Vol. 174, No. 5
JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 1641-1646
0021-9193/92/051641-06$02.00/0 Copyright © 1992, American Society for Microbiology
Expression of Doxorubicin-Daunorubicin Resistance Genes in Different Anthracycline-Producing Mutants of Streptomyces peucetius ANNA L. COLOMBO,'* MARIA M. SOLINAS,2 GIOVANNI PERINI,2 GIUSEPPE BIAMONTI2* GIUSEPPE ZANELLA,' MARINELLA CARUSO,' FRANCESCA TORTI,' SILVIA FILIPPINI,1 AUGUSTO INVENTI-SOLARI,' AND LUISA GAROFANO' Dipartimento Biotecnologie, Unita Screening, Farmitalia Carlo Erba, Milan,' and Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, Pavia,2 Italy Received 11 July 1991/Accepted 7 December 1991
Two DNA fragments, ricl and ric2, were isolated from the Streptomyces peucetius 7600 mutant, which produces daunorubicin and doxorubicin, on the basis of their abilities to confer doxorubicin and daunorubicin resistance to Streptomyces lividans. These two fragments are unrelated by restriction mapping and do not show any homology by Southern analysis, yet both of them increase the level of resistance 10-fold in transformed S. lividans. Functional analysis revealed that icl also contains two genes of daunorubicin biosynthesis: one coding for the aklavinone C-il hydroxylase and the other corresponding to the putative dnrR2 regulatory gene of wild-type S. peucetius ATCC 29050 (K. J. Stutzman-Engwall, S. L. Otten, and C. R. Hutchinson, J. Bacteriol. 174:144-154, 1992). Northern (RNA) blot experiments, performed with a ricl fragment containing daunorubicindoxorubicin resistance gene(s), revealed a transcript of about 2,100 nucleotides that is present only during the phase of anthracycline metabolite production. The amount of this transcript is higher in strain 7600 than in strain 7900, a mutant which produces 5-fold more daunorubicin and 10-fold less doxorubicin than 7600. Furthermore, two 7900-derived blocked mutants, 8600 and 9700, do not express the 2,100-nucleotide transcript in spite of the absence of gross rearrangements in the ricl region such as occur with the 7900 parental strain.
Studies of the Streptomyces genes involved in the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), the two most widely employed agents in antitumor therapy, can be useful to produce specific intermediates for subsequent biotransformation into new analogs (3, 5, 11, 17, 21, 24). Furthermore, a better understanding of the mechanism conferring resistance to the drugs in the producing organism may lead to the construction of improved strains. These considerations prompted us to investigate the genes involved in the biosynthesis of and in the resistance to these drugs. Doxorubicin was initially obtained from Streptomyces peucetius subsp. caesius ATCC 27952, a mutant from a wild-type strain previously isolated (1, 2, 4, 13, 25). During a program of strain improvement undertaken in our laboratories, we isolated several mutants of ATCC 27952 that accumulate anthracyclines in different ratios (7). One mutant, S. peucetius 7600, efficiently converts daunorubicin to doxorubicin via C-14 hydroxylation, while another mutant, S. peucetius 7900, produces fivefold more daunorubicin and tenfold less doxorubicin than 7600. Both of these mutants are more resistant to doxorubicin than to daunorubicin. We have cloned two fragments (ricl and ric2) containing doxorubicin-daunorubicin resistance genes from S. peucetips 7600. The level of expression of the resistance gene present in ricl was compared for S. peucetius 7600, S. peucetius 7900, and two different blocked mutants, derived from 7900 (S. peucetius 8600 and 9700), which do not produce detectable amounts of anthracycline metabolites and are more sensitive to doxorubicin and daunorubicin than the parental strain.
MATERIALS AND METHODS Bacterial strains and plasmid. All S. peucetius strains which appear in this work were isolated in Farmitalia Carlo Erba laboratories. S. peucetius subsp. caesius ATCC 27952 (2), a daunorubicin and doxorubicin producer, is a mutant derived from wild-type strain S. peucetius ATCC 29050 (8) isolated in 1963. All the other mutant strains were obtained by several cycles of mutagenesis with UV light, N-methyl-N'-nitro-Nnitrosoguanidine, and nitrosomethylurethane applied singly or in combination accordingly to standard procedures (7, 19). S. peucetius 7600 converts daunorubicin to doxorubicin at high levels and was obtained from S. peucetius ATCC 27952. S. peucetius 7100 is a blocked mutant obtained from S. peucetius ATCC 27952. S. peucetius 7800, derived from S. peucetius 7600, is a yellow mutant which lacks aklavinone C-11 hydroxylation and is sensitive to doxorubicin and daunorubicin. S. peucetius 7900 was obtained by a fusion of strains 7600 and 7100 and produces more daunorubicin (fivefold) and less doxorubicin (tenfold) than S. peucetius 7600. S. peucetius 8600 and 9700 are blocked mutants derived from 7900. Streptomyces lividans TK23 and pIJ702 were obtained from D. A. Hopwood (John Innes Institute,
Norwich, United Kingdom). Biochemicals and chemicals. Thiostrepton was obtained from Behring. Aklavinone, doxorubicin, and daunorubicin were obtained from Farmitalia Carlo Erba. Restriction enzymes and other recombinant DNA materials were purchased from Boehringer. All other chemicals and biochemicals were obtained from Sigma. Media, growth conditions, and protoplast formation. Cultures for preparation of Streptomyces spore stocks were grown on R2YE agar medium (9) at 28°C for 8 to 10 days. Protoplast and chromosomal/plasmid DNAs were produced
* Corresponding authors. 1641
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COLOMBO ET AL.
GK B lI Ite,
C
K
I
I
S I
B
B
I
I
BS
5
P I
I
I
I
S
S
I
I
P
K
I
Ii
ric 1
3 .5 kb BamHI genomic fragment
KTI, L
B I
I
C
,.
,
K I
ric 2
lkb II
FIG. 1. Restriction maps of the ricl and ric2 fragments. Restriction enzyme abbreviations: B, BamHI; C, ClaI; G, Bglll; K, Kpnl; L, Bcll; M, MboI; P, PstI; S, SphI; T, SstI. 111m Region of homology with dnrR2; SS, fragment conferring daunorubicin and doxorubicin resistance. ric2 region utilized as a probe in Southern blot experiments. The interrupted bar ( E] ) represents the conserved 3.5-kb BamHI fragment. ,
from Streptomyces strains grown in YEME medium (9) supplemented with 0.5% glycine at 28°C. Streptomyces protoplasts were regenerated on R2YE agar. S. lividans and S. peucetius transformants were selected and maintained on thiostrepton at 100 and 25 ,ug/ml, respectively. Conditions used for protoplast formation were essentially as described by Hopwood et al. (9) for S. lividans TK23 and as described by Lampel and Strohl (14) for S. peucetius 7800. Detection of anthracycline metabolites and bioconversion experiments. S. peucetius cells were grown in 25-ml culture flasks at 30°C with shaking at 300 rpm in the fermentation medium described by McGuire et al. (16). After 5 days, cultures were acidified by adding 25 mg of oxalic acid per ml, incubated at 30°C overnight, and extracted with an equal volume of acetonitrile. The extract was filtered, and the filtrate was analyzed by reverse-phase high-performance liquid chromatography (HPLC). Bioconversion expenments were performed by using whole cells of S. peucetius 8600 and S. lividans TK23(ricl) in the fermentation process. Aklavinone (100 ,ug/ml) and E-rhodomycinone (50 ,ug/ml) were added to 96-h cultures, and after 24 h, the products were extracted and HPLC analyzed. Analysis of doxorubicin and daunorubicin resistance. S. lividans and S. peucetius were tested for doxorubicin and daunorubicin resistance on R2YE agar plates. Approximately 106 spores were streaked onto plates containing 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, and 750 p.g of doxorubicin per ml and daunorubicin at the same concentrations (from 0 to 150 jj.g/ml). DNA and RNA extraction. DNA was isolated from S. peucetius as described by Hopwood et al. (9). S. peucetius RNA was extracted from 3 to 5 g of mycelium grown in the fermentation medium (16) by homogenization in a 25-ml Corex tube containing 10 ml of GIT solution (4 M guanidine thiocyanate salt, 25 mM sodium acetate [pH 6.0], 120 mM 2-mercaptoethanol) and 10 g of 0.5-mm-diameter glass balls. Cells were vortexed for a total of 10 min, with stops of 30 s in ice every minute. After centrifugation at 0°C (7,000 x g for 15 min), RNA was separated from DNA and purified as described by Maniatis et al. (15). Southern and Northern (RNA) hybridization analysis. For RNA gels, 10 ,ug of total RNA was lyophilized, resuspended in 15 RI of sample buffer (50% formamide, 6% formaldehyde, 20 mM NaH2PO4-Na2HPO4 [pH 7.0], 5% glycerol, 0.02% bromophenol blue, 0.02% xylene cyanol, 0.2 mg of ethidium
bromide per ml) and incubated for 10 min at 68°C. RNA was electrophoresed onto a 1.5% agarose-6% formaldehyde gel. At the end of the run, the gel was photographed on a UV transilluminator and then transferred to a Zetabind (Cuno Laboratory Products) membrane. Southern and Northern blot hybridizations were performed as described by Maniatis et al. (15) and by Rave et al. (20), using Zetabind (Cuno) filters. Processing of filters was as recommended by the supplier. The prehybridization and hybridization solution was 4x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-4x Denhardt's solution-50% formamide-0.5% sodium dodecyl sulfate (SDS)-7.5 mM Na4P2O-r12.5 mM NaH2PO4-0.3 mg of tRNA per ml. The probe was labelled at a specific activity of 109 cpm/,Lg with the Multiprime DNA labelling system (Amersham) and was added to the hybridization solution at a concentration of 2 x 106 cpm/ml. Southern hybridizations were carried out at 42°C, while Northern hybridizations were carried out at 52°C. After an overnight hybridization, filters were washed twice for 30 min each in 2x SSC-1% SDS at room temperature, once in 2x SSC-1% SDS at 68°C, and once in 0.2x SSC-0.1% SDS at 68°C. In order to rehybridize the filters, probes were stripped away by boiling the filters for 10 min in 10 mM Tris HCI (pH 7.5)-l mM EDTA-1% SDS. RESULTS Cloning of doxorubicin and daunorubicin resistance genes. Chromosomal DNA of S. peucetius 7600 was partially digested with MboI, and DNA fragments in the range of 4 to 6 kb were recovered from an agarose gel and cloned into the BglII site of the pIJ702 vector (12). The ligation mixture was used to transform S. lividans TK23, which is sensitive to doxorubicin (MIC, 20 ,ug/ml). Transformants were selected with thiostrepton (100 p.g/ml) or with thiostrepton (100 ,ug/ml) and doxorubicin (500 ,ug/ ml). Two thiostrepton- and doxorubicin-resistant clones out of 3,000 thiostrepton-resistant colonies were isolated, and the recombinant plasmids extracted from them were named ricl (5.7-kb insert) and ric2 (4.4-kb insert). Both plasmids were able to confer doxorubicin and thiostrepton resistance when used to retransform S. lividans TK23. Physical and functional characterization of ric1 and ric2. The restriction maps of ricl and ric2 are completely different (Fig. 1). To rule out the possibility that the observed differences were due to rearrangements during cloning, we
VOL. 174, 1992
DOXORUBICIN-DAUNORUBICIN RESISTANCE IN S. PEUCETIUS
A 1 2 3 4 5
B 12 3 4 5
6
DECAKETIDE (not pigmented) 6
V AKLAVINONE (yellow)
x,
23.1 9.46.54.3-
23.1 9.4 6.5 -
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4.3 -
C-1 1 Hydroxylation e RHODOMYCINONE
2.3 2.0 -
(violet)
C-10 C-1 0 C-13 C-1 3 FIG. 2. (A) Southern blot analysis of 10 ,ug of DNA isolated from different S. peucetius strains and digested with BamHI as described in Materials and Methods. Lanes: 1, ATCC 27952 DNA; 2, 7600 DNA; 3,7900 DNA; 4,8600 DNA; 5,9700 DNA; 6,7100 DNA. The blot was probed with the BgIII-ClaI fragment of ric2. (B) The same blot probed with two ricl fragments (BglII-PstI and PstI-PstI) mixed together in the hybridization solution. The sizes (kilobases) and positions of X HindIII molecular weight markers are indicated.
CARMINOMYCIN
(red)
Den nethylation Dec;:arboxylation HydiIroxylation OxidJation
11-DEOXYCARMINOMYCIN
C-4 Methylation
(yellow)
DAUNORUBICIN 11-DEOXYDAUNORUBICIN (yellow) C-14 Hydroxylation
(red) performed Southern blot analysis on ricl DNA by using the BglII-ClaI DNA fragment of ric2 as a probe (Fig. 1). No significant hybridization was observed (data not shown). This result is in accord with the different hybridization pattern observed when each of these DNAs was used to probe genomic DNA from S. peucetius 7600 digested with BamHI (Fig. 2A and B) and suggests that doxorubicindaunorubicin resistance is conferred by at least two different genes. Both ricl and ric2 increased the level of resistance to doxorubicin and daunorubicin in S. lividans 10-fold over the level obtained with the pIJ702 vector alone (Table 1). We suspect that the partial resistance detectable after transforTABLE 1. Doxorubicin and daunorubicin MICs for strains cited in this work MIC
Strain
TK23 TK23(pIJ702) TK23(ricl) TK23(ric2) 7100 7600 7900 7800
7800(ricl) 7800(ric2) 8600 9700
(pg/ml)a of:
Doxorubicin
Daunorubicin
20 60 500 500 10 250 250 10 500 500 40 40
5 10 100 100 5 30 50 5
a Drugs were added to solid medium as described in ods. b ND, not determined.
NDb ND 10 10
Materials and Meth-
DOXORUBICIN (red)
1 1-DEOXYDOXORUBICIN (yellow)
FIG. 3. Proposed biosynthetic pathway for daunorubicin and doxorubicin.
mation with the vector could be due to the thiostrepton resistance gene. The effect of ricl and ric2 was also analyzed in the yellow mutant 7800 (derived from S. peucetius 7600), which is sensitive to doxorubicin (MIC, 10 ,ug/ml) and produces only yellow anthracyclines lacking hydroxylation in C-11 (Fig. 3). When used to transform strain 7800, both fragments gave doxorubicin-resistant clones (Table 1), but only the ricl transformants produced red pigments instead of the typical yellow ones. HPLC analysis of the anthracycline metabolites produced by the ricl-transformed clones confirmed that ricl complemented the block in C-li hydroxylation (Table 2). Bioconversion experiments with S. lividans TK23(ricl) transformants confirmed the presence of the C-11 hydroxylation gene in ricl. In these experiments, aklavinone (100 ,ugIml) was added to liquid cultures of S. lividans TK23 transformed with ricl or ric2 and with the vector (pIJ702) as a control. Only clones transformed with ricl were able to convert the added aklavinone to e-rhodomycinone (an 80% conversion was observed) (data not shown). These data indicate that the doxorubicin and daunorubicin resistance gene in ricl is linked to one of the genes of the daunorubicin biosynthetic pathway. By subcloning ricl restriction fragments into pIJ702, we could map more precisely the resistance gene; in fact, we
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COLOMBO ET AL. TABLE 2. Metabolites synthesized by S. peucetius 7600 and 7800 and by different 7800 transformants Strain'
7600 7800 (yellow mutant) 7800(pIJ702) + thio 7800(ricl) + thio 7800(ricl) + thio + doxorubicin
11-Deoxy-
11-Deoxy-
daunorubicin
doxorubicin
0 183 51 0 0
0 71 17 0 0
Concn (4g/ml)b of: D u Daunorubicin drodaunorubicin
D
11-Deoxydihy-
65 0 0 44 60
0 49 3 0 0
u
Doxorubicin
Dihydrodaunorubicin
21 0 0 8 17
13 0 0 12 10
aConcentration of thiostrepton (thio) was 25 pg/ml, and that of doxorubicin was 50 pg/mi. b Metabolite concentration present in the medium after 5 days of fermentation as determined by HPLC.
found that transformation of TK23 with a 2.6-kb PstI-KpnI DNA fragment (Fig. 1) made S. lividans resistant to daunorubicin and doxorubicin. The ricl clone also appears to contain a third gene that may have a regulatory function, since an S. lividans TK23(ricl) transformant strain overproduced a blue pigment usually produced only at low levels in S. lividans (10). By means of Southern blot hybridization, we found that the 1.8-kb BamHI fragment of ricl (Fig. 1) hybridizes to the putative dnrR2 regulatory gene (23) from S. peucetius ATCC 29050 (data not shown). Hybridization of the icl and ric2 fragments to total DNAs of different S. peucetius mutants. In our laboratory, we isolated a set of mutants obtained from S. peucetius ATCC 27952. A variety of mutagenic agents (UV light, nitrosomethylurethane, N-methyl-N'-nitro-N-nitrosoguanidine) applied singly or in combination and known for their different modes of action were employed, and individual colonies were scored on the basis of two main characteristics: altered colony morphology and pigmentation (7). In order to further improve the production of daunorubicin and doxorubicin, the isolated mutants were subsequently utilized in cell fusion experiments. Here we present the results obtained with the fusion between the producing mutant 7600 and the nonproducing mutant 7100. One fusion product, selected on the basis of enhanced red pigment production and called 7900, produced fivefold more daunorubicin and tenfold less doxorubicin than strain 7600. Two nonproducing blocked mutants, 8600 and 9700, with significantly reduced resistances to doxorubicin and daunorubicin (Table 1), were obtained from 7900 by chemical mutagenesis. We analyzed these strains by Southern blot to determine if they contained major rearrangements affecting the ricl and ric2 regions. As shown in Fig. 2, two different hybridization patterns exist; the first is typical of strain ATCC 27952 and of mutant 7600, and the second is detectable both in 7100 and in all the postfusion mutants. We suppose that the striking difference between mutant 7100 and ATCC 27952 is due both to the mutagenesis protocol utilized and to the already described considerable natural variability of ATCC 27952 (7). The result of Southern analysis indicates that in the postfusion mutants most, if not all, of the analyzed region derives from strain 7100. If this is the case, one should hypothesize that other genes (i.e., for regulatory proteins) outside the considered region may play a role in determining the 7900 phenotype. If we focus our attention on the hybridization pattern of ric2 (Fig. 2A), it is quite evident that the ric2 region was particularly affected by the mutagenesis that eventually led to mutant 7100. In fact, not only the size of the recognized band but also the strength of hybridization differs from that
observed in wild-type ATCC 27952, indicating gross rearrangements of this region. The result of ricl hybridization (Fig. 2B) is particularly interesting; in fact, the 3.5-kb BamHI fragment containing most of the resistance gene (Fig. 1) is the only one among the recognized bands which is unchanged in all the analyzed strains. This result supports the idea that this region plays a key role in both the biosynthesis of and the resistance to anthracycline. Transcription of the ricl resistance gene in different S. peucetius mutants. We investigated the expression of the resistance gene in mutant strains that produce different amounts of daunorubicin and doxorubicin and yet show the same genomic organization in the region of this gene (Fig. 2B). Northern blot analysis was performed with the 2.6-kb PstI-KpnI probe (Fig. 1) on total RNA extracted from a 48-h fermentation culture of producing (7900) and nonproducing (8600 and 9700) strains. It should be noted that at 48 h, strain 7900 produces a substantial amount of the antitumor drug. As shown in Fig. 4, an RNA with a size of about 2,100 nucleotides (nt) is detected only for S. peucetius 7900. No hybridization was observed with RNA from the nonproduc-
0 o
'
00
0 ' c%D 0
0
a
2100 -_
EtBr
FIG. 4. Northern blot analysis of 2 ,g of RNA from strain 7900 (producing) and strains 8600 and 9700 (nonproducing). RNA was extracted after 48 h of growth (see Materials and Methods). The blot was probed with the PstI-KpnI fragment of ricl. The position of the 2,100-nt transcript is indicated. In the lower panel is shown an ethidium bromide staining of the gel before blotting.
DOXORUBICIN-DAUNORUBICIN RESISTANCE IN S. PEUCETIUS
VOL. 174, 1992 1
2
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3
seem to parallel the production of anthracyclines. Moreover, in strain 7900, the level of the 2,100-nt transcript at 48 h is low (Fig. 6) and never attains the level observed for strain 7600, even though a certain increase in the hybridization signal is observed at 72 h (Fig. 6). This is somewhat surprising in view of the fact that strain 7900 produces fivefold more daunorubicin than strain 7600. This point will be further addressed in Discussion.
2100 nt --w-
DISCUSSION
EtBr
FIG. 5. Northern blot analysis of 2 ,ug of RNA from producing strain 7600. RNA was extracted after 8 (lane 1), 24 (lane 2), 48 (lane 3), 72 (lane 4), and 96 (lane 5) h of growth. The blot was probed with the PstI-KpnI fragment of ricl. The position of the 2,100-nt transcript is indicated. In the lower panel is shown an ethidium bromide staining of the gel before blotting.
ing mutant strains 8600 and 9700. It is worth noting that both these mutants, although lacking ricl mRNA and probably having an inactive ric2 region (see previous section), show a limited but significant resistance to doxorubicin-daunorubicin (Table 1), suggesting that another still unidentified gene could be involved in the resistance phenotype. RNAs in strains 7600 and 7900 were also compared at different times during anthracycline metabolite production by means of Northern blot hybridizations on total RNA with the PstI-KpnI probe (2.6 kb) (Fig. 5 and 6). The quantification of loaded RNA was obtained by staining the ribosomal RNAs with ethidium bromide (see Materials and Methods). In strain 7600 (Fig. 5), no signal was detectable at 8 h of growth, even after overexposure (not shown). On the other hand, a prominent band with an apparent size of 2,100 nt is present at 48 h, and its intensity increases at 72 and 96 h. The ladder of lower-molecular-weight bands is probably due to partial RNA degradation, since all the hybridization signals
1
2 l 00 nt
_
2
3
jS
EtBr
FIG. 6. Northern blot analysis of 2 ,ug of RNA from producing strains 7900 and 7600. RNA was extracted after 76 h (lane 1) and 48 h (lane 2) of growth for strain 7900 and after 48 h of growth for strain 7600 (lane 3). The blot was probed with the PstI-KpnI fragment of ricl. The position of the 2,100-nt transcript is indicated. In the lower panel is shown an ethidium bromide staining of the gel before
blotting.
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In the past few years, many genes involved in antibiotic biosynthesis in Streptomyces spp. have been isolated. As a general rule, it has been observed that biosynthetic genes are clustered together, with one or more genes conferring resistance to the produced drug (6). We thus decided to search for a doxorubicin-daunorubicin resistance gene(s) as a first step towards cloning the doxorubicin production genes. We succeeded in isolating two different DNA fragments, ricl and ric2, that when introduced into S. lividans by transformation are able to make it resistant to doxorubicin-daunorubicin. Further analysis showed that the ricl fragment also contains a gene able to convert aklavinone to e-rhodomycinone via hydroxylation at C-11. The two activities (resistance and hydroxylation) are associated with different genes, since the resistance phenotype can be conferred by a 2.6-kb PstI-KpnI fragment that, on the other hand, is unable to cause hydroxylation of aklavinone. Our results, therefore, indicate that ricl contains a resistance gene associated with a biosynthetic gene presumably belonging to a single daunorubicin gene cluster as recently described by C. R. Hutchinson and coworkers (18, 22). The results obtained from Southern analysis of five mutant strains, two of which were derived directly from ATCC 27952 (mutants 7600 [producing] and 7100 [nonproducing]) and the others of which were obtained from a cell fusion between the former two, revealed that ric2 sequences isolated from strain 7600 were drastically affected by the mutagenesis that eventually led to mutant 7100. The fact that one postfusion strain (7900) is a good producer and shows a level of resistance to daunorubicin and doxorubicin comparable to that observed with 7600 (Table 1) suggests that ric2 is not necessary to give the daunorubicin resistance phenotype. It is conceivable that ric2 belongs to another anthracycline production cluster present in the original strain, ATCC 27952. In contrast to ric2, some portions of the ricl region are highly conserved in all five strains, although the overall pattern of hybridization is clearly different in 7100 and in the postfusion strains compared with 7600 and ATCC 27952. In fact, a 3.5-kb BamHI band is present in all five genomes (Fig. 2B) regardless of whether they are daunorubicin producers (ATCC 27952, 7600, and 7900) or nonproducers (7100 and blocked mutants 8600 and 9700). It is interesting to observe (Fig. 1) that the 3.5-kb BamHI band overlaps most of the 2.6-kb PstI-KpnI fragment that is able to give doxorubicin and daunorubicin resistance, thus explaining the observed high conservation of this region. The PstI-KpnI fragment utilized as a probe in Northern experiments hybridized to a prominent transcript of 2,100 nt that is probably encoded by the resistance gene. Furthermore, Northern blot experiments with different portions of the PstI-KpnI fragment allowed us to restrict the hybridization to the BamHI-KpnI region, whose size closely matches that of the detected RNA
1646
COLOMBO ET AL.
(data not shown). This result suggests that one border of the resistance gene is at the immediate left of the BamHI site. The hypothesis that the 2,100-nt RNA is encoded by the resistance gene is in accord with the observation that the blocked mutants 8600 and 9700 completely lack the 2,100-nt specific transcript (Fig. 4). On the other hand, this messenger is present in both 7600 and 7900, and its level increases concomitantly with daunorubicin production. In fact, Northern blot analysis of RNA extracted from strain 7600 at different times of culture showed that this messenger is undetectable at a very early time (8 h) and appears in parallel with the drug production, suggesting a regulation at the transcriptional level. One possibility is that the transcription of the 2,100-nt RNA is under the control of a regulatory protein expressed and/or active only during the phase of anthracycline production. It is reasonable to speculate that the absence or the mutation of this factor is responsible for the phenotypes of 7100 and of the two blocked mutants (8600 and 9700). Finally, we would like to comment on the fact that the steady-state amount of the 2,100-nt transcript is significantly higher in 7600 than in 7900 (Fig. 4), even though the latter produces fivefold more daunorubicin. To explain this apparent paradox, two hypotheses can be advanced. One possibility is that the amount of the 2,100-nt messenger observed for strain 7900 is already sufficient to give resistance. Alternatively, we propose that another resistance gene, yet to be found, is expressed in strain 7900. In support of the latter hypothesis is the observation that the nonproducing strain 8600, with an undetectable level of the 2,100-nt RNA, is nevertheless partially resistant to daunorubicin-doxorubicin (Table 2). ACKNOWLEDGMENTS We thank C. R. Hutchinson (University of Wisconsin, Madison) for the genomic fragments from S. peucetius ATCC 29050 and for discussion; D. A. Hopwood (John Innes Institute, Norwich, United Kingdom) and S. Riva (Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale della Ricerche) for helpful suggestions; S. Merli (Farmitalia Carlo Erba) for the different S. peucetius mutants; and U. Breme (Farmitalia Carlo Erba) for HPLC analysis. G.P. was supported by a fellowship from the Italian Association for Cancer Research (A.I.R.C.). REFERENCES 1. Arcamone, F. 1981. Doxorubicin. Med. Chem. Ser. Monogr. 17:25-31. 2. Arcamone, F., G. Cassinelli, G. Fantini, A. Grein, P. Orezzi, C. Pol, and C. Spalla. 1969. 14-Hydroxydaunomycin, a new antitumor antibiotic from Streptomyces peucetius var. caesius. Biotechnol. Bioeng. 11:1101-1110. 3. Cassinelli, G., A. Grein, P. Masi, A. Suarato, L. Bernardi, F. Arcamone, A. Di Marco, A. M. Casazza, G. Pratesi, and C. Soranzo. 1978. Preparation and biological evaluation of 4-0demethyldaunorubicin (carminomycin I) and of its 13-dihydroderivate. J. Antibiot. 31:178-184. 4. Cassinelli, G., and P. Orezzi. 1963. Daunomicina: un nuovo antibiotico ad attivita citostatica. Isolamento e proprieta. G. Microbiol. 11:167-174. 5. Crespi-Perellino, N., A. Grein, S. Merli, A. Minghetti, and C. Spalla. 1982. Biosynthetic relationships among daunorubicin, doxorubicin and 13-dihydrodaunorubicin in Streptomyces peucetius. Experientia 38:1455-1456. 6. Esser, K., and G. Dohmen. 1987. Drug resistance genes and their use in molecular cloning. Process Biochem. 22:144-148.
J. BACTERIOL. 7. Grein, A. 1981. Development of biosynthetic anthracyclines of the daunorubicin group by genetic and fermentation studies. Process Biochem. 16:34-35. 8. Grein, A., C. Spalla, A. Di Marco, and G. Canevazzi. 1963. Descrizione e caratterizzazione di un attinomicete (Streptomyces peucetius sp. nova) produttore di una sostanza ad attivita antitumorale: la daunorubicina. G. Microbiol. 11:109-118. 9. Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. M. Kieser, D. J. Lydiate, C. P. Smith, J. M. Ward, and H. Schrempf. 1985. Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich, United
Kingdom. 10. Horinouchi, S., and T. Beppu. 1984. Production in large quantities of actinorhodin and undecylprodigiosin induced by asfB in Streptomyces lividans. Agric. Biol. Chem. 48:2131-2133. 11. Hutchinson, C. R. 1987. The impact of genetic engeneering on the commercial production of antibiotics by Streptomyces and related bacteria. Appl. Biochem. Biotechnol. 16:169-190. 12. Katz, E., J. Thompson, and D. A. Hopwood. 1983. Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J. Gen. Microbiol. 129:2703-2714. 13. Komiyama, T., M. Yasue, T. Oki, and T. Inui. 1977. Baumycins, new antitumor antibiotics related to daunomycin. J. Antibiot. 30:619-620. 14. Lampel, J. S., and W. R. Strohl. 1986. Transformation and transfection of anthracycline-producing streptomycetes. Appl. Environ. Microbiol. 51:126-131. 15. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 16. McGuire, J. C., B. K. Hamilton, and R. J. White. 1979. Approaches to development of the daunorubicin fermentation. Process Biochem. 14:2-5. 17. Oki, T., Y. Takatsuki, H. Tobe, A. Yoshimoto, T. Takeuchi, and H. Umezawa. 1981. Microbial conversion of daunomycin, carminomycin I and feudomycin A to adriamycin. J. Antibiot. 34:1229-1231. 18. Otten, S. L., K. J. Stutzman-Engwall, and R. C. Hutchinson. 1990. Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and Streptomyces peucetius subsp. caesius. J. Bacteriol. 172:3427-3434. 19. Randazzo, R., G. Sciandrello, A. Carere, M. Bignami, A. Velcich, and G. Sermonti. 1976. Localized mutagenesis in Streptomyces coelicolor A3(2). Mutat. Res. 36:291-302. 20. Rave, N., R. Crkvenjakov, and H. Boedtker. 1979. Identification of procollagen mRNAs transfered to diazobenzyloximothyl paper from formaldehyde agarose gel. Nucleic Acids Res. 6:35593567. 21. Strohl, W. R., P. L. Bartel, N. C. Connors, C. B. Zhu, D. C. Dosch, J. M. Beale, Jr., H. G. Floss, K. Stutzman-Engwall, S. L. Otten, and C. R. Hutchinson. 1989. Biosynthesis of natural and hybrid polyketides by anthracycline-producing streptomycetes, p. 68-84. In C. L. Hershberger, S. W. Queener, and G. Hegeman (ed.), Genetics and molecular biology of industrial microorganisms. American Society for Microbiology, Washington, D.C. 22. Stutzman-Engwall, K. J., and C. R. Hutchinson. 1989. Multigene families for anthracyclines: antibiotic production in Streptomyces peucetius. Proc. Natl. Acad. Sci. USA 86:3135-3139. 23. Stutzman-Engwall, K. J., S. L. Otten, and C. R. Hutchinson. 1992. Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J. Bacteriol. 174:144-154. 24. White, R. J., and R. M. Stroshane. 1984. Daunorubicin and adriamycin: properties, biosynthesis, and fermentation, p. 569594. In E. J. Vandamme (ed.), Biotechnology of industrial antibiotics, vol. 22. Marcel Dekker, Inc., New York. 25. Yoshimoto, A., and T. Oki. 1980. Microbial conversion of anthracyclinone to daunomycin by blocked mutants of Streptomyces coeruleorubidus. J. Antibiot. 33:1158-1166.
JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 1641-1646
0021-9193/92/051641-06$02.00/0 Copyright © 1992, American Society for Microbiology
Expression of Doxorubicin-Daunorubicin Resistance Genes in Different Anthracycline-Producing Mutants of Streptomyces peucetius ANNA L. COLOMBO,'* MARIA M. SOLINAS,2 GIOVANNI PERINI,2 GIUSEPPE BIAMONTI2* GIUSEPPE ZANELLA,' MARINELLA CARUSO,' FRANCESCA TORTI,' SILVIA FILIPPINI,1 AUGUSTO INVENTI-SOLARI,' AND LUISA GAROFANO' Dipartimento Biotecnologie, Unita Screening, Farmitalia Carlo Erba, Milan,' and Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, Pavia,2 Italy Received 11 July 1991/Accepted 7 December 1991
Two DNA fragments, ricl and ric2, were isolated from the Streptomyces peucetius 7600 mutant, which produces daunorubicin and doxorubicin, on the basis of their abilities to confer doxorubicin and daunorubicin resistance to Streptomyces lividans. These two fragments are unrelated by restriction mapping and do not show any homology by Southern analysis, yet both of them increase the level of resistance 10-fold in transformed S. lividans. Functional analysis revealed that icl also contains two genes of daunorubicin biosynthesis: one coding for the aklavinone C-il hydroxylase and the other corresponding to the putative dnrR2 regulatory gene of wild-type S. peucetius ATCC 29050 (K. J. Stutzman-Engwall, S. L. Otten, and C. R. Hutchinson, J. Bacteriol. 174:144-154, 1992). Northern (RNA) blot experiments, performed with a ricl fragment containing daunorubicindoxorubicin resistance gene(s), revealed a transcript of about 2,100 nucleotides that is present only during the phase of anthracycline metabolite production. The amount of this transcript is higher in strain 7600 than in strain 7900, a mutant which produces 5-fold more daunorubicin and 10-fold less doxorubicin than 7600. Furthermore, two 7900-derived blocked mutants, 8600 and 9700, do not express the 2,100-nucleotide transcript in spite of the absence of gross rearrangements in the ricl region such as occur with the 7900 parental strain.
Studies of the Streptomyces genes involved in the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), the two most widely employed agents in antitumor therapy, can be useful to produce specific intermediates for subsequent biotransformation into new analogs (3, 5, 11, 17, 21, 24). Furthermore, a better understanding of the mechanism conferring resistance to the drugs in the producing organism may lead to the construction of improved strains. These considerations prompted us to investigate the genes involved in the biosynthesis of and in the resistance to these drugs. Doxorubicin was initially obtained from Streptomyces peucetius subsp. caesius ATCC 27952, a mutant from a wild-type strain previously isolated (1, 2, 4, 13, 25). During a program of strain improvement undertaken in our laboratories, we isolated several mutants of ATCC 27952 that accumulate anthracyclines in different ratios (7). One mutant, S. peucetius 7600, efficiently converts daunorubicin to doxorubicin via C-14 hydroxylation, while another mutant, S. peucetius 7900, produces fivefold more daunorubicin and tenfold less doxorubicin than 7600. Both of these mutants are more resistant to doxorubicin than to daunorubicin. We have cloned two fragments (ricl and ric2) containing doxorubicin-daunorubicin resistance genes from S. peucetips 7600. The level of expression of the resistance gene present in ricl was compared for S. peucetius 7600, S. peucetius 7900, and two different blocked mutants, derived from 7900 (S. peucetius 8600 and 9700), which do not produce detectable amounts of anthracycline metabolites and are more sensitive to doxorubicin and daunorubicin than the parental strain.
MATERIALS AND METHODS Bacterial strains and plasmid. All S. peucetius strains which appear in this work were isolated in Farmitalia Carlo Erba laboratories. S. peucetius subsp. caesius ATCC 27952 (2), a daunorubicin and doxorubicin producer, is a mutant derived from wild-type strain S. peucetius ATCC 29050 (8) isolated in 1963. All the other mutant strains were obtained by several cycles of mutagenesis with UV light, N-methyl-N'-nitro-Nnitrosoguanidine, and nitrosomethylurethane applied singly or in combination accordingly to standard procedures (7, 19). S. peucetius 7600 converts daunorubicin to doxorubicin at high levels and was obtained from S. peucetius ATCC 27952. S. peucetius 7100 is a blocked mutant obtained from S. peucetius ATCC 27952. S. peucetius 7800, derived from S. peucetius 7600, is a yellow mutant which lacks aklavinone C-11 hydroxylation and is sensitive to doxorubicin and daunorubicin. S. peucetius 7900 was obtained by a fusion of strains 7600 and 7100 and produces more daunorubicin (fivefold) and less doxorubicin (tenfold) than S. peucetius 7600. S. peucetius 8600 and 9700 are blocked mutants derived from 7900. Streptomyces lividans TK23 and pIJ702 were obtained from D. A. Hopwood (John Innes Institute,
Norwich, United Kingdom). Biochemicals and chemicals. Thiostrepton was obtained from Behring. Aklavinone, doxorubicin, and daunorubicin were obtained from Farmitalia Carlo Erba. Restriction enzymes and other recombinant DNA materials were purchased from Boehringer. All other chemicals and biochemicals were obtained from Sigma. Media, growth conditions, and protoplast formation. Cultures for preparation of Streptomyces spore stocks were grown on R2YE agar medium (9) at 28°C for 8 to 10 days. Protoplast and chromosomal/plasmid DNAs were produced
* Corresponding authors. 1641
1642
J. BACTERIOL.
COLOMBO ET AL.
GK B lI Ite,
C
K
I
I
S I
B
B
I
I
BS
5
P I
I
I
I
S
S
I
I
P
K
I
Ii
ric 1
3 .5 kb BamHI genomic fragment
KTI, L
B I
I
C
,.
,
K I
ric 2
lkb II
FIG. 1. Restriction maps of the ricl and ric2 fragments. Restriction enzyme abbreviations: B, BamHI; C, ClaI; G, Bglll; K, Kpnl; L, Bcll; M, MboI; P, PstI; S, SphI; T, SstI. 111m Region of homology with dnrR2; SS, fragment conferring daunorubicin and doxorubicin resistance. ric2 region utilized as a probe in Southern blot experiments. The interrupted bar ( E] ) represents the conserved 3.5-kb BamHI fragment. ,
from Streptomyces strains grown in YEME medium (9) supplemented with 0.5% glycine at 28°C. Streptomyces protoplasts were regenerated on R2YE agar. S. lividans and S. peucetius transformants were selected and maintained on thiostrepton at 100 and 25 ,ug/ml, respectively. Conditions used for protoplast formation were essentially as described by Hopwood et al. (9) for S. lividans TK23 and as described by Lampel and Strohl (14) for S. peucetius 7800. Detection of anthracycline metabolites and bioconversion experiments. S. peucetius cells were grown in 25-ml culture flasks at 30°C with shaking at 300 rpm in the fermentation medium described by McGuire et al. (16). After 5 days, cultures were acidified by adding 25 mg of oxalic acid per ml, incubated at 30°C overnight, and extracted with an equal volume of acetonitrile. The extract was filtered, and the filtrate was analyzed by reverse-phase high-performance liquid chromatography (HPLC). Bioconversion expenments were performed by using whole cells of S. peucetius 8600 and S. lividans TK23(ricl) in the fermentation process. Aklavinone (100 ,ug/ml) and E-rhodomycinone (50 ,ug/ml) were added to 96-h cultures, and after 24 h, the products were extracted and HPLC analyzed. Analysis of doxorubicin and daunorubicin resistance. S. lividans and S. peucetius were tested for doxorubicin and daunorubicin resistance on R2YE agar plates. Approximately 106 spores were streaked onto plates containing 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, and 750 p.g of doxorubicin per ml and daunorubicin at the same concentrations (from 0 to 150 jj.g/ml). DNA and RNA extraction. DNA was isolated from S. peucetius as described by Hopwood et al. (9). S. peucetius RNA was extracted from 3 to 5 g of mycelium grown in the fermentation medium (16) by homogenization in a 25-ml Corex tube containing 10 ml of GIT solution (4 M guanidine thiocyanate salt, 25 mM sodium acetate [pH 6.0], 120 mM 2-mercaptoethanol) and 10 g of 0.5-mm-diameter glass balls. Cells were vortexed for a total of 10 min, with stops of 30 s in ice every minute. After centrifugation at 0°C (7,000 x g for 15 min), RNA was separated from DNA and purified as described by Maniatis et al. (15). Southern and Northern (RNA) hybridization analysis. For RNA gels, 10 ,ug of total RNA was lyophilized, resuspended in 15 RI of sample buffer (50% formamide, 6% formaldehyde, 20 mM NaH2PO4-Na2HPO4 [pH 7.0], 5% glycerol, 0.02% bromophenol blue, 0.02% xylene cyanol, 0.2 mg of ethidium
bromide per ml) and incubated for 10 min at 68°C. RNA was electrophoresed onto a 1.5% agarose-6% formaldehyde gel. At the end of the run, the gel was photographed on a UV transilluminator and then transferred to a Zetabind (Cuno Laboratory Products) membrane. Southern and Northern blot hybridizations were performed as described by Maniatis et al. (15) and by Rave et al. (20), using Zetabind (Cuno) filters. Processing of filters was as recommended by the supplier. The prehybridization and hybridization solution was 4x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-4x Denhardt's solution-50% formamide-0.5% sodium dodecyl sulfate (SDS)-7.5 mM Na4P2O-r12.5 mM NaH2PO4-0.3 mg of tRNA per ml. The probe was labelled at a specific activity of 109 cpm/,Lg with the Multiprime DNA labelling system (Amersham) and was added to the hybridization solution at a concentration of 2 x 106 cpm/ml. Southern hybridizations were carried out at 42°C, while Northern hybridizations were carried out at 52°C. After an overnight hybridization, filters were washed twice for 30 min each in 2x SSC-1% SDS at room temperature, once in 2x SSC-1% SDS at 68°C, and once in 0.2x SSC-0.1% SDS at 68°C. In order to rehybridize the filters, probes were stripped away by boiling the filters for 10 min in 10 mM Tris HCI (pH 7.5)-l mM EDTA-1% SDS. RESULTS Cloning of doxorubicin and daunorubicin resistance genes. Chromosomal DNA of S. peucetius 7600 was partially digested with MboI, and DNA fragments in the range of 4 to 6 kb were recovered from an agarose gel and cloned into the BglII site of the pIJ702 vector (12). The ligation mixture was used to transform S. lividans TK23, which is sensitive to doxorubicin (MIC, 20 ,ug/ml). Transformants were selected with thiostrepton (100 p.g/ml) or with thiostrepton (100 ,ug/ml) and doxorubicin (500 ,ug/ ml). Two thiostrepton- and doxorubicin-resistant clones out of 3,000 thiostrepton-resistant colonies were isolated, and the recombinant plasmids extracted from them were named ricl (5.7-kb insert) and ric2 (4.4-kb insert). Both plasmids were able to confer doxorubicin and thiostrepton resistance when used to retransform S. lividans TK23. Physical and functional characterization of ric1 and ric2. The restriction maps of ricl and ric2 are completely different (Fig. 1). To rule out the possibility that the observed differences were due to rearrangements during cloning, we
VOL. 174, 1992
DOXORUBICIN-DAUNORUBICIN RESISTANCE IN S. PEUCETIUS
A 1 2 3 4 5
B 12 3 4 5
6
DECAKETIDE (not pigmented) 6
V AKLAVINONE (yellow)
x,
23.1 9.46.54.3-
23.1 9.4 6.5 -
1643
4.3 -
C-1 1 Hydroxylation e RHODOMYCINONE
2.3 2.0 -
(violet)
C-10 C-1 0 C-13 C-1 3 FIG. 2. (A) Southern blot analysis of 10 ,ug of DNA isolated from different S. peucetius strains and digested with BamHI as described in Materials and Methods. Lanes: 1, ATCC 27952 DNA; 2, 7600 DNA; 3,7900 DNA; 4,8600 DNA; 5,9700 DNA; 6,7100 DNA. The blot was probed with the BgIII-ClaI fragment of ric2. (B) The same blot probed with two ricl fragments (BglII-PstI and PstI-PstI) mixed together in the hybridization solution. The sizes (kilobases) and positions of X HindIII molecular weight markers are indicated.
CARMINOMYCIN
(red)
Den nethylation Dec;:arboxylation HydiIroxylation OxidJation
11-DEOXYCARMINOMYCIN
C-4 Methylation
(yellow)
DAUNORUBICIN 11-DEOXYDAUNORUBICIN (yellow) C-14 Hydroxylation
(red) performed Southern blot analysis on ricl DNA by using the BglII-ClaI DNA fragment of ric2 as a probe (Fig. 1). No significant hybridization was observed (data not shown). This result is in accord with the different hybridization pattern observed when each of these DNAs was used to probe genomic DNA from S. peucetius 7600 digested with BamHI (Fig. 2A and B) and suggests that doxorubicindaunorubicin resistance is conferred by at least two different genes. Both ricl and ric2 increased the level of resistance to doxorubicin and daunorubicin in S. lividans 10-fold over the level obtained with the pIJ702 vector alone (Table 1). We suspect that the partial resistance detectable after transforTABLE 1. Doxorubicin and daunorubicin MICs for strains cited in this work MIC
Strain
TK23 TK23(pIJ702) TK23(ricl) TK23(ric2) 7100 7600 7900 7800
7800(ricl) 7800(ric2) 8600 9700
(pg/ml)a of:
Doxorubicin
Daunorubicin
20 60 500 500 10 250 250 10 500 500 40 40
5 10 100 100 5 30 50 5
a Drugs were added to solid medium as described in ods. b ND, not determined.
NDb ND 10 10
Materials and Meth-
DOXORUBICIN (red)
1 1-DEOXYDOXORUBICIN (yellow)
FIG. 3. Proposed biosynthetic pathway for daunorubicin and doxorubicin.
mation with the vector could be due to the thiostrepton resistance gene. The effect of ricl and ric2 was also analyzed in the yellow mutant 7800 (derived from S. peucetius 7600), which is sensitive to doxorubicin (MIC, 10 ,ug/ml) and produces only yellow anthracyclines lacking hydroxylation in C-11 (Fig. 3). When used to transform strain 7800, both fragments gave doxorubicin-resistant clones (Table 1), but only the ricl transformants produced red pigments instead of the typical yellow ones. HPLC analysis of the anthracycline metabolites produced by the ricl-transformed clones confirmed that ricl complemented the block in C-li hydroxylation (Table 2). Bioconversion experiments with S. lividans TK23(ricl) transformants confirmed the presence of the C-11 hydroxylation gene in ricl. In these experiments, aklavinone (100 ,ugIml) was added to liquid cultures of S. lividans TK23 transformed with ricl or ric2 and with the vector (pIJ702) as a control. Only clones transformed with ricl were able to convert the added aklavinone to e-rhodomycinone (an 80% conversion was observed) (data not shown). These data indicate that the doxorubicin and daunorubicin resistance gene in ricl is linked to one of the genes of the daunorubicin biosynthetic pathway. By subcloning ricl restriction fragments into pIJ702, we could map more precisely the resistance gene; in fact, we
1644
J. BACTERIOL.
COLOMBO ET AL. TABLE 2. Metabolites synthesized by S. peucetius 7600 and 7800 and by different 7800 transformants Strain'
7600 7800 (yellow mutant) 7800(pIJ702) + thio 7800(ricl) + thio 7800(ricl) + thio + doxorubicin
11-Deoxy-
11-Deoxy-
daunorubicin
doxorubicin
0 183 51 0 0
0 71 17 0 0
Concn (4g/ml)b of: D u Daunorubicin drodaunorubicin
D
11-Deoxydihy-
65 0 0 44 60
0 49 3 0 0
u
Doxorubicin
Dihydrodaunorubicin
21 0 0 8 17
13 0 0 12 10
aConcentration of thiostrepton (thio) was 25 pg/ml, and that of doxorubicin was 50 pg/mi. b Metabolite concentration present in the medium after 5 days of fermentation as determined by HPLC.
found that transformation of TK23 with a 2.6-kb PstI-KpnI DNA fragment (Fig. 1) made S. lividans resistant to daunorubicin and doxorubicin. The ricl clone also appears to contain a third gene that may have a regulatory function, since an S. lividans TK23(ricl) transformant strain overproduced a blue pigment usually produced only at low levels in S. lividans (10). By means of Southern blot hybridization, we found that the 1.8-kb BamHI fragment of ricl (Fig. 1) hybridizes to the putative dnrR2 regulatory gene (23) from S. peucetius ATCC 29050 (data not shown). Hybridization of the icl and ric2 fragments to total DNAs of different S. peucetius mutants. In our laboratory, we isolated a set of mutants obtained from S. peucetius ATCC 27952. A variety of mutagenic agents (UV light, nitrosomethylurethane, N-methyl-N'-nitro-N-nitrosoguanidine) applied singly or in combination and known for their different modes of action were employed, and individual colonies were scored on the basis of two main characteristics: altered colony morphology and pigmentation (7). In order to further improve the production of daunorubicin and doxorubicin, the isolated mutants were subsequently utilized in cell fusion experiments. Here we present the results obtained with the fusion between the producing mutant 7600 and the nonproducing mutant 7100. One fusion product, selected on the basis of enhanced red pigment production and called 7900, produced fivefold more daunorubicin and tenfold less doxorubicin than strain 7600. Two nonproducing blocked mutants, 8600 and 9700, with significantly reduced resistances to doxorubicin and daunorubicin (Table 1), were obtained from 7900 by chemical mutagenesis. We analyzed these strains by Southern blot to determine if they contained major rearrangements affecting the ricl and ric2 regions. As shown in Fig. 2, two different hybridization patterns exist; the first is typical of strain ATCC 27952 and of mutant 7600, and the second is detectable both in 7100 and in all the postfusion mutants. We suppose that the striking difference between mutant 7100 and ATCC 27952 is due both to the mutagenesis protocol utilized and to the already described considerable natural variability of ATCC 27952 (7). The result of Southern analysis indicates that in the postfusion mutants most, if not all, of the analyzed region derives from strain 7100. If this is the case, one should hypothesize that other genes (i.e., for regulatory proteins) outside the considered region may play a role in determining the 7900 phenotype. If we focus our attention on the hybridization pattern of ric2 (Fig. 2A), it is quite evident that the ric2 region was particularly affected by the mutagenesis that eventually led to mutant 7100. In fact, not only the size of the recognized band but also the strength of hybridization differs from that
observed in wild-type ATCC 27952, indicating gross rearrangements of this region. The result of ricl hybridization (Fig. 2B) is particularly interesting; in fact, the 3.5-kb BamHI fragment containing most of the resistance gene (Fig. 1) is the only one among the recognized bands which is unchanged in all the analyzed strains. This result supports the idea that this region plays a key role in both the biosynthesis of and the resistance to anthracycline. Transcription of the ricl resistance gene in different S. peucetius mutants. We investigated the expression of the resistance gene in mutant strains that produce different amounts of daunorubicin and doxorubicin and yet show the same genomic organization in the region of this gene (Fig. 2B). Northern blot analysis was performed with the 2.6-kb PstI-KpnI probe (Fig. 1) on total RNA extracted from a 48-h fermentation culture of producing (7900) and nonproducing (8600 and 9700) strains. It should be noted that at 48 h, strain 7900 produces a substantial amount of the antitumor drug. As shown in Fig. 4, an RNA with a size of about 2,100 nucleotides (nt) is detected only for S. peucetius 7900. No hybridization was observed with RNA from the nonproduc-
0 o
'
00
0 ' c%D 0
0
a
2100 -_
EtBr
FIG. 4. Northern blot analysis of 2 ,g of RNA from strain 7900 (producing) and strains 8600 and 9700 (nonproducing). RNA was extracted after 48 h of growth (see Materials and Methods). The blot was probed with the PstI-KpnI fragment of ricl. The position of the 2,100-nt transcript is indicated. In the lower panel is shown an ethidium bromide staining of the gel before blotting.
DOXORUBICIN-DAUNORUBICIN RESISTANCE IN S. PEUCETIUS
VOL. 174, 1992 1
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3
seem to parallel the production of anthracyclines. Moreover, in strain 7900, the level of the 2,100-nt transcript at 48 h is low (Fig. 6) and never attains the level observed for strain 7600, even though a certain increase in the hybridization signal is observed at 72 h (Fig. 6). This is somewhat surprising in view of the fact that strain 7900 produces fivefold more daunorubicin than strain 7600. This point will be further addressed in Discussion.
2100 nt --w-
DISCUSSION
EtBr
FIG. 5. Northern blot analysis of 2 ,ug of RNA from producing strain 7600. RNA was extracted after 8 (lane 1), 24 (lane 2), 48 (lane 3), 72 (lane 4), and 96 (lane 5) h of growth. The blot was probed with the PstI-KpnI fragment of ricl. The position of the 2,100-nt transcript is indicated. In the lower panel is shown an ethidium bromide staining of the gel before blotting.
ing mutant strains 8600 and 9700. It is worth noting that both these mutants, although lacking ricl mRNA and probably having an inactive ric2 region (see previous section), show a limited but significant resistance to doxorubicin-daunorubicin (Table 1), suggesting that another still unidentified gene could be involved in the resistance phenotype. RNAs in strains 7600 and 7900 were also compared at different times during anthracycline metabolite production by means of Northern blot hybridizations on total RNA with the PstI-KpnI probe (2.6 kb) (Fig. 5 and 6). The quantification of loaded RNA was obtained by staining the ribosomal RNAs with ethidium bromide (see Materials and Methods). In strain 7600 (Fig. 5), no signal was detectable at 8 h of growth, even after overexposure (not shown). On the other hand, a prominent band with an apparent size of 2,100 nt is present at 48 h, and its intensity increases at 72 and 96 h. The ladder of lower-molecular-weight bands is probably due to partial RNA degradation, since all the hybridization signals
1
2 l 00 nt
_
2
3
jS
EtBr
FIG. 6. Northern blot analysis of 2 ,ug of RNA from producing strains 7900 and 7600. RNA was extracted after 76 h (lane 1) and 48 h (lane 2) of growth for strain 7900 and after 48 h of growth for strain 7600 (lane 3). The blot was probed with the PstI-KpnI fragment of ricl. The position of the 2,100-nt transcript is indicated. In the lower panel is shown an ethidium bromide staining of the gel before
blotting.
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In the past few years, many genes involved in antibiotic biosynthesis in Streptomyces spp. have been isolated. As a general rule, it has been observed that biosynthetic genes are clustered together, with one or more genes conferring resistance to the produced drug (6). We thus decided to search for a doxorubicin-daunorubicin resistance gene(s) as a first step towards cloning the doxorubicin production genes. We succeeded in isolating two different DNA fragments, ricl and ric2, that when introduced into S. lividans by transformation are able to make it resistant to doxorubicin-daunorubicin. Further analysis showed that the ricl fragment also contains a gene able to convert aklavinone to e-rhodomycinone via hydroxylation at C-11. The two activities (resistance and hydroxylation) are associated with different genes, since the resistance phenotype can be conferred by a 2.6-kb PstI-KpnI fragment that, on the other hand, is unable to cause hydroxylation of aklavinone. Our results, therefore, indicate that ricl contains a resistance gene associated with a biosynthetic gene presumably belonging to a single daunorubicin gene cluster as recently described by C. R. Hutchinson and coworkers (18, 22). The results obtained from Southern analysis of five mutant strains, two of which were derived directly from ATCC 27952 (mutants 7600 [producing] and 7100 [nonproducing]) and the others of which were obtained from a cell fusion between the former two, revealed that ric2 sequences isolated from strain 7600 were drastically affected by the mutagenesis that eventually led to mutant 7100. The fact that one postfusion strain (7900) is a good producer and shows a level of resistance to daunorubicin and doxorubicin comparable to that observed with 7600 (Table 1) suggests that ric2 is not necessary to give the daunorubicin resistance phenotype. It is conceivable that ric2 belongs to another anthracycline production cluster present in the original strain, ATCC 27952. In contrast to ric2, some portions of the ricl region are highly conserved in all five strains, although the overall pattern of hybridization is clearly different in 7100 and in the postfusion strains compared with 7600 and ATCC 27952. In fact, a 3.5-kb BamHI band is present in all five genomes (Fig. 2B) regardless of whether they are daunorubicin producers (ATCC 27952, 7600, and 7900) or nonproducers (7100 and blocked mutants 8600 and 9700). It is interesting to observe (Fig. 1) that the 3.5-kb BamHI band overlaps most of the 2.6-kb PstI-KpnI fragment that is able to give doxorubicin and daunorubicin resistance, thus explaining the observed high conservation of this region. The PstI-KpnI fragment utilized as a probe in Northern experiments hybridized to a prominent transcript of 2,100 nt that is probably encoded by the resistance gene. Furthermore, Northern blot experiments with different portions of the PstI-KpnI fragment allowed us to restrict the hybridization to the BamHI-KpnI region, whose size closely matches that of the detected RNA
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COLOMBO ET AL.
(data not shown). This result suggests that one border of the resistance gene is at the immediate left of the BamHI site. The hypothesis that the 2,100-nt RNA is encoded by the resistance gene is in accord with the observation that the blocked mutants 8600 and 9700 completely lack the 2,100-nt specific transcript (Fig. 4). On the other hand, this messenger is present in both 7600 and 7900, and its level increases concomitantly with daunorubicin production. In fact, Northern blot analysis of RNA extracted from strain 7600 at different times of culture showed that this messenger is undetectable at a very early time (8 h) and appears in parallel with the drug production, suggesting a regulation at the transcriptional level. One possibility is that the transcription of the 2,100-nt RNA is under the control of a regulatory protein expressed and/or active only during the phase of anthracycline production. It is reasonable to speculate that the absence or the mutation of this factor is responsible for the phenotypes of 7100 and of the two blocked mutants (8600 and 9700). Finally, we would like to comment on the fact that the steady-state amount of the 2,100-nt transcript is significantly higher in 7600 than in 7900 (Fig. 4), even though the latter produces fivefold more daunorubicin. To explain this apparent paradox, two hypotheses can be advanced. One possibility is that the amount of the 2,100-nt messenger observed for strain 7900 is already sufficient to give resistance. Alternatively, we propose that another resistance gene, yet to be found, is expressed in strain 7900. In support of the latter hypothesis is the observation that the nonproducing strain 8600, with an undetectable level of the 2,100-nt RNA, is nevertheless partially resistant to daunorubicin-doxorubicin (Table 2). ACKNOWLEDGMENTS We thank C. R. Hutchinson (University of Wisconsin, Madison) for the genomic fragments from S. peucetius ATCC 29050 and for discussion; D. A. Hopwood (John Innes Institute, Norwich, United Kingdom) and S. Riva (Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale della Ricerche) for helpful suggestions; S. Merli (Farmitalia Carlo Erba) for the different S. peucetius mutants; and U. Breme (Farmitalia Carlo Erba) for HPLC analysis. G.P. was supported by a fellowship from the Italian Association for Cancer Research (A.I.R.C.). REFERENCES 1. Arcamone, F. 1981. Doxorubicin. Med. Chem. Ser. Monogr. 17:25-31. 2. Arcamone, F., G. Cassinelli, G. Fantini, A. Grein, P. Orezzi, C. Pol, and C. Spalla. 1969. 14-Hydroxydaunomycin, a new antitumor antibiotic from Streptomyces peucetius var. caesius. Biotechnol. Bioeng. 11:1101-1110. 3. Cassinelli, G., A. Grein, P. Masi, A. Suarato, L. Bernardi, F. Arcamone, A. Di Marco, A. M. Casazza, G. Pratesi, and C. Soranzo. 1978. Preparation and biological evaluation of 4-0demethyldaunorubicin (carminomycin I) and of its 13-dihydroderivate. J. Antibiot. 31:178-184. 4. Cassinelli, G., and P. Orezzi. 1963. Daunomicina: un nuovo antibiotico ad attivita citostatica. Isolamento e proprieta. G. Microbiol. 11:167-174. 5. Crespi-Perellino, N., A. Grein, S. Merli, A. Minghetti, and C. Spalla. 1982. Biosynthetic relationships among daunorubicin, doxorubicin and 13-dihydrodaunorubicin in Streptomyces peucetius. Experientia 38:1455-1456. 6. Esser, K., and G. Dohmen. 1987. Drug resistance genes and their use in molecular cloning. Process Biochem. 22:144-148.
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