fragment carrying the rifG-I genes from pRM043 and the
6.0-kb PacI-HindIII fragment carrying the
rifK-N, rifJ, actII-orf4 genes and
pactI-III promoters from pHGF7504 were
sequentially cloned into pRM5 to replace the resident act
genes. The resulting construct was designated pHGF7604 (Fig.
4).
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pHGF7604-derived Constructs-- To examine the functional activities of the rif genes in pHGF7604, the coding region of each individual gene was partially deleted or modified by the elimination of a unique restriction site. First, the unique AscI site on the N terminus of the rifG gene in pHGF7604 was cut, blunt-ended, and religated to generate pHGF7606. This created a new BssHII site and led to a frameshift mutation in the rifG gene. pHGF7607 and pHGF7608 were constructed directly from pHGF7604 by the deletion of a 624-bp XhoI or a 224-bp ApaI fragment located in the rifL and rifM coding regions, respectively. The 832-bp NcoI-PmlI fragment covering the C terminus of the rifN gene and the 505-bp NcoI-SpeI fragment containing the entire rifJ coding region, respectively, were deleted in pHGF7604 by double cutting with NcoI and PmlI, or SpeI, blunt-ending and religation to form pHGF7609 and pHGF7610. Using the same strategy, the 0.8-kb XhoI-EcoRI fragment containing the coding region of the rifI C terminus was removed from pHGF7604 and pHGF7605, to create pHGF7612 and pHGF7611, respectively. pHGF7613 was constructed from pHGF7604 by the elimination of the 2.4-kb PacI-BstBI fragment carrying the coding sequences of the rifG-I genes.
Assay for AHBA Production
The AHBA produced in the bacterial cultures was detected through the ability to restore rifamycin B production to the AHBA nonproducing rifK mutant, A. mediterranei HGF003 (33). First, A. mediterranei HGF003 and the test bacterial strains were grown separately in YMG medium and the modified R5 medium (without sucrose) (35). When the cultures had reached the stationary phase, the HGF003 and the test cultures were mixed in a 3 to 1 ratio and culturing was continued for 3 more days. The mixed cultures were then harvested and analyzed for rifamycin B production by HPLC.
AHBA production was assayed quantitatively by the inverse
isotope-dilution procedure described by Kim et al. (32). In
general, 40 µg of [7-13C]AHBA (90% 13C)
was added to the crude S. coelicolor cell extract (2.5 ml), which was then silylated using 100 µl of SIGMA-SIL-A. The gas chromatography mass spectrometry analysis was carried out on a Hewlett
Packard 5890 gas chromatograph connected to a Hewlett Packard 5971A
mass selective detector under conditions as described previously (32).
Enzymatic AHBA formation in cell-free extracts of A. mediterranei mutants was assayed as described by Kim et al. (32).
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RESULTS |
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Effects of rifG, rifH, and rifI Genes on Rifamycin B Production-- Based on DNA and deduced peptide sequence analysis, the products of rifG and rifH are homologous to a DHQ synthase and a plant-type DAHP synthase involved in the shikimate biosynthetic pathway, respectively. The rifI gene product is similar to a shikimate or quinate dehydrogenase in the shikimate biosynthesis and quinate utilization pathways in bacteria, plants, and fungi. These three genes are located side by side and immediately upstream of rifK, the AHBA synthase gene. To relate their functions to AHBA biosynthesis, we constructed a mutant of A. mediterranei, HGF009, in which a 2496-bp NotI DNA fragment carrying the C terminus of rifG, the entire rifH, and the N terminus of rifI had been deleted from the genome. The mutant construction showed that there is an endogenous promoter located upstream of rifG since all isolated primary tranformants that arose by the expected single-crossover integration either upstream of rifG or downstream of rifI had maintained the ability to produce a normal amount of rifamycin B (data not shown). HGF009 is able to grow on SM medium and produces rifamycin B, but HPLC analysis showed that the yield has decreased to 1-2% of that of the wild type strain S699 (Table III). Full rifamycin B productivity can be restored by supplementation of the culture with AHBA.
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To further examine the functional role of each of the three genes in rifamycin biosynthesis individually, A. mediterranei mutants HGF005 and HGF008 were constructed. In both HGF005 and HGF008, four additional nucleotides were introduced either at the unique XhoI or AscI site, resulting in frameshift mutations at the N terminus of the rifI and rifG genes, respectively (see "Materials and Methods"). Both mutants show a similar growth rate, either in SM or YMG medium, as the wild type strain S699. Neither the truncation of the rifI gene in HGF005 nor that of the rifG gene in HFG008 causes a significant change, if any, in the production of rifamycin B compared with the wild type strain S699 (Table III). Thus, the reduced rifamycin production by mutant HGF009 must be due largely to the absence of rifH.
The Products of rifL, rifM, and rifN Genes Are Essential for AHBA Formation-- Located downstream of the rifK gene are three other genes, rifL, rifM, and rifN, that form a subcluster and potentially an operon sharing a transcription unit starting at rifK or possibly earlier (33, 34). The rifL gene encodes a 359-amino acid protein (37.91 kDa), and its initiation codon appears to be located 73 bases downstream of the rifK gene. To probe the function of the rifL product in AHBA and rifamycin B biosynthesis, the replacement vector pRM05 (see "Materials and Methods") was transformed and integrated into A. mediterranei S699. Mutant RM01, in which a 624-bp XhoI DNA fragment has been deleted from the rifL gene, was isolated after a serial screening for the loss of resistance to hygromycin. RM01 is unable to produce rifamycin B, but production can be restored to wild type levels by supplementation of the culture with AHBA (Table III).
The rifM gene encodes a 232-amino acid protein (24.84 kDa) and is located 14 bases downstream of rifL, and 34 bases upstream of the rifN gene, which codes for a 293-amino acid protein (29.78 kDa). The mutants RM04 and RM05 carry the inactivated rifM and rifN genes, respectively, in which four additional nucleotides were introduced at the unique MluI or BsrGI site, leading to a frameshift mutation in the N terminus of rifM and rifN, respectively. As observed above with RM01, there is no detectable rifamycin B production in either mutant and AHBA supplementation restores rifamycin B productivity to wild type levels (Table III). Incubation of the crude cell-free extracts of the mutants with the known pathway intermediates, aDAHP, aDHQ, or aDHS, showed that both the RM04 and RM05 strains were able to convert aDAHP to AHBA with efficiency comparable to that of the wild-type strain S699 (Table III). However, cell-free extracts of strain RM01 failed to convert aDAHP or aDHQ into AHBA, and gave only poor conversion of aDHS, 10% compared with about 90% for the wild-type strain (32).
The rifJ Gene Is Involved in Rifamycin B
Biosynthesis--
The rifJ gene is located 26 kb downstream
of the rifK gene (Fig. 2). The deduced peptide it encodes is
closely related to type II DHQ dehydratases found in the biosynthetic
shikimate and the quinate utilization pathways. rifJ is
likely to be part of the rifamycin biosynthetic gene cluster, since
rpoB and rpoC, genes coding for the
DNA-dependent RNA polymerase - and
'-subunits respectively, have been located 2.3 kb downstream of the
rifJ gene (34). The product of the rpoB gene was
shown to confer resistance to high doses of rifamycin B or rifampicin
when transferred to E. coli and
Mycobacteria.4 As
antibiotic biosynthetic gene clusters in Actinomycetes typically include one or more genes that confer resistance to the antibiotic produced, the rif gene cluster is assumed to extend to the
rpo genes, and thus include rifJ. The
rifJ gene product may catalyze the conversion of aDHQ to
aDHS through the elimination of a water molecule.
To probe for the involvement of rifJ in AHBA formation, the rifJ-inactivated suicide vector pRM051 (see "Materials and Methods") was introduced and integrated into A. mediterranei S699. After serial propagation in hygromycin-free YMG medium, the rifJ-inactivated mutant XZ01 was isolated. In it the functional rifJ gene has been replaced with a mutated version carrying four additional bases, GATC, at the unique BglII site, which causes a frameshift mutation at the N terminus. The elimination of rifJ does not abolish antibiotic production in the XZ01 strain completely, but results in a reduction to ~10% of the wild type level (Table III). Full rifamycin B production can be restored by supplementation with AHBA (Table III). Mutant XZ01 shows similar growth rates in SM and YMG media as the wild type strain S699.
No Detectable Effect of the orf9 Gene on Rifamycin B Production-- The gene product of the orf9 gene is related to a dNTP-hexose aminotransferase and has been proposed to be responsible for the formation of an aminodeoxyhexose nucleotide (34). However, since no glycosylated rifamycin has been identified in the cultures of A. mediterranei S699, one cannot exclude the possibility that this gene product might play a role in the introduction of the amino group in the AHBA biosynthetic pathway. Thus, the orf9-inactivated suicide vector pHGFAT108 was constructed (see "Materials and Methods") and introduced into A. mediterranei S699. After serial culturing, the orf9 gene was then replaced through a second homologous recombination with the truncated version in which an 897-bp PstI DNA fragment has been deleted. The resulting mutant MM01 showed no significant phenotypic change in either the growth pattern or rifamycin B production (Table III).
Production of AHBA in S. coelicolor-- The actII-orf4 gene product has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes in S. coelicolor (46, 47). The actIII-actI intergenic region, as the regulatory target for the actII-orf4 protein, carries a pair of divergently arranged promoters for the early biosynthetic steps to build the polyketide backbone of actinorhodin. The development of a regulatory expression system employing the transcription regulator (the actII-orf4 gene product) and the pathway-specific promoters (pactIII-actI) has already served to generate many novel products of aromatic and macrolide polyketide synthases (44, 48, 49). To verify the functional specificity for each candidate gene in the AHBA biosynthetic pathway, a pNEB193-based actII-orf4/pactIII-actI cassette vector, pHGF7505, was constructed (see "Materials and Methods"). This actII-orf4/pactIII-actI promoter cassette (Fig. 4) with different multiple cloning sites allows for cloning of the target genes to be regulated by a pactI or pactIII promoter for tight transcriptional control. The putative AHBA biosynthetic genes defined as described above are located at two separate regions of the rif gene cluster and may be organized into three separate transcription units (Fig. 2). The 0.5-kb NcoI-HindIII PCR-amplified DNA fragment carrying the entire rifJ gene and a putative ribosome binding site, GGAGG, was connected to the NcoI site, 64 bases downstream of the stop codon of the rifN gene. As described under "Materials and Methods," the fused 4.5-kb NdeI-XbaI DNA fragment containing the rifK-N and rifJ genes, and the 3.4-kb PacI-EcoRI DNA fragment carrying the rifG-I genes, were relocated into the actII-orf4/pactIII-actI promoter cassette at the corresponding restriction sites. The resulting E. coli-Streptomyces shuttle vector pHGF7604, which carries the actII-orf4 regulatory gene and eight rif genes (rifG-N and -J) under the control of the pactIII-actI promoters, was then transformed into S. coelicolor YU105. Unlike S. coelicolor YU105, which is unable to produce AHBA or AHBA-derived ansamycins, S. coelicolor YU105/pHGF7604 transformants can restore rifamycin B production in the AHBA nonproducing rifK mutant, A. mediterranei HGF003, and they produce a significant amount of AHBA (350-400 mg/liter of culture) (Table IV).
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AHBA Production Is Repressed by the Presence of the rifI Gene-- The specific functional roles of the rif genes in pHGF7604 in AHBA formation were further examined in S. coelicolor YU105. First, individual rif genes were removed from pHGF7604 by deleting a region of the DNA fragment or by eliminating restriction sites. The resulting nine new constructs, pHGF7605 to pHGF7613 (Table I), were then transformed into S. coelicolor YU105 to assay for AHBA production. Table IV summarizes the rif gene compositions and the AHBA yields of pHGF7604 and the nine pHGF7604-derived constructs in S. coelicolor YU105. There was no AHBA detectable in the three cultures of S. coelicolor YU105 transformed with pHGF7607, pHGF7608, or pHGF7609. These results are consistent with those from the corresponding three genomic mutants of A. mediterranei, rifamycin-deficient strains RM01, -04, and -05, and confirm that the rifL, -M, and -N gene products are indeed absolutely essential for the formation of AHBA.
As mentioned above, A. mediterranei HGF008 and HGF005, the rifG- and rifI-knockout mutants, did not show a significant change in rifamycin B production compared with the wild type. However, the absence of these genes from the AHBA gene cassette clearly changes the AHBA production profile in S. coelicolor YU105. Reproducibly, 20-25% more AHBA is detected in the cultures transformed with pHGF7612, lacking the rifI gene, than in those carrying pHGF7604. Removal of the product of the rifI gene is not detrimental but seems to enhance AHBA production. In contrast, the product of the rifG gene plays a significant role in maintaining the AHBA productivity in S. coelicolor YU105, since the yield decreased to only 10% in the cultures of pHGF7606 transformants. These opposing effects are further confirmed by the moderate yields detected in the cultures of transformants with pHGF7611, in which both the rifG and rifI genes have been removed.
Deletion of the rifH gene causes a similarly severe effect on AHBA production in S. coelicolor YU105 as on the production level of rifamycin B in A. mediterranei seen in mutant HGF009. The pHGF7605 transformants, in which both the rifH and rifI genes have been deleted, produce less than 0.2% of the amount of AHBA of the pHGF7612 transformants which lack only the rifI gene. When the rifG gene is also removed, AHBA production is completely abolished in cultures of the pHGF7605 transformants.
Eliminating the product of the rifJ gene decreases the yield
of AHBA to 6-8% in the cultures of the pHGF7610 transformants. This
result is comparable to the one observed with the genomic rifJ mutant, A. mediterranei XZ01, which lost
about 90% of its rifamycin B productivity.
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DISCUSSION |
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The biosynthesis of AHBA has been investigated through target-directed mutagenesis in A. mediterranei S699 and heterologous expression in S. coelicolor YU105 of specific genes from the rifamycin biosynthetic gene cluster. Three genes homologous to shikimate biosynthesis pathway genes, the rifG, -H, and -J genes, and four not shikimate pathway-related genes, the rifK (33), -L, -M, and -N genes, were identified as being necessary and sufficient for AHBA formation. The observation that all these genes were required for AHBA production in the heterologous host when expressed under the control of an external promoter/regulator system suggests that none of these genes have a regulatory function in AHBA formation by the natural producer, A. mediterranei. Rather, all of them should encode proteins with catalytic functions in the biosynthetic pathway. The requirement for seven proteins for AHBA formation, particularly the involvement of the rifL, -M, and -N gene products, points to a degree of complexity of the pathway not predicted by our previous hypothesis.
Functions of the rifG, rifH, and rifJ Gene Products-- The products of the rifG, -H, and -J genes have been shown to be functionally active and, as predicted by our original hypothesis, to play a key role in providing the substrate aDHS for AHBA formation in the rifamycin producer, A. mediterranei S699. It is worth noting that all three mutants, HGF005, -8, and -9, showed no detectable growth defects in both the YMG and SM media. This implies that the products of the rifG, -H, and -J genes are probably not directly involved in any other, essential shikimate pathway-related metabolic processes in the cell. The fact that all three mutants kept producing rifamycin B at some level suggests that the defects in rifamycin production caused by the gene inactivations can be at least in part functionally compensated by the presence of the corresponding shikimate pathway isoenzymes in the strain, due to some overlap in substrate specificity. For example, the DHQ synthase from E. coli can cyclize aDAHP to produce aDHQ (32). The heterologously expressed RifG protein from S. lividans 1326 catalyzes the cyclization of DAHP as well as aDAHP as substrate.5 We have detected and partially purified a second type II DHQ dehydratase, a homologue of the rifJ gene product, from A. mediterranei S699 (50). Recently, two additional DAHP synthase isoenzymes and their encoding genes have been identified and cloned from A. mediterranei.6 Interestingly, one of the deduced peptide sequences is closely related to the product of the rifH gene and to type II DAHP synthases from higher plants.
The functional substitution and cross-talk between the products of the rifG, -H, and -J genes, and their corresponding normal shikimate pathway homologues also occurred in the expression of the rif genes to form AHBA in S. coelicolor YU105. However, judging from the amounts of AHBA and rifamycin B produced (Tables III and IV), it is evident that the isoenzymes from S. coelicolor YU105 and from A. mediterrainei S699 display different levels of competence to replace the functions of the rifG, -H, and -J gene products. Notably, and perhaps significantly, in both systems, the complementation of the rifH mutation by endogenous DAHP synthases is less efficient. It is not clear whether the greater competence for complementation observed in the A. mediterranei mutants is accidental or has evolved due to a long term molecular adaptation between substrates and the enzymes exposed to the rifamycin-producing environment.
Functional Role of the rifI Product-- Although its functional role is not obvious, the presence of the rifI gene in the rif gene cluster of A. mediterranei S699 is unlikely to be an evolutionary accident. First, the recombinant protein expressed from the rifI gene in E. coli had the ability to catalyze the 3-dehydrogenation of shikimate, aminoshikimate, and aminoquinate, but not quinate (51). The present work showed that AHBA production in S. coelicolor YU105 was significantly repressed by the presence of the rifI gene in the expression vectors, pHGF7604 and -6 (Table IV). Since the rifI gene is part of the same transcription unit as the rifG and rifH genes, it should be functionally expressed and produce enzymatically active protein in A. mediterranei S699; yet, its disruption had no effect on rifamycin B production. This result recalls the previous observation (32) that there is no increase in the production of rifamycin B by supplementation of wild-type A. mediterranei S699 with AHBA, suggesting that rifamycin B synthesis in the cell is not limited by the production of AHBA.
The earlier AHBA feeding experiments had indicated that AHBA is very stable in the cells of A. mediterranei and is maintained throughout the whole fermentation process without substantial degradation. It is not known whether the accumulation of AHBA could lead to inhibition or suppression of regular shikimate pathway-related enzyme functions or cause any other cellular toxicity, but this possibility might provide an explanation for the presence of the rifI gene. The product of the rifI gene may act in the conversion of aDHQ and/or aDHS to 3-deoxy-3-aminoquinic acid and/or 5-deoxy-5-aminoshikimic acid, and closely regulate an AHBA-precursor reservoir to prevent the uncontrolled accumulation of AHBA in the cell.
Functions of the Products of the rifL, rifM, and rifN Genes-- As the enzyme AHBA synthase (the product of the rifK gene) described previously (33), the products of the rifL, -M, and -N genes are also absolutely essential for the biosynthesis of AHBA, yet they are not related to any shikimate pathway enzymes. It is noteworthy that the location and arrangement of all AHBA synthase genes identified relative to other genes involved in AHBA biosynthesis in their respective gene clusters varies (34, 52, 53).7 However, the close association between the AHBA synthase gene and the rifL and -M gene homologues is conserved in all the AHBA biosynthesis gene clusters analyzed. This suggests that there are functional interactions between these gene products. The results of the cell-free experiments with the rifL, -M, and -N mutants suggest that the rifM and -N gene products act at an early stage of AHBA biosynthesis, probably in or prior to the formation of aDAHP. The product of the rifL gene may also be related to the formation of aDAHP, but it could also or additionally affect the enzymatic activity of AHBA synthase.
The Nitrogen Source in aDAHP Formation-- The available data do not support the original suggestion that the nitrogen of AHBA originates from the amide nitrogen of glutamine through the action of an amidohydrolase, which acts in concert with a DAHP synthase to generate aDAHP. Therefore, a different source and mode of introduction of the nitrogen must be identified. The product of the orf9 gene is homologous to pyridoxal phosphate (PLP)-dependent transaminases and was suspected to carry out this function of introducing the nitrogen into a precursor of AHBA. This has been ruled out by the mutagenesis experiment, which showed no effect of the inactivation of orf9 on rifamycin B production. The fact that the coexpression of rifG-H, -K-N, and -J resulted in production of AHBA in S. coelicolor YU105 further suggests that the nitrogen-introducing activity must reside on these seven rif genes, although we cannot exclude the possibility that a protein from S. coelicolor YU105 has taken over this function. It is worth noting that the deduced peptide sequence of the AHBA synthase gene (RifK) shows homology to the products of a series of genes implicated primarily in dehydration/deoxygenation as well as transamination reactions in deoxysugar biosynthesis (33). The recombinant AHBA synthase can bind the cofactor pyridoxamine phosphate as well as PLP, and the PLP form of the enzyme can be converted to the pyridoxamine phosphate form by incubation with the amino donor, glutamate.8 Based on these observations, it is speculated that the AHBA synthase, possibly together with the rifL, -M, and/or -N gene products, may have an additional function to introduce the nitrogen into an intermediate in AHBA biosynthesis. Consistent with this hypothesis, no accumulation of aDHS, aDHQ, or their likely aromatization product, protocatechuic acid, was observed in the rifK mutant of A. mediterranei (33).9 Plausible scenarios for such a second function of rifK could involve its joint action with rifL either to convert a keto group into the corresponding imine by transamination (RifK) and oxidation (RifL), or to replace a hydroxy with an amino group by oxidation to the ketone (RifL) and transamination (RifK).
Pathway of AHBA Formation-- The results of this study define which of the rif biosynthetic genes are necessary for formation of the AHBA starter unit of rifamycin. This sets the stage for the further definition of the AHBA biosynthetic pathway at the enzymatic level. The transfer of the ability to produce AHBA to the heterologous host, S. coelicolor, through the expression cassette built into vector pHGF7604 also provides an important tool both for further analysis of the AHBA pathway and for the heterologous expression of other genes from the biosynthetic gene clusters of AHBA-derived antibiotics.
The demonstrated involvement of rifG, -H,
-J, and -K in AHBA formation is consistent with
and lends credence to the originally proposed sequence of reactions
leading from aDAHP via aDHQ and aDHS to AHBA (Fig. 1). However, the
part of the original hypothesis dealing with the formation of aDAHP may
require some modification to accommodate a different mode of nitrogen
introduction as well as the requirement for three additional genes,
rifL, -M, and -N, in the pathway. The
formation of aDAHP may involve a very different way of generating a
nitrogen-containing precursor, presumably the imine of erythrose
4-phosphate or erythrose, for the condensation reaction catalyzed by
the rifH gene product, one that is mediated by the action of
the RifK-N proteins. Alternatively, the nitrogen may be introduced at
a later stage, again by a process that involves the action of the
rifK-N gene products.
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ACKNOWLEDGEMENT |
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We thank Dr. Yuemao Shen for help in developing the HPLC assays.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Research Grant AI 20264 (to H. G. F. and T. Y.), Deutsche Forschungsgemeinschaft Research Grant Le260/15-2 (to E. L.), and NATO Collaborative Research Grant Sa 5-2-05 (CRG 960 515) (to E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF040570, AF040571, and AF335989.
§ To whom correspondence may be addressed: Dept. of Chemistry, Box 351700, University of Washington, Seattle, WA 98195-1700. Tel.: 206-543-3791; Fax: 206-543-8318; E-mail: yu@u.washington.edu.
¶ Present address: Gesellschaft für Biotechnologische Forschung, D-38124 Braunschweig, Germany.
Present address: Inst. für Biotechnologie,
Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
** Present address: Inst. für Organische Chemie, Universität Hannover, D-30167 Hannover, Germany.
Permanent address: Dept. of Chemical Engineering, Inje
University, Obang-dong 607, Kimhae, Kyongnam 621-749, Korea.
¶¶ To whom correspondence may be addressed: Dept. of Chemistry, Box 351700, University of Washington, Seattle, WA 98195-1700. Tel.: 206-543-0310; Fax: 206-543-8318; E-mail: floss@chem.washington.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009667200
2 R. Müller and M. Taylor, unpublished results.
3 C. Khosla, personal communication.
4 E. Pogosova-Agadjanyan and T.-W. Yu, unpublished data.
5 R. Müller and H. G. Floss, unpublished results.
6 L.-Y. Kuan and T.-W. Yu, unpublished results.
7 T.-W. Yu, D. Hoffmann, D. Clade, E. Zeistner, and H. G. Floss, unpublished results.
8 T.-W. Yu and C.-G. Kim, unpublished results.
9 T.-W. Yu and Y. Shen, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: AHBA, 3-amino-5-hydroxybenzoic acid; DHQ, 3-dehydroquinic acid; DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate; aDAHP, 3,4-dideoxy-4-amino-D-arabino-heptulosonate 7-phosphate; aDHQ, 5-deoxy-5-amino-3-dehydroquinic acid; aDHS, 5-deoxy-5-amino-3-dehydroshikimic acid; kb, kilobase pair(s); bp, base pair(s); ORF, open reading frame; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; PLP, pyridoxal phosphate.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Anderson, M. G., Kibby, J. J., Rickards, R. W., and Rothchild, J. M. (1980) J. Chem. Soc. Chem. Commun. 1227-1278 |
2. | Ghisalba, O., and Nüesch, J. (1981) J. Antibiot. (Tokyo) 34, 64-71[Medline] [Order article via Infotrieve] |
3. | Becker, A. M., Herlt, A. J., Hilton, G. L., Kibby, J. J., and Rickards, R. W. (1983) J. Antibiot. (Tokyo) 36, 1323-1328[Medline] [Order article via Infotrieve] |
4. | Kibby, J. J., McDonald, I. A., and Rickards, R. W. (1980) J. Chem. Soc. Chem. Commun. 768-769 |
5. | Rinehart, K. L., Jr., and Shield, L. S. (1976) Fortschr. Chem. Org. Naturst. 33, 231-307[Medline] [Order article via Infotrieve] |
6. | Deboer, C., Meulman, P. A., Wnuk, R. J., and Peterson, D. H. (1970) J. Antibiot. (Tokyo) 23, 442-447[Medline] [Order article via Infotrieve] |
7. | Sasaki, K., Rinehart, K. L., Jr., Slomp, G., Grostic, M. F., and Olson, E. C. (1970) J. Am. Chem. Soc. 92, 7591-7593[Medline] [Order article via Infotrieve] |
8. | Damberg, M., Russ, P., and Zeeck, A. (1982) Tetrahedron Lett. 59-62 |
9. | Sugita, M., Natori, Y., Sasaki, T., Furihata, K., Shimazu, A., Seto, H., and Otake, N. (1982) J. Antibiot. (Tokyo) 35, 1460-1466[Medline] [Order article via Infotrieve] |
10. | Kupchan, S. M., Komoda, Y., Court, W. A., Thomas, G. J., Smith, R. M., Karim, A., Gilmore, G. J., Haltiwanger, R. J., and Bryan, R. F. (1972) J. Am. Chem. Soc. 94, 1354-1356[Medline] [Order article via Infotrieve] |
11. | Kupchan, S. M., Branfman, A. R., Sneden, A. T., Verma, A. K., Dailey, R. C., Jr., Komoda, Y., and Nagao, Y. (1975) J. Am. Chem. Soc. 97, 5294-5295[Medline] [Order article via Infotrieve] |
12. | Wani, M. C., Taylor, H. L., and Wall, M. E. (1973) J. Chem. Soc. Chem. Commun. 390 |
13. | Higashide, E., Asai, M., Ootsu, K., Tanida, S., Kozai, Y., Hasegawa, T., Kishi, T., Sugino, Y., and Yoneda, M. (1977) Nature 270, 721-722[Medline] [Order article via Infotrieve] |
14. | Williams, T. H. (1975) J. Antibiot. (Tokyo) 28, 85-86[Medline] [Order article via Infotrieve] |
15. | Rinehart, K. L., Jr., and Antosz, F. J. (1972) J. Antibiot. (Tokyo) 25, 71-73[Medline] [Order article via Infotrieve] |
16. | Sensi, P., Margalith, P., and Timbal, M. T. (1959) Farmaco. Ed. Sci. 14, 146-147 |
17. | Oppolzer, W., and Prelog, V. (1973) Helv. Chim. Acta 56, 2287-2314[Medline] [Order article via Infotrieve] |
18. | Sepkowitz, K. A., Rafalli, J., Riley, L., Kiehn, T. E., and Armstrong, D. (1995) Clin. Microbiol. Rev. 8, 180-199[Abstract] |
19. | Shibata, M., Hasegawa, T., and Higashide, E. (1971) J. Antibiot. (Tokyo) 24, 810-816[Medline] [Order article via Infotrieve] |
20. | Karlsson, A., Sartori, G., and White, R. J. (1974) Eur. J. Biochem. 47, 251-256[Medline] [Order article via Infotrieve] |
21. | White, R. J., and Martinelli, E. (1974) FEBS Lett. 49, 233-236[CrossRef][Medline] [Order article via Infotrieve] |
22. | Hornemann, U., Kehrer, J. P., and Eggert, J. H. (1974) J. Chem. Soc. Chem. Commun. 1045-1046 |
23. | Hornemann, U., Eggert, J. H., and Honor, D. P. (1980) J. Chem. Soc. Chem. Commun. 11-13 |
24. | Ghisalba, O., and Nüesch, J. (1978) J. Antibiot. (Tokyo) 31, 202-214[Medline] [Order article via Infotrieve] |
25. | Ghisalba, O., and Nüesch, J. (1978) J. Antibiot. (Tokyo) 31, 215-225[Medline] [Order article via Infotrieve] |
26. | Ghisalba, O., Fuhrer, H., Richter, W. J., and Moss, S. (1981) J. Antibiot. (Tokyo) 34, 58-63[Medline] [Order article via Infotrieve] |
27. | Gygax, D., Ghisalba, O., Treichler, H., and Nüesch, J. (1990) J. Antibiot. (Tokyo) 43, 324-326[Medline] [Order article via Infotrieve] |
28. | Harber, A., Johnson, R. D., and Rinehart, K. L., Jr. (1977) J. Am. Chem. Soc. 99, 3541-3544[Medline] [Order article via Infotrieve] |
29. | Besanzon, G. S., and Vining, L. C. (1971) Can. J. Biochem. 49, 911-918[Medline] [Order article via Infotrieve] |
30. | Meier, R.-M., and Tamm, C. (1992) J. Antibiot. (Tokyo) 45, 400-410[Medline] [Order article via Infotrieve] |
31. | Kim, C.-G., Kirschning, A., Bergon, P., Ahn, Y., Wang, J. J., Shibuya, M., and Floss, H. G. (1992) J. Am. Chem. Soc. 114, 4941-4943 |
32. | Kim, C.-G., Kirschning, A., Bergon, P., Zhou, P., Su, E., Sauerbrei, B., Ning, S., Ahn, Y., Breuer, M., Leistner, E., and Floss, H. G. (1996) J. Am. Chem. Soc. 118, 7486-7491[CrossRef] |
33. | Kim, C.-G., Yu, T.-W., Fryhle, C. B., Handa, S., and Floss, H. G. (1998) J. Biol. Chem. 272, 6030-6040[CrossRef] |
34. | August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79[Medline] [Order article via Infotrieve] |
35. | Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Schrempf, H. (1985) Genetic Manipulation of Streptomyces: A Laboratory Manual , The John Innes Foundation, Norwich, United Kingdom |
36. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
37. | Loos, H., Sahm, H., and Sprenger, G. A. (1993) FEMS Microbiol. Lett. 107, 293-298[Medline] [Order article via Infotrieve] |
38. | Schaferjohann, J., Yoo, J. G., Kusian, B., and Bowien, B. (1993) J. Bacteriol. 175, 7329-7340[Abstract] |
39. | Angell, S., Schwartz, E., and Bibb, M. J. (1992) Mol. Microbiol. 6, 2833-2844[Medline] [Order article via Infotrieve] |
40. | Spath, C., Kraus, A., and Hillen, W. (1997) J. Bacteriol. 179, 7603-7605[Abstract] |
41. | Lydiate, D. J., Malpartida, F., and Hopwood, D. A. (1985) Gene (Amst.) 35, 223-235[CrossRef][Medline] [Order article via Infotrieve] |
42. | Yu, T.-W., and Hopwood, D. A. (1995) Microbiology 141, 2779-2791[Abstract] |
43. | Vieira, J., and Messing, J. (1987) Methods Enzymol. 153, 3-11[Medline] [Order article via Infotrieve] |
44. | McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., and Khosla, C. (1993) Science 262, 1546-1550[Medline] [Order article via Infotrieve] |
45. | Eads, J. C., Beeby, M., Scapin, G., Yu, T.-W., and Floss, H. G. (1999) Biochemistry 38, 9840-9849[CrossRef][Medline] [Order article via Infotrieve] |
46. | Gramajo, H. C., Takano, E., and Bibb, M. J. (1993) Mol. Microbiol. 7, 837-845[Medline] [Order article via Infotrieve] |
47. |
Arias, P.,
Fernández-Moreno, M.,
and Malpartida, F.
(1999)
J. Bacteriol.
181,
6958-6968 |
48. | Kao, C. M., Katz, L., and Khosla, C. (1994) Science 265, 509-512[Medline] [Order article via Infotrieve] |
49. |
McDaniel, R.,
Thamchaipenet, A.,
Gustafsson, C.,
Fu, H.,
Betlach, M.,
and Ashley, G.
(1999)
Proc. Nat. Acad. Sci. U. S. A.
96,
1846-1851 |
50. | Ning, S. (1996) Studies on the Biosynthesis of Rifamycin by Amycolatopsis mediterranei.Ph.D. thesis , University of Washington, Seattle, WA |
51. | Müller, M., Müller, R., Yu, T.-W., and Floss, H. G. (1998) J. Org. Chem. 63, 9753-9755[CrossRef] |
52. |
Chen, S.,
von Bamberg, D.,
Hale, V.,
Breuer, M.,
Hardt, B.,
Müller, R.,
Floss, H. G.,
Reynolds, K. A.,
and Leistner, E.
(1999)
Eur. J. Biochem.
261,
98-107 |
53. | Mao, Y., Varoglu, M., and Sherman, D. H. (1999) Chem. Biol. 6, 251-263[CrossRef][Medline] [Order article via Infotrieve] |
54. | Takano, E., White, J., Thompson, C. J., and Bibb, M. J. (1995) Gene (Amst.) 166, 133-137[CrossRef][Medline] [Order article via Infotrieve] |