Conjugation and transformation of Streptomyces species by tylosin resistance

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1 FEMS Microbiology Letters 186 (2000) 319^325 Conjugation and transformation of Streptomyces species by tylosin resistance Roberto Fouces, Marta Rodr guez, Encarnaciön Mellado, Bruno D ez, Josë Luis Barredo * A è rea de Biotecnolog a, Antibiöticos S.A., Avenida de Antibiöticos 59^61, Leön, Spain Received 24 January 2000; received in revised form 20 March 2000; accepted 28 March 2000 Abstract The tlrb gene from Streptomyces fradiae has been cloned and used to construct bifunctional Streptomyces^Escherichia coli shuttle vectors carrying the antibiotic resistance genes to kanamycin^neomycin, thiostrepton and tylosin as selection markers. In the same way, the tlrb gene was subcloned in plasmids including the apramycin resistance gene and the orit sequence from the plasmid pset152 to facilitate conjugation of Streptomyces spores. The usefulness of the tlrb gene as tylosin resistance marker was ascertained in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor, but not in Streptomyces clavuligerus. The tlrb gene constitutes a useful selection marker when high-frequency of conjugation/transformation is not required or as secondary marker in recombinant Streptomyces species where thiostrepton and kanamycin have been utilized for primary selection. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Streptomyces; Tylosin; Resistance; Marker; Conjugation; Transformation 1. Introduction * Corresponding author. Tel.: +34 (987) ; Fax: +34 (987) ; jbarredo@antibioticos.it Streptomyces fradiae produces tylosin, an antibiotic used in veterinary practice. Studies on the biosynthetic genes of tylosin-producing strains have revealed that biosynthetic and self-resistance genes are closely linked in the genome [1]. S. fradiae has been reported to possess four tylosin resistance genes: tlra, tlrb, tlrc and tlrd. tlra [2] and tlrd [3] encode methyltransferases responsible for the methylation of a speci c adenine residue in the 23S rrna. Constitutive expression of tlrd causes induction of tlra gene expression. The tlrc gene is located at the right end of the tylosin biosynthetic cluster and codes for an ATPbinding protein involved in tylosin e ux [4]. The tlrb gene is at the left edge of the tylosin biosynthetic cluster [5] and encodes a protein with high similarity to the product of the myra gene from Micromonospora griseorubida [6]. Neither myra nor tlrb gene products seem to belong to the MLS-resistance phenotype family. The TlrB protein inactivates macrolides in the presence of S-adenosyl-methionine, but the modi ed product(s) has not been characterized. Since this protein is capable of modifying tylosin and various of its biosynthetic precursors, it might reduce the nal yield of tylosin in fermentations [7]. A number of antibiotic resistance markers are available for Streptomyces: thiostrepton (tsr), kanamycin^neomycin (kmr), apramycin (amr), geneticin, viomycin, hygromycin, bleomycin, chloramphenicol, etc. Furthermore, the abovementioned antibiotic resistance determinants have been used for the construction of Escherichia coli^streptomyces shuttle vectors [8,9]. Similarly, vectors for intergeneric conjugation between E. coli and Streptomyces have been developed [10^11]. They contain the 760-bp orit fragment for conjugation, but require the transfer functions to be supplied in trans by the E. coli donor strain. Some of the plasmids include the attachment site (attp) and the integrase (int) function of the temperate phage PC31 to facilitate the site-speci c integration of the vector at the attb site of the Streptomyces chromosome. In this work, we report the construction of bifunctional E. coli^streptomyces shuttle vectors containing tsr and tylosin resistance (tlr) as selection markers for Streptomyces and kmr as marker for both E. coli and Streptomyces. Likewise, intergeneric conjugation between Streptomyces and E. coli using the tlrb gene as selection marker and / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (00)

2 320 R. Fouces et al. / FEMS Microbiology Letters 186 (2000) 319^325 conjugation between S. clavuligerus and E. coli are demonstrated. 2. Materials and methods 2.1. Microbial strains, plasmids and microbiological procedures S. fradiae ATCC was used as source of DNA. Escherichia coli DH5K [12] was used for subcloning purposes, E. coli WK6 for ssdna puri cation, E. coli ET12567(pUZ8002) [13] for conjugation assays and E. coli LE392 [12] for library construction. pbluescript I KS(+), pbluescript II SK(+) and pbc KS(+) phagemids (Stratagene) were utilized for routine subcloning and ssdna preparation with the helper phage M13K07. pulvk99 [14] was used as E. coli^streptomyces spp. shuttle vector and pset152 [10] as intergeneric conjugation vector. Protoplast transformation of S. lividans, S. parvulus and S. coelicolor was performed according to Hopwood et al. [15], whereas S. clavuligerus was transformed as described by Garc a-dom nguez et al. [16]. palf250 and palf295 were propagated in S. lividans JI 1326 prior to be transformed into S. fradiae ATCC 19609, S. parvulus DSM 40048, S. coelicolor DSM or S. clavuligerus ATCC Streptomyces transformants were selected on R5 plates supplemented with thiostrepton (50 Wg ml 31 ), kanamycin (50 Wg ml 31 ) or tylosin (200 Wg ml 31 for S. lividans and S. parvulus, 20Wg ml 31 for S. coelicolor and 10 Wg ml 31 for S. clavuligerus). Plasmids palf297 and palf298 were mobilized from E. coli to S. lividans or S. clavuligerus essentially according to the protocol previously described for S. lividans and S. coelicolor [13]. Cultures of E. coli ET12567(pUZ8002) including the plasmids palf297 or palf298 were mixed with spores of Streptomyces and the mixture was spread on agar plates containing MS+10 mm MgCl 2 [17] or MTD (MM [15]+trace element solution [15]+dextrin (2.5 g l 31 ) instead of glucose). Nalidixic acid (500 Wg) and either apramycin (1 mg in MS) or tylosin (1 mg in MTD) were overlaid on the surface of the plates. The exconjugant colonies were tested in minimal medium [15] supplemented with apramycin (50 Wg ml 31 ) or tylosin (50 Wg ml 31 ). converted in ssdna by standard procedures [12] and sequenced by the dideoxynucleotide method using Sequenase 2.0 (Amersham). Deaza-nucleotides and/or high annealing temperature (45³C) were employed to eliminate compression problems. Sequence analyses were performed with Dnastar and Winstar packages. The sequence corresponding to the tlrb gene has been deposited in the Gen- Bank under the accession number AF Results and discussion 3.1. tlrb gene cloning In order to isolate the tylosin gene cluster, a genetic library of S. fradiae was constructed in VGEM12 and screened with the oligonucleotide 5P-GCTCGATGTAGA- GATCG-3P, designed according to the sequence of the 5P region of the previously described tylf gene [18]. The four recombinant phages characterized by restriction mapping and Southern analysis shared common restriction fragments corresponding to the same genomic region. A common 5.5-kb BglII fragment was subcloned and sequenced, identifying an ORF of 840 bp (72.6% G+C) encoding a deduced protein of 280 residues, 30.4 kda and a pi of 8.1. This ORF corresponded to the tlrb gene previously described [6,7], but four di erent amino acids were found at positions 78, 81, 82 and 83 when it was compared to the gene described by Wilson and Cundli e [7]. A purine rich sequence that could conform the RBS (5P-GAAGG- GA-3P) is present 13 bp upstream from the translation initiation site ATG. Furthermore, the sequence 5P- CGCCCCCCCGGGGGGCG-3P, that could potentially form a loop involved in the end of transcription, is located 8 bp downstream of the translation end codon. The deduced protein showed high similarity to the macrolide-resistance determinant encoded by the myra gene from the mycinamicin producer M. griseorubida [6] Construction of a genomic library, DNA sequencing and Southern analysis The construction of the genomic library of S. fradiae was previously described [6]. Nucleic acid puri cation and manipulation were performed by standard procedures [12,15] with minor modi cations. Southern blotting was carried out according to standard procedures [12,15] using the non-radioactive method DIG-DNA Labeling and Detection Kit (Boehringer-Mannheim). Sequencing clones were constructed with the kit `Erase a base' (Promega), Fig. 1. Restriction map of the DNA fragments including the tlrb gene (1.2 kb KpnI^SacII and 3.5 kb SacI) used to construct the plasmids palf250, palf295, palf297 and palf298. The tlrb gene confers tylosin resistance, the ddca gene encodes a penicillin binding protein (PBP) and the tyln gene codes for a glycosyltransferase. The gray box at the 3P end of ddca corresponds to the polylinker of pbluescript I KS(+). The organization of the tylosin biosynthetic gene cluster has been previously described [7].

3 R. Fouces et al. / FEMS Microbiology Letters 186 (2000) 319^ Fig. 2. Combined physical and genetic map of palf250, palf295, palf297 and palf298. Map distances and the location of restriction sites are indicated in kb. A single EcoRI site useful for subcloning is located at position 0 of palf250 and palf295. The single BamHI site located inside the tlrb gene at position 5.7 of palf250 is useful for routine subcloning and selection of transformants for tsr or kmr and tylosin sensitivity. The genetic maps were compiled from the data of the progenitors: pulvk99 [18], pset152 [11] and tlrb gene [7] Construction of plasmids carrying the tlrb gene Two DNA fragments of 1.2 kb KpnI^SacII and 3.5 kb SacI, both containing the tlrb gene (Fig. 1), were puri ed from the 5.5-kb BglII fragment and subcloned in the bifunctional E. coli^streptomyces plasmid pulvk99 [14] rendering palf250 and palf295, respectively (Fig. 2). These plasmids include tsr and tlr as selection markers in Streptomyces and kmr as marker for both E. coli and Streptomyces. Furthermore, palf250 and palf295 contain an EcoRI single site for routine subcloning located at position 0 and two independent replication origins: ColE1 for E. coli and pij101 for Streptomyces. Additionally, palf250 carries a single BamHI site located inside the tlrb gene (position 5.7) useful for routine subcloning and selection of transformants for tsr or kmr and tylosin sensitivity. Likewise, the 1.2 kb KpnI^SacII fragment including the tlrb gene was blunt-ended and subcloned in the EcoRV single site of pset152 [10] rendering the plasmids palf297 and palf298 (both orientations. Fig. 2). These plasmids contain amr and tlr as selection markers in Streptomyces, the 760-bp orit fragment from the plasmid RK2, the bacteriophage PC31 integration functions and the ColE1 replication origin for E. coli. They ful lled the requirements for mobilization by mating in Streptomyces species, showing stable replication both in E. coli and Streptomyces.

4 322 R. Fouces et al. / FEMS Microbiology Letters 186 (2000) 319^325 Table 1 Transformation of Streptomyces species using tylosin as selection marker S. lividans S. parvulus S. coelicolor S. clavuligerus Tylosin MIC (Wg ml 31 ) a Tylosin for selection (Wg ml 31 ) b Resistance conferred (Wg ml 31 ) c 250 to s ^ ^50 0 Transformation frequency (%) d a Tylosin minimal inhibitory concentration (MIC) in R5 medium. b Tylosin concentration used for transformant selection. c Range of tylosin resistance conferred by palf250 or palf295. d Number of tylosin-resistant transformants obtained with palf250 or palf295 divided by the number of thiostrepton-resistant transformants obtained with same plasmids and expressed as percentage Transformation of Streptomyces species The MIC of tylosin for S. lividans, S. parvulus, S. coelicolor and S. clavuligerus was determined in R5 medium (Table 1). Attempts to transform the last three species with palf250 or palf295 isolated from E. coli were always unsuccessful, probably due to the presence of an e cient restriction system in these microorganisms [8]. To overcome this problem, palf250 and palf295 were puri ed from S. lividans, analyzed for plasmid integrity and used to transform S. parvulus, S. coelicolor and S. clavuligerus. Transformants were selected on R5 plates supplemented with tylosin, obtaining clones able to grow in the tylosin ranges shown in Table 1. Therefore, the tlrb gene can be used as transformation marker in Streptomyces species. S. clavuligerus was checked in a tylosin range from 10 to 100 Wg ml 31 and no transformants were obtained at all. Moreover, tsr transformants of S. clavuligerus were unable to grow on inhibitory concentrations (10^ 20 Wg ml 31 ) of tylosin. The transformation frequency using tlr as selection marker was lower than the obtained by tsr in the three species tested. The percentage of tylosin-resistant transformants obtained with palf250 or palf295 with respect to the thiostrepton-resistant transformants obtained with same plasmids was around 50% for S. lividans, 30% for S. parvulus and 20% for S. coelicolor (Table 1). There is a direct correlation between the tylosin MIC in the parental untransformed strains (higher in S. lividans and S. parvulus) and the level of tylosin resistance of these strains transformed with palf250 or palf295 (Table 1). The resistance di erences observed could be related to the transport of tylosin inside the cells and to the level of expression of the tlrb gene. MIC values to tylosin (1^ 2.5 Wg ml 31 ) and erythromycin (5^10 Wg ml 31 ) described for S. lividans OS456 in NE medium [7] were lower than the obtained for S. lividans JI 1326 (Tables 1 and 2) probably due to the use of di erent culture media (R5, MS or MTD) and strains Erythromycin and lincomycin resistance assays of tlrb transformants The ability of the tlrb gene to confer erythromycin or lincomycin resistance was tested in R5 medium. Previously, erythromycin MICs were determined for S. lividans (25 Wg ml 31 ) and S. parvulus (200 Wg ml 31 ) and lincomycin MIC was resolved for S. parvulus (100 Wg ml 31 ). Transformants of both species with the plasmids palf250 or palf295, selected by tsr, were unable to grow in presence of the above mentioned erythromycin or lincomycin MICs. According to these results, the tlrb gene is unable to confer neither erythromycin nor lincomycin resistance. The myra gene does not belong to the MLS resistance phenotype family and, as occurs with the tlrb gene, it is unable to confer erythromycin resistance. Moreover, the TlrB protein does not confer resistance to lincomycin, but it is principally e ective against tylosin, spiramycin and mycinamicin [7]. According to this data and to the fact that the TlrB protein is able to inactivate tylosin in the presence of S-adenosyl-methionine [7], we conclude that the tlrb gene does not belong to the MLS group of resistance determinants in contrast to the previously described [19]. Table 2 Conjugation of S. lividans and S. clavuligerus in MS and MTD media S. lividans S. clavuligerus Conjugation in MS medium Tylosin MIC (Wg ml 31 ) a s 100 s 50 Apramycin for selection (Wg ml 31 ) b Conjugation frequency by amr d 8U U10 35 Conjugation in MTD medium Tylosin MIC (Wg ml 31 ) a Tylosin for selection (Wg ml 31 ) b Resistance conferred (Wg ml 31 ) c 50^200 0 Conjugation frequency by amr d ^ 1U10 34 Conjugation frequency by tlr e 2U a Tylosin minimal inhibitory concentration (MIC) in MS and MTD media. b Apramycin or tylosin concentration used for exconjugant selection. c Range of tylosin resistance conferred by palf297 or palf298. d Number of exconjugants per initial number of recipient spores obtained with palf297 or palf298 by amr. e Number of exconjugants per initial number of recipient spores obtained with palf297 or palf298 by tlr.

5 R. Fouces et al. / FEMS Microbiology Letters 186 (2000) 319^ Fig. 3. Southern analysis of S. clavuligerus conjugated with the plasmid pset152 using as probe BamHI-digested pset152. (A) Genomic DNA of three randomly chosen exconjugants digested with PstI (lanes 2, 3 and 4), HindIII (lanes 6, 7 and 8) or SalI (lanes 10, 11 and 12). As expected, the PstI digestion generated two bands of 2.4 and 0.8 kb corresponding to pset152, together with two additional bands of 2.5 and s 25 kb corresponding to speci c integration by att. HindIII digestion rendered four bands of s 25, 4.5, 0.9 and 0.8 kb. The last two bands agree with pset152, whereas the rst two correspond to the integrated region. SalI digestion generates ve bands of 4.2, 2.4, 1.6, 0.9 and 0.7 kb. A further band of 0.4 kb corresponding to pset152 did not appear. The bands of 2.4, 0.9 and 0.7 kb coincide with pset152 and the bands of 4.2 and 1.6 kb correspond to the integrated zone. (B^D) The genomic DNA of an exconjugant digested with EcoRI generates two bands of s 25 and 5.8 kb (lane 17), digested with SacI originates three bands of 9, 5.3 and 0.7 kb (lane 18) and digested with NcoI gives rise to two bands of 5.8 and 1.8 kb (lane 20), respectively. Lanes 5, 9, 13 and 16, unconjugated parental strain. Lanes 1, 14, 15, 19 and 21, molecular mass markers. (E) Restriction map of the integrated pset Conjugation of S. lividans and S. clavuligerus Intergeneric conjugation between E. coli and several species of the order Actinomycetales including the genera Actinomadura, Arthrobacter, Micromonospora, Nocardia, Rodococcus and 16 strains of the genus Streptomyces has been described [11]. Based on conjugation vectors with amr, we have developed the plasmids palf297 and palf298 able to facilitate intergeneric conjugation between E. coli and Streptomyces. Since tylosin MIC values for S. lividans and S. clavuligerus were higher in MS than in R5 or MTD media (Tables 1 and 2), Streptomyces exconjugants were selected on MS+10 mm MgCl 2 + apramycin (50 Wg ml 31 ) or MTD+tylosin (50 Wg ml 31 ).

6 324 R. Fouces et al. / FEMS Microbiology Letters 186 (2000) 319^325 In spite of the conjugation frequency (expressed as exconjugants per initial number of recipient spores) of S. lividans was higher by amr in MS (8U10 32 ) than by tlr in MTD (2U10 35 ), this result demonstrates the possibility to use tlr as selection marker in S. lividans conjugation. Likewise, the conjugation frequency obtained by amr in MS was higher in S. lividans (8U10 32 ) than in S. clavuligerus (3U10 35 ). In contrast to S. lividans, exconjugants of S. clavuligerus were obtained by amr (3U10 35 in MS and 1U10 34 in MTD), but not by tlr. S. lividans amr exconjugants were able to grow in MTD supplemented with 50 Wg ml 31 of tylosin, whereas amr exconjugants of S. clavuligerus were unable to grow on inhibitory concentrations (20 Wg ml 31 ) of tylosin (Table 2), con rming the inability of the tlrb gene to confer tylosin resistance to S. clavuligerus. In order to characterize the integration of pset152 at the att site in the genome of S. clavuligerus, Southern analysis of three randomly chosen amr exconjugants was performed (Fig. 3). The DNA of the exconjugants was digested with PstI, HindIII or SalI and hybridized to Bam- HI digested pset152. Identical hybridization patterns were obtained for every exconjugant tested (Fig. 3A). Likewise, EcoRI (Fig. 3B), SacI (Fig. 3C) or NcoI (Fig. 3D) digestions of the DNA of one of the exconjugants were hybridized, in separate experiments, with the same probe above described. The patterns obtained were coincident with the expected (Fig. 3E). These results con rm monocopy directed integration at the att site of the genome, avoiding potential gene inactivation events arising when random integration occurs. Additionally we show that conjugation mediated by the transfer machinery of the plasmid pset152 is an e cient and reliable method for the introduction of foreign DNA into S. clavuligerus. The higher level of tylosin resistance reached in S. lividans by transformation (250 to s 3500 Wg ml 31 ) compared to conjugation (50^200 Wg ml 31 ) is probably due to the multicopy presence of the tlrb gene when using the autonomously replicating plasmids palf250 or palf295, whereas monocopy integration is obtained by conjugation with palf297 or palf298. The lower frequency obtained by tlr in transformation and conjugation experiments could be due to ine cient expression of the tlrb gene in the host microorganisms. Additional promoter replacement studies will be performed in order to improve transcription. However, tylosin presents some interesting characteristics that make it attractive as selection marker: absence of spontaneous resistant colonies, low price and high water solubility avoiding the need of organic solvents. In this way, tylosin resistance can be used as selection marker for routine transformation or conjugation experiments when high-frequency is not required or as secondary marker in recombinant Streptomyces species where tsr, kmr or amr have been utilized for primary selection. Further improvement of the transformation and conjugation technology and tlrb gene expression should be made in order to increase frequency. Acknowledgements The authors thank M. Sandoval, J.A. Gonzälez, M.T. Garc a and P. Merino for their excellent technical assistance. R.F. and M.R. were supported by a fellowship of the Spanish Ministry of Education and Science. References [1] Beckmann, R.J., Cox, K. and Seno, E.T. (1989) A cluster of tylosin biosynthetic genes is interrupted by a structurally unstable segment containing four repeated sequences. In: Genetics and Molecular Biology of Industrial Microorganisms (Hershberger, C.L., Queener, S.W. and Hegeman, G., Eds.), pp. 176^186. 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