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DNA Sequencing and Transcriptional Analysis of the Kasugamycin

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DNA Sequencing and Transcriptional Analysis of the Kasugamycin J. Antibiot. 59(1): 18–28, 2006 THE JOURNAL OF ORIGINAL ARTICLE ANTIBIOTICS DNA Sequencing and Transcriptional Analysis of the Kasugamycin Biosynthetic Gene Cluster from Streptomyces kasugaensis M338-M1 Souichi Ikeno, Daisuke Aoki, Masa Hamada, Makoto Hori, Kayoko S. Tsuchiya Received: August 23, 2005 / Accepted: December 4, 2005 © Japan Antibiotics Research Association Abstract Streptomyces kasugaensis M338-M1 produces the aminoglycoside antibiotic kasugamycin (KSM). We Introduction previously cloned, sequenced and characterized the KSM acetyltransferase, transporter, and some of the biosynthetic The genus Streptomyces is a group of Gram-positive bacteria genes from this strain. To identify other potential genes in a that produce a wide variety of secondary metabolites chromosome walk experiment, a 6.8-kb EcoRI-PstI region including antibiotics and other pharmacologically active immediately downstream from the KSM transporter genes agents. The biosynthetic pathways and genetic regulation was sequenced. Five open reading frames (designated as that result in the production of these compounds are poorly kasN, kasO, kasP, kasQ, kasR) and the 5Ј region of understood and need to be studied to develop the ability to kasA were found in this region. The genes are apparently increase antibiotic productivity and to design and produce co-transcribed as bicistrons, all of which are co-directional new antibiotics, potentially with hybrid structures that except for the kasPQ transcript. Homology analysis of would result in more effective properties, particularly the deduced products of kasN, kasP, kasQ and kasR against resistant infective agents. revealed similarities with known enzymes: KasN, D-amino Kasugamycin (KSM) is an aminoglycoside antibiotic acid oxidase from Pseudomonas aeruginosa (35% identity); produced by Streptomyces kasugaensis M338-M1 [1] KasP, F420-dependent H4MPT reductase from Streptomyces that is effective against Pyricularia oryzae Cavara and is lavendulae (33% identity); KasQ, UDP-N-acetylglucosamine widely used in agriculture in Japan to prevent rice blast 2-epimerase from Streptomyces verticillus (45% identity); disease. A gene encoding an enzyme that inactivates the Ј and KasR, NDP-hexose 3,4-dehydratase from Streptomyces antibiotic by acetylation of the 2 -NH2 of KSM was cloned cyanogenus (38% identity); respectively. A gel retardation from Streptomyces kasugaensis MB273-C4, another assay showed that KasT, a putative pathway-specific KSM producing strain, and named kac (JP. A-05-23187, regulator for this gene cluster, bound to the upstream region Hirasawa et al. 1993). In Streptomycetes, genes required for of kasN and to the intergenic region of kasQ-kasR, biosynthesis of an antibiotic are usually clustered and suggesting that the expression of these operons is under the linked to a gene or genes for self-protection from its own control of the regulator protein. antibiotic. Genes associated with regulation of antibiotic biosynthesis are also often found in clusters. Starting with Keywords kasugamycin, biosynthetic gene cluster, identification of KSM acetyltransferase (kac338) [2], we Streptomyces kasugaensis found three KSM biosynthetic genes (kasC, kasD, and kasJ) [3, 4], three KSM transporter genes (kasK, kasL, and S. Ikeno (Corresponding author), D. Aoki, M. Hori, K. S. M. Hamada: Institute of Microbial Chemistry, 3-14-23 Tsuchiya: Showa Pharmaceutical University, 3-3165 Higashi Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan Tamagawagakuen, Machida-shi, Tokyo 194-8543, Japan. E-mail: [email protected] 19 Table 1 Strains and plasmids Strains and plasmids Genotype and genetic construct Source or reference Strains S. kasugaensis M338-M1 Kasugamycin producing strain 1) E. coli Ϫ ϩ DH5af80lacZDM15 D(lacZYA-argF) U169 deoR recA1 endA1 hidR17(rK , mK ) phoA TOYOBO supE44 l-thi-1 gyrA96 relA1 Ϫ ϩ TH2 supE44 hsdS20(rB , mB ) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 TaKaRa thi-1 trpR624 Ј Ϫ ϩ BL21(DE3) F ompT (rB , mB ) Novagen Plasmids pUC118 Cloning vector. Ampr. 3.1-kb. TaKaRa pKF 3 Cloning vector. Sms, Cmr. 2.2-kb. TaKaRa pET-32a(ϩ) Expression vector derived from pBR322, containing trxA [1-109] and His-tag expressed from T7 promoter for construction of Trx hybrid proteins. Ampr. 5.7-kb. Novagen pSKE 4 pKF 3 derivative containing 8.4-kb KpnI-KpnI fragment. 11-kb. 5) pET-KasT pET-32a(ϩ) derivative containing 1.0-kb NcoI-BamHI fragment. Trx-KasT expression plasmid. 6.7-kb. 4) kasM) [5] and a putative regulatory gene (kasT) [4] in the DNA Sequencing and Sequence Analysis upstream region of kac338. Isolation of genomic DNA from S. kasugaensis M338-M1 In the present paper, we report the nucleotide sequence and cloning of the region downstream from KSM of a 6.8-kb region downstream from the KSM transporter transporter genes were described previously [2, 5]. The genes and propose the existence of KSM biosynthetic nucleotide sequence of both strands was determined using genes in that segment of the chromosome. We suggest that an ALFredTM DNA sequencer (Amersham Biosciences). these genes are transcribed as three bicistronic transcripts Sequencing reactions were carried out with Cy5TM and that these operons are under the control of KasT. AutoCycleTM Sequencing Kit (Amersham Biosciences) according to the supplier’s instructions. M13-40 universal primer, M13-reverse primer and some synthesized Materials and Methods oligonucleotide primers (labeled with Cy5, purchased from Amersham Biosciences) were used as sequencing primers. Strains, Growth Conditions, and Plasmids Amino acid sequences of the corresponding proteins were Characteristics of bacterial strains and plasmids used in this deduced from the nucleotide sequences of appropriate work are summarized in Table 1. S. kasugaensis M338-M1 regions of DNA using DNASIS-Mac version 3.7 (Hitachi is maintained at the Institute of Microbial Chemistry, Software Engineering). FramePlot 2.3.1 [6] was used to Tokyo, Japan. E. coli TH2 (TaKaRa), E. coli DH5a search for open reading frames (ORF). Amino acid (TOYOBO), E. coli BL21(DE3) (STRATAGENE), pKF 3 sequences of potential gene products were compared with (TaKaRa), pUC118 (TaKaRa) and pET-32a(ϩ) (Novagen) the SWISS-PROT and PIR databases using FASTA [7] and were of commercial origin. All other plasmids were BLAST [8]. Nucleotide sequence data reported in this produced in the present study. paper have been deposited in the DDBJ, EMBL and S. kasugaensis M338-M1 was grown in MR medium GenBank nucleotide sequence databases with the accession (KSM-producing medium) [5] with shaking at 27°C for 72 number AB120043. hours. E. coli TH2 transformants were grown at 37°C in L- broth containing 12 mg/ml chloramphenicol and 50 mg/ml Isolation of Total RNA and Northern Blot Analysis streptomycin. E. coli DH5a and BL21(DE3) transformants Isolation of total RNA from S. kasugaensis M338-M1 were grown at 37°C in Luria-Bertani (LB) medium was as described previously [5]. The RNA sample was containing 50 mg/ml ampicillin. quantified by absorbance at 260 nm and a portion was 20 Fig. 1 Restriction map of the kasugamycin biosynthetic gene cluster from Streptomyces kasugaensis M338-M1. The gray region of the restriction map is the subject of the present paper. The black region has been previously reported [4, 5]. The open arrows indicate the deduced genes and direction of transcription. The start codon (ATG) of kasA overlaps the stop codon (TGA) of the adjacent kasR. Abbreviations: B, BamHI; E, EcoRI; K, KpnI; P, PstI; Sa, SacI; Sp, SphI. electrophoresed (10 mg/lane) on a 1.2% agarose gel and products were electrophoresed on a 0.6% agarose gel and transferred to a cellulose nitrate membrane (Schleicher visualized using ethidium bromide staining. & Schuell). Hybridization was carried out at 42°C for 16 hours in hybridization buffer (50% formamide, Determination of Transcription Start Sites 900 mM NaCl, 6 mM EDTA, 90 mM Tris-HCl [pH 7.5], The 5Ј end of kasNO mRNA was determined by the 5Ј 10ϫDenhardt’s, 0.1% SDS, 100 mg/ml Salmon testis RACE method with a 5Ј-Full RACE Core Set (TaKaRa) DNA), followed by washing at 68°C for 1 hours with according to the manufacturer’s instructions [9]. RT 2ϫSSC (300 mM NaCl, 30 mM trisodium citrate). The reaction was performed in a 15 ml solution containing 811 bp SphI-SacI fragment, which corresponds to the 200 pmol of 5Ј-phosphorylated primer 3Ј-PKasN (5Ј- internal region of the kasN structural gene, was used as ATCGCCCACCACTGCCACGTCCACA-3Ј), 5 U of AMV probe (Fig. 1). The DNA fragment was labeled with [a- reverse transcriptase XL and 5 mg of total RNA, and 32P]dCTP using a Random Primer DNA Labeling Kit incubated at 50°C for 1 hour. A negative control lacked (TaKaRa). reverse transcriptase. The first PCR mixture contained 20 pmol each of 5Ј-RACE15a (5Ј-CTCCGCCCCGCAC- RT-PCR CACCCCT-3Ј) and 3Ј-RACE15a (5Ј-GGAGGTGTCACG- RT-PCR was conducted as described previously [5]. 3Ј- GGACGGTCTT-3Ј). The reaction was carried out for 15 pKS17 (5Ј-CAGTCGGACTCCCTGATGCCCAGGA-3Ј), cycles with denaturation for 30 seconds at 98°C and 3Ј-Q1 (5Ј-ACGGAACTCGCCCCGAAGCCATCAA-3Ј) annealing and extension for 2 minutes at 72°C. The and 3Ј-ABAM (5Ј-CCGGATCCTCACCGCTGGCCACA- resulting PCR product (1 ml) was used as a template for a CGCCACTTCGTA-3Ј) were used as cDNA synthesis second PCR amplification with primers 5Ј-RACE15b (5Ј- primers for kasNO, kasPQ and kasRA transcripts, CCCTGCAGAAAGGAAGACGGCCCGATGA-3Ј) and 3Ј- respectively. Three sets of primers [5Ј-pKS16b (5Ј- RACE15b (5Ј-CCGTCGACCCGGCCACCGCATGCGA- AGAACAGGTGGCTCAGCCCCGCGGA-3Ј)/3Ј-pKS17, AGA-3Ј) under the same conditions as described above. 5Ј-kasP (5Ј-CGGCAGTGCCAAGTCGCCGGTCAGC-3Ј)/ The major 230 bp product was digested with SalI and PstI 3Ј-Q1 and 5Ј-RT20 (5Ј-ATCGTGTCCGGCGCGACCT- (recognition sites are underlined in the primers), cloned TCGTCA-3Ј)/3Ј-ABAM] were used in subsequent PCR into pUC118, and the DNA sequence of the product was amplification with 30 cycles of 30 seconds denaturation at determined. 98°C and 2 minutes annealing/extension at 72°C.
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