Annals of Microbiology, 57 (2) 259-263 (2007) Cloning and sequencing of partial segment of cholesterol oxidase encoding gene from Streptomyces luridus Mohammad Saeid HEJAZI1,2*, Kamal KAZEMI TABAR3, Reza AZARBAIJANI3, Laleh ZERESHKI NOBAR3 1Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, 2Drug Applied Research Center and Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz; 3Higher Education Complex of Agricultural and Natural Resource Science, University of Mazandaran, Sari, Iran Received 18 October 2006 / Accepted 18 April 2007 ABSTRACT - Cloning, sequencing and analysis of partial segment of cholesterol oxidase (cho) encoding gene from Streptomyces luridus using PCR technique is described. The primers used for PCR were designed on the basis of inter-species homology, and the pTZ57R/T vector was used for molecular cloning. The sequencing results lead to identification of the oxidoreductase domain of cho enzyme with 818 nucleotide length encoding 272 amino acids, which was submitted in NCBI with DQ480365 accession number. The submitted sequence includes complete FAD-linked reductase C terminal domain with 170 amino acids size and N-terminal of GMC-oxidoreductase domain of cho enzyme. Key words: cholesterol oxidase, Streptomyces luridus, cloning and sequencing. INTRODUCTION The insecticide effect of cho on boll weevil larvae and the other pests is attributed to the oxidation of cholesterol in the midget epithelial membrane, resulting in physical and Cholesterol oxidase (cho) is a flavin adenine dinucleotide functional disruption of the membrane (Purcell et al., (FAD)-dependent enzyme that catalyses the oxidation of 1993). Considering this point, identification and evaluation cholesterol (5-cholesten-3-ß-ol) to 4-cholesten-3-one with of new cho enzymes in order to find more potent enzymes the reduction of oxygen to hydrogen peroxide (Stadtman et still retains its importance. Hereby, cloning, sequencing al., 1954). Streptomycetes produce both intracellular and and characterisation of oxidoreductase domain of cho extracellular cho (Murooka et al., 1986). This enzyme is encoding gene from Streptomyces luridus is reported. associated with the lipid bilayer and has an important role in bacterial metabolism and pathogenesis (Rodrigueza et al., 1995). The enzyme has been isolated and sequenced MATERIALS AND METHODS from several microorganisms including Streptomyces species (Murooka et al., 1986; Ishizaki et al., 1989). Bacterial cells. Streptomyces luridus (ATCC 19782; PTCC Previous studies on this group of bacteria showed that the 1134) was obtained from Persian Type Culture Collection expression level and activity of cho enzyme varies in dif- (PTCC) in Tehran. DH5α strain of Escherichia coli was used ferent species of Streptomyces (Nishiya et al., 1997; in transformation step of cloning process as the competent Pollegioni et al., 1999). This enzyme is industrially impor- cells. tant and useful for the clinical determination of total or free serum cholesterol by coupling with related enzymes (Allain Culture medium and growth conditions. The bacterial et al., 1974) and also for biological control of pests (Corbin cells were grown in YEME (ISP2) medium [agar 20 g, malt et al., 1994, 2001). Moreover, cho as a secreted bacterial extract 10 g, yeast extract 4 g and glucose 4 g per litre] at enzyme has lethal effect on the boll weevil (Anthonomus incubation temperature of 26 °C for 24 until 48 h. grandis grandis Boheman) and on several species of lepi- dopteran cotton insect pests including tobacco budworm Enzymes and chemicals. Proteinase K (Sigma, USA), Taq (Heliothis virescens), corn earworm (Helicoverpa zea), and DNA polymerase, T4 DNA Ligase, SacI & HindIII, RNase pink bollworm (Pectinophora gossypiella) (Perlak et al., with BSA, T/A cloning kit (Fermentas, Blue Russia), 1990; Greenplate et al., 1995, 1997; Sharma et al., 2000). agarose gel extraction kit (Qiagen, USA), were used for DNA manipulation. * Corresponding author. Phone: + 98 (411) 3372256; Fax: + 98 (411) 3344798; E-mail: [email protected], Genomic DNA extraction. DNA extraction was performed [email protected] using “Genomic DNA isolation” protocol (Corbin et al., 260 M.S. Hejazi et al. 1994) with some modifications. Briefly, cells were pul- RNaseA for 1 h at 37 °C. The equal volume of chloro- verised in liquid nitrogen and then suspended in solution I form/phenol was added and centrifuged. The plasmid DNA [Tris 10 mM (pH 7.4), EDTA 1 mM, sodium dodecyl sul- present in the supernatant was precipitated using 2 volume phate (SDS) 0.5%, Proteinase K 0.1 mg/ml] and lysed by of absolute ethanol, and final washing was performed using incubation at 37 °C for 1 h. Then, the solution II [0.8 M 70% ethanol centrifuge. NaCl and 1% CTAB] was added to the lysates, and incu- In order to confirm the presence of the insert into bated at 65 °C for 20 min and genomic DNA extraction was pTZ57R/T vector, extracted plasmids were double digested done with equal volume of CHCl3-isoamylalcohol (24:1). with HindIII and SacI restriction enzymes. The extracted Nucleic acid was precipitated from the aqueous phase with plasmids were sequenced by Macrogen Co. 0.6 volume of isopropanol and purified using ethanol 70%. Software and websites for bioinformatics analysis. Authenticity of studied bacterium. In order to confirm The blast search (Blastx) engine at National Centre for the authenticity and purity of studied strain “S. luridus”, a Biotechnology Information (NCBI) (http://www.ncbi.nlm. single colony of the bacteria was isolated and grown. Then, nih.gov/Blast) was used to compare the identified the partial segment of 16S rDNA gene for about 1070 bp sequence in this study with homologous proteins with long was amplified using PCR technique in the presence of known structures. Also the “Protparam” tool from the specific StrepB (5´-AAGCCCTGGAAACGGGGT-3´) and ExPASy (Expert Protein Analysis System) website was used StrepF (5´-CGTGTGCAGCCCAAGACA-3´) primers (Rintala for analysing physicochemical parameters of protein. The et al., 2001). The amplified fragment cloned into pTZ57R/T Prosite, Prodom, Pfam, and Interproscan tools from the vector and sequenced (Macrogen Co., S. Korea). ExPASy website were used to distinguish the blocks, domains and protein family of this segment of cho proteins PCR amplification. In order to amplify cho encoding gene using alignment and comparison with known similar two oligonucleotide primers COF-4 (5´ ATCTACGGCAA- sequences and structures. CAACCACG 3´) and COR-5 (5´ ATGGTCACGAA(GC)GGGT TGA 3´) were designed. It should be noted that primer COF4 was a specific primer while primer COR5 was a RESULTS AND DISCUSSION degenerate one. These primers were designed on the basis of similarity within the DNA sequences of cho genes from Amplification and sequencing of 16S rDNA various Streptomyces species. Considering the location of In order to verify the identity of the studied strain as “S. designed primers on the selected genes, it was expected luridus”, a 1070 bp region of 16S rRNA gene was that the primers would amplify a fragment of cho with sequenced and aligned with the submitted 16S rDNA about 818 bp size. The PCR was performed in the reaction sequence from this strain of Streptomyces with AB184150 mixture (50 µl) containing 50 pmol each of COF-4 and accession number. The comparison result showed that the COR-5 primers, dNTPs at 0.2 mM each, 1.5 mM MgCl2, 0.5 amplified 16S rDNA segment had 100% similarity with the ng/µl S. luridus genomic DNA as the template DNA and 4 submitted 16S rDNA gene from S. luridus confirming the U Taq DNA polymerase. The reaction mixture was subject- authenticity of the studied strain S. luridus especially at ed to hot start amplification composed of 35 cycle of poly- 640, 800 and 1100 nucleotide positions (Rintala et al., merisation consisting of 1 min denaturation at 96 °C, 35 s 2001). annealing at 54 °C, and 45 s extension at 72 °C, followed by a final extension time of 72 °C for 10 min. PCR product of 818 bp corresponding to cho was isolated from agarose 1234 gel using the DNA agarose gel extraction kit after gel elec- trophoresis. DNA manipulation. The amplified DNA fragment was lig- ated into pTZ57R/T cloning vector using T/A cloning kit. E. coli strain DH5a cells were transformed with the ligation product using heat shock protocol. These bacterial cells had been made competent using CaCl2 and heat shock method. Colony screening was carried out using blue/white screen- 3000 bp ing method on LB agar medium in the presence of ampi- cillin (100 µg/ml), X-Gal and IPTG. Then, 5 white colonies were selected and cultured in 3-5 ml LB liquid medium that supplemented with 100 µg/ml ampicillin, and incubated for 1000 bp 12-16 h at 37 °C at 200 rpm. In order to extract plasmid DNA using alkaline lysis protocol, overnight culture (3 ml of LB) was centrifuged and the pellet was resuspended in the following buffer (50 mM glucose, 25 mM Tris-HCl, 10 mM 250 bp EDTA, pH 8.0), 200 µl of lyses buffer (0.2 N NaOH, 1% SDS) was added and held 5 min on ice. 300 µl of cooled (4 °C) precipitation solution (5 M potassium acetate 60 ml, glacial acetic acid 11.5 ml and water 28.5 ml) was added FIG. 1 -Double digestion results using SacI and HindIII to the mixture, inverted and kept on ice. The tubes were enzymes. Lane 1: DNA ladder (1 kb); lanes 2, 3 and 4: centrifuged and the supernatants were treated with the released DNA fragment with 818 bp size. Ann. Microbiol., 57 (2), 259-263 (2007) 261 Amplification and sequencing of cho encoding gene Sequences belonged to the vector backbone were from Streptomyces luridus removed and the results showed that the exact length of The PCR amplification of cho gene using designed primers the DNA is 818 bp encoding 272 amino acids.