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Journal of Bacteriology, January 2003, p. 592-600, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.592-600.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Accumulation of S-Adenosyl-L-Methionine Enhances Production of Actinorhodin but Inhibits Sporulation in Streptomyces lividans TK23

Dong-Jin Kim,1 Jung-Hyun Huh,1 Young-Yell Yang,1 Choong-Min Kang,1 In-Hyung Lee,2 Chang-Gu Hyun,1 Soon-Kwang Hong,1 and Joo-Won Suh1*

Institute of Bioscience and Biotechnology and Department of Biological Science, Myongji University, Yongin 449-728,1 Food and Life Science Major, School of Techno Science, Kookmin University, Seoul 136-702, Korea2

Received 19 June 2002/ Accepted 2 September 2002


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ABSTRACT
 
S-Adenosyl-L-methionine synthetase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. Despite extensive research with many organisms, its role in Streptomyces sp. remains unclear. In the present study, the putative SAM-s gene was isolated from a spectinomycin producer, Streptomyces spectabilis. The purified protein from the transformed Escherichia coli with the isolated gene synthesized SAM from L-methionine and ATP in vitro, strongly indicating that the isolated gene indeed encoded the SAM-s protein. The overexpression of the SAM-s gene in Streptomyces lividans TK23 inhibited sporulation and aerial mycelium formation but enhanced the production of actinorhodin in both agar plates and liquid media. Surprisingly, the overexpressed SAM was proven by Northern analysis to increase the production of actinorhodin through the induction of actII-ORF4, a transcription activator of actinorhodin biosynthetic gene clusters. In addition, we found that a certain level of intracellular SAM is critical for the induction of antibiotic biosynthetic genes, since the control strain harboring only the plasmid DNA did not show any induction of actII-ORF4 until it reached a certain level of SAM in the cell. From these results, we concluded that the SAM plays important roles as an intracellular factor in both cellular differentiation and antibiotic production in Streptomyces sp.


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INTRODUCTION
 
Streptomycetes are gram-positive free-living soil bacteria that produce a wide variety of secondary metabolites, including >70% of all known antibiotics (22). They are also characterized by morphological differentiation, manifested by the formation of aerial mycelium on a solid medium (7, 8, 17). The activation of antibiotic production is often coupled to morphological development, although the multiple and coordinated regulation of secondary metabolism is poorly understood (5, 9).

Streptomyces coelicolor is an excellent model system for studying the regulation of antibiotic production, as well as morphological differentiation, since its genetic background is well characterized with its whole genome sequence identified (1, 31). It produces at least four distinctive antibiotics, of which the blue-pigmented polyketide actinorhodin and red-pigmented undecylprodigiosin are produced in stationary phase of cell growth (10). The biosynthetic gene clusters for these antibiotics have been isolated and that for actinorhodin (the act cluster) has been particularly well characterized (26). Although much progress has been made in elucidating both the gene clusters of antibiotic biosynthesis and their regulations (6), little is known about the physiological and metabolic signals that trigger antibiotic production and/or morphological differentiation. However, it has been reported that ppGpp [GDP 2'(3')-diphosphate] acts as a positive regulator that triggers the onset of actinorhodin production through the activation of actII-ORF4 transcription in S. coelicolor (4). In addition, Hara et al. reported that A-factor (2-isocapryloyl-R-hydroxy-methyl-{gamma}-butyrolactone) served at a very low concentration as a multifunctional signaling molecule, which induced streptomycin production and sporulation, as well as the enhancement of self-resistance to streptomycin (14, 15).

In Streptomyces lividans, a species closely related to S. coelicolor, all of the genetic components for actinorhodin biosynthesis have also been identified, but actinorhodin is not produced under usual growth conditions (32). Because of this trait, the focus of study in this strain has been on how actinorhodin biosynthesis is initiated and regulated.

In the course of studying the gene cluster and the mechanism for the biosynthesis of the aminoglycoside antibiotic spectinomycin in Streptomyces spectabilis, which is another relative of S. lividans, we isolated a genomic region that included the putative S-adenosyl-L-methionine synthetase (SAM-s) gene. Since spectinomycin has two methyl groups and SAM-s may provide SAM as a methyl donor, we were interested in whether the putative SAM-s gene could affect antibiotic production. SAM synthesized from methionine and ATP via SAM-s plays important roles in the primary and secondary metabolism of the cell (37). It is well known that it functions as a methyl or methylene donor in many cellular reactions and as a precursor for the synthesis of spermidine (37). Recent studies have revealed its novel functions, such as an intracellular factor involved in morphological differentiation in both eukaryotic and prokaryotic organisms (16, 28, 29). However, it is not known whether SAM is involved in cellular differentiation of Streptomyces sp.

Based on the fact that SAM could function as a signal molecule in cellular differentiation, in addition to its role as a methyl group donor, we tested whether SAM could affect morphological and physiological differentiations in S. lividans. The present report describes the finding that SAM provided by either the overexpression of SAM-s from S. spectabilis or the exogenous addition can lead to actinorhodin production as well as morphological differentiation in S. lividans. In addition, we here show that SAM itself enhances actinorhodin production through activating a transcriptional factor, actII-ORF4, thereby increasing the expression of genes in the actinorhodin biosynthetic gene clusters.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. Escherichia coli DH5{alpha}F' and E. coli BL21 were used as hosts for plasmid manipulation and protein expression, respectively. pGEM-7zf, pBluescript KS(+) and pUC19 plasmids were also used for routine subcloning work (34). The S. lividans TK23 (spc-1 SLP2- SLP3-) strain was used as the host for the manipulation of Streptomyces-E. coli shuttle vector pWHM3 (18, 41). The SAM-s gene was isolated from S. spectabilis ATCC 27741. The Streptomyces and E. coli strains and the plasmids used are listed in Table 1.


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  		TABLE 1. Strains and plasmids used in this study

Culture conditions. E. coli DH5{alpha}F' was maintained on Luria-Bertani (LB) agar plates and cultured in LB broth at 37°C with agitation. Procedures for the manipulation of Streptomyces and general recombinant DNA manipulation have been described elsewhere (18, 34). Protoplasts of S. lividans TK23 were transformed by the procedure of Hunter (19). The antibiotic thiostrepton was used at a concentration of 50 µg/ml in agar plate cultures and at 10 µg/ml in broth cultures. The actinorhodin concentration was determined as described by Liao et al. (25). The mycelium cell paste was dried at 70°C to determine the dry cell weight as an indication of growth. The actinorhodin production medium was previously described by Liao et al. (25) and supplemented with 5% glycerol and 0.6 mM phosphate (24). R2YE broth was made according to the recipe of Hopwood et al. (18). Fifty milliliters of the inoculated medium in a 250-ml baffled flask was cultured at 28°C by using a rotary shaker (250 rpm), unless specified otherwise.

To test the effect of the exogenous SAM treatment, S. lividans protoplast cells were treated four times with 1 µmol of SAM at 12-h intervals on R1R2 agar media and observed for their relative effects on antibiotic production after 3 and 5 days.

Isolation of SAM-s gene from S. spectabilis. A cosmid library of S. spectabilis genomic DNA was constructed in pDW103, an E. coli-Streptomyces shuttle cosmid (20). Each of 1,000 ampicillin-resistant clones from the genomic library was separately inoculated into 1.5-ml Eppendorf tubes containing 500 µl of LB broth and ampicillin (100 µg/µl). After overnight growth, 30 µl from 50 distinct tubes was combined in a new 1.5-ml tube to give a total of 20 samples. The purified plasmid DNA from each of these 20 samples was suspended in 100 µl of distilled water and submitted to PCR screening. The primers for this screen were SAM-F (5'-CGXCTXTTTACCTXGAGTC-3') and SAM-R (5'-TGCCXGTGAAGCCGCCGGCGTG-3'), which were designed to amplify a 1.1-kb cassette harboring the partial SAM-s gene. These degenerate PCR primers were designed based on the similarities found among Mycobacterium tuberculosis (AE007015), Bacillus subtilis (AF008220), Staphylococcus aureus (U36379), and E. coli (AP002563). Sample 4 revealed a positive PCR fragment of the appropriate size, and the subsequent second PCR screen of these 50 samples revealed a single positive clone. By successive Southern hybridization and subcloning experiments, a 4.0-kb BamHI fragment that contained the SAM-s gene was isolated and cloned into E. coli plasmid pBluescript SK(+) to give pBle-SAM-s.

Assay of SAM-s activity. The 1.2-kb fragment containing the SAM-s gene was generated by PCR with the forward primer 5'-AAGCTTAACCACAGGGAG-3' and the reverse primer 5'-CTGACGCACTGACCTACC-3' from the cosmid vector. The PCR product (1.2 kb) was excised by HindIII and BamHI and then cloned into the pET28a expression vector to generate pETmetK. The plasmid pETmetK was then introduced into E. coli BL21. This strain was treated with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) to induce expression of the metK gene and grown overnight at 37°C in LB medium containing 100 µg of kanamycin/ml.

Cells for the enzyme preparation were grown with aeration at 37°C in LB medium to stationary phase. A total of 20 mg of harvested cells was resuspended in 20 ml of 100 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA. Lysozyme was added to 50 µg/ml, and the suspension was incubated at room temperature for 30 min. After phenylmethylsulfonyl fluoride was added to a final concentration of 0.1 mM, the cells were lysed by sonication in an ice bath, and the lysate was centrifuged for 15 min at 12,000 rpm. A 10-ml portion of supernatant containing the soluble His-tagged MetK protein was loaded on a nickel chromatography column, and 1 ml of purified protein solution was obtained. The 10 ml of the protein solution was mixed with reaction mixture.

The assay mixture contained (in a 1-ml final volume [pH 8.0]) 100 mM Tris-HCl, 200 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 5 mM ATP, 5 mM L-methionine, and 10 to 50 µl of enzyme solution. A blank control was prepared simultaneously without methionine. The reactions were carried out at 30°C for 30, 60, or 120 min and were stopped by the addition of 1 ml of cold 6% HClO4 or 50 mM EDTA. The mixture was centrifuged, and the supernatant was neutralized with KOH (~200 µl).

This solution was analyzed by thin-layer chromatography (TLC) by using a silica gel plate which was developed by using n-butanol-acetic acid-H2O (60:15:25). The reaction mixture was also characterized by high-performance liquid chromatography (HPLC) on a C18 reversed-phase HPLC column with a gradient elution system. Mobile phase A was 0.1 M NaH2PO4 acetonitrile (98:2 [vol/vol]) and contained 8 x 10-3 M octanesulfonic acid sodium salt (OSA). The pH was adjusted to 2.65 with 10.5 ml of 3 M H3PO4. It was usually filtered under a vacuum with a 0.45-µm-pore-size Millipore HA filter before the addition of acetonitrile, followed by mixing with acetonitrile and degassing in an ultrasonic bath. Mobile phase B was a mixture of 0.15 M NaH2PO4 and 260 ml of acetonitrile and contained OSA at 8 x 10-3 M. It was filtered under a vacuum with a 0.5-µm-pore-size Millipore FHUP filter and kept for 10 min in an ultrasonic bath before use. A linear gradient was used starting with 85% of eluent A and 15% of eluent B and ending with final concentrations of 30% eluent A and 100% eluent B.

Electron microscopy. The spores and hyphae of S. lividans TK23 strain were observed by scanning electron microscopy after grown for 5 days on R1R2 agar medium (19, 25). For the preparation of the specimens, agar blocks were fixed with 1% osmium tetroxide for 12 h and then dehydrated by freeze-drying. Each specimen was sputter coated with platinum-gold and examined under a Hitachi S4000 scanning electron microscope.

Assay of actinorhodin. Actinorhodin production medium contained the following: glycerol, 50 g; glutamic acid, 5 g; morpholinepropanesulfonic acid, 21 g; MgSO4 · 7H2O, 200 mg; CaCl2 · 2H2O, 100 mg; NaCl, 100 mg; KH2PO4, 82 mg; FeSO4 · 7H2O, 9 mg (per liter); and trace elements at a final pH of 6.5 (18, 25). Fifty milliliters of the medium was contained in a 250-ml baffled flask and incubated at 28°C at 250 rpm. The medium was inoculated with spores and mycelia from the R2YE agar plate cultures of the recombinant strains of S. lividans TK23. To prepare vegetative inoculate, the cells from a R2YE agar were added to 50 ml of R2YE medium in a 250-ml baffled flask. The cultures were incubated for 7 days at 28°C at 250 rpm; the mycelia obtained by centrifugation were washed with distilled water, resuspended in the original volume of water, and used to inoculate the production medium. Actinorhodin content and growth were determined by the method of Liao et al. (25).

Northern analysis for metK and actII-ORF4. The isolation of total RNA from Streptomyces and Northern analysis were described in detail elsewhere (18). RNA was isolated from S. lividans liquid cultures at the following five time points: lag phase, early log phase, log phase, late log phase, and stationary phase. The RNA concentration was determined based on UV absorbance at 260 nm and verified by determining the amount of 5S RNA. The probe for Northern analysis of the actII-ORF4 gene was generated from the S. lividans chromosome by PCR with the forward primer 5'-GGCGCAGATGAGATTCAACTTATT-3' and the reverse primer 5'-CTACACGAGCACCTTCTCACCGTT-3'. The 790-bp PCR product contained the upstream region (7 bp) of the structural gene actII-ORF4, actII-ORF4 (767 bp), and the downstream region (16 bp). Amplified PCR product was then digested with NaeI, and the resulting 224-bp fragment contained the upstream region (7 bp) of the structural gene. The fragment was labeled with [{gamma}-32P]ATP (3,000 Ci/mmol; DuPont-NEN) by using a hexanucleotide priming kit (Amersham) and used for Northern analysis.

Nucleotide sequence accession number The nucleotide sequence of the SAM-s gene has been submitted to the GenBank database and assigned accession no. AF117274.


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RESULTS
 
Cloning of the SAM-s gene from S. spectabilis. Recent research on B. subtilis and Schizosaccharomyces pombe has shown that the intracellular level of SAM affects their sporulation processes (16, 29). Since it had not been known whether SAM was involved in cellular differentiation in Streptomyces spp., we wanted to test the possibility that the change in the intracellular level of SAM in S. lividans affects its sporulation.

To address the possible role(s) of SAM, we first amplified the DNA fragment corresponding to the SAM-s gene from S. spectabilis by PCR with two oligonucleotide primers designed from well-conserved amino acid sequences present in the many known SAM-s genes. A 3.9-kb genomic DNA fragment that hybridized with the PCR fragment was subcloned and sequenced. The deduced amino acid sequence of an open reading frame showed significant homology to the many known SAM-s genes, as shown in Fig. 1. The highest similarity was to the metK gene of its close relative S. coelicolor and M. tuberculosis, with 75% identity. It also contained major domains that are considered important for the biochemical functions of SAM-s.



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  		FIG. 1. Amino acid sequence alignments of MetK proteins. Amino acid sequences, shown in single-letter code, were aligned by using programs available online (http://prodes.toulouse.inra.fr/multalin/multalin.html and http://www.ncbi.nlm.nihgov/PubMed/). The amino acid sequences are from S. spectabilis (Ss), S. coelicolor (Sc), M. tuberculosis (Mt), B. subtilis (Bs), S. cerevisiae (Se), and Homo sapiens (Hs). The solid underline indicates putative ATP-binding sites, and the dotted underline indicates two putative metal-binding sites.

In order to examine whether the clone encodes SAM-s, the 1.2-kb PCR fragment was cloned and expressed in E. coli. The gene product, a 40-kDa protein was purified from E. coli by using nickel column chromatography. As shown in Fig. 2A, when it was incubated with ATP and methionine, the purified 40-kDa protein produced in vitro a substance which had the same Rf value as chemically synthesized SAM. In contrast, the reaction mixture with the cell extracts from E. coli harboring only plasmid or just buffer did not bring forth any peaks corresponding to SAM. When the reaction mixture was analyzed through HPLC, the peak corresponding to SAM was found only when the purified protein was added to the reaction mixture with ATP and methionine (Fig. 2B). Along with the TLC analysis, these results strongly indicated that the cloned PCR fragment from S. spectabilis encoded SAM-s, which catalyzed the synthesis of SAM from ATP and methionine. Based on the results presented here and the high degree of similarity with many known SAM-s genes in other organisms, we named the isolated gene metK.



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  		FIG. 2. The isolated gene product has SAM-s activity in vitro. When incubated with ATP and L-methionine, the protein purified from E. coli catalyzed the production of SAM. (A) TLC analysis of the reaction products. Lanes: 1, standard synthetic SAM; 2, reaction product with purified SAM-s, ATP, and L-methionine; 3, reaction product with E. coli cell extract harboring vector without SAM gene; 4, reaction product with ATP and L-methionine. The black arrow indicates the position of the SAM spot with an Rf of 0.66. (B) HPLC analysis of reaction products. Curves: 1, reaction product with purified SAM-s, ATP, and L-methionine; 2, reaction product with E. coli cell extract harboring vector with no SAM gene; 3, reaction product with ATP and L-methionine; 4, standard synthetic SAM (retention time = 19.6 min).

Overexpression of SAM-s in S. lividans affects antibiotic production and the sporulation process. To study the functions of the gene product of the cloned metK gene from S. spectabilis in cellular differentiation, we subcloned it into the pWHM3 plasmid and expressed it in S. lividans, which is an actinorhodin producer. Since the genetic manipulative system in S. spectabilis has yet to be established and not much is known about the gene cluster for spectinomycin production, we employed the S. lividans system, which is very closely related to our S. spectabilis and relatively easy to genetically deal with. It also contains abundant information for its cellular differentiation and antibiotic production. Therefore, we decided to determine the physiological functions of the metK gene product in S. lividans TK23 as a model system.

As shown in Fig. 3, the expression of the metK gene from S. spectabilis in S. lividans inhibited cellular differentiation, particularly the sporulation process. When sporulation was compared on agar plates, transformants harboring the SAM-s gene showed retarded spore formation, which is evident in the reduced white color of the spore. Surprisingly, transformants harboring the SAM-s gene also revealed a much higher level of actinorhodin (blue pigment) compared to the control strain containing only the vector plasmid. These results strongly suggested that the introduction of the SAM-s gene from S. spectabilis inhibited morphological differentiation but at the same time stimulated the production of actinorhodin in S. lividans.



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  		FIG. 3. Expression of the SAM-s gene from S. spectabilis enhances actinorhodin production but inhibits sporulation. Cells were grown on R1R2 agar plates with 50-µg/ml concentrations of thiostrepton at 28°C for 5 days. (A) The front side of the plate showing sporulation; (B) the reverse side of the plate showing actinorhodin production. Subpanels: 1, cells with SAM-s gene overexpression; 2, cells with empty vector (pWHM3); 3, wild-type S. lividans TK23 cells.

To elucidate further how overexpression of the metK gene affects the sporulation process in S. lividans, we observed three different strains under the microscope: wild-type S. lividans, transformants with the metK gene, and transformants with vector only (Fig. 4). Transformants with only plasmid developed normal aerial mycelium and spores that cannot be distinguished from those of wild-type cells. In contrast, cells that contained the metK gene gave rise not only to abnormally elongated aerial mycelium but also to the unusually swollen spore at the end of the cell, which indicated that SAM produced by the metK gene product delayed or inhibited the sporulation process in S. lividans, directly or indirectly. These results are consistent with the reports that in B. subtilis the treatment of SAM prevented spore formation (29).



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  		FIG. 4. Overexpression of the metK gene results in inhibition of aerial mycelium and formation of swollen spores in S. lividans TK23. (A) Wild-type S. lividans TK23 cells; (B) S. lividans TK23 cells harboring only vector; (C) S. lividans TK23 cells that have the SAM-s gene in pSAM-s. All strains were grown on an R1R2 agar plate at 28°C for 5 days.

When the production of actinorhodin was measured in a liquid medium, cells containing the metK gene in pSAM-s plasmid showed a higher level of actinorhodin production than cells with vector only, although their growth rates were almost identical (Fig. 5A). In addition, the antibiotic production showed the maximum level in stationary phase of growth. In contrast, transformants with plasmid only showed a low level of actinorhodin production, especially in the late stationary phase, which is considered to be a result of the accumulation of SAM in the cell. From these results, we concluded that the expression of SAM-s in S. lividans, most likely through the production of SAM, inhibited the sporulation process and strongly enhanced actinorhodin production.



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  		FIG. 5. Introduction of the metK gene into S. lividans TK23 enhances actinorhodin biosynthesis without affecting cell growth. Cells were grown in actinorhodin production medium with 10 µg of thiostrepton/ml at 28°C for 168 h. Cell growth (A) and the production of actinorhodin (B) of S. lividans TK23 with SAM overexpression or empty vector in actinorhodin production medium were evaluated over time. Symbols: {blacksquare}, cells harboring SAM-s gene; {blacklozenge}, cells harboring empty vector. DCW, dry cell weight.

Exogenous SAM also has the same effects on sporulation and antibiotic production in S. lividans. The results presented above indicated that SAM-s gene expression had influences on both sporulation and antibiotic production in S. lividans. However, they did not provide any clues as to how SAM-s functions and, in particular, as to whether the catalytic product SAM itself activated actinorhodin biosynthesis. To address this issue, we compared the effects of the exogenous addition of SAM on both the sporulation process and antibiotic production in S. lividans. Cells were treated four times with 1 µmol of SAM at 12-h intervals and observed for their relative effects on antibiotic production after 3 and 5 days. As shown in Fig. 6, cells treated with SAM showed severe impairment upon sporulation from the third day of incubation onward and clear enhancement of actinorhodin production over a 5-day incubation period. In contrast, the inhibition of sporulation, as well as the stimulation of actinorhodin production, was not observed in cells treated with SAM's metabolic products, S-adenosylhomocysteine (SAH) and homocysteine, until the end of a 5-day incubation, a result which was the same as that obtained with pure-water treatment (data not shown). These results suggested that the effect of introducing SAM-s on sporulation and antibiotic production was due to the direct effect of SAM, which is the direct catalytic product of the enzyme and not of its metabolic processing to SAH or homocysteine.



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  		FIG. 6. Exogenous SAM, not SAH or homocysteine, is responsible for inhibition of sporulation and stimulation of actinorhodin production in S. lividans TK23. (A and B) Cells treated with 1 µmol of SAM; (C and D) cells treated with water. After four treatments with SAM or water at 12-h intervals, the cells in panels A and C were cultured for 3 days, whereas the cells in panels B and D were cultured for 5 days at 28°C.

Expression of the SAM-s gene activates the transcription of the actII-ORF4 activator gene in S. lividans. SAM could activate actinorhodin biosynthesis in the S. lividans cell through the direct induction of actinorhodin biosynthetic genes or the indirect activation of regulator(s) involved in controlling the induction of actinorhodin biosynthetic genes. Since it is well known that in S. lividans the gene product of actII-ORF4 activates the transcription of actinorhodin biosynthetic gene clusters (12) and we also found two putative SAM binding motifs in ActII-ORF4 (D.-J. Kim et al., unpublished data), we decided to test whether the introduction of SAM by SAM-s expression could lead to the induction of actII-ORF4 in vivo. To do this, we introduced the SAM-s gene into S. lividans and analyzed the transcripts of actII-ORF4 by Northern analysis. In addition, we checked the amount of intracellular SAM over the incubation time to analyze the correlation between the transcriptional level of the actII-ORF4 gene and SAM levels. The transcription level of actII-ORF4 was highest at 88 h of growth (Fig. 7A), a finding consistent with the results shown in Fig. 5. The fact that the control strain harboring only plasmid DNA did not show any transcription of actII-ORF4 indicated that the expression of the SAM-s gene, most probably through the production of SAM, induced the transcription of actII-ORF4 directly or indirectly. These results also suggested that SAM induced the transcription of genes for actinorhodin biosynthesis via the induction of the actII-ORF4 activator gene, thereby increasing antibiotic production.



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  		FIG. 7. Introduction of SAM-s elevates the intracellular level of SAM, which in turn activates the transcription of the actII-ORF4. Cells were grown in actinorhodin production medium with 10 µg of thiostrepton/ml at 28°C for 124 h. (A) Northern analysis of the actII-ORF4 gene in cells with or without the SAM-s gene. The transcript levels of the metK and actII-ORF4 genes were determined with 100 µg of RNA. (B) Intracellular levels of SAM during the incubation period. The black bars represent the levels of SAM in cells harboring pSAM-s, whereas the shaded bars represent the levels of SAM in cells harboring only plasmid. Intracellular concentrations of SAM were determined by HPLC.

When the amount of intracellular SAM was quantified over the incubation time, it showed a maximum level at 64 h, which coincided with the transcription pattern of actII-ORF4 (Fig. 7B). Interestingly, however, the strain with only plasmid did not show any transcription of actII-ORF4 or actinorhodin production until the end of a 124-h incubation period even though it accumulated ca. 660 nmol of SAM/g (wet cell weight) inside the cell. This suggests that there exists a certain basal level of intracellular SAM that has to be accumulated before it starts to exert its effects on sporulation and antibiotic production.

In the Northern analysis (Fig. 7A), we have shown that the SAM-s transcriptional level at the early growth phase of wild-type cells is more active, but it diminished as cells entered into the later stage. In contrast, the intracellular level of SAM showed a gradual increase as cells grew. Currently, we do not understand why there exists the discrepancy between the transcriptional level of SAM-s and the intracellular SAM level. However, one possible explanation might be the active consumption of intracellular SAM for growth at an early stage and, as cells enter into the later stages of growth, the growth rate is reduced and intracellular SAM could be accumulated in the cell.


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DISCUSSION
 
In this study, we have demonstrated that SAM provided by either the overexpression of the SAM-s gene or the exogenous addition of SAM leads to enhanced production of actinorhodin, as well as to the inhibition of the sporulation process in S. lividans. In previous reports, it has been known that the accumulated SAM could inhibit cellular differentiation in bacterial systems such as B. subtilis (28, 29). However, the present study is the first to show that SAM indeed can induce antibiotic production but inhibit morphological differentiation in Streptomyces spp. Interestingly, Okamoto et al. recently found that overexpression of the SAM-s gene also led to high production of actinorhodin in S. coelicolor, a finding that strongly supports the role of SAM in antibiotic production in streptomycetes (30). Therefore, in addition to the biochemical role as a methyl donor in the cell, it is now clear that SAM also functions as an effector in both physiological differentiation (antibiotic synthesis) and morphological differentiation (sporulation) in S. lividans.

In both prokaryotes and eukaryotes, SAM has been known to function as a methyl donor (38). It donates its methyl group to various proteins, nucleic acids, and polysaccharides in their methylation reactions, as well as to many metabolites in intermediary metabolism (27). Therefore, SAM plays very important roles as a methyl donor in many of the primary and secondary metabolisms of the cell (27, 41). In addition to its role in metabolism, SAM has also been reported to be involved in the morphological differentiations of both prokaryotes and eukaryotes (16, 28, 29). For example, the high level of SAM in B. subtilis or Saccharomyces cerevisiae inhibits their sporulation and cellular differentiation. However, it was not clear whether the intracellular level of SAM has any effect on sporulation and/or secondary metabolism in Streptomyces. Therefore, in the present study, we tested the effect of SAM provided by SAM-s gene expression on S. lividans cell, for which a genetic system is well established and cellular differentiation studies are actively under way. We speculated that depletion of SAM by deleting SAM-s would be lethal to the cell, since it is indispensable to both primary and secondary metabolisms of the cell (27, 37, 41). Instead, SAM was overproduced by introduction of the SAM-s gene into S. lividans. Accumulation of SAM turned out to repress the formation of aerial mycelium and the maturation of spores. In addition, to our surprise, the increased SAM in the cell highly enhanced the production of actinorhodin, a well-characterized secondary metabolite of S. lividans (Fig. 5).

To test whether the changes in sporulation and antibiotic production were due to SAM produced by overexpression of the SAM-s gene or simply side effects of expression of the metK gene, we examined the effect of exogenous SAM. When protoplast cells of S. lividans were treated with SAM, the same effects on sporulation and actinorhodin production were observed as were seen in the case of SAM-s overexpression (Fig. 6). However, we could not exclude the possibility that the metabolic products of SAM, such as SAH and homocysteine, may also affect the production of actinorhodin and sporulation. SAM-s is the enzyme responsible for the synthesis of SAM by using L-methionine and ATP (13). Most of the SAM generated in the cell is used in transmethylation reactions in which methyl groups are added to compounds and SAM is converted to SAH, which is a potent competitive inhibitor of SAM (39). SAH hydrolase then catalyzes a reversible reaction, which converts SAH to homocysteine and adenosine (3, 27, 35). Therefore, these metabolites of SAM could also affect sporulation and antibiotic production instead of SAM itself. However, when SAH and homocysteine were used to treat protoplast cells of S. lividans, we found no difference in either sporulation or antibiotic production, which strongly suggested that SAM itself produced by the overexpression of SAM-s indeed inhibited the sporulation process and increased actinorhodin production.

Then, what is the mechanism by which the production of actinorhodin was dramatically increased in the stationary phase as shown in Fig. 5. One of the possibilities was that SAM donates its methyl group to some of the biosynthetic intermediates of actinorhodin, thereby facilitating antibiotic production. However, according to the biosynthetic pathway of actinorhodin, it seems very unlikely that SAM directly serves as a substrate and enhances antibiotic production, since the methyl moiety of actinorhodin originates from acetyl coenzyme A (2). In the biosynthetic machinery of the polyketide antibiotic family, biosynthetic enzymes are encoded by clusters of genes functioning in a sequential manner, as in fatty acid biosynthesis. Genes for actinorhodin biosynthesis are also in a gene cluster, including actI through actVII (23). It has been shown that the gene product of actII-ORF4 in the cluster activated the transcription of actI, which encodes the first enzyme of the pathway (2). Interestingly, it has been also reported that ppGpp served as a signal molecule to enhance actinorhodin production via activation of actII-ORF4 (4). Therefore, based on our findings, in which SAM increased actinorhodin production, we postulated that SAM could also act as a signal molecule to activate transcription of actII-ORF4, thereby enhancing actinorhodin biosynthesis.

To address this possibility, the transcription level of actII-ORF4 was examined through Northern analysis, and the internal SAM level was also determined by HPLC (Fig. 7). These results suggested that a certain basal level of SAM is required to induce actII-ORF4 transcription, which then activates transcription of the genes in the cluster, thereby increasing actinorhodin biosynthesis. However, we still do not know how SAM activates transcription of the actII-ORF4 gene. SAM could activate actII-ORF4 directly by binding to it or indirectly by activating other unknown intermediate molecules even though ActII-ORF4 seems to have SAM-binding motifs (42). It will be interesting to see if SAM could make any cross-linked complex with actII-ORF4 in vitro and in vivo.

Another interesting fact in antibiotic production was that the production of undecylprodigiosin, as well as that of actinorhodin, was also enhanced by the accumulation of intracellular SAM (data not shown). Undecylprodigiosin biosynthesis is known to require at least 18 genes, but the precise biochemical mechanisms have not been elucidated (11, 33, 36). It seems that RedD, a pathway-specific transcriptional positive activator in undecylprodigiosin biosynthesis, has putative SAM binding motifs like ActII-ORF4 (42).

How does SAM affect both cellular differentiation and antibiotic production? We currently do not know how overexpressed SAM in the cell inhibits the sporulation process, but it could exert its effect through the action of the BldD repressor. bldD mutants are known to be defective in the formation of aerial mycelium, and BldD acts as a repressor of genes such as bldN and whiG (both encoding sigma factors), which are involved in cell growth and differentiation (21). Interestingly, BldD protein turned out to have a SAM binding motif in the middle of its sequence (J.-H. Huh et al., unpublished data). Therefore, it is possible to postulate that BldD bound to SAM overexpressed by SAMs could not release its repression for genes required for cellular differentiation thereby impeded the sporulation process. It will be interesting to determine whether a mutant strain harboring a defective SAM binding motif in BldD would show different cellular differentiation.


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ACKNOWLEDGMENTS
 
We greatly appreciate the invaluable help and contributions of J. H. Roe and S. Y. Oh for transcript analyses. We also thank K. Chater, E. Malpartida, and K. Ochi for helpful discussions.

This work was supported by a grant from the Science and Technology Policy Institute of Korea (Biotech 2000 program; project no. M10015000015-01A21000410), by MOST and KOSEF (RRC program), and by the Ministry of Education's Brain Korea 21 Project.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Science, Myongji University, San 38-2, Nam-dong, Yongin 449-728, Korea. Phone: 82-31-330-6190. Fax: 82-31-336-0870. E-mail: jwsuh{at}mju.ac.kr. Back


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Journal of Bacteriology, January 2003, p. 592-600, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.592-600.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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