Department of Microbiology and Biological
Process Technology Institute, University of Minnesota, Minneapolis,
Minnesota 55455
Mitomycin C (MC) is an antitumor antibiotic derived
biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA),
D-glucosamine, and carbamoyl phosphate. A gene
(mitA) involved in synthesis of AHBA has been identified
and found to be linked to the MC resistance locus, mrd, in
Streptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid protein that has
71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as with two additional
AHBA synthases from related ansamycin antibiotic-producing
microorganisms. Gene disruption and site-directed mutagenesis of the
S. lavendulae chromosomal copy of mitA
completely blocked the production of MC. The function of
mitA was confirmed by complementation of an S. lavendulae strain containing a K191A mutation in MitA with AHBA.
A second gene (mitB) encoding a 272-amino-acid protein
(related to a group of glycosyltransferases) was identified immediately downstream of mitA that upon disruption resulted in
abrogation of MC synthesis. This work has localized a cluster of key
genes that mediate assembly of the unique mitosane class of natural products.
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INTRODUCTION |
Streptomyces spp. are
filamentous gram-positive soil bacteria with a nucleotide base
composition greater than 70 mol% G+C (53). They produce a
wide array of biologically active compounds, including over two-thirds
of the commercially important natural-product metabolites (1,
10). Genetic information accumulated over the past 15 years has
demonstrated that genes encoding enzymes for natural product assembly
are clustered on the Streptomyces genome (38). In
addition, one or more pathway-specific transcriptional regulatory genes
and at least one resistance gene are typically found within the
antibiotic biosynthetic gene cluster (14). Among the
strategies for cloning antibiotic biosynthetic genes, heterologous
hybridization with gene probes based on highly conserved biosynthetic-enzyme amino acid sequences has been very effective (25, 49, 56).
Streptomyces lavendulae produces the clinically important
antitumor antibiotic mitomycin C (MC) (22). MC has become
one of the most effective drugs against non-small-cell lung carcinoma, as well as other soft tumors (24). The molecule has an
unusual structure, comprised of aziridine, pyrrolizidine,
pyrrolo-(1,2a)-indole, and amino-methylbenzoquinone rings to give the
mitosane nucleus (58). A significant amount of information
on the biosynthesis of MC has accumulated since 1970. The mitosane core
was shown to be derived from the junction of an
amino-methylbenzoquinone (mC7N unit) and hexosamine
(C6N unit) (27) (Fig.
1). The C6N unit consists of
carbons 1, 2, 3, 9, 9a, and 10, with the aziridine nitrogen derived
intact from D-glucosamine (29).
The mC7N unit in MC and the ansamycins is derived from
3-amino-5-hydroxybenzoic acid (AHBA) (8, 33). AHBA was first
shown to be incorporated into the ansamycin antibiotic actamycin
(32). Subsequently, it was confirmed as an efficient
precursor for rifamycin (21), geldanamycin (46),
ansamitocin (23), ansatrienin (59), streptovaricin (54), and naphthomycin A (37).
Anderson et al. demonstrated that [carboxy-13C]AHBA could
be efficiently and specifically incorporated into the C-6 methyl group
of porfiromycin, which contains the same mitosane core as MC
(3). 14C-labeled precursor feeding studies with
D-glucose, pyruvate, and D-erythrose indicated
that de novo biosynthesis of AHBA resulted directly from the shikimate
pathway. However, no incorporation into the mC7N unit of
either MC (27) or the ansamycin antibiotics (15)
was found from labeling studies with shikimic acid, the shikimate
precursor 3-dehydroquinic acid, or the shikimate-derived amino acids.
These results led to the hypothesis of a modified shikimate pathway, in
which a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase-like enzyme catalyzes the conversion to
3,4-dideoxy-4-amino-D-arabino-heptulosonic acid-7-phosphate
(aminoDAHP) to give the ammoniated shikimate pathway (34).
Kim et al. provided strong support for this new variant of the
shikimate pathway by showing that aminoDAHP,
5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and
5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) could be efficiently
converted into AHBA by a cell extract of Amycolatopsis
mediterranei (the rifamycin producer), in contrast to the normal
shikimate pathway intermediate DAHP, which was not converted (34,
35). Recently, the AHBA synthase gene (rifK) from
A. mediterranei has been cloned, sequenced, and functionally characterized (36).
Since AHBA is a biosynthetic precursor for MC, we decided to use
rifK as a probe to identify a corresponding gene from
S. lavendulae that may be linked with one of the previously
characterized MC resistance genes (4, 50). A 3.8-kb
BamHI fragment from the S. lavendulae genome was
identified, and its nucleotide sequence revealed three open reading
frames (ORFs). One ORF (mitA) showed high similarity to
previously identified AHBA synthase genes (36), while a
second (mitB) showed sequence similarity to several
procaryotic and eucaryotic glycosyltransferases. The involvement of
both of these genes in MC biosynthesis was demonstrated by gene
disruption, site-directed mutagenesis, and subsequent isolation of
mutants blocked in antibiotic biosynthesis. MC production was restored when the mitA mutant strain was cultured in the presence of
exogenous AHBA.
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MATERIALS AND METHODS |
Strains and culture conditions.
Escherichia
coli DH5
was grown in either Luria broth or tryptic soy broth
(TSB) (Difco) as liquid medium or agar plates. E. coli
DH5F', the host for harvesting single-stranded DNA, was grown at
37°C on TBG (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 55 mM
K2HPO4, and 20 mM glucose). E. coli S17-1 (39), used for conjugation, was grown in TSB with 10 µg of streptomycin/ml. S. lavendulae was grown in TSB or
on R5T plates (containing [grams per liter] sucrose, 121.2;
K2SO4, 0.3; MgCl2 · 6H2O, 11.92; glucose, 11.8; yeast extract, 5.89; Casamino
Acids, 0.12; agar, 25.9; and 2.35 ml of trace elements
[26]; after the mixture was autoclaved, 0.5%
KH2PO4 [11.8 ml], 5 M CaCl2
[4.71 ml], and 1 N NaOH [8.25 ml] were added). For MC production,
S. lavendulae was grown in Nishikohri medium (containing
[grams per liter] glucose, 15; soluble starch, 5; NaCl, 5;
CaCO3, 3; and yeast extract, 5) for 72 h from a 1%
(vol/vol) inoculum of frozen mycelia. Pulse feeding of AHBA to the
disruption mutant, MV100, and the site-directed mutant, MV102, was done
with feedings of 2.5 mg of a 20-mg/ml solution of the sodium salt of
AHBA (pH 7.1) in three pulses at 24, 43, and 57 h of growth of a
culture that was harvested at 76 h.
DNA preparation and amplification.
Isolation and
purification of DNA was performed by standard methods (47).
S. lavendulae NRRL 2564 genomic DNA was isolated by using
the modified Chater protocol (26). Plasmid DNA was isolated
from E. coli by using the alkaline-sodium dodecyl sulfate method.
pDHS2002 was constructed as follows. The 1.1-kb thiostrepton resistance
gene (tsr) fragment was removed from pDHS5000 by
SmaI-BamHI digestion, blunt ended with the large
fragment of DNA polymerase (Gibco BRL), and ligated to MscI
restriction enzyme-digested pDHS7601 to yield pDHS2001. MscI
digestion of pDHS7601 resulted in the removal of 155 nucleotides at the
C terminus of the mitA gene, and ligation of the blunt-ended
BamHI site of the tsr adjacent to the
MscI site of pDHS7601 resulted in regeneration of the
BamHI site in pDHS2001. The 4.9-kb
EcoRI-HindIII fragment from pDHS2001 containing the tsr-disrupted mitA gene was
removed and ligated into
EcoRI-HindIII-digested pKC1139 to yield pDHS2002.
Primer-mediated site-directed mutagenesis was employed to construct
pDHS2015 containing a K191A mutation in mitA. Primer 1 (5'-GGCAAGGCATGCGAGGGTCGC-3') and primer 2 (5'-TTCCAGAACGGCGCCCTGATGACCGCCGGC-3') were used to amplify
the 691-bp fragment of the 5' end of mitA. The 3' end of
mitA was amplified with primer 3 (5'-GCCGGCGGTCATCAGGGCGCCGTTCTGGAA-3') and primer 4 (5'-TCAGAATTCGGATCCGAGGGCCGGAGT-3') to generate a 1,151-bp
band. A second round of PCR was performed, with the overlapping 691- and 1,151-bp units as the initial templates, with primer 1 and primer 4 to afford a 1.8-kb fragment. The final product, containing mutagenized
mitA, was digested with EcoRI-SphI and ligated to the 2.1-kb HindIII-SphI fragment
from pDSH2004 and the EcoRI-HindIII-digested
pKC1139 to yield pDSH2015. The site-directed mutation of MitA K191A in
pDHS2015 was confirmed by sequencing with forward primer
(5'-ACCTACTGCCTCGATGCC-3') and reverse primer (5'-CTGATCCTTCAAGCG-3').
The mitB disruption vector pDHS7702 was constructed as
follows. pDHS7601 was digested with BstBI, blunt ended, and
ligated with the 1.4-kb neomycin resistance gene fragment from pFD666 (17) (ApaL1-HindIII digestion;
blunt ended). The 5.2-kb EcoRI-HindIII fragment from the resulting construct, pDHS7701, was subcloned into
pKC1139 to create pDHS7702.
DNA library construction and screening.
S. lavendulae
NRRL 2564 genomic DNA was partially digested with Sau3AI,
and a fraction containing 30- to 50-kb fragments was recovered by
sucrose gradient centrifugation and ligated into the calf intestinal
alkaline phosphatase-treated BglII site of the E. coli-Streptomyces shuttle vector pNJ1 (55) and then
packaged with the Packagene Lambda DNA packaging system (Promega). The cosmid library was constructed by transfecting E. coli
DH5, and colonies that appeared on the Luria broth plates containing
100 µg of ampicillin/ml were transferred to a BioTrace NT
nitrocellulose blotting membrane (Gelman Sciences). Colony
hybridization was performed as specified by the manufacturer. A
PCR-amplified 0.7-kb DNA fragment from plasmid pKN108 (Table
1) was used to screen the library. The
primers used for PCR were 5'-GCGTCCGTGCTGCGCGCGCA-3' and
5'-TGCGCGCGCAGCACGGACGC-3'. The cosmids from the positive colonies were confirmed by Southern blot hybridization, and a 1.7-kb
AflIII-BamHI fragment from pDHS3001 containing
the mitomycin resistance determinant (mrd) (50)
was used as a probe to establish genetic linkage.
DNA sequencing and analysis.
Deletion subclones from
pDHS7601 were made with the exonuclease III Erase-a-Base system
(Promega). Sequencing was accomplished with the PRISM dye terminator
cycle-sequencing ready reaction kit (Applied Biosystems) and analyzed
on an Applied Biosystems 377 DNA sequencer at the University of
Minnesota Advanced Genetic Analysis Center. For generating
single-stranded DNA, deletion subclones in pUC119 were transformed into
E. coli DH5F', and M13K07 helper phage (Gibco BRL) was
used. Nucleotide sequence data were analyzed with Wisconsin Genetics
Computer Group software (version 9.0) (18) and GeneWorks
software version 2.51 (Oxford Molecular Group).
Conjugation from E. coli S17-1 to S. lavendulae.
To transfer plasmid from E. coli S17-1 to
S. lavendulae, the procedure of Bierman et al.
(12) was used with the following modification. A single
colony of E. coli S17-1/pDHS2002 was used to inoculate 2 ml
of TSB containing 100 µg of apramycin/ml and 10 µg of
streptomycin/ml. Following overnight incubation at 37°C, a 1:100
inoculation was made into TSB with 100 µg of apramycin/ml and 10 µg
of streptomycin/ml. This culture was grown for 3 h at 37°C, and
the cells were washed twice with TSB and resuspended in 2 ml of TSB to
provide the donor E. coli culture. The recipient S. lavendulae culture was generated by inoculating 9 ml of TSB with 1 ml of frozen wild-type culture. Following overnight (16-h) incubation
at 29°C, the culture was homogenized by sonication, and 2 ml of this
culture was used to inoculate 18 ml of TSB. Following overnight growth
at 29°C and sonication treatment to homogenize the culture, a 1-ml
inoculum was placed in 9 ml of TSB. This culture was grown for 3 h, and the mycelia were washed with TSB and resuspended in 2 ml of TSB
to provide the stock recipient culture.
The donor and recipient cultures were mixed together in 9:1, 1:1, and
1:10 donor/recipient ratios, and 100 µl of the cell mixture was
spread on AS1 plates (5). The plates were incubated overnight at 29°C and overlaid with 1 ml of water containing a suspension of 500 µg each of thiostrepton, apramycin, and nalidixic acid/ml. For the pKC1139 control, only apramycin and nalidixic acid
were overlaid, while for pDHS7702, 500 µg of kanamycin/ml was used
instead of thiostrepton. S. lavendulae exconjugants appeared in approximately 11 to 13 days at a frequency ranging from
10
7 to 105. pKC1139 has a
temperature-sensitive Streptomyces replication origin, which
is unable to replicate at temperatures above 34°C (41),
while the S. lavendulae host grows well at 42°C. Thus, after the conjugants were propagated at 39°C for several generations, double-crossover mutants were readily generated. The presence of the
plasmid was determined by transformation of E. coli DH5 with plasmid extracts from S. lavendulae transconjugants.
Double-crossover selection procedure.
A single colony of
S. lavendulae/pDHS2002 grown on R5T plates (50 µg
[each] of thiostrepton and apramycin/ml) was used to inoculate TSB
broth containing 20 µg of thiostrepton/ml. After 72 h of
incubation at 39°C, 104, 105, and
106 diluted aliquots were used to inoculate R5T plates
containing 50 µg of thiostrepton/ml. Following 48 h of growth at
39°C, 84 colonies were picked randomly and each colony was patched
out on separate R5T plates containing 50 µg of thiostrepton/ml and 50 µg of apramycin/ml. One of the 84 colonies displayed the
double-crossover phenotype of thiostrepton resistance and apramycin
sensitivity. The integration of the tsr-disrupted
mitA gene and the loss of plasmid pDHS2002 were confirmed by
Southern hybridization analysis.
MitA K191A site-directed mutants (MV102) were selected by propagating
MV100/pDHS2015 on R5T plates for two generations at 37°C. The
colonies were replicated to plates containing 50 µg of
thiostrepton/ml and plates without antibiotics. Of the 108 colonies
replicated in the first round, one had the correct
(thiostrepton-sensitive) phenotype. To confirm the K191A mutation, the
mitA gene was amplifed from the chromosome with primers 1 and 4. Mutation of the conserved lysine codon (AAG) to an alanine codon
(GCC) was verified with the same sequencing primers employed to confirm
the correct construction of pDHS2015. The alanine codon was observed in
both the forward and reverse sequence data.
Mutants for mitB (MM101) were selected as follows: S. lavendulae/pDHS7702 was propagated on R5T plates for five
generations at 39°C before single colonies were replicated on R5T
plates as described above. Of the 300 colonies tested, 12 clones
displayed the correct phenotype (kanamycin resistance and apramycin
sensitivity). The genotypes of selected mitB mutants were
confirmed by Southern blot hybridization of S. lavendulae
genomic DNA.
Analysis of MC production.
All cultures intended for MC
extraction were grown in Nishikohri medium (42) for a period
of 72 h. In all cases, a wild-type S. lavendulae
culture was grown concurrently with the mutant cultures to provide MC
production reference points. A 72-h, 50-ml culture (250-ml flask) of
the MitA K191A MV102 mutant strain was supplemented with 125 µl of a
20-mg/ml solution of the sodium salt of AHBA (pH. 7.05) at 24, 43, and
55 h. In each case, the culture broth was separated from mycelia
by centrifugation and then extracted three times with equal volumes of
ethyl acetate. The ethyl acetate extracts were pooled, and the solvent
was removed by vacuum to provide the crude broth extract. The
preliminary screen for MC production involved thin-layer chromatography
(TLC) on silica gel plates (Whatman K6) eluted with 9:1
chloroform-methanol. Production of MC was monitored by high-performance
liquid chromatography (HPLC) (C18 reverse-phase column)
with a gradient of 80% 50 mM Tris buffer (pH 7.2)-20% methanol to
40% 50 mM Tris buffer (pH 7.2)-60% methanol with the UV detector set
to 363 nm.
Bioassay detection of MC was performed by loading a 1-cm disk with
fractions eluting at the mitomycin retention time from HPLC injections
of wild-type, MV100, pKC1139 vector control crude extracts and MC
standards. The disks were placed on antibiotic medium no. 2 agar plates
(Difco) with Bacillus subtilis spores added directly to the
medium. The plates were incubated overnight at 29°C and examined for
zones of inhibition. To confirm the production of MC by MV102 in the
presence of exogenous AHBA, the fraction eluting at the MC retention
time was collected, dried down, desalted, and submitted for desorption
ionization mass spectrometric analysis on a Bio-Ion 20R DS-MS
instrument (Applied Biosystems). The MC (molecular weight, 334)-sodium
(molecular weight, 23) adduct peak, [M+Na]+, of 357 was
diagnostic for the presence of MC in the AHBA-supplemented culture.
Nucleotide sequence accession number.
The GenBank accession
number for mitABC is AF115779.
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RESULTS |
The mrd and ahbas genes are linked
in the S. lavendulae genome.
Southern blot analysis
with the A. mediterranei AHBA synthase gene
(rifK) probe (36) showed a single 3.8-kb band
that hybridized with BamHI-digested S. lavendulae
genomic DNA (Fig. 2). Subsequently, a
S. lavendulae genomic DNA library was constructed with the
E. coli-Streptomyces shuttle cosmid pNJ1 (55). Of
the 5,000 colonies screened, 21 positive clones were identified, with 6 of them hybridizing with the mrd gene probe (none hybridized
with the mcr gene probe [reference 4 and
data not shown]). Restriction enzyme mapping and reciprocal
hybridization of the cosmid clones established that the mrd
and S. mediterranei AHBA synthase homologous genes were
~20 kb apart in the S. lavendulae genome. The 3.8-kb
BamHI fragment bearing a putative S. lavendulae
AHBA synthase gene was subcloned, and its nucleotide sequence was
determined.
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FIG. 2.
Southern hybridization and restriction enzyme map of the
mrd and rifK hybridizing regions from S. lavendulae. (A) Southern hybridization with the rifK
gene probe (36). Lane 1, A. mediterranei ATCC
27643 genomic DNA digested with BamHI; lane 2, S. lavendulae NRRL 2564 genomic DNA digested with BamHI
revealed a 3.8-kb hybridization band. (B) Physical map showing the
mitA, mitB, and mitC genes. The
locations of mrd and rifK hybridizing genes in
cosmid pDHS7529 are indicated by solid bars. E,
EcoRI; B, BamHI. The sequenced 3.8-kb
BamHI fragment containing mitA, mitB,
and mitC is enlarged (wide arrows). The thin arrows below
show sites of resistance gene integration for disruption experiments.
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Three ORFs identified within the 3.8-kb BamHI
fragment.
Three ORFs (mitA, mitB, and
mitC) were identified within the sequenced 3.8-kb
BamHI fragment (Fig. 2 and 3).
mitA comprises 1,164 nucleotides and starts from an ATG
(position 579 of the sequenced fragment) that is preceded by a
potential ribosome binding site (RBS), GAAAGG. The deduced product of
the mitA gene encodes a hydrophilic protein of 388 amino
acids with a predicted mass of 41,949 Da and a calculated pI of 5.62. A
BLAST (2) search showed that the predicted MitA protein has
high sequence similarity (~71% identity and 80% similarity) to
AHBA synthases, from the rifamycin producer, A. mediterranei (36), and other ansamycin-producing actinomycetes, including Actinosynnema pretiosum
(ansamitocin) and Streptomyces collinus (naphthomycin A and
ansatrienin) (Fig. 4). A conserved
pyridoxal phosphate (PLP) coenzyme binding motif (GX3DX7AX8EDX14GX13KX4-5geGGX19G)
including the conserved lysine residue (boldface and underlined) can
also be found in these four proteins (45).
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FIG. 3.
Nucleotide sequence of the 3.8-kb DNA fragment
containing mitABC. The deduced gene products are indicated
in the one-letter code under the DNA sequence. Possible RBSs are boxed.
The presumed translational start site and direction of transcription
for each ORF are indicated by a labeled arrow. Restriction enzyme sites
are marked by arrowheads.
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FIG. 4.
Alignment of MitA with three other AHBA synthases. The
deduced amino acid sequence comparison from AHBA synthase genes derived
from S. lavendulae (mitA), S. collinus
(Z54208), A. pretiosum (I39657), and A. mediterranei (I39657) is shown with the conserved lysine in the
PLP-binding motif in boldface and underlined. Residues conserved in all
sequences are shaded.
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The mitB gene is predicted to start at a GTG (position 1879)
that is preceded by a presumed RBS (GGAACG). This gene encodes a
272-amino-acid protein with a deduced mass of 28,648 Da and a deduced
pI of 6.06. Database sequence homology searches revealed that the
protein product of mitB shows local sequence similarity to a
group of O-glycosyltransferases involved in polysaccharide biosynthesis. One segment of 70 amino acid residues at the N terminus of MitB has 43% similarity (36% identity) to the two
glycosyltransferases SpsL and SpsQ from Sphingomonas sp.
strain S88 and ExoO from Rhizobium meliloti, involved in
polysaccharide (S88) and succinoglycan biosynthesis, respectively
(7, 60). Another 60 amino acid residues located at the C
terminus displayed 30% identity with UDP-GalNAc-polypeptide N-acetylgalactosaminyltransferase from Mus
musculus and Homo sapiens (9).
The third ORF, mitC, starts from the ATG at position 2694, which is coupled to the stop codon, TGA, of mitB, and
encodes a putative protein of 260 amino acids with a predicted
molecular mass of 27,817 Da and a pI of 10.45. Database searches with
the deduced protein product showed significant similarity over the first 90 amino acids (38% identity and 40% similarity) to the lmbE gene product (unknown function) from
Mycobacterium leprae (U15183).
Insertional disruption of the mitA and mitB
genes in S. lavendulae.
To test the dependence of functional
mitA and mitB genes for MC biosynthesis, gene
disruption constructs were generated for subsequent isolation of the
corresponding S. lavendulae isogenic mutant strains.
The mitA disruption construct was made by replacing a 155-bp
fragment between the two MscI sites (located at the C
terminus of the mitA gene in pDHS7601) with the 1.1-kb
SmaI-BamHI fragment containing a thiostrepton
resistance gene from pDHS5000 (Fig. 5A).
This replacement regenerated a BamHI site at the junction, and the resulting construct was then subcloned into the E. coli-Streptomyces conjugative shuttle plasmid pKC1139, followed by
conjugation into S. lavendulae. A double-crossover mutant
strain (MV100) was selected based on the expected phenotype
(thiostrepton resistant and apramycin sensitive) and further confirmed
by Southern blot hybridization. Genomic DNAs from wild-type S. lavendulae and MV100 were digested with BamHI and
SphI and hybridized with the 4.9-kb
EcoRI-HindIII tsr-disrupted
mitA fragment from pDHS2001. As expected, the 4.0-kb SphI-hybridized band in the wild-type strain was shifted to
4.9 kb in MV100, whereas the 3.8-kb BamHI hybridization band
in the wild type was converted to two bands (2.2 and 2.5 kb) in the
mutant (Fig. 5B).
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FIG. 5.
Southern blot analysis of the mitA mutant
strain MV100. (A) Construction of mitA disruption mutant and
restriction map of the wild type and mitA disruption mutant
showing expected band sizes in the Southern blot. The maps are not
drawn to scale. (B) S. lavendulae genomic DNAs from the wild
type (lanes 1 and 2) and the double-crossover mutant MV100 (lanes 3 and
4) were digested with BamHI (lanes 1 and 3) and
SphI (lanes 2 and 4), respectively. The 4.9-kb
EcoRI-HindIII fragment from pDHS2001
containing tsr-disrupted mitA was used as the
probe.
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The mitB gene was disrupted by inserting a neomycin
resistance gene (aphII) into the BstBI site
(located at the 5' end of mitB) (Fig.
6A). Transconjugants were selected on
kanamycin-apramycin plates, and a double-crossover mutant strain
(MM101) with a kanamycin-resistant, apramycin-sensitive phenotype was
identified and subsequently confirmed by Southern blot hybridization.
As expected, the 3.8-kb BamHI hybridization band in
wild-type S. lavendulae was shifted to 5.2 kb in MM101,
whereas a 5.2-kb SacI hybridization band was shifted to 6.6 kb (Fig. 6B).
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FIG. 6.
(A) Construction of mitB disruption mutant
and restriction map of the wild type and mitB disruption
mutant showing expected band sizes in the Southern blot. (B) Southern
blot analysis of mitB mutant MM101. S. lavendulae
genomic DNAs from the wild type (lanes 1 and 3) and mitB
mutant MM101 (lanes 2 and 4) were digested with BamHI (lanes
1 and 2) and SacI (lanes 3 and 4). The DNA probe was the
3.8-kb BamHI fragment insert from pDHS7601. (B) Southern
blot hybridization of S. lavendulae genomic DNA of the
double-crossover disruption mutant showing the expected hybridization
bands.
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mitA- and mitB-disrupted strains (MV100 and
MM101) are blocked in MC biosynthesis.
The growth characteristics
and morphologies of MV100 and MM101 in liquid medium and on agar plates
were identical to those of wild-type S. lavendulae. HPLC was
used to quantify production of MC in MV100 and MM101 (Fig.
7A), and culture extracts were used in a
biological assay to test for the presence of the drug (Fig. 7B).
Injection of 1 mg of wild-type S. lavendulae culture extract
gave a peak in the HPLC that eluted with the same retention time as the
MC standard. Upon injection of 1 mg of culture extract from the
mitA- or mitB-disrupted strains (MV100 and
MM101), no MC peak was observed. To corroborate the lack of production
of MC, the HPLC eluant obtained from the MV100 culture extracts was collected over the retention time range determined for MC. This eluant
completely lacked biological activity against B. subtilis (the MC target strain), while the fraction collected from the same
retention time region of wild-type S. lavendulae and the vector control strain culture extracts showed substantial levels of
biological activity (Fig. 7B).
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FIG. 7.
Chemical analysis and biological activities of extracts
from S. lavendulae wild-type and mutant strains. (A) HPLC
analysis of authentic mitomycin C standard, mitomycin C production in
the wild-type S. lavendulae, wild type with vector control,
and mitA and mitB disruption mutants of S. lavendulae. For each analysis, 1 mg of crude extract was injected;
1 µg of MC was injected as a standard. (B) B. subtilis
bioassay of MC production in mitA disruption mutant strain
of S. lavendulae. Filter discs: 1, 100-µg injection of
wild type (collected from 12.5 to 13.5 min); 2, 100-µg injection of
mitA disruption mutant (collected from 12.5 to 13.5 min); 3, 100-µg injection of the wild type containing the vector (collected
from 12.5 to 13.5 min); 4, 1 µg of MC collected from HPLC from 12.5 to 13.5 min; 5, Tris buffer negative control; 6) methanol solvent
negative control.
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It is important to note that the presence of the vector pKC1139 in
S. lavendulae reduced the percentage of MC in the total crude extract while simultaneously increasing the total amount of
material extractable by ethyl acetate. The combination of these two
effects reduces the absolute amount of MC by approximately 25% in the
vector control culture crude extract compared to that in the wild-type
crude extract.
Exogenous AHBA can restore MC production in the MC-deficient MitA
K191A mutant.
Although complementation of MV100 (the
mitA insertional disruptant) was attempted by providing
exogenous AHBA in the culture medium, MC production was not restored as
measured by HPLC or biological assay. A polar effect on genes
downstream of tsr-disrupted mitA in MV100
appeared likely, since supplying mitA in trans on a medium-copy-number plasmid (MV103) also failed to restore MC production. Therefore, site-directed mutagenesis was employed to
generate a MitA K191A mutant, resulting in strain MV102. Kim et al. had
demonstrated that the AHBA synthase from A. mediterranei is
PLP dependent and catalyzes the aromatization of aminoDHS
(36). Thus, the nitrogen of conserved lysine 191 is supposed
to form a Schiff base with the PLP cofactor. Replacement of lysine 191 with alanine prevents binding of the cofactor and eliminates enzymatic activity. Replacement of the AGG encoding lysine 191 in wild-type S. lavendulae with a GCC codon in MV102 was confirmed by
nucleotide sequence analysis. As expected, MV102 did not produce MC;
however, when the culture medium was supplemented with exogenous AHBA, MC production was restored, as determined by MS ([M+Na]+ = 357), HPLC, and TLC analysis (Table 2).
| |
DISCUSSION |
An effective strategy for the identification of natural product
biosynthetic gene clusters in actinomycetes has included cloning of
antibiotic resistance genes followed by investigation of adjacent DNA
for the presence of structural and regulatory genes (13, 20, 40,
57). Although linkage of antibiotic resistance and biosynthetic
genes appears to be a general feature in procaryotes, a growing number
of examples involve the existence of multiple-resistance loci that may
be linked or unlinked to the biosynthetic gene cluster (16, 49,
51). The identification and characterization of two genetically
unlinked resistance loci (4, 50) for MC created a dilemma
for mounting an effective search for the MC biosynthetic gene cluster.
However, the use of the AHBA synthase gene from A. mediterranei provided an effective probe for identifying cosmid clones bearing a linked MC resistance gene. Thus, the isolation of
several cosmid clones from an S. lavendulae genomic DNA
library that hybridized to both the A. mediterranei AHBA
synthase gene and the S. lavendulae mrd gene indicated that
the MC biosynthetic gene cluster resided on DNA adjacent to
mrd. DNA sequence analysis of the 3.8-kb BamHI
fragment revealed three ORFs whose deduced protein sequences
corresponded to an AHBA synthase, a glycosyltransferase, and an
lmbE-like product.
As determined by precursor feeding experiments, the mitosane core is
formed through the condensation of AHBA and D-glucosamine (27). AHBA is thought to be derived from the ammoniated
shikimate pathway from PEP and E4P, in which the last step from
aminoDHS to AHBA is catalyzed by AHBA synthase (Fig. 1) (35,
36). Meanwhile, the reaction of attaching an activated sugar
residue to a core compound is usually catalyzed by a group of enzymes
called glycosyltransferases as specified by macrolide, glycopeptide
antibiotic, and polysaccharide biosynthesis (31, 43, 52,
60). In principle, the condensation of AHBA with
D-glucosamine can be initiated in two ways (Fig. 1). One
would involve the formation of the C-8a-C-9 bond by an electrophilic
aromatic alkylation or acylation. A second possibility would be
formation of a Schiff base between the nitrogen of AHBA and the
D-glucosamine C-1 aldehyde, followed by ring closure at C-8a-C-9. In either case, a C- or
N-glycosyltransferase instead of an
O-glycosyltransferase is expected. Although previously
described glycosyltransferases display a high degree of sequence
divergence (60), the mechanistic similarity with
O-glycosyl transfer may suggest that mitB encodes
a N-glycosyltransferase that initiates the formation of the
mitosane system by linking glucosamine to AHBA. The mitA and
mitB genes and their corresponding products are likely
candidates to mediate formation of AHBA and the mitosane ring system,
respectively. However, the possible function of the lmbE-like protein remains unclear, since its current role
within the lincomycin biosynthetic pathway of Streptomyces
lincolnensis is not known (44).
The involvement of AHBA synthase (encoded by mitA) and the
putative glycosyltransferase (encoded by mitB) in MC
biosynthesis was established by gene disruption to create mutants with
MC biosynthesis blocked. This required development of a method to
introduce DNA into S. lavendulae NRRL 2564, since the strain
remains refractory to traditional Streptomyces protoplast-
and electroporation-mediated transformation procedures. The modified
protocol of Bierman et al. (12) was used to effect efficient
conjugative transfer into S. lavendulae by using the
E. coli-Streptomyces shuttle plasmid pKC1139. This result is
significant because it has enabled the development of an effective
system for analyzing in detail the genes involved in MC biosynthesis.
The function of mitA was probed by providing strains MV100
and MV102 with exogenous AHBA in the culture medium. Despite repeated attempts to complement MV100, MC production was not restored as measured by HPLC or biological assay. It is believed that insertion of
the tsr gene into mitA resulted in disruption of
biosynthetic genes immediately downstream, since supplying
mitA in trans on a medium-copy-number plasmid
also failed to restore MC production to MV100. This putative polar
effect was eliminated by generating the MitA K191A mutant strain MV102.
Providing exogenous AHBA to this mutant strain of S. lavendulae restored production of MC as shown by TLC, HPLC, and
mass spectrometry. When MV102 was grown in the absence of AHBA there
was no detectable production of MC. The ability of AHBA to complement
the mutant MitA protein further supports the function of MitA as an
AHBA synthase, as indicated by the database protein sequence alignment
and previous studies of rifK (36).
Although our studies have focused on the identification of genes
involved in the early steps of MC biosynthesis, further
characterization of the genes and functions that mediate individual
steps in the pathway will provide information required to understand
more fully the details of gene regulation, molecular assembly, and
cellular resistance for this important molecule.
We sincerely thank Heinz G. Floss, University of Washington, for
providing the rifamycin AHBA synthase gene (rifK) probe.
This work was supported in part by the Biological Process Technology
Institute Seed Grant Program, University of Minnesota (to D.H.S.). M.V.
was the recipient of a Postdoctoral Fellowship from the National Cancer
Institute (training grant CA09138).
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Abstract
Introduction
