Document Type : Brief Report
Department of Biology, Faculty of Sciences, University of Isfahan, P.O. Box 81746-73441, Isfahan, I.R. Iran
Actinomycetes have been known as producers of secondary metabolites including antibacterial and antifungal antibiotics, anticancer drugs, natural herbicides, and immunosuppressive agents (Challis and Hopwood, 2003; Paradkar et al., 2001). Streptomyces clavuligerus produces a number of b-lactam compounds, including cephamycin C, clavulanic acid and at least four other recognized clavam metabolites (Liras and Martin, 2006; Brown et al., 1976). Clavulanic acid is a clinically significant inhibitor of b-lactamases, while the other clavam metabolites produced by S. clavuligerus demonstrate weak antibacterial and antifungal activities (Chater, 2006; Tahlan et al., 2004). Several other Streptomyces spp. have also been determined as producer of clavulanic acid (Dawn et al., 2005; Jensen and Paradkar, 1999). b-lactams have been used extensively for treatment of various bacterial infections for more than half a century (Thykaer and Nielsen, 2003; Demain, 2000). Perhaps, commercial products such as AugmentinTM and TimentinTM are composed of clavulanic acid together with amoxycillin and ticarcillin, respectively (Jensen and Paradkar, 1999).
Antibiotic production in Strepomyces species is regulated by a variety of physiological and nutritional conditions and is also harmonized with morphological development of the organism (Paradkar et al., 1998). Antibiotic production is under the control of an array of regulatory signals that are organized in a hierarchical manner (Martin and Liras, 1989; Chater and Bibb, 1997). At the bottom of this hierarchy are the pathway-specific transcriptional regulators that are normally located within the antibiotic biosynthetic gene cluster (Paradkar et al., 1998).
The clavulanic acid gene cluster is situated immediately downstream of the cephamycin gene cluster, and together they form a super-cluster (Hodgson et al., 1995; Aidoo et al., 1994). The regulatory gene, claR, is located at the downstream end of this cluster (Mellado et al., 2002; Khaleeli and Townsend, 2000). The claR gene of S. clavuligerus encodes a LysR-type regulatory protein, ClaR, which controls clavulanic acid biosynthesis (Perez-Redondo et al., 1998). ClaR is a protein with 431 amino acids, which shows a significant degree of homology with several transcriptional activators of the LysR family. This protein contains two helix-turn-helix (HTH) motifs in the amino and carboxyl terminal regions. Amplification of the claR gene in multicopy plasmids results in a threefold increase in clavulanic acid production (Perez-Redondo et al., 1998).
In this work, two new recombinant constructs which carry the claR regulatory gene are presented. These vectors share a few distinguished features with their original plasmid. Their integration into the genomic DNA could be the most impressive one. These integrative vectors are really useful tools for site-directed mutagenesis and gene replacement strategies in different strains of Streptomyces.
For this purpose, Escherichia coli XL1-blue was used as a recipient for high-frequency plasmid transformation. E. coli strains were grown at 37°C on Luria-Bertani (LB) agar media (containing, per liter; 10 g of tryptone, 5 g of bacto-yeast extract, 10 g of NaCl and 17 g of agar; pH 7.5) supplemented with ampicillin (100 µg/ml), when required. E. coli competent cells were prepared by using the calcium chloride method (Sambrook and Russel, 2001) and subsequently transformed by the pMA::hyg plasmid. The clavulanic acid producing strains, S. clavuligerus DSM 738 and DSM 41826 (Reading and Cole, 1977) were used in this study. These strains were grown at 28°C on glucose yeast malt extract (GYME) media (containing per liter, 4 g of yeast extract, 4 g of glucose, 10 g of malt extract, 2 g of CaCO3 and 12 g of agar; pH 7.2). A suspension of Streptomyces spores was prepared in 20% (v/v) glycerol and stored at -20°C (Kieser, et al., 2000). Cultures for isolation of chromosomal DNA were prepared by inoculating 100 ml of yeast extract-malt extract medium (YEME; Kieser et al., 2000) with 100 ml of spore suspension. The YEME medium was supplemented with (per liter) 3 g of malt extract, 5 g of bacteriological peptone, 3 g of yeast extract, 10 g of glucose and 340 g of sucrose. Inoculated flasks were shaken on a rotary shaker at 120 rpm for 3 days, at 28°C (Reading and Cole, 1977). All media were sterilized by autoclaving at 121°C for 15 min. The ampicillin antibiotic was used for selection of the transformed colonies.
Genome extractions from the two S. clavuligerus strains DSM738 and DSM41826, cultured in YEME medium were carried out by using the cetyl trimethyl ammonium bromide (CTAB) method (Cullings, 1992). Forward and reverse primers were designed using Oligo® software (Version 5.0, Rychlik, 2007). The entire coding region of the claR gene was considered for primer selection. For the post-amplification procedure in the next steps, specially cloning of the claR gene in the vector of interest, restriction enzyme recognition sites were added to the 5´ ends of the designed primers. Finally the claR gene was amplified by PCR using the following oligonucleotide pair: forward claR2F (5´-ATTCTAGACGCTCAGCCGGACATCC-3´) and reverse claR2R (5´-AAGGATCCAGGAGAATCCGAAGAGC-3´). The restriction sites for XbaI and BamHI are underlined, respectively. The nucleotide concentrations for PCR amplification were adapted to the 70% GC content of Streptomyces. The PCR conditions were as follows: 3 µl of 10X PCR buffer without MgSO4 (200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA); 4 ml of MgSO4, 25 mM; 1.5 ml of dNTP mixture, 10 mM; 2 µl of pure dimethyl sulfoxide (DMSO); 0.75 ml Pfu polymerase, 2.5 u/ml; 1 ml of every up- and down-stream primer, 20 pM; 1 ml of Chromosomal DNA, 100 ng/ml (DSM738 and DSM41826), and up to 25 ml ddH2O. The amplification steps were as follows: hot start at 95°C for 5 min; 33 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, primer extension at 72°C for 4 min, and a final extension at 72°C for 15 min. In this main PCR reaction where claR2 primers were used, a 1500 bp claR gene fragment was produced (Fig. 1). For confirmation of this amplified fragment, RFLP-PCR was performed. Hence AluI was chosen in accordance with the restriction map of the claR gene sequence. AluI cuts the amplified claR fragment at position 726 and leaves two fragments with different sizes (726 and 774 pb). Restriction endonuclease digestion of DNA samples was carried out according to the manufacturer’s procedure (Fermentas, Germany). The integrity of the amplified fragment was then confirmed using nested PCR. The isolated claR fragment was then used as a template in a nested PCR reaction, whilst in the nested PCR reaction the nested primers claRnF and claRnR had been used (Fig. 2).
The amplified fragments in the main PCR reaction had preferred restriction sites for XbaI and BamHI. The presence of these sites was appropriate for the insertion of the fragment directly into the digested vector, pMA::hyg, following the double digestion procedure. pMA::hyg is a plasmid specifically designed for site-directed mutagenesis in Streptomyces, as it exists as a multicopy plasmid in E. coli, however, it does not have any origin of replication for Streptomyces. Therfore, this plasmid must be integrated into the genome in order to propagate. The multiple cloning site of this vector contains the recognition sites for BamHI, XbaI, SalI, PstI and HindIII. The two recognition sites for BamHI and XbaI are located adjacent to each other; therefore to double cut it properly, BamHI and XbaI digestion of this plasmid should be carried out separately. pMA::hyg was initially cut with BamHI and then gel purified. The single cut plasmid was then cut again with XbaI and subsequently gel purified. The double digested claR gene was directly ligated into the cut pMA::hyg plasmid using BamHI/XbaI restriction sites. DNA ligation was carried out using 1 unit (1 µl) of T4 DNA ligase according to supplier’s protocol. Eighty nano-grams of the new recombinant vector was added to E. coli competent cells and the transformation procedure was carried out according to the CaCl2 protocol (Sambrook and Russel, 2001). The recombinant colonies were selected according to their resistance to ampicillin. Finally, the presence of colonies on selective LBA media showed the success of the transformation procedure. To ensure the ligation of claR and the pMA::hyg vector and the success of their transformation, insert check analysis was carried out by using the colony-PCR method. So claR from the transformed E. coli cells was successfully amplified, indicating the presence of this gene in the new recombinant strains (data not shown). Recombinant vectors were isolated from transformed E. coli cells using the Holmes-Quigley method (Sambrook and Russel, 2001). Preparation of ligation mixture and transformation was performed separately for the two different claR fragments which were isolated from two different strains of S. clavuligerus. Two new recombinant vectors were initially confirmed and named as pFclaR (claR gene from S. clavuligerus DSM738 in the pMA::hyg vector) and pGclaR (claR gene from S. clavuligerus DSM41826 in the pMA::hyg vector). These new constructed vectors were then isolated from recombinant colonies. The main PCR reaction was repeated again using the constructed vectors and SmarTag polymerase. A 1500 bp fragment corresponding to the claR gene was obtained.
The whole amplified fragment from just S. clavuligerus DSM 41826 was then fully sequenced. DNA sequencing was performed by using the ABI system. The identified DNA sequence was initially compared with all known DNA sequences by using the Blast program and more detailed comparison was performed by using the Blast2seq program. The Blast program by itself indicated sequence similarity between the claR gene from S. clavuligerus DSM 738 and that of S. clavuligerus DSM41826. Finally, sequencing analyses confirmed that claR was successfully amplified and inserted into the vector (in strain DSM41826). It was also free from any unwanted mutation which might interfere with appropriate gene expression.
It has been shown that the ClaR protein is a transcriptional regulator of the late steps in clavulanic acid production in S. clavuligerus (Perez-Redondo, 1998). Amplification of the claR gene in multicopy plasmids results in a threefold increase in clavulanic acid production and in a five- to six fold increase of alanylclavam biosynthesis, whereas cephamycin production is significantly reduced (Hung et al., 2006). Paradkar, et al., in 1998 excised a 1.9 kb BglII fragment located immediately downstream from orf-7 from the cosmid K6L2 (Aidoo et al., 1994) and subcloned it into the sequencing vectors pUC119 and pUC118. Both strands of this fragment were then sequenced. The results indicated were a complete ORF, designated as claR. The sequence data, related to the claR gene from S. clavuligerus DSM738 of this study, have been submitted to the DDBJ/EMBL/GenBank databases under the accession number U87786.
Isolation, confirmation, structural determination and cloning of the claR gene were the main targets of this study. The claR regulatory gene was isolated from two strains of S. clavuligerus (DSM 738 and DSM 41826) and cloned into pMA::hyg, a multicopy vector. The correct structure of each new construct was completely confirmed using colony PCR, Nested-PCR and RFLP-PCR. Sequencing analysis of the claR gene revealed that these genes were amplified and subcloned free from any mutation which is essential for correct expression of the gene. In addition, the sequence of the claR gene from S. clavuligerus DSM 41826 was determined for the first time in this study and will be submitted to the DDBJ/EMBL/GenBank databases in the near future.
Moreover, the pMA::hyg vector has notable characteristics as a unique practical tool in Streptomyces molecular studies (Fig. 3). The newly constructed vectors in this study also share those features; such as functioning like a shuttle vector, being a multicopy plasmid in E. coli, and an integrative plasmid in Streptomyces. Moreover, they are extremely appropriate tools for site-directed mutagenesis and gene replacement strategies. They also contain the chloramphenicol resistant gene CLA (Fig. 3). The presence of the hygromycin resistant hyg gene (Fig. 3) in pMA::hyg in addition to the ampicillin resistant BLA gene (Fig. 3) also makes it an efficient system for eukaryotic gene expression studies. In future studies, production of different mutant forms of claR can be carried out using these constructs. The new and mutant forms of the claR gene could be then be used to transform Streptomyces using gene replacement strategies (via these new constructs).
This study was performed at the University of Isfahan and financially supported by the Graduate Studies Offices.