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1 Department of Biological Engineering, Inha University, Incheon 402-751, Korea
2 Life Sciences Institute and Departments of Medicinal Chemistry, Chemistry, and Microbiology & Immunology, University of Michigan, Ann Arbor, MI 48109-2216, USA
Correspondence
Eung-Soo Kim
eungsoo{at}inha.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Hopwood, 1988; Strauch et al., 1991; Myles, 2003; Hranueli et al., 2005). It has been well documented that secondary metabolite production generally occurs at the onset of the stationary phase of growth of Streptomyces spp. and correlates temporally with the formation of aerial mycelium in cultures grown on the surface of solid media (Chater & Bibb, 1997; Martin et al., 2000; Bibb, 2005). Previous studies have revealed that the regulation of secondary metabolite production in Streptomyces spp. involves a complex regulatory network in response to nutritional and environmental factors, operating at several layers of control. Some of these affect only antibiotic production, whereas others affect both antibiotic production and morphological differentiation, suggesting that the two processes share some elements of genetic control (Chater & Bibb, 1997), while others are unique.
Among this regulatory network, the genes working at the proximal level usually reside within the respective biosynthetic gene cluster and are pathway-specific regulatory genes that only affect a single secondary metabolite biosynthetic pathway. The best-characterized pathway-specific regulatory proteins, including ActII-ORF4 for actinorhodin biosynthesis from Streptomyces coelicolor A3(2) (Arias et al., 1999; Wietzorrek & Bibb, 1997) and DnrI for doxorubicin biosynthesis from Streptomyces peucetius (Sheldon et al., 2002), belong to the so-called Streptomyces antibiotic regulatory proteins (SARPs) family (Wietzorrek & Bibb, 1997). These regulatory proteins contain a unique OmpR-like DNA-binding domain that is quite different from the typical domain with a helix–turn–helix (HTH) motif (Sheldon et al., 2002; Wietzorrek & Bibb, 1997). Unlike the SARP family (typically between 277 and 665 aa), another important, less-studied transcriptional family of regulators has recently been found in several macrolide antibiotic pathways (De Schrijver & De Mot, 1999), which are relatively large in size (872 to 1 159 aa) and have an N-terminal ATP-binding domain represented by discernible Walker A and B motifs (Walker et al., 1982) and a C-terminal LuxR-type DNA-binding domain (Henikoff et al., 1990). Regulators belonging to this so-called LAL (large ATP-binding regulators of the LuxR) family, whose prototype member is the Escherichia coli MalT, involved in the uptake and catabolism of maltodextrins (Richet & Raibaud, 1989), have been identified and characterized in several macrolide antibiotic pathways, including PikD for pikromycin from Streptomyces venezuelae (Wilson et al., 2001), RapH for rapamycin from Streptomyces hygroscopicus (Aparicio et al., 1996; Molnár et al., 1996) and NysRI/RIII for nystatin from Streptomyces nouresi (Brautaset et al., 2000).
Tautomycetin (TMC), a secondary metabolite produced by Streptomyces sp. CK4412, is a novel activated T-cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene, whose chemical structure is identical to a previously reported antifungal compound produced by Streptomyces griseochromogenes (Cheng et al., 1989). Inhibition of T cell proliferation by TMC was observed at concentrations 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A (CsA) (Shim et al., 2002). Due to its promising characteristics, TMC, whose mechanism of action is different from CsA or FK506, has been identified as a new drug candidate with potent T-cell-specific immunosuppressive activity (Shim et al., 2002). Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412 originating from Jeju Island, Korea, and demonstrated its identity by gene disruption analysis (Choi et al., 2007). The TMC biosynthetic gene cluster revealed two ORFs encoding a typical modular polyketide synthetase (PKS) gene as well as 12 ORFs located at both flanking regions, the deduced functions of which were consistent with TMC biosynthesis (Fig. 1).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) (Shim et al., 2002). The strain was cultivated at 28 °C in either R2YE or YEME liquid medium (Kieser et al., 2000). E. coli DH5
was used for DNA cloning and plasmid propagation. E. coli XL-1 Blue MR was used for cosmid library construction. E. coli ET12567/pUZ8002 (dam2 dcm2 hrdM) was used as the transient host for E. coli–Streptomyces conjugation (Choi et al., 2004). All E. coli strains were cultured at 37 °C in Luria broth or on Luria agar, supplemented with the appropriate antibiotics when needed (Kieser et al., 2000).
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) (Choi et al., 2007). DNA sequences for tmcN were assembled using BLAST searches on the National Center for Biotechnology Information (NCBI) server, and the ORF was subsequently predicted using the web-based program FramePlot 2.3.2 (http://www.nih.go.jp/
jun/cgi-bin/frameplot.pl).
Construction of a tmcN mutant via chromosomal gene disruption.
The tmcN gene encoding a putative LuxR family regulator with an ATP-binding site located downstream of the PKS tmcB gene was inactivated using a PCR-targeted gene-disruption system (Gust et al., 2003). An apramycin-resistance gene/oriT cassette for the replacement of tmcN was amplified using the pIJ773 as a template (Gust et al., 2003) using the following primers: forward primer (5'-tcagatccgtctcgcttcg tgtgctcgtcaccgtgccgttATTCCGGGGATCCGTCGACC-3') and reverse primer (5'-acgaatatcgccggcatctgaccacgttcgaggtcacggtTGTAGGCTGGAGCTGCTTC-3'). The lower-case type represents 40 nt homologous extensions to the DNA regions inside tmcN. This cassette was introduced into E. coli BW25113/pIJ790 containing pTMC2290. Gene replacement in tmcN was confirmed by restriction analysis of the mutated pTMC2290 (pTMC2290
tmcN). pTMC2290tmcN was introduced into Streptomyces sp. CK4412 by conjugation from E. coli ET12567/pUZ8002. After incubation at 28 °C for 16 h, each plate was overlaid with 1 ml sterile water containing apramycin at a final concentration of 50 mg ml–1 and nalidixic acid at a final concentration of 25 mg ml–1. Incubation was continued at 28 °C until conjugants appeared. The double-crossover exconjugants were selected using a standard AprR/KanS method (Gust et al., 2003), followed by the confirmation of both Streptomyces sp. CK4412 and Streptomyces sp. CK4412-002 (tmcN mutant of Streptomyces sp. CK4412) genomic DNAs by both PCR and Southern blot hybridization. Three different PCR primers for the confirmation of the double-crossover recombinants were tmcN test primer F (5-AAGCTTAGTCGTCGTGGGTCGCCGGGCTGGACA-3), tmcN test R (5'-AAAGCGTCATCCTGCTCAA-3) and oriT test primer F (5'-GAATTCAGCGTGACATCATTCTGTGG-3'), which is in the aprR/oriT cassette. (Fig. 3a).
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C31, attP); the neomycin-resistance gene originating from pFDNEO-S plasmid (Denis & Brzezinski, 1991) was cloned into the EcoRV restriction site in pSET152 (Bierman et al., 1992). Finally, neomycin was used for selection of the recombinant Streptomyces sp. CK4412-002/tmcN– strain, which was already resistant to apramycin due to tmcN gene disruption.
Isolation of total RNA and gene expression analysis by RT-PCR.
Streptomyces sp. CK4412 and Streptomyces sp. CK4412-002 were grown for 72 h in R2YE medium, and the cultures were washed twice with 1 vol. sterile water. The mycelia were subsequently harvested by centrifugation and immediately frozen by immersion in liquid nitrogen. The frozen mycelia were then broken by shearing in a mortar, and the frozen lysate was added to buffer RLT (Qiagen) in the presence of 1 % β-mercaptoethanol. RNeasy mini spin columns were used for RNA isolation according to the manufacturer's instructions. RNA preparations were treated with DNase I (Qiagen) to eliminate possible chromosomal DNA contamination.
DNase I-treated RNA (7 µg) was used as a template for reverse transcription at 50 °C with an AVM Reverse Transcriptase XL (TaKaRa) and random hexamers. The conditions for cDNA synthesis were as follows: 30 °C for 10 min, 50 °C for 1 h, 99 °C for 2 min, 5 °C for 5 min. The resulting cDNA was used for PCR amplification under the following conditions: 25 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 68 °C for 35 s. Each primer pair of TMC biosynthetic genes was carefully designed to generate a PCR product of approximately 150 to 200 bp using a genscript site (http://www.genscript.com/ssl-bin/app/primer). The complete RT-PCR primer pair sequences are listed in Supplementary Table S1; the RNA samples that had not been subjected to RT reaction were used for PCR as negative controls.
HPLC quantification and antifungal bioassay for TMC.
For HPLC analysis, culture broth supernatants were extracted with equal volumes of chloroform. The extracts were dried using a rotoevaporator and then resuspended in methanol. Extracts were fractionated by HPLC using isocratic conditions of methanol/water/buffer (1 % diethylamine/formic acid, pH 7.3) 75 : 15 : 10 on a Genesis C18 4 µm column and with UV detection at 273 nm. TMC production was also evaluated by a biological assay against Aspergillus niger, using a paper disc containing the same culture broth extract as used in the HPLC assay. The paper disc was placed on top of A. niger that had been incubated on ME medium (0.05 % malt extract, 0.05 % glucose, 0.001 % peptone in 1 l double-distilled H2O) for 6 h at 30 °C, followed by measurement of the inhibition zone after overnight incubation at 30 °C.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), often found in secondary metabolite gene clusters of Gram-positive bacteria (Haydock et al., 2005). Further characterization of the tmcN gene product via database-assisted in silico analysis revealed that it encodes a protein of 1 029 aa and shows 33 % identity at the amino acid level to ThcG, a regulator of the LuxR family with an ATP-binding site in Rhodococcus erythropolis (De Schrijver & De Mot, 1999), 35 % identity to a LuxR regulatory protein from Frankia sp. EAN1pec (Copeland et al., unpublished data) and 30 % identity to a putative transcriptional regulator from Streptomyces ambofaciens (Choulet et al., 2006).
Notably, the predicted amino acid sequence of TmcN shows two highly conserved domains: a putative HTH motif in the C-terminal region typically found in various bacterial DNA-binding proteins (Pabo & Sauer, 1992) and Walker A and B nucleoside triphosphate binding motifs at the N-terminal region (Walker et al., 1982; Fig. 2). The latter have also been found in polyketide biosynthetic regulatory proteins, including PikD for pikromycin from S. venezuelae (Wilson et al., 2001), RapH for rapamycin from S. hygroscopicus (Aparicio et al., 1996; Molnár et al., 1996) and NysRI/RIII for nystatin from S. nouresi (Brautaset et al., 2000). Since all these proteins were previously assigned to the LAL-family (Demain & Fang, 1995), tmcN is also presumed to encode a LAL-family pathway-specific regulatory protein involved in TMC biosynthesis from Streptomyces sp. CK4412. Interestingly, however, TmcN exhibited relatively low amino acid identities to the following Streptomyces LAL-type regulators: 17 % with PikD, 16 % with RapH and 17 % with NysRI/RIII. In addition, tmcN contains two rare TTA leucine codons (codon 81, codon 375), which indicates its dependence on bldA, the structural gene for tRNAUUA. This may be a further indication of a regulatory role of TmcN in TMC biosynthesis, since most of the known TTA-containing genes specify regulatory or resistance proteins associated with antibiotic biosynthesis gene clusters (Leskiw et al., 1991; Li et al., 2007).
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tmcN, which was introduced into Streptomyces sp. CK4412 by conjugative gene transfer (Fig. 3a). Construction of the tmcN mutant (named Streptomyces sp. CK4412-002) generated by PCR-targeted disruption was confirmed by both PCR and Southern hybridization analysis. The expected 1.43 kb PCR-amplified band was observed in genomic DNA samples isolated from Streptomyces sp. CK4412, and a band of the expected size (1.53 kb) was observed in genomic DNA samples isolated from Streptomyces sp. CK4412-002 (Fig. 3b). A PCR-amplified fragment of the expected size (0.47 kb), amplified using an alternative PCR primer pair designed to detect an apramycin resistance gene/oriT cassette, was observed only in the Streptomyces sp. CK4412-002 (Fig. 3b). Moreover, two different theoretically calculated BamHI digestion patterns were observed in Southern hybridization with Streptomyces sp. CK4412 and Streptomyces sp. CK4412-002 (Fig. 3c), implying that tmcN was specifically disrupted as expected.
Loss of TMC production by tmcN disruption and rescue by tmcN complementation
Fermentation broths of both Streptomyces sp. CK4412 and Streptomyces sp. CK4412-002 grown under conditions optimal for TMC production were extracted with chloroform, followed by an antifungal bioassay and HPLC quantification for the presence of TMC. Although very weak antifungal activity was detected, presumably due to either residual TMC or some other unconfirmed metabolites produced by Streptomyces sp. CK4412, the significantly reduced antifungal activity against A. niger, as well as the absence of TMC in extracts of Streptomyces sp. CK4412-002 under the same culture conditions, provide strong evidence that tmcN plays an essential regulatory role in TMC biosynthesis (Fig. 4).
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), into which the coding region of tmcN and 144 bp of the DNA region upstream of the putative translational start point was cloned, resulting in pSETNEOtmcN. Also, a neomycin-resistance gene, originated from plasmid pFDNEO-S (Denis & Brzezinski, 1991), was inserted into the multiple cloning site (MCS) of parental pSET152 to select the recombinant strains bearing complementation plasmids. Both HPLC and the bioassay confirmed that TMC productivity and antifungal activity were restored to significant levels in the Streptomyces sp. CK4412-002 mutant strain carrying pSETNEOtmcN (Fig. 4), implying that the absence of TMC productivity from Streptomyces sp. CK4412-002 strains was due to a lack of a TMC-specific positive regulatory gene, tmcN. Moreover, the wild-type Streptomyces sp. CK4412 strain containing an extra copy of tmcN via chromosomal integration of pSETNEOtmcN led to an approximately 5.5-fold increase in TMC biosynthesis (Fig. 4a).
Transcriptional control of TMC biosynthetic pathway genes
Total RNA samples were prepared from the Streptomyces sp. CK4412 wild-type and the Streptomyces sp. CK4412-002 mutant after 72 h of growth and used as a template for gene expression analysis by RT-PCR. Primers for RT-PCR were specific to sequences within tmc genes (Table 2) and were designed to produce cDNAs of approximately 200 bp. A primer pair designed to amplify a cDNA of the rRNA gene was used as an internal control. Transcripts were analysed from the 14 genes located within the tmc cluster, including tmcN, after 25 PCR cycles. This analysis was carried out at least three times for each primer pair. In the RT-PCR analysis, the transcripts of all 14 genes were detected in the Streptomyces sp. CK4412 wt, while the transcription pattern in the Streptomyces sp. CK4412-002 was significantly reduced for most of the tmc genes within the cluster, except for tmcN (Fig. 5). Interestingly, the transcripts of the putative PKS operon containing the translationally coupled genes tmcA and tmcB were apparently much less affected by the absence of the tmcN gene in the Streptomyces sp. CK4412-002 (Fig. 5). The absence of the tmcM transcript is believed to be a polar effect derived from tmcN disruption in the Streptomyces sp. CK4412-002 due to translational coupling of these two genes, even though the PCR-targeting method is designed to limit this effect. Real-time RT-PCR analysis also confirmed that the absence of tmcN reduced transcripts of the PKS operon less significantly (only 33 % reduction), while both flanking operons were severely affected by the absence of tmcN (88 % reduction for tmcC operon and 63 % reduction for tmcJ operon, respectively) (see Supplementary Fig. S1, available with the online version of this paper). These results strongly suggest that TmcN might activate all three putative TMC operons at different levels, either directly or indirectly. Taken together, these data demonstrate that tmcN encodes a LAL-family pathway-specific activator of the TMC biosynthetic pathway in Streptomyces sp. CK4412.
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; Aparicio et al., 1996; Molnár et al., 1996; Brautaset et al., 2000), and the study described in this report has revealed that TmcN exerts its effects at different levels on individual operons in this large biosynthetic gene cluster. Gene disruption and complementation studies have confirmed its role as a positive regulator, and the ability to increase production of this important metabolite by adding an additional copy of tmcN into the chromosome demonstrates the value of this strategy for strain improvement. Currently, one of the key factors for further development of TMC as a medicinal agent is the limited amount of this natural product available through fermentation methods. Thus, this initial study of TmcN paves the way for further analysis of this growing class of regulatory factors and offers a new approach to improving access to TMC for detailed biological studies, target identification and analogue development.
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ACKNOWLEDGEMENTS |
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Edited by: P. R. Herron
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REFERENCES |
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Received 24 March 2008;
revised 3 July 2008;
accepted 3 July 2008.
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