- Email: [email protected]
Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095
Microbiol. Res. (2001) 156, 239–246 http://www.urbanfischer.de/journals/microbiolres
Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095 Hardeep Kaur1, Jesus Cortes2, Peter Leadlay3, Rup Lal1 1 2 3
Department of Zoology, University of Delhi, Delhi 110007, India Bioprocessing Unit, Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Cambridge, UK
Accepted: May 5, 2001
Abstract The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.
Corresponding author: R. Lal e-mail: [email protected] 0944-5013/01/156/03-239
$15.00/0
Key words: Rifamycin – A. mediterranei – rif PKS
Introduction Rifamycin is a clinically important antibiotic produced by the actinomycete Amycolatopsis mediterranei. The antibiotic is extensively used against tuberculosis and leprosy and several other mycobacterial infections. Because of its extensive demand, A. mediterranei has been subjected to an extensive strain improvement program, leading to the development of current industrial strains (Ghisalba et al. 1984; Lal et al. 1995, 1996). Nevertheless, the rapidly rising incidence of multidrug-resistant Mycobacterium tuberculosis (causative agents of tuberculosis) worldwide has led to an alarming resurgence of tuberculosis, especially among immuno-compromised persons and in underdeveloped parts of the world. Rapid developments in molecular genetics of Streptomyces has further motivated several groups to study the molecular genetics of rifamycin biosynthesis in A. mediterranei (August et al. 1998; Schupp et al. 2000; Lal et al. 2000). Recently, the rifamycin biosynthetic gene cluster has been cloned and characterized (August et al. 1998; Tang et al. 1998; Schupp et al. 1998) and partial success has been achieved in the development of cloning vectors and transformation methods (Lal et al. 1991, 1998; Khanna et al. 1998; Tuteja et al. 2000). Microbiol. Res. 156 (2001) 3
239
Although the rifamycin biosynthetic gene cluster has been independently cloned from A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998), there are several other strains of A. mediterranei, reported to be isolated from various soils, and their successors are in use for the commercial production of rifamycin. The variability of these strains in terms of the rifamycin biosynthetic gene cluster remains to be determined. The availability of A. mediterranei strains with a new or slightly different DNA sequence of the rifamycin biosynthetic gene cluster (rif gene cluster) could provide new genetic material for manipulating these genes for the production of analogs, which are required to combat the problem of multidrug-resistant mycobacteria.
Material and methods Bacterial strains, plasmids and culture conditions. Different strains of A. mediterranei used in the present study are listed in Table 1. These were grown in TYN or YM – agar medium at 28°C as described earlier (Lal et al. 1998). Saccharopolyspora erythraea was maintained in YM medium under conditions as described for A. mediterranei. E. coli cultures were maintained on LB. DNA preparations and manipulations. The high-molecular-weight DNA of different strains of A. mediterranei and the erythromycin producer S. erythraea MTCC1136 were isolated by Kirby’s method (Hopwood et al. 1985). Plasmid DNA isolation, agarose gel electrophoresis, restriction enzyme digestions, restriction mapTable 1. Bacterial strains used in the study. Organism Amycolatopsis mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei Saccharopolyspora erythraea Escherichia coli
Strain
Source or reference
40773 43303 14 17 F1/24 ans-13 T-195 ans 13, thi-8 46095 46096
DSM DSM MTCC MTCC Ciba Geigy Ciba Geigy DSM DSM
1136 GM 2163
MTCC New England Biolabs
DSM Deutsche Stammsammlung für Mirkoorganismen und Zellkulturen GmbH, Germany; MTCC Microbial Type Culture Collection, Chandigarh, India; Ciba Geigy (Now Novartis), Switzerland. 240
Microbiol. Res. 156 (2001) 3
ping, alkaline phosphatase treatment, T4 polynucleotide kinease treatment, blunt end cloning of PCR DNA and ligations were performed according to standard procedures (Sambrook et al. 1989). Identification of novel A. mediterranei strains by PCR. In order to detect a novel strain with striking differences in the nucleotide and protein sequences of the rifamycin biosynthetic cluster, eight different strains of A. mediterranei, that have been reported to produce rifamycin or its analogs, were selected. The genomic DNAs of A. mediterranei strains were amplified by using oligonucleotide primers designed from the known sequence of the rifamycin biosynthetic cluster of A. mediterranei S699. The primers were designed from the loading domain (LD) of the PKS and rif K gene that encodes 3-amino-5 hydroxybenzoic acid synthase regions of the rifamycin biosynthetic gene cluster (August et al. 1998) (Accession no. AF040570). The primers flanked the loading domain sequence at position 3884–3912 and 4924–4947 (C) and rif K gene sequence at position 58288–58316 and 59302–59324 (C) (Assession no. AF040570). The amplification was performed using a Stratagene Robocycler (USA) by the standard protocol. PCR was carried out in a total volume of 50 µl, at a temperature of 55°C and for 25 cycles of extension. Each PCR reaction consisted of: genomic DNA of A. mediterranei, 1 µl (1 µg); primer I, 3 µl (0.5 µg/µl); primer II, 3 µl (0.5 µg/µl); dNTP, 5 µl (10 mM of each dNTP); buffer (10×), 5 µl; DMSO, 5 µl; millipore water, 27 µl and enzyme (Pfu), 1 µl (2.5 U/µl). The PCR products from different strains of A. mediterranei were then analyzed on 0.8% (w/v) agarose gels. Screening by using dEBS II (6 deoxy erythronolide B synthase) DNA as a probe. The BamHI-digested genomic DNA of different strains of A. mediterranei were hybridized using [α32P]dATP-labeled dEBS II DNA as a probe. On careful analysis of the DNA profiling of all the strains, it was observed that the strain A. mediterranei DSM 46095 gave a different pattern of bands along with a strong signal corresponding to 4.0 kb. The DNA fragment of 4.0 kb. was cloned in E. coli Bluescript vector pBSc SK (+). For this purpose, the high-molecular-weight DNA of A. mediterranei DSM 46095 was digested with BamHI. The 4-kb DNA fragment was electroeluted onto an NA 45 DEAE membrane (Schleicher und Schuell, Germany) and ligated to BamHI-linearized pBSc SK (+). The ligation mix was transferred to E. coli JM 101, and transformants were selected on LBplates containing X-Gal, IPTG and ampicillin. Transformants were screened by colony hybridization using [α32P]dATP-labeled dEBS II DNA as a probe. A clone which gave a positive signal was selected and its plasmid DNA (pRIF H6) was isolated,
digested with BamHI and the homology of the cloned DNA fragment to dEBS II was confirmed by Southern blot hybridization under stringent conditions. Construction of genomic libraries of A. mediterranei DSM 46095 and A. mediterranei F1/24. Based on the PCR amplification and hybridization against dEBS II of the genomic DNAs of different strains of A. mediterranei, strains were divided into two groups. The first group consisted of seven strains with identical DNA amplification patterns. Into the second group fell A. mediterranei DSM 46095 which gave an amplification product smaller than expected with the primers from the LD and rif K region. Thus, genomic libraries of two strains, A. mediterranei DSM 46095 and F1/24, representing each group were made. The genomic DNA of these two strains were partially digested with Sau3AI and size fractionated on a sucrose density gradient (10%: 40%). DNA fragments of 35 to 45 kb were ligated to the BamHI-digested pWE 15 cosmid vector and the ligation mixture was packaged into lambda phage particles by using the in vitro packaging kit from Stratagene and transfected into E. coli XL-1 Blue MR. To evaluate the quality of the library, around 20 colonies were analyzed which were found to contain DNA inserts of approximately 40 – 45 kb. Screening of the genomic library of A. mediterranei. The cosmid libraries of the two strains A. mediterranei DSM 46095 and F1/24 were screened by using the dig11-UTP (Boehringer Mannheim, Germany) randomly labeled 4.0 kb DNA insert of pRIFH6 or the dig-11UTP-labeled PCR-amplified DNA from the loading domain and the rif K region of the strain A. mediterranei F1/24 as probe at stringent conditions (65°C), followed by stringent wash with 0.1 × SSC and 0.1% SDS for 20 min/65 °C. The tentative clones selected after screening of the library were further confirmed by Southern blot hybridization. DNA sequencing and analysis. An automated ABI 800 DNA sequencer was used for sequencing cloned DNA fragments. DNA sequencing of the 4-kb fragment of pRIFH6 cloned in pBSc SK (+) was carried out by using universal primers. Several subclones were prepared to complete the sequence. The anomalies in the sequence along with certain gaps were filled with a number of oligonucleotide primers, which were synthesized using the sequence information obtained from the sequence of subclones. The ends of the cloned DNA on cosmids were sequenced by using T3 and T7 universal primers. The nucleotide sequences were analyzed using the Wisconsin Sequence Analysis Package programs, Genetics Computer Group (GCG, Madison, WI, USA). The DNA and amino acid sequences of potential gene products were compared with those in the databank by means of BLAST program (Altschul et al. 1990).
Results PCR amplification of the loading domain and rif K region of different strains of A. mediterranei PCR amplifications of genomic DNA using primers from the loading domain and rif K gene were performed with all eight strains of A. mediterranei (Table 1). Interestingly, the sizes of PCR products were identical (1 kb) in seven out of eight strains screened (Fig. 1). However, in one of the strains, A. mediterranei DSM 46095, the amplified product was found to be smaller (around 0.9 kb).
Fig. 1. Amplification of genomic DNA of various strains of A. mediterranei. (Lanes 1, 10, 1 kb ladder; lanes 2–9, amplified products of DSM 46096; 46095; T-195; F1/24; MTCC 17; MTCC14; DSM 43304; DSM 40773, respectively. Using oligo nucleotide primers from (A) loading domain, and (B) rif K region of rif biosynthetic gene cluster. Microbiol. Res. 156 (2001) 3
241
DNA hybridization of dEBS II with genomic DNA of different strains of A. mediterranei
Cloning of the 4.0 kb fragment from A. mediterranei DSM 46095
The genomic DNA of different A. mediterranei strains, when hybridized against the dEBS II DNA probe initially under low stringency (42 °C), showed a similar pattern of weak signals except for A. mediterranei DSM 46095 in which not only the pattern of bands was different but also there was a strong signal corresponding to approximately 4.0 kb (Fig. 2. lane 7).
The Bam HI-digested genomic DNA of A. mediterranei DSM 46095 in the region around 4.0 kb was ligated to BamHI-digested vector pBSc SK (+) and the ligation mixture was transferred into E. coli JM101. Several transformants containing recombinant plasmids were then screened by colony hybridization using a [α32P]dATP-labeled dEBS II DNA fragments as probe. One clone harbouring a 4.0-kb DNA fragment of A. mediterranei DSM 46095 gave a positive signal on colony hybridization with dEBS II. The plasmid from this clone was termed pRIFH6. Subsequent DNA-DNA hybridization of the cloned DNA insert under stringent conditions confirmed its homology to dEBS II (Fig. 3A and B, lane 4). Complete DNA sequencing of the 4.0-kb insert of pRIF H6
Fig. 2. Southern blot showing hybridization of BamHI-digested genomic DNA of different strains of A. mediterranei [α32P]dATP-labeled dEBS II. Lanes 1, λ EcoRI/HindIII size marker indicated in kb; lanes 2–9, BamHI-digested genomic DNAs of A. mediterranei DSM 40773, MTCC 14, 17, F1/24, T-195, DSM 46095, 46096, and Saccharopolyspora erythraea MTCC 1136, respectively; lane 10, electroeluted dEBSII fragment of pDB26 (arrow indicates strong signal corresponding to 4.0 kb in A. mediterranei DSM 46095).
Fig. 3. Southern blot showing hybridization of cloned DNA fragments of A. mediterranei DSM 46095 probed with [α32P]dATP-labeled dEBS II (A) lanes 1, 7, λ size markers indicated in kb; lane 2, plasmid pDB26; lane 3, -ve controlpBSc with 4.0 kb BamHI-digested insert with no homology to dEBSII; lane 4, pRIFH6 BamHI; lane 5, pBSc SK(+) BamHI; lane 6, dEBS II fragment of pDB26, (B) Autoradiogram of (A). 242
Microbiol. Res. 156 (2001) 3
The 4.0-kb insert of plasmid pRIF H6 was subcloned in pUC13. Various oligonucleotide primers designed on the basis of partial sequence information available were used to fill the gaps in the DNA sequence of the subclones. The sequence of the entire 4.0-kb insert of pRIF was compared to the rifPKS sequence of A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998). The comparison showed nearly 90% DNA-DNA and 92% proteinprotein similarity between the DNA sequence of the cloned fragment in pRIFH6 and the sequence of the rifamycin biosynthetic genes, thus suggesting that the insert DNA probably represents a part of the rif PKS of A. mediterranei DSM 46095 (Fig. 4). Upon sequence alignment with the known sequences of A. mediterranei S699, the sequence of the 4.0-kb insert of pRIFH6 (accession no. AF 262754) was found to cover the region from position 10246–14170 bp of rif PKS. This sequence represents the acyl carrier protein (ACP) of module 2, the entire module 3 with its ketosynthase (KS), propionate specific acyl transferase (AT), the acyl carrier protein (ACP) and the keto synthase (KS) of module 4. The cloned fragment, therefore, appears to represent partly the rif A gene of the biosynthetic cluster (Fig. 4). Cloning, partial sequencing and analysis of the rif PKS and the post PKS gene cluster from A. mediterranei DSM 46095 and F1/24 The genomic libraries of A. mediterranei DSM 46095 and F1/24 in the cosmid vector pWE15 were screened with the dig-UTP-labeled 4.0-kb DNA insert of pRIF H6 and the PCR products of the loading domain and rif K as probes. Out of nearly 2000 cosmid clones screened
Fig. 4. Amino acid sequence alignment of the 4.0-kb DNA insert of the presumptive rifamycin biosynthetic gene cluster of A. mediterranei DSM 46095 (accession number AF 262754) (top row) with that of A. mediterranei S699 (accession number AF040570) (bottom row). Position of modules and corresponding active sites of the domains in rif PKS of A. mediterranei DSM 46095 (acp- acyl carrier protein, ks-β keto acyl synthase, at- acyl transferase, prop- propionate) are also shown Comparison has been made with the sequence of rif PKS from 3883–18090 nucleotides (accession number AF040570).
in each case, nine in case, of A. mediterranei DSM 46095 and eighteen in case of A. mediterranei F1/24 gave strong positive signals. Restriction analysis of the positive clones showed an insert size of nearly 30– 40 kb. Sequencing of the ends of the cloned DNA fragments confirmed their position in the rifamycin biosynthetic gene cluster. The inserts of these cosmid clones were found to be overlapping and the presumptive rifamycin biosynthetic cluster was found to be covered by two clones numbered 2 and 6 in case of DSM 46095 and 13, 21 and 24 in case of F1/24 (Fig. 5). Further sequence comparison of the
ends of the cloned fragments of the cosmids showed 92% protein and 90% DNA homology of the insert DNA of A. mediterranei DSM 46095 to that of the known sequence of rif PKS of strains A. mediterranei S699 and LBGA 3136 (August et al. 1998, Schupp et al. 1998). This confirms the novel status of the rif biosynthetic gene cluster of A. mediterranei DSM 46095. However, DNA sequence comparison between the end of the cloned DNA of A. mediterranei F1/24 with that of A. mediterranei S699 (August et al. 1998) revealed nearly 100% sequence identity (data not shown). Microbiol. Res. 156 (2001) 3
243
Fig. 5. Schematic representation of the position of the inserts in the cosmid clones of the entire PKS and non-PKS gene cluster of A. mediterranei DSM 46095 and F1/24. Rifamycin biosynthetic gene cluster of A. mediterranei S699 (August et al. 1998) is also shown.
Discussion The rapid increase in information on the molecular genetics of modular polyketide synthases (type I PKSs) has brought a revolution in our understanding of the rules that govern the synthesis of erythromycin, rapamycin and rifamycin (Lal et al. 2000). As a result, the modules and domains in the type I PKSs can be deleted, added or modified. These permutations and combinations may thus lead to synthesis of a number of analogs of a particular polyketide. Success has been partially achieved in the manipulation of the erythromycin biosynthetic gene cluster and a number of erythromycin analogs have been recently generated. However, two of the major requirements for the production of analogs in a particular target strain through this approach are: the availability of a complete biosynthetic gene cluster and analogous modules or domains on suitable vectors (Lal et al. 2000). The availability of an A. mediterranei strain with putative novel rifamycin biosynthetic gene clusters as observed in this study could, therefore, provide novel genetic material for engineering rifamycin biosynthetic genes and related type I PKSs. In order to confirm the variability of rif gene clusters, eight strains of A. mediterranei were hence screened using two methods, i.e. PCR amplification with specific oligonucleotide pri244
Microbiol. Res. 156 (2001) 3
mers and hybridization against dEBS II. The results obtained made it possible to classify the eight strains of A. mediterranei into two different categories. The first category was represented by seven strains in which the banding pattern after hybridization of the genomic DNA of each strain with dEBS II was almost similar indicating that these strains are similar as far as the rifamycin biosynthetic gene cluster is concerned. In the second category, the strain DSM 46095 was included in which the PCR product as well as the DNA banding pattern after hybridization with dEBS II were different from the strains of the first group. In order to confirm this, a 4.0-kb DNA fragment that presumably represented part of the rif biosynthetic gene cluster, was cloned from this strain (DSM 46095) and sequenced. DNA sequencing results showed 8% genetic variation at the level of amino acids as compared to A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998). These results were further confirmed by the data obtained from partial sequencing of cloned DNA fragments on cosmid clone # 2 and 6 that represented the putative rif (rifamycin) PKS of this strain. The differences in the amino acid sequence between the presumptive rif PKS of A. mediterranei DSM 46095 (accession no AF 262754) and the published sequence of the rif biosynthetic gene cluster of A. mediterranei
S699 (August et al. 1998) and LBGA 3136 (Schupp et al. 1998) were more stark in the so-called “linker regions” which is consistent with the general ideas on the way these multienzymes are organized (Bevitt et al. 1992). As the constituent modules of a particular PKS are remarkably tolerant towards diverse incoming acyl chains (Lal et al. 2000), it will be interesting to use this sequence to design engineering experiments based on cutting and splicing in these linker regions. The role of linker regions in combinatorial chemistry has recently been highlighted (Lal et al. 2000). Besides showing significant homology with rif PKS, the cloned 4.0-kb DNA of A. mediterranei DSM 46095 was also found to be homologous to other type I PKS systems including the erythromycin ery A gene II & III ORF (81%), rapamycin PKS (80%), tylactone synthase starter and 1-7 modules (80%), Streptomyces sp. fkb A gene and partial fkb D gene encoding FK506 PKS and cytochrome P-450-9-deoxo-FK506 hydroxylase (79%) and Sorangium cellulosum sor PKS (90%). The similarity of the rif PKS cluster of the strain A. mediterranei DSM 46095 to that of other type I PKSs of Streptomyces is not unexpected, however, its homology to sor PKS, associated with the synthesis of the antibiotic soraphen A, is an interesting result since the antibiotic soraphen A is produced by a myxobacterium, Sorangium cellulosum (Schupp et al. 1995). Although the DNA sequences of the rif biosynthetic gene cluster of the seven strains of A. mediterranei of the first category have not been determined, our results indicate that they have similar rifamycin biosynthetic gene clusters. The biosynthetic gene glusters of these strains is also expected to be similar to the ones reported for A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998). Also, on the basis of partial sequence analysis of rif PKS from A. mediterranei DSM 46095, it can be suggested that the limits of modules and domains of the putative rif PKS gene cluster in this strain might be more or less similar to that of rif PKS of strain A. mediterranei S699 (August et al. 1998, Tang et al. 1998). The putative rifamycin PKS gene cluster of A. mediterranei DSM 46095 (that has been shown to occupy 54 kb in A. mediterranei S699), therefore, should have five closely packed large ORFs in the same direction and ten well defined rifamycin PKS modules. Only the complete nucleotide sequence of the rifamycin biosynthetic gene cluster of the strain A. mediterranei DSM 46095 will yield more information on the structural organization. However, the isolation of the entire rif biosynthetic cluster from strain A. mediterranei DSM 46095 and grouping of eight A. mediterranei strains into two classes now forms the basis for venturing into the field of genetic engineering in order to produce hybrid or novel derivatives of rifamycins.
Acknowledgements Thanks are due to T. Schupp for mutant strains of A. mediterranei, R. Eichenlaub and K. H. Gartemann for helping in DNA amino acid sequence analysis and P. K. Ghosh for valuable suggestions. The project is supported by grants from the Department of Biotechnology, Government of India. Hardeep Kaur gratefully acknowledges University Grants Commission, Govt. of India, for providing the research fellowships.
References Altschul, S. F., Gish, W., Müller, W., Myers, E. W., Lipman, D. J. (1990): Basic local alignment search tool. J. Mol. Biol. 215, 403–410. August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T. W., Taylor, M., Hoffmann, D., Kim, C. G., Zhang, X., Hutchinson, C. R., Floss, H. G. (1998): Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster for Amycolatopsis mediterranei S699. Chem. Biol. 5, 69–79. Bevitt, D. G., Cortes, J., Haydock, S. F., Leadlay, P. F. (1992): 6-deoxyerythronolide B-synthase 2 from Saccharopolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the mutifunctional enzyme. Eur. J. Biochem. 204, 39–49. Ghisalba, O., Auden, J. A. L., Schupp, T., Nuesch, J. (1984): The rifamycins: properties, biosynthesis and fermentation. In : Biotechnology of Industrial Antibiotics (Ed. Vandamme, E. J.), Marcel Decker Inc., New York, 281– 327. Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., Schrempf, H. (1985) : Genetic manipulation of Streptomyces. A Laboratory Manual. John Innes Foundation, Norwich. Khanna, M., Dua, M., Lal, R. (1998): Selection of suitable marker genes for the development of cloning vectors and electroporation in different strains of Amycolatopsis mediterranei. Microbiol. Res. 153, 205–211. Lal, R., Lal, S., Grund, E., Eichenlaub, R. (1991) : Construction of a hybrid plamid capable of replication in Amycolatopsis mediterranei. Appl. Environ. Microbiol. 57, 665–671. Lal, R., Khanna, M., Kaur, H., Srivastava, N., Tripathi, K. K., Lal, S. (1995): Rifamycins Strain improvement programme. CRC Critical Rev. Microbiol. 21, 19–30. Lal, R., Khanna, R., Kaur, H., Khanna, M., Dhingra, N., Lal, S., Gartemann, K. H., Eichenlaub, R., Ghosh, P. K. (1996): Engineering antibiotic producers to overcome the limitations of strain improvement programme. CRC Critical Rev. Microbiol. 22, 201–255. Lal, R., Khanna, R., Dhingra, N., Khanna, M., Lal, S. (1998): Development of an improved cloning vector and transformation system in Amycolatopsis mediterranei (Nocardia mediterranei). J. Antibiot. 51, 161–169. Lal, R., Kumari, R., Kaur, H., Khanna, R., Dhingra, N., Tuteja, D. (2000): Regulation and manipulation of gene clusters encoding type-I PKSs. Trends Biotechnol. 18, 264–274. Microbiol. Res. 156 (2001) 3
245
Sambrook, J., Fritsch, E. F., Maniatis, T. (1989): Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor. N. Y. Schupp, T., Toupet, C., Cluzel, B., Neff, S., Hill, S., Beck, J. J., Ligon, J. M. (1995) : A Sorangium cellulosum (Myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: Cloning, characterization and homology to polyketide synthase genes from actinomycetes. J. Bacteriol. 177, 3673–3679. Schupp, T., Toupet, C., Engel, N., Goff, S. (1998): Cloning and sequence analysis of the putative rifamycin polyketide
246
Microbiol. Res. 156 (2001) 3
synthase gene cluster from Amycolatopsis mediterranei. FEMS Microbiol. Lett. 159, 201–207. Tang, L., Yoon, Y. J., Choi, C.-Y., Hutchinson, C. R. (1998): Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei. Gene 216, 255–265. Tuteja, D., Dua, M., Khanna, R., Dhingra, N., Khanna, M., Kaur, H., Saxena, D. M., Lal, R. (2000): The importance of homologus recombination in the generation of large deletions in hybrid plasmids in A. mediterranei. Plasmid 43, 1–11.
Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095 Hardeep Kaur1, Jesus Cortes2, Peter Leadlay3, Rup Lal1 1 2 3
Department of Zoology, University of Delhi, Delhi 110007, India Bioprocessing Unit, Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Cambridge, UK
Accepted: May 5, 2001
Abstract The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.
Corresponding author: R. Lal e-mail: [email protected] 0944-5013/01/156/03-239
$15.00/0
Key words: Rifamycin – A. mediterranei – rif PKS
Introduction Rifamycin is a clinically important antibiotic produced by the actinomycete Amycolatopsis mediterranei. The antibiotic is extensively used against tuberculosis and leprosy and several other mycobacterial infections. Because of its extensive demand, A. mediterranei has been subjected to an extensive strain improvement program, leading to the development of current industrial strains (Ghisalba et al. 1984; Lal et al. 1995, 1996). Nevertheless, the rapidly rising incidence of multidrug-resistant Mycobacterium tuberculosis (causative agents of tuberculosis) worldwide has led to an alarming resurgence of tuberculosis, especially among immuno-compromised persons and in underdeveloped parts of the world. Rapid developments in molecular genetics of Streptomyces has further motivated several groups to study the molecular genetics of rifamycin biosynthesis in A. mediterranei (August et al. 1998; Schupp et al. 2000; Lal et al. 2000). Recently, the rifamycin biosynthetic gene cluster has been cloned and characterized (August et al. 1998; Tang et al. 1998; Schupp et al. 1998) and partial success has been achieved in the development of cloning vectors and transformation methods (Lal et al. 1991, 1998; Khanna et al. 1998; Tuteja et al. 2000). Microbiol. Res. 156 (2001) 3
239
Although the rifamycin biosynthetic gene cluster has been independently cloned from A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998), there are several other strains of A. mediterranei, reported to be isolated from various soils, and their successors are in use for the commercial production of rifamycin. The variability of these strains in terms of the rifamycin biosynthetic gene cluster remains to be determined. The availability of A. mediterranei strains with a new or slightly different DNA sequence of the rifamycin biosynthetic gene cluster (rif gene cluster) could provide new genetic material for manipulating these genes for the production of analogs, which are required to combat the problem of multidrug-resistant mycobacteria.
Material and methods Bacterial strains, plasmids and culture conditions. Different strains of A. mediterranei used in the present study are listed in Table 1. These were grown in TYN or YM – agar medium at 28°C as described earlier (Lal et al. 1998). Saccharopolyspora erythraea was maintained in YM medium under conditions as described for A. mediterranei. E. coli cultures were maintained on LB. DNA preparations and manipulations. The high-molecular-weight DNA of different strains of A. mediterranei and the erythromycin producer S. erythraea MTCC1136 were isolated by Kirby’s method (Hopwood et al. 1985). Plasmid DNA isolation, agarose gel electrophoresis, restriction enzyme digestions, restriction mapTable 1. Bacterial strains used in the study. Organism Amycolatopsis mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei A. mediterranei Saccharopolyspora erythraea Escherichia coli
Strain
Source or reference
40773 43303 14 17 F1/24 ans-13 T-195 ans 13, thi-8 46095 46096
DSM DSM MTCC MTCC Ciba Geigy Ciba Geigy DSM DSM
1136 GM 2163
MTCC New England Biolabs
DSM Deutsche Stammsammlung für Mirkoorganismen und Zellkulturen GmbH, Germany; MTCC Microbial Type Culture Collection, Chandigarh, India; Ciba Geigy (Now Novartis), Switzerland. 240
Microbiol. Res. 156 (2001) 3
ping, alkaline phosphatase treatment, T4 polynucleotide kinease treatment, blunt end cloning of PCR DNA and ligations were performed according to standard procedures (Sambrook et al. 1989). Identification of novel A. mediterranei strains by PCR. In order to detect a novel strain with striking differences in the nucleotide and protein sequences of the rifamycin biosynthetic cluster, eight different strains of A. mediterranei, that have been reported to produce rifamycin or its analogs, were selected. The genomic DNAs of A. mediterranei strains were amplified by using oligonucleotide primers designed from the known sequence of the rifamycin biosynthetic cluster of A. mediterranei S699. The primers were designed from the loading domain (LD) of the PKS and rif K gene that encodes 3-amino-5 hydroxybenzoic acid synthase regions of the rifamycin biosynthetic gene cluster (August et al. 1998) (Accession no. AF040570). The primers flanked the loading domain sequence at position 3884–3912 and 4924–4947 (C) and rif K gene sequence at position 58288–58316 and 59302–59324 (C) (Assession no. AF040570). The amplification was performed using a Stratagene Robocycler (USA) by the standard protocol. PCR was carried out in a total volume of 50 µl, at a temperature of 55°C and for 25 cycles of extension. Each PCR reaction consisted of: genomic DNA of A. mediterranei, 1 µl (1 µg); primer I, 3 µl (0.5 µg/µl); primer II, 3 µl (0.5 µg/µl); dNTP, 5 µl (10 mM of each dNTP); buffer (10×), 5 µl; DMSO, 5 µl; millipore water, 27 µl and enzyme (Pfu), 1 µl (2.5 U/µl). The PCR products from different strains of A. mediterranei were then analyzed on 0.8% (w/v) agarose gels. Screening by using dEBS II (6 deoxy erythronolide B synthase) DNA as a probe. The BamHI-digested genomic DNA of different strains of A. mediterranei were hybridized using [α32P]dATP-labeled dEBS II DNA as a probe. On careful analysis of the DNA profiling of all the strains, it was observed that the strain A. mediterranei DSM 46095 gave a different pattern of bands along with a strong signal corresponding to 4.0 kb. The DNA fragment of 4.0 kb. was cloned in E. coli Bluescript vector pBSc SK (+). For this purpose, the high-molecular-weight DNA of A. mediterranei DSM 46095 was digested with BamHI. The 4-kb DNA fragment was electroeluted onto an NA 45 DEAE membrane (Schleicher und Schuell, Germany) and ligated to BamHI-linearized pBSc SK (+). The ligation mix was transferred to E. coli JM 101, and transformants were selected on LBplates containing X-Gal, IPTG and ampicillin. Transformants were screened by colony hybridization using [α32P]dATP-labeled dEBS II DNA as a probe. A clone which gave a positive signal was selected and its plasmid DNA (pRIF H6) was isolated,
digested with BamHI and the homology of the cloned DNA fragment to dEBS II was confirmed by Southern blot hybridization under stringent conditions. Construction of genomic libraries of A. mediterranei DSM 46095 and A. mediterranei F1/24. Based on the PCR amplification and hybridization against dEBS II of the genomic DNAs of different strains of A. mediterranei, strains were divided into two groups. The first group consisted of seven strains with identical DNA amplification patterns. Into the second group fell A. mediterranei DSM 46095 which gave an amplification product smaller than expected with the primers from the LD and rif K region. Thus, genomic libraries of two strains, A. mediterranei DSM 46095 and F1/24, representing each group were made. The genomic DNA of these two strains were partially digested with Sau3AI and size fractionated on a sucrose density gradient (10%: 40%). DNA fragments of 35 to 45 kb were ligated to the BamHI-digested pWE 15 cosmid vector and the ligation mixture was packaged into lambda phage particles by using the in vitro packaging kit from Stratagene and transfected into E. coli XL-1 Blue MR. To evaluate the quality of the library, around 20 colonies were analyzed which were found to contain DNA inserts of approximately 40 – 45 kb. Screening of the genomic library of A. mediterranei. The cosmid libraries of the two strains A. mediterranei DSM 46095 and F1/24 were screened by using the dig11-UTP (Boehringer Mannheim, Germany) randomly labeled 4.0 kb DNA insert of pRIFH6 or the dig-11UTP-labeled PCR-amplified DNA from the loading domain and the rif K region of the strain A. mediterranei F1/24 as probe at stringent conditions (65°C), followed by stringent wash with 0.1 × SSC and 0.1% SDS for 20 min/65 °C. The tentative clones selected after screening of the library were further confirmed by Southern blot hybridization. DNA sequencing and analysis. An automated ABI 800 DNA sequencer was used for sequencing cloned DNA fragments. DNA sequencing of the 4-kb fragment of pRIFH6 cloned in pBSc SK (+) was carried out by using universal primers. Several subclones were prepared to complete the sequence. The anomalies in the sequence along with certain gaps were filled with a number of oligonucleotide primers, which were synthesized using the sequence information obtained from the sequence of subclones. The ends of the cloned DNA on cosmids were sequenced by using T3 and T7 universal primers. The nucleotide sequences were analyzed using the Wisconsin Sequence Analysis Package programs, Genetics Computer Group (GCG, Madison, WI, USA). The DNA and amino acid sequences of potential gene products were compared with those in the databank by means of BLAST program (Altschul et al. 1990).
Results PCR amplification of the loading domain and rif K region of different strains of A. mediterranei PCR amplifications of genomic DNA using primers from the loading domain and rif K gene were performed with all eight strains of A. mediterranei (Table 1). Interestingly, the sizes of PCR products were identical (1 kb) in seven out of eight strains screened (Fig. 1). However, in one of the strains, A. mediterranei DSM 46095, the amplified product was found to be smaller (around 0.9 kb).
Fig. 1. Amplification of genomic DNA of various strains of A. mediterranei. (Lanes 1, 10, 1 kb ladder; lanes 2–9, amplified products of DSM 46096; 46095; T-195; F1/24; MTCC 17; MTCC14; DSM 43304; DSM 40773, respectively. Using oligo nucleotide primers from (A) loading domain, and (B) rif K region of rif biosynthetic gene cluster. Microbiol. Res. 156 (2001) 3
241
DNA hybridization of dEBS II with genomic DNA of different strains of A. mediterranei
Cloning of the 4.0 kb fragment from A. mediterranei DSM 46095
The genomic DNA of different A. mediterranei strains, when hybridized against the dEBS II DNA probe initially under low stringency (42 °C), showed a similar pattern of weak signals except for A. mediterranei DSM 46095 in which not only the pattern of bands was different but also there was a strong signal corresponding to approximately 4.0 kb (Fig. 2. lane 7).
The Bam HI-digested genomic DNA of A. mediterranei DSM 46095 in the region around 4.0 kb was ligated to BamHI-digested vector pBSc SK (+) and the ligation mixture was transferred into E. coli JM101. Several transformants containing recombinant plasmids were then screened by colony hybridization using a [α32P]dATP-labeled dEBS II DNA fragments as probe. One clone harbouring a 4.0-kb DNA fragment of A. mediterranei DSM 46095 gave a positive signal on colony hybridization with dEBS II. The plasmid from this clone was termed pRIFH6. Subsequent DNA-DNA hybridization of the cloned DNA insert under stringent conditions confirmed its homology to dEBS II (Fig. 3A and B, lane 4). Complete DNA sequencing of the 4.0-kb insert of pRIF H6
Fig. 2. Southern blot showing hybridization of BamHI-digested genomic DNA of different strains of A. mediterranei [α32P]dATP-labeled dEBS II. Lanes 1, λ EcoRI/HindIII size marker indicated in kb; lanes 2–9, BamHI-digested genomic DNAs of A. mediterranei DSM 40773, MTCC 14, 17, F1/24, T-195, DSM 46095, 46096, and Saccharopolyspora erythraea MTCC 1136, respectively; lane 10, electroeluted dEBSII fragment of pDB26 (arrow indicates strong signal corresponding to 4.0 kb in A. mediterranei DSM 46095).
Fig. 3. Southern blot showing hybridization of cloned DNA fragments of A. mediterranei DSM 46095 probed with [α32P]dATP-labeled dEBS II (A) lanes 1, 7, λ size markers indicated in kb; lane 2, plasmid pDB26; lane 3, -ve controlpBSc with 4.0 kb BamHI-digested insert with no homology to dEBSII; lane 4, pRIFH6 BamHI; lane 5, pBSc SK(+) BamHI; lane 6, dEBS II fragment of pDB26, (B) Autoradiogram of (A). 242
Microbiol. Res. 156 (2001) 3
The 4.0-kb insert of plasmid pRIF H6 was subcloned in pUC13. Various oligonucleotide primers designed on the basis of partial sequence information available were used to fill the gaps in the DNA sequence of the subclones. The sequence of the entire 4.0-kb insert of pRIF was compared to the rifPKS sequence of A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998). The comparison showed nearly 90% DNA-DNA and 92% proteinprotein similarity between the DNA sequence of the cloned fragment in pRIFH6 and the sequence of the rifamycin biosynthetic genes, thus suggesting that the insert DNA probably represents a part of the rif PKS of A. mediterranei DSM 46095 (Fig. 4). Upon sequence alignment with the known sequences of A. mediterranei S699, the sequence of the 4.0-kb insert of pRIFH6 (accession no. AF 262754) was found to cover the region from position 10246–14170 bp of rif PKS. This sequence represents the acyl carrier protein (ACP) of module 2, the entire module 3 with its ketosynthase (KS), propionate specific acyl transferase (AT), the acyl carrier protein (ACP) and the keto synthase (KS) of module 4. The cloned fragment, therefore, appears to represent partly the rif A gene of the biosynthetic cluster (Fig. 4). Cloning, partial sequencing and analysis of the rif PKS and the post PKS gene cluster from A. mediterranei DSM 46095 and F1/24 The genomic libraries of A. mediterranei DSM 46095 and F1/24 in the cosmid vector pWE15 were screened with the dig-UTP-labeled 4.0-kb DNA insert of pRIF H6 and the PCR products of the loading domain and rif K as probes. Out of nearly 2000 cosmid clones screened
Fig. 4. Amino acid sequence alignment of the 4.0-kb DNA insert of the presumptive rifamycin biosynthetic gene cluster of A. mediterranei DSM 46095 (accession number AF 262754) (top row) with that of A. mediterranei S699 (accession number AF040570) (bottom row). Position of modules and corresponding active sites of the domains in rif PKS of A. mediterranei DSM 46095 (acp- acyl carrier protein, ks-β keto acyl synthase, at- acyl transferase, prop- propionate) are also shown Comparison has been made with the sequence of rif PKS from 3883–18090 nucleotides (accession number AF040570).
in each case, nine in case, of A. mediterranei DSM 46095 and eighteen in case of A. mediterranei F1/24 gave strong positive signals. Restriction analysis of the positive clones showed an insert size of nearly 30– 40 kb. Sequencing of the ends of the cloned DNA fragments confirmed their position in the rifamycin biosynthetic gene cluster. The inserts of these cosmid clones were found to be overlapping and the presumptive rifamycin biosynthetic cluster was found to be covered by two clones numbered 2 and 6 in case of DSM 46095 and 13, 21 and 24 in case of F1/24 (Fig. 5). Further sequence comparison of the
ends of the cloned fragments of the cosmids showed 92% protein and 90% DNA homology of the insert DNA of A. mediterranei DSM 46095 to that of the known sequence of rif PKS of strains A. mediterranei S699 and LBGA 3136 (August et al. 1998, Schupp et al. 1998). This confirms the novel status of the rif biosynthetic gene cluster of A. mediterranei DSM 46095. However, DNA sequence comparison between the end of the cloned DNA of A. mediterranei F1/24 with that of A. mediterranei S699 (August et al. 1998) revealed nearly 100% sequence identity (data not shown). Microbiol. Res. 156 (2001) 3
243
Fig. 5. Schematic representation of the position of the inserts in the cosmid clones of the entire PKS and non-PKS gene cluster of A. mediterranei DSM 46095 and F1/24. Rifamycin biosynthetic gene cluster of A. mediterranei S699 (August et al. 1998) is also shown.
Discussion The rapid increase in information on the molecular genetics of modular polyketide synthases (type I PKSs) has brought a revolution in our understanding of the rules that govern the synthesis of erythromycin, rapamycin and rifamycin (Lal et al. 2000). As a result, the modules and domains in the type I PKSs can be deleted, added or modified. These permutations and combinations may thus lead to synthesis of a number of analogs of a particular polyketide. Success has been partially achieved in the manipulation of the erythromycin biosynthetic gene cluster and a number of erythromycin analogs have been recently generated. However, two of the major requirements for the production of analogs in a particular target strain through this approach are: the availability of a complete biosynthetic gene cluster and analogous modules or domains on suitable vectors (Lal et al. 2000). The availability of an A. mediterranei strain with putative novel rifamycin biosynthetic gene clusters as observed in this study could, therefore, provide novel genetic material for engineering rifamycin biosynthetic genes and related type I PKSs. In order to confirm the variability of rif gene clusters, eight strains of A. mediterranei were hence screened using two methods, i.e. PCR amplification with specific oligonucleotide pri244
Microbiol. Res. 156 (2001) 3
mers and hybridization against dEBS II. The results obtained made it possible to classify the eight strains of A. mediterranei into two different categories. The first category was represented by seven strains in which the banding pattern after hybridization of the genomic DNA of each strain with dEBS II was almost similar indicating that these strains are similar as far as the rifamycin biosynthetic gene cluster is concerned. In the second category, the strain DSM 46095 was included in which the PCR product as well as the DNA banding pattern after hybridization with dEBS II were different from the strains of the first group. In order to confirm this, a 4.0-kb DNA fragment that presumably represented part of the rif biosynthetic gene cluster, was cloned from this strain (DSM 46095) and sequenced. DNA sequencing results showed 8% genetic variation at the level of amino acids as compared to A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998). These results were further confirmed by the data obtained from partial sequencing of cloned DNA fragments on cosmid clone # 2 and 6 that represented the putative rif (rifamycin) PKS of this strain. The differences in the amino acid sequence between the presumptive rif PKS of A. mediterranei DSM 46095 (accession no AF 262754) and the published sequence of the rif biosynthetic gene cluster of A. mediterranei
S699 (August et al. 1998) and LBGA 3136 (Schupp et al. 1998) were more stark in the so-called “linker regions” which is consistent with the general ideas on the way these multienzymes are organized (Bevitt et al. 1992). As the constituent modules of a particular PKS are remarkably tolerant towards diverse incoming acyl chains (Lal et al. 2000), it will be interesting to use this sequence to design engineering experiments based on cutting and splicing in these linker regions. The role of linker regions in combinatorial chemistry has recently been highlighted (Lal et al. 2000). Besides showing significant homology with rif PKS, the cloned 4.0-kb DNA of A. mediterranei DSM 46095 was also found to be homologous to other type I PKS systems including the erythromycin ery A gene II & III ORF (81%), rapamycin PKS (80%), tylactone synthase starter and 1-7 modules (80%), Streptomyces sp. fkb A gene and partial fkb D gene encoding FK506 PKS and cytochrome P-450-9-deoxo-FK506 hydroxylase (79%) and Sorangium cellulosum sor PKS (90%). The similarity of the rif PKS cluster of the strain A. mediterranei DSM 46095 to that of other type I PKSs of Streptomyces is not unexpected, however, its homology to sor PKS, associated with the synthesis of the antibiotic soraphen A, is an interesting result since the antibiotic soraphen A is produced by a myxobacterium, Sorangium cellulosum (Schupp et al. 1995). Although the DNA sequences of the rif biosynthetic gene cluster of the seven strains of A. mediterranei of the first category have not been determined, our results indicate that they have similar rifamycin biosynthetic gene clusters. The biosynthetic gene glusters of these strains is also expected to be similar to the ones reported for A. mediterranei S699 (August et al. 1998) and A. mediterranei LBGA 3136 (Schupp et al. 1998). Also, on the basis of partial sequence analysis of rif PKS from A. mediterranei DSM 46095, it can be suggested that the limits of modules and domains of the putative rif PKS gene cluster in this strain might be more or less similar to that of rif PKS of strain A. mediterranei S699 (August et al. 1998, Tang et al. 1998). The putative rifamycin PKS gene cluster of A. mediterranei DSM 46095 (that has been shown to occupy 54 kb in A. mediterranei S699), therefore, should have five closely packed large ORFs in the same direction and ten well defined rifamycin PKS modules. Only the complete nucleotide sequence of the rifamycin biosynthetic gene cluster of the strain A. mediterranei DSM 46095 will yield more information on the structural organization. However, the isolation of the entire rif biosynthetic cluster from strain A. mediterranei DSM 46095 and grouping of eight A. mediterranei strains into two classes now forms the basis for venturing into the field of genetic engineering in order to produce hybrid or novel derivatives of rifamycins.
Acknowledgements Thanks are due to T. Schupp for mutant strains of A. mediterranei, R. Eichenlaub and K. H. Gartemann for helping in DNA amino acid sequence analysis and P. K. Ghosh for valuable suggestions. The project is supported by grants from the Department of Biotechnology, Government of India. Hardeep Kaur gratefully acknowledges University Grants Commission, Govt. of India, for providing the research fellowships.
References Altschul, S. F., Gish, W., Müller, W., Myers, E. W., Lipman, D. J. (1990): Basic local alignment search tool. J. Mol. Biol. 215, 403–410. August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T. W., Taylor, M., Hoffmann, D., Kim, C. G., Zhang, X., Hutchinson, C. R., Floss, H. G. (1998): Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster for Amycolatopsis mediterranei S699. Chem. Biol. 5, 69–79. Bevitt, D. G., Cortes, J., Haydock, S. F., Leadlay, P. F. (1992): 6-deoxyerythronolide B-synthase 2 from Saccharopolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the mutifunctional enzyme. Eur. J. Biochem. 204, 39–49. Ghisalba, O., Auden, J. A. L., Schupp, T., Nuesch, J. (1984): The rifamycins: properties, biosynthesis and fermentation. In : Biotechnology of Industrial Antibiotics (Ed. Vandamme, E. J.), Marcel Decker Inc., New York, 281– 327. Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., Schrempf, H. (1985) : Genetic manipulation of Streptomyces. A Laboratory Manual. John Innes Foundation, Norwich. Khanna, M., Dua, M., Lal, R. (1998): Selection of suitable marker genes for the development of cloning vectors and electroporation in different strains of Amycolatopsis mediterranei. Microbiol. Res. 153, 205–211. Lal, R., Lal, S., Grund, E., Eichenlaub, R. (1991) : Construction of a hybrid plamid capable of replication in Amycolatopsis mediterranei. Appl. Environ. Microbiol. 57, 665–671. Lal, R., Khanna, M., Kaur, H., Srivastava, N., Tripathi, K. K., Lal, S. (1995): Rifamycins Strain improvement programme. CRC Critical Rev. Microbiol. 21, 19–30. Lal, R., Khanna, R., Kaur, H., Khanna, M., Dhingra, N., Lal, S., Gartemann, K. H., Eichenlaub, R., Ghosh, P. K. (1996): Engineering antibiotic producers to overcome the limitations of strain improvement programme. CRC Critical Rev. Microbiol. 22, 201–255. Lal, R., Khanna, R., Dhingra, N., Khanna, M., Lal, S. (1998): Development of an improved cloning vector and transformation system in Amycolatopsis mediterranei (Nocardia mediterranei). J. Antibiot. 51, 161–169. Lal, R., Kumari, R., Kaur, H., Khanna, R., Dhingra, N., Tuteja, D. (2000): Regulation and manipulation of gene clusters encoding type-I PKSs. Trends Biotechnol. 18, 264–274. Microbiol. Res. 156 (2001) 3
245
Sambrook, J., Fritsch, E. F., Maniatis, T. (1989): Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor. N. Y. Schupp, T., Toupet, C., Cluzel, B., Neff, S., Hill, S., Beck, J. J., Ligon, J. M. (1995) : A Sorangium cellulosum (Myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: Cloning, characterization and homology to polyketide synthase genes from actinomycetes. J. Bacteriol. 177, 3673–3679. Schupp, T., Toupet, C., Engel, N., Goff, S. (1998): Cloning and sequence analysis of the putative rifamycin polyketide
246
Microbiol. Res. 156 (2001) 3
synthase gene cluster from Amycolatopsis mediterranei. FEMS Microbiol. Lett. 159, 201–207. Tang, L., Yoon, Y. J., Choi, C.-Y., Hutchinson, C. R. (1998): Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei. Gene 216, 255–265. Tuteja, D., Dua, M., Khanna, R., Dhingra, N., Khanna, M., Kaur, H., Saxena, D. M., Lal, R. (2000): The importance of homologus recombination in the generation of large deletions in hybrid plasmids in A. mediterranei. Plasmid 43, 1–11.