FEMS Microbiology Letters 70 (1990) 15-18 15 Published by Elsevier

FEMSLE 04034

Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains

Jos6 A. Gil , Luis M. Criado, Teresa Alegre and Juan F. Mar t in

Area de Microbiologia, Facultad de Biologla. Universidad de Lebn, Lebn, Spain

Received 7 February 1990 Revision received 26 February 1990

Accepted 27 February 1990

Key words: Streptomyces 8riseus; Candicidin; pab Gene; Hybridization

1. SUMMARY

A p-aminobenzoic synthase gene (pabS) from Streptomyces griseus IMRU 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 hy- bridize 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 IMRU 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 IMRU 3570 [3-5] but cloning studies with this strain are still hampered by the difficulty of proto- plasting, 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 DNAs 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 IMRU 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.

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3. MATERIALS A N D METHODS

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/ l i ter) and zinc sulphate (0.5 raM) (SPG medium) [7]. The cultures were incubated at 32°C in 250 mi triple- baffled 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 YED medium con- raining yeast extract and glucose (10 g/ l i ter 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 genomic DNA digests and the pab gene

Species Strain Antibiotic Hybridi- zation with pab

S. griseus IMRU 3570 Candicidin + S. acrimycini Jl 2236 Candicidin + $. coelicolor J 1 2 1 5 9 Candicidin + S. coelicolor JI l 157 Candicidin + S. allms G Candicidin + S. griseus JI 2212 Candicidin + S. parvulus ATCC 12434 Actinomycin - S. glaucescens ETH 22794 Tetracenomycin - S. ambofaciens ATCC 15154 Spiramicin $. antibioticus ATCC 11891 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 spectrophotometri- cally taking into account the highest absorption peak (380 nm) after measuring an absorption spectrum of the polyene [5]. p-aminoace- tophenone and mycosamine were released by hy- drolysis of candicidin and purified as described by Liu et al. [8] and Martin and McDaniel [9], respec- tively.

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 mM D T r , 10 mM MgCI, 20 mM glutamine and 30~ (v /v ) glycerol and l0 mM 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 CsCl- ethidium 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/fl ier; glycine, 5 g/ l i ter ; glucose, 5 g/ l i ter , and MgCI 2, 10.25 g / l i te 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 DISCUSSION

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 ~/i ,~/1

1.4Kb I LT l~ chromosomal DNA I j 3.5 Kb fragment used

as radiactive probe

1 2 3 4 5 6 7 8 9 1 0 1 1

! 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 streptomy- ceres. S. a/bus G DNA was digested with BamH! because such strain contained the Sail RM system. Lane and orsanisms as follows: 1, ~ Hindlll; 2, S. a/bus G; 3, S. coelica/or A3 (2); 4, S. clava/igwus NRRL 3585; 5, S. gr/seu~ IMRU 3570; 6, S. gr/seus ATCC 10137; 7, S. griseu.v Jl 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 DNAs were electrophoresed, transferred to nitrocellulose and hybridized with the 3.5 kb BamHl 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 BamHl fragments of S. griseus used as a probe [5,6].

There was a unique 4.5 kb BamHl fragment in the D N A from S. albus G which hybridizes with

the probe and is similar to the fragment obtained when DNA from S. griseus IMRU 3570 was digested with BamHl 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 posi- tive 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 antibio- tics. These results clearly suggest that the pabS gene is involved specifically in candicidin bio- synthesis. 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 per- centage (30~) of polyene producer strains ob- tained 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 produc- tion 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 Malpart ida 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 tech- niques for Streptomyces was very inefficient; the t ransformation frequency using pIJ101 derivatives (pIJ699 or pIJ702) was in the range of 1 -5 trans- fo rman t s / / tg DNA. The efficiency did not im- prove 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 eff ic ient ly (104-105 t r ans fo r - m a n t s / / t g DNA) with pIJ101 derivatives. Several genes, including the pabS gene and the saf gene (A. Daza, personal communicat ion) 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.

A C K N O W L E D G E M E N T S

This work was supported by a grant of the CICYT (1062/81), Madrid. We thank S.J. Lucania

for samples of th ios t repton and 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. Mart in and M.I. Corrales.

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