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Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains
FEMS MicrobiologyLetters 70 (1990) 15-18 Published by Elsevier
15
FEMSLE 04034
Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains Jos6 A. Gil, Luis M. Criado, T e r e s a Alegre a n d J u a n F. M a r t i n
Area de Microbiologia,Facultadde Biologla. Universidadde Lebn, Lebn, Spain Received7 February 1990 Revision received26 February 1990 Accepted 27 February 1990 Key words: Streptomyces 8riseus; Candicidin; pab Gene; Hybridization
1. S U M M A R Y A p-aminobenzoic synthase gene (pabS) from Streptomyces griseus I M R U 3570 involved in candicidin production was used as probe to find new aromatic polyene producing Streptomyces strains. The pab gene hybridizes with 6 out of 16 Streptomyces strains, and those strains which hybridize turned out to be polyene producers. Such strains w,~re never before described as polyene producers.
2. I N T R O D U C T I O N Candicidin is a polyene (heptaene) macrolide antifungal antibiotic produced by Streptomyces griseus I M R U 3570 [I]. Its structure contains an aromatic moiety (p-aminoacetophenone) derived from p-aminobenzoic acid (PABA) and the aminosugar mycosamine (3-amino-3,6-dideoxy-D-
Coerespondence to: Juan F. Martin, Area de Microbiologia. Facultad de Biologia, Universidad de Le6n, Campus de Vegazana, s/n, 24071 Lebn, Spain.
mannopyranose) attached glycosidically to the macrolide ring [2]. We have been studying the biosynthesis and regulation of candicidin production by S. griseus I M R U 3570 [3-5] but cloning studies with this strain are still hampered by the difficulty of protoplasting, regeneration and transformation by plasmid DNA. In order to find a candicidin-pro~ ducing strain more amenable to recombinant D N A work we decided to look for candicidin producing strains by hybridizing chromosomal D N A s from different Streptomyces strains with the cloned pab gene from S. griseus. Gil and Hopwood [6] showed that the pab gene from S. griseus hybridized only with S. griseus I M R U 3570, S. griseus 38A and S. fradiae ATCC 10745 but not with S. lividans 1326, S. coelicolor A3 (2), S. clavuligerus ATCC 27064, S. parvulus ATCC 12534 or S. griseus 52.2. In this work we described that by increasing the number of Streptomyces strains we found clear hybridization with 6 different Streptomyces. When these strains, never before described as polyene producers, were studied for candicidin production, they produced aromatic polyene antibiotics.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
3. MATERIALS A N D M E T H O D S 3.1. Bacterial strains and plasmids Streptomyces strains are described in Table I. Plasmid pIJ819 is a pBR322 derivative carrying the pabS gene from StrepWmyces griseus I M R U 3570 in a 3.5 kb BamHI fragment [6]. 3.2. Growth conditions Streptomyces strains were grown aerobically in a medium supporting high antibiotic production which contained starch-free soya peptone (Staley 4S, USA) (25 g/liter), glucose (65 g/liter) and zinc sulphate (0.5 raM) (SPG medium) [7]. The cultures were incubated at 32°C in 250 mi triplebaffled flasks containing 50 ml SPG medium, in a Gallenkamp orbital shaking incubator at 225 rpm. The production flasks were inoculated with 5 ml of a 24 h inoculum grown in Y E D medium conraining yeast extract and glucose (10 g/liter each). 3.3. Extraction of candicidin and isolation of p-amino acetophenone and mycosamine Candicidin was extracted and purified from the culture broth at the end of the fermentation in SPG (5 days after inoculatioii) according to ~he
Table 1 Summary of hybridization between genomicDNA digests and the pab gene Species
Strain
Antibiotic
Hybridization with pab S. griseus IMRU 3570 Candicidin + S. acrimycini Jl 2 2 3 6 Candicidin + $. coelicolor J 1 2 1 5 9 Candicidin + S. coelicolor JI l 157 Candicidin + S. allms G Candicidin + S. griseus JI 2 2 1 2 Candicidin + S. parvulus ATCC 12434 Actinomycin S. glaucescens ETH 22794 Tetracenomycin S. ambofaciens ATCC15154 Spiramicin $. antibioticus ATCC11891 Oleendomycin S. venezuelae Chlorampheni¢ol S. ctavuligems NRRL 3585 Cefamicin S. lactaradurans NRRL 3802 Cefamicin S. griseus ATCC 10137 Streptomycin S. coelicolor A3(2) Actinorhodin S. Iividans Jl 1326 -
method described by Liu et al. [8]. Candicidin production was determinated spectrophotometrically taking into account the highest absorption peak (380 nm) after measuring an absorption spectrum of the polyene [5]. p-aminoacetophenone and mycosamine were released by hydrolysis of candicidin and purified as described by Liu et al. [8] and Martin and McDaniel [9], respectively. 3. 4. Preparation of cell-free extracts Cells grown in SPG were harvested 24 h after inoculation coinciding with the highest activity of the PABA synthase and resuspended in D M G buffer containing 1 m M D T r , 10 mM MgCI, 20 m M glutamine and 30~ ( v / v ) glycerol and l0 m M potassium buffer, pH 6.8. Cells were disrupted in D M G buffer by sonication and PABA synthase was assayed as described previously [10]. 3.5. DNA isolation and manipulation Chromosomal D N A from Streptomyces was isolated by the procedure 3 of Hopwcod et al. [11]. Plasmid D N A was isolated from E. coil or Streptomyces by a scaled version of the method of Kieser [12] followed by centrifugation in a CsClethidium bromide gradient. Restriction enzymes, agarose gel electrophoresis, transfer of D N A to nitrocellulose, labeling of D N A fragments and hybridization conditions were as described by Hopwood et al. [11]. After hybridization the filters were washed twice in 2 × SSC at 65°C. 3.6. Pro£oplast transformation Protoplasts from the different Streptomyces strains were obtained as described by Hopwood et al. [11], except for S. acrimycini which was grown in SH medium (which contains casamino acid, 24 g/flier; glycine, 5 g/liter; glucose, 5 g/liter, and MgCI 2, 10.25 g / l i t e r [13]. Transformants were selected by overlaying the regeneration plates after 18 h of growth with 2,5 ml of soft nutrient agar (Difco) containing 500 .ag/rnl Thioestrepton. 4. RESULTS A N D D I S C U S S I O N 4.1. Distribution of the pab gene in Streptomyces In order to find new strains carrying the pabS gene we isolated chromosomal D N A from the
,f~/º
,~/i
1.4KbI I
~/i
,~/1
LTl~
j
chromosomalDNA 3.5 Kbfragmentused as radiactiveprobe
1 2 34567891011
!
t w~ 4P
- ~4k
1.710) 1AKb
Fig. 1. Southern hybridization of the 3.5 kb BamHl fragment from S. gr/seu.f IMRU 3570 containing the pab gene to Sail restriction digests of 8enomic DNA from various streptomyceres. S. a/bus G DNA was digestedwith BamH! because such strain contained the Sail RM system. Lane and orsanisms as follows: 1, ~ Hindlll; 2, S. a/bus G; 3, S. coelica/orA3 (2); 4, S. clava/igwus NRRL 3585; 5, S. gr/seu~ IMRU 3570; 6, S. gr/seus ATCC 10137; 7, S. griseu.vJl 2212; 8, S. acrimycini J1 2236; 9, S. cocli¢olor Jl 1157; 10, S. IWidans J! 1326. and 11. S. lactamdurans NRRL 3802.
strains described in Table 1. These I)NAs were digested with Sail except for S. albusG D N A which was digested with BamHl. Digested D N A s were electrophoresed, transferred to nitrocellulose and hybridized with the 3.5 kb B a m H l fragment from plJ819 containing the pab gene from S. griseus I M R U 3570. As can be observed in Fig. 1, three Sail bands of 1.4, 1.7 and 2.5 kb that hybridize with the probe were found in S. griseus I M R U 3570, S. acrimycini 2236, S. coelicolor 1157 and S. gr/seus 2212. Similar hybridization pattern was found with D N A from S. coelicolor 2159 digested with Sail (data not shown). Two internal Sail sites are known to occur within the 3.5 kb B a m H l fragments of S. griseus used as a probe [5,6]. There was a unique 4.5 kb B a m H l fragment in the D N A from S. albus G which hybridizes with
the probe and is similar to the fragment obtained when D N A from S. griseus I M R U 3570 was digested with B a m H l and hybridized with the 3.5 kb fragment from plJS19 [6]. These results clearly indicate that there is a high degree of homology between the probe and the target sequences. In contrast, D N A digestions from S. lividans 1326, '3. coelicolor 2280 and other Streptomyces strains described in Table 1 failed to hybridize with the probe. The strains which hybridize with the pab gene showed high levels of PABA synthase (4-15 U / r a g protein depending of the strain) and those which do not hybridize have no detectable level of PABA synthase activity. 4.2. Production of candicidin by strains giving positive hybridization The Streptomyces strains which hybridized with the pab gene were grown in SPG medium for 5 days. After fermentation candieidin production was measured and all of them produced polyene antibiotics which were indistinguishable from candicidin after measuring the typical heptaene absorption spectrum of the extracted antibiotic (betwe¢,~ 360 and 430 pr~,,~, and after '~l~n layer chromatography of the purified antibiotic. The antibiotic produced by the strains which hybridized with the pab gene contained an aromatic moiety of p-aminoacetopbenone, the a,-ninosugar mycos-ami,¢, a,-,~ ~,'as active against Candida utilis" it was, therefore, identified as candicidin. The homologous pabS 8en¢ is only present in Streptomyces species which produce candicidin or related aromatic heptaene antibiotics. These results clearly suggest that the pabS gene is involved specifically in candicidin biosynthesis. A totally different pab gene, which appears to be involved in folic acid biosynthesis has been isolated in our laboratory (T. Alegre, J.A. Gil and J.F. Martin, unpublished). The high percentage (30~) of polyene producer strains obtained in this study is not surprising. Pisano et al. [14] found 22 actinomyc~tes strains out of 165 isolated from stuarine sediments which produce antifungal compounds. Nineteen of 22 isolates were associated with polyene moieties and the heptaenes was the most common group found.
The use of a gene involved in antibiotic production as a probe to look for new producer strains has been successfuly demonstrated in the case of candicidin. This strategy was based on the finding by Malpartida et al. [15] that a gene involved in the biosynthesis of a polyketide antibiotic might be used as hybridization probe for the isolation of biosynthetic genes of related antibiotics.
4.3. Transformation of Streptomyces strains by plasmid DNA The aim of this work was to discover new producers of candicidin to use them as host for genetic studies. Protoplasting and regeneration of S. griseus I M R U 3570 using the standard techniques for Streptomyces was very inefficient; the transformation frequency using pIJ101 derivatives (pIJ699 or pIJ702) was in the range of 1 - 5 transf o r m a n t s / / t g DNA. The efficiency did not improve using D N A from one of the transformants. S. griseus 2212 and S. acrimycini 2236 were t r a n s f o r m e d efficiently ( 1 0 4 - 1 0 5 t r a n s f o r m a n t s / / t g D N A ) with pIJ101 derivatives. Several genes, including the pabS gene and the saf gene (A. Daza, personal communication) from S. griseus I M R U 3570, have been introduced efficiently into S. acrimycini and were stably maintained and expressed. The transformation of S. acrimycini by pIJ101 derivatives has been described previously [161. Several mutants from S. acrimycini blocked in ¢andicidin biosynthesis are available and they are being used as recipient to clone genes involved in candicidin production different from the pabS.
ACKNOWLEDGEMENTS This work was supported by a grant of the C I C Y T (1062/81), Madrid. We t h a n k S.J. Lucania
for samples of t h i o s t r e p t o n a n d David A. Hopwood and M.R. Rodicio for the gift of the Streptomyces strains used in this work. We acknowledge the excellent technical assistance of M.P. Puertas, B. Martin and M.I. Corrales.
REFERENCES [1] Lechevalier,H., Acker, R.F., Corke, C.T., Haenseler, C.M. and Waksman, S.A. (1953) Mycologia 45,151-171. [2] Zielinski, J., Sorowy.Borowski, H., Golik, J., Gumieniak, J., Ziminski, T., Kolodziejczyk, P., Pawlak, J. and Borowski, E. (1979) Tetrahedron Lett. 20,1791-1794. [3] Gil, J.A., Liras, P., Naharro, G., Villanneva, J.R. and Martin, J.F. (1980) J. Gen. Microbiol. 118,189-195. [4] Liras, P., Villanueva,J.R. and Martin, J.F. (1977) J. Gen. Microbiol. 102, 269-277. [5] Rebollo, A., Gil, J.A., Liras, P., Asturias, J.A. and Martin, J.F. (1989) Gene 79, 47-58. [6] Gil, J.A. and Hopwood, D.A. (1983) Gene 25,119-132. [7] Martin, J.F. and McDaniel, L.E. (1975) BiotechnoL Bioeng. 17, 208-214. [8] Liu, C.M., McDaniel, L.E. and Schaffner, C.P. (1972) J. Antibiot. 25,116-121. [9] Martin, J.F. and McDaniel, L.E. (1975) Antimicrob. Agents Chemother. 8, 200-208. [10] Gil, J.A., Naharro, G., Villanueva, J.R. and Martin, J.F. (1985) J. Gen. Microbiol. 131,1279-1287. [11] Hopwood, D.A., Bibb, M.J., Chafer, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, DJ., Smith, C.F., Ward, J.M. and Schrempf, H. (1985) Genetic Manipulation of gtreptomyces.A Laboratory Manual. The John Innes Foundation. [12] Kieser, T. (1984) Plasmid 12,19-36. [13] Shaw, W.V. and Hopwood, D.A. (1976) J. Gen. Microbiol. 94,159-166. [14] Pisano, M.A., Sommer, M.J. and Bren, B.P. (1987) Appl. Microbiol. Biotechnol. 27, 214-217. [15] Malpartida, F., Hallam, S.E., Kieser, H.M., Motamedi, H., Hutchinson, C.R., Butler, M.J., Sugden, D.A., Warren, M., McKillop, C., Bailey, C.R., Humphreys, G.O. and Hopwood, D.A. (1987) Nature 325, 818-821. [16l Kieser, T., Hopwood, D.A., Wright, H.M. and Thompson, C.J. (1982) MoL Gen. Genet. 185, 223-238.
15
FEMSLE 04034
Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains Jos6 A. Gil, Luis M. Criado, T e r e s a Alegre a n d J u a n F. M a r t i n
Area de Microbiologia,Facultadde Biologla. Universidadde Lebn, Lebn, Spain Received7 February 1990 Revision received26 February 1990 Accepted 27 February 1990 Key words: Streptomyces 8riseus; Candicidin; pab Gene; Hybridization
1. S U M M A R Y A p-aminobenzoic synthase gene (pabS) from Streptomyces griseus I M R U 3570 involved in candicidin production was used as probe to find new aromatic polyene producing Streptomyces strains. The pab gene hybridizes with 6 out of 16 Streptomyces strains, and those strains which hybridize turned out to be polyene producers. Such strains w,~re never before described as polyene producers.
2. I N T R O D U C T I O N Candicidin is a polyene (heptaene) macrolide antifungal antibiotic produced by Streptomyces griseus I M R U 3570 [I]. Its structure contains an aromatic moiety (p-aminoacetophenone) derived from p-aminobenzoic acid (PABA) and the aminosugar mycosamine (3-amino-3,6-dideoxy-D-
Coerespondence to: Juan F. Martin, Area de Microbiologia. Facultad de Biologia, Universidad de Le6n, Campus de Vegazana, s/n, 24071 Lebn, Spain.
mannopyranose) attached glycosidically to the macrolide ring [2]. We have been studying the biosynthesis and regulation of candicidin production by S. griseus I M R U 3570 [3-5] but cloning studies with this strain are still hampered by the difficulty of protoplasting, regeneration and transformation by plasmid DNA. In order to find a candicidin-pro~ ducing strain more amenable to recombinant D N A work we decided to look for candicidin producing strains by hybridizing chromosomal D N A s from different Streptomyces strains with the cloned pab gene from S. griseus. Gil and Hopwood [6] showed that the pab gene from S. griseus hybridized only with S. griseus I M R U 3570, S. griseus 38A and S. fradiae ATCC 10745 but not with S. lividans 1326, S. coelicolor A3 (2), S. clavuligerus ATCC 27064, S. parvulus ATCC 12534 or S. griseus 52.2. In this work we described that by increasing the number of Streptomyces strains we found clear hybridization with 6 different Streptomyces. When these strains, never before described as polyene producers, were studied for candicidin production, they produced aromatic polyene antibiotics.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
3. MATERIALS A N D M E T H O D S 3.1. Bacterial strains and plasmids Streptomyces strains are described in Table I. Plasmid pIJ819 is a pBR322 derivative carrying the pabS gene from StrepWmyces griseus I M R U 3570 in a 3.5 kb BamHI fragment [6]. 3.2. Growth conditions Streptomyces strains were grown aerobically in a medium supporting high antibiotic production which contained starch-free soya peptone (Staley 4S, USA) (25 g/liter), glucose (65 g/liter) and zinc sulphate (0.5 raM) (SPG medium) [7]. The cultures were incubated at 32°C in 250 mi triplebaffled flasks containing 50 ml SPG medium, in a Gallenkamp orbital shaking incubator at 225 rpm. The production flasks were inoculated with 5 ml of a 24 h inoculum grown in Y E D medium conraining yeast extract and glucose (10 g/liter each). 3.3. Extraction of candicidin and isolation of p-amino acetophenone and mycosamine Candicidin was extracted and purified from the culture broth at the end of the fermentation in SPG (5 days after inoculatioii) according to ~he
Table 1 Summary of hybridization between genomicDNA digests and the pab gene Species
Strain
Antibiotic
Hybridization with pab S. griseus IMRU 3570 Candicidin + S. acrimycini Jl 2 2 3 6 Candicidin + $. coelicolor J 1 2 1 5 9 Candicidin + S. coelicolor JI l 157 Candicidin + S. allms G Candicidin + S. griseus JI 2 2 1 2 Candicidin + S. parvulus ATCC 12434 Actinomycin S. glaucescens ETH 22794 Tetracenomycin S. ambofaciens ATCC15154 Spiramicin $. antibioticus ATCC11891 Oleendomycin S. venezuelae Chlorampheni¢ol S. ctavuligems NRRL 3585 Cefamicin S. lactaradurans NRRL 3802 Cefamicin S. griseus ATCC 10137 Streptomycin S. coelicolor A3(2) Actinorhodin S. Iividans Jl 1326 -
method described by Liu et al. [8]. Candicidin production was determinated spectrophotometrically taking into account the highest absorption peak (380 nm) after measuring an absorption spectrum of the polyene [5]. p-aminoacetophenone and mycosamine were released by hydrolysis of candicidin and purified as described by Liu et al. [8] and Martin and McDaniel [9], respectively. 3. 4. Preparation of cell-free extracts Cells grown in SPG were harvested 24 h after inoculation coinciding with the highest activity of the PABA synthase and resuspended in D M G buffer containing 1 m M D T r , 10 mM MgCI, 20 m M glutamine and 30~ ( v / v ) glycerol and l0 m M potassium buffer, pH 6.8. Cells were disrupted in D M G buffer by sonication and PABA synthase was assayed as described previously [10]. 3.5. DNA isolation and manipulation Chromosomal D N A from Streptomyces was isolated by the procedure 3 of Hopwcod et al. [11]. Plasmid D N A was isolated from E. coil or Streptomyces by a scaled version of the method of Kieser [12] followed by centrifugation in a CsClethidium bromide gradient. Restriction enzymes, agarose gel electrophoresis, transfer of D N A to nitrocellulose, labeling of D N A fragments and hybridization conditions were as described by Hopwood et al. [11]. After hybridization the filters were washed twice in 2 × SSC at 65°C. 3.6. Pro£oplast transformation Protoplasts from the different Streptomyces strains were obtained as described by Hopwood et al. [11], except for S. acrimycini which was grown in SH medium (which contains casamino acid, 24 g/flier; glycine, 5 g/liter; glucose, 5 g/liter, and MgCI 2, 10.25 g / l i t e r [13]. Transformants were selected by overlaying the regeneration plates after 18 h of growth with 2,5 ml of soft nutrient agar (Difco) containing 500 .ag/rnl Thioestrepton. 4. RESULTS A N D D I S C U S S I O N 4.1. Distribution of the pab gene in Streptomyces In order to find new strains carrying the pabS gene we isolated chromosomal D N A from the
,f~/º
,~/i
1.4KbI I
~/i
,~/1
LTl~
j
chromosomalDNA 3.5 Kbfragmentused as radiactiveprobe
1 2 34567891011
!
t w~ 4P
- ~4k
1.710) 1AKb
Fig. 1. Southern hybridization of the 3.5 kb BamHl fragment from S. gr/seu.f IMRU 3570 containing the pab gene to Sail restriction digests of 8enomic DNA from various streptomyceres. S. a/bus G DNA was digestedwith BamH! because such strain contained the Sail RM system. Lane and orsanisms as follows: 1, ~ Hindlll; 2, S. a/bus G; 3, S. coelica/orA3 (2); 4, S. clava/igwus NRRL 3585; 5, S. gr/seu~ IMRU 3570; 6, S. gr/seus ATCC 10137; 7, S. griseu.vJl 2212; 8, S. acrimycini J1 2236; 9, S. cocli¢olor Jl 1157; 10, S. IWidans J! 1326. and 11. S. lactamdurans NRRL 3802.
strains described in Table 1. These I)NAs were digested with Sail except for S. albusG D N A which was digested with BamHl. Digested D N A s were electrophoresed, transferred to nitrocellulose and hybridized with the 3.5 kb B a m H l fragment from plJ819 containing the pab gene from S. griseus I M R U 3570. As can be observed in Fig. 1, three Sail bands of 1.4, 1.7 and 2.5 kb that hybridize with the probe were found in S. griseus I M R U 3570, S. acrimycini 2236, S. coelicolor 1157 and S. gr/seus 2212. Similar hybridization pattern was found with D N A from S. coelicolor 2159 digested with Sail (data not shown). Two internal Sail sites are known to occur within the 3.5 kb B a m H l fragments of S. griseus used as a probe [5,6]. There was a unique 4.5 kb B a m H l fragment in the D N A from S. albus G which hybridizes with
the probe and is similar to the fragment obtained when D N A from S. griseus I M R U 3570 was digested with B a m H l and hybridized with the 3.5 kb fragment from plJS19 [6]. These results clearly indicate that there is a high degree of homology between the probe and the target sequences. In contrast, D N A digestions from S. lividans 1326, '3. coelicolor 2280 and other Streptomyces strains described in Table 1 failed to hybridize with the probe. The strains which hybridize with the pab gene showed high levels of PABA synthase (4-15 U / r a g protein depending of the strain) and those which do not hybridize have no detectable level of PABA synthase activity. 4.2. Production of candicidin by strains giving positive hybridization The Streptomyces strains which hybridized with the pab gene were grown in SPG medium for 5 days. After fermentation candieidin production was measured and all of them produced polyene antibiotics which were indistinguishable from candicidin after measuring the typical heptaene absorption spectrum of the extracted antibiotic (betwe¢,~ 360 and 430 pr~,,~, and after '~l~n layer chromatography of the purified antibiotic. The antibiotic produced by the strains which hybridized with the pab gene contained an aromatic moiety of p-aminoacetopbenone, the a,-ninosugar mycos-ami,¢, a,-,~ ~,'as active against Candida utilis" it was, therefore, identified as candicidin. The homologous pabS 8en¢ is only present in Streptomyces species which produce candicidin or related aromatic heptaene antibiotics. These results clearly suggest that the pabS gene is involved specifically in candicidin biosynthesis. A totally different pab gene, which appears to be involved in folic acid biosynthesis has been isolated in our laboratory (T. Alegre, J.A. Gil and J.F. Martin, unpublished). The high percentage (30~) of polyene producer strains obtained in this study is not surprising. Pisano et al. [14] found 22 actinomyc~tes strains out of 165 isolated from stuarine sediments which produce antifungal compounds. Nineteen of 22 isolates were associated with polyene moieties and the heptaenes was the most common group found.
The use of a gene involved in antibiotic production as a probe to look for new producer strains has been successfuly demonstrated in the case of candicidin. This strategy was based on the finding by Malpartida et al. [15] that a gene involved in the biosynthesis of a polyketide antibiotic might be used as hybridization probe for the isolation of biosynthetic genes of related antibiotics.
4.3. Transformation of Streptomyces strains by plasmid DNA The aim of this work was to discover new producers of candicidin to use them as host for genetic studies. Protoplasting and regeneration of S. griseus I M R U 3570 using the standard techniques for Streptomyces was very inefficient; the transformation frequency using pIJ101 derivatives (pIJ699 or pIJ702) was in the range of 1 - 5 transf o r m a n t s / / t g DNA. The efficiency did not improve using D N A from one of the transformants. S. griseus 2212 and S. acrimycini 2236 were t r a n s f o r m e d efficiently ( 1 0 4 - 1 0 5 t r a n s f o r m a n t s / / t g D N A ) with pIJ101 derivatives. Several genes, including the pabS gene and the saf gene (A. Daza, personal communication) from S. griseus I M R U 3570, have been introduced efficiently into S. acrimycini and were stably maintained and expressed. The transformation of S. acrimycini by pIJ101 derivatives has been described previously [161. Several mutants from S. acrimycini blocked in ¢andicidin biosynthesis are available and they are being used as recipient to clone genes involved in candicidin production different from the pabS.
ACKNOWLEDGEMENTS This work was supported by a grant of the C I C Y T (1062/81), Madrid. We t h a n k S.J. Lucania
for samples of t h i o s t r e p t o n a n d David A. Hopwood and M.R. Rodicio for the gift of the Streptomyces strains used in this work. We acknowledge the excellent technical assistance of M.P. Puertas, B. Martin and M.I. Corrales.
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