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Correspondence: Kozo Ochi, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Tel.: +81 29 838 8125; fax: +81 29 838 7996; e-mail: email@example.com
Despite their importance in the chemical industry, the significance of rare earths in biology has been largely overlooked. Here, it is reported that the rare earth, scandium (Sc), causes antibiotic overproduction by 2–25-fold when added at a low concentration (10–100 μM) to cultures of Streptomyces coelicolor A3(2) (actinorhodin producer), Streptomyces antibioticus (actinomycin producer), and Streptomyces griseus (streptomycin producer). Not just for enhancement of antibiotic production, scandium was also effective in activating the dormant ability to produce actinorhodin in Streptomyces lividans. The effects of scandium were exerted at the level of transcription of pathway-specific positive regulatory genes, as demonstrated by marked up-regulation of actII-ORF4 in S. coelicolor cells exposed to this element. The bacterial alarmone, guanosine 5′-diphosphate 3′-diphosphate, was essential for actinorhodin overproduction provoked by scandium.
Rare earth is a general term for 17 elements that includes scandium (Sc), yttrium (Y), and the lanthanides [15 elements from lanthanum (La) to lutetium (Lu)]. Each of the lanthanides is characterized by a 4-f electron (Bunzli & Choppin, 1989). Rare-earth elements have been widely used in high-technology products, such as permanent magnets, fluorescent materials, and new ceramics, and they are currently being used in computers, mobile telephones, plasma displays, magneto-optical disks, high-powered lasers, fluorescent lamps, and hybrid cars. In addition, they are used in the glass, petroleum, and nuclear industries, as well as in various medical fields. Despite their importance in ‘physics’ and ‘chemistry’, the significance of rare earths in biology has been largely overlooked. During the course of studying the effects of rare earths on bacterial physiology, it was found that they can elicit bacterial capabilities, and thus exert evident effects on secondary metabolism in Streptomyces spp., the typical soil microorganisms that often produce antibiotics. This paper describes the physiology of scandium, focusing mainly on Streptomyces coelicolor A3(2), the genetically best studied of the actinomycetes.
Materials and methods
Bacterial strains and culture conditions
The strains of Streptomyces spp. used to study the effect of scandium are all prototrophic wild-type strains, which produce actinorhodin, streptomycin, or actinomycin. Bacillus subtilis 168 is a standard strain frequently used for studying sporulation. Cultivation was performed at 30 or 25°C with rotary shaking at 200 r.p.m. GYM and SPY media were described previously (Ochi, 1987). SYM-1 and SYM-2 media contained 0.4% and 3% soluble starch instead of glucose in GYM, respectively.
Determination of antibiotics and guanosine 5′-diphosphate 3′-diphosphate (ppGpp)
Actinorhodin produced in the medium was measured according to the method described by Kieser et al. (2000). Actinomycin and streptomycin were assayed as described previously (Ochi, 1987; Hosoya et al., 1998). The intracellular pool size of ppGpp was determined using HPLC as described previously (Ochi, 1987).
Molecular biology procedures
Plasmids were introduced into S. coelicolor strains by intergeneric transfer from Escherichia coli (Kieser et al., 2000). Reverse transcriptase-PCR (RT-PCR) analysis of actII-ORF4 expression was performed as described previously (Saito et al., 2006).
Scandium chloride (ScCl3·6H2O; purity >95%) and other rare earths (all chloride salts) were purchased from Wako Pure Chemical (Osaka).
Results and discussion
Scandium enhances actinorhodin production by S. coelicolor
Streptomyces coelicolor A3(2) produces a deep blue-pigmented polyketide antibiotic, actinorhodin (Act), when the cells enter the stationary growth phase (Chater & Bibb, 1997; Kieser et al., 2000; Bibb, 2005; Saito et al., 2006). It was found that addition of scandium (ScCl3·6H2O) markedly enhanced actinorhodin production when cells were grown on GYM agar medium for 4 days (Fig. 1). Scandium was effective at concentrations 20–100 μM, with the optimal concentration of 70 μM, as determined using plates containing fixed concentrations of scandium. Aerial mycelium formation (and thus sporulation) was also enhanced with 50–200 μM scandium, whereas growth was inhibited completely by 1 mM scandium. Similar experiments were conducted in liquid culture to allow quantitative evaluation of the efficacy of scandium, where actinorhodin production by S. coelicolor was increased by more than 20-fold by addition of scandium (Table 1). Streptomyces lividans, a close relative of S. coelicolor A3(2), normally does not produce actinorhodin, although this organism, like S. coelicolor A3(2), has a complete set of genes for actinorhodin biosynthesis. Scandium was effective even for actinorhodin production by S. lividans (Fig. 1 and Table 1). Thus, scandium not only enhances actinorhodin production in S. coelicolor but also activates the normally dormant actinorhodin production in S. lividans. The other rare earths, yttrium (Y), lanthanum (La), cerium (Ce), and europium (Eu), also enhanced actinorhodin production, whereas copper (Cu), zinc (Zn), manganese (Mn), nickel (Ni), and cobalt (Co) (all chloride salts) were ineffective (data not shown). It is reported that Sc3+ competes with Fe3+ for its uptake within cells (Plaha & Rogers, 1983). Therefore, the addition of scandium might result in iron deficiency and thus cause antibiotic overproduction. In relation to this concept, iron limitation is shown to actually enhance the production of actinorhodin by S. coelicolor (Coisne et al., 1999). However, the observed positive effects of scandium were not abolished by addition of FeCl3 (tested at concentrations from 1 to 200 μM), eliminating the possibility mentioned above.
Table 1. Effect of scandium on actinorhodin production by Streptomyces coelicolor and Streptomyces lividans
The principal regulator of actinorhodin production in S. coelicolor is the availability of the pathway-specific transcriptional regulatory protein ActII-ORF4, a threshold concentration of which is required for efficient transcription of its cognate biosynthetic structural genes (Gramajo et al., 1993). As expected, scandium enhanced actII-ORF4 transcription in S. coelicolor and S. lividans (Fig. 2), implying that the effects of scandium were exerted at the level of initiation of secondary metabolism rather than the level of biosynthesis.
ppGpp is essential for act overproduction provoked by scandium
Bacteria exert the stringent response, a general and ubiquitous response to nutrient starvation in prokaryotes. ppGpp, which is synthesized on the ribosome in response to amino acid limitation, plays a central role in the stringent repsonse (Cashel et al., 1996; Braeken et al., 2005). It is hypothesized that actinorhodin overproduction observed with addition of scandium may be due to an increase in the ability to accumulate ppGpp, which has been shown to play a key role as a bacterial alarmone in initiating the onset of secondary metabolism in bacteria (Ochi et al., 1997; Hesketh et al., 2001; Inaoka et al., 2003; Gomez-Escribano et al., 2006). Unexpectedly, however, the addition of scandium did not cause an increase in ppGpp pool size in S. coelicolor; rather, it reduced the ppGpp pool size just after addition of scandium (Fig. 3), although scandium at the concentration used in this experiment (50 μM) did not affect growth per se. Nevertheless, the basal-level ppGpp was essential for actinorhodin overproduction provoked by scandium, as scandium showed no positive effect on actinorhodin production in strain KO-490, whose ability to accumulate ppGpp has been entirely abolished due to deletion of the relA gene encoding ppGpp synthetase I (Fig. 4a). This conclusion was further confirmed by the restoration of actinorhodin production by KO-490 by forced expression of relA' (C-terminal-truncated relA gene) (Fig. 4b), whose expression is known to confer the ability to synthesize ppGpp in a ribosome-independent manner (Hesketh et al., 2001).
Effects of scandium on other Streptomyces spp.
Scandium was effective for antibiotic production by other Streptomyces spp. Actinomycin production by Streptomyces antibioticus (Fig. 5a) and Streptomyces parvulus (data not shown) was enhanced by twofold, and streptomycin production by Streptomyces griseus was enhanced by nearly fourfold (Fig. 5b) when strains were cultivated in SYM-1 or SYM-2 medium, although growth was inhibited completely at 1 mM scandium.
Streptomyces griseus is able to produce greater amounts of streptomycin when cultured in SPY medium containing a high concentration (1%) of MgSO4 (14). Scandium no longer inhibited the growth of S. griseus in SPY medium even at the high concentration of 5 mM, apparently due to the presence of large amounts of MgSO4. Nonetheless, scandium was still effective in increasing streptomycin production in this medium (Fig. 5c), indicating that the observed positive effect of scandium on antibiotic production was not simply due to a secondary effect of growth inhibition.
Bacillus subtilis 168 (the standard strain often used for the study of sporulation) produces an antibiotic bacilysin, whose production is regulated cooperatively by ppGpp and GTP (Inaoka et al., 2003). Scandium did enhance the production of bacilysin by fivefold when added at 100 μM (data not shown), indicating the efficacy of scandium in bacteria other than streptomycetes.
Differential susceptibility of microorganisms to scandium
The scandium complex of enterochelin exerts its inhibitory effect on the growth of Klebsiella pneumoniae (Rogers et al., 1980) and E. coli (Rogers et al., 1982), although the mechanism underlying the inhibitory effect is not fully understood (Plaha & Rogers, 1983). The microorganisms examined were susceptible to scandium at high concentrations. Streptomyces spp. (S. coelicolor and S. lividans), typical soil bacteria, were most susceptible [minimum inhibitory concentration (MIC)=1 mM], while Mycobacterium smegmatis (MIC=1.5 mM) and E. coli, Staphylococcus aureus, and B. subtilis (MICs=3 mM) were less susceptible. In contrast to bacteria, the eukaryotic microorganisms Saccharomyces cerevisiae (MIC=5 mM) and Aspergillus oryzae (MIC=15 mM) were relatively resistant to scandium. Similar results were obtained when susceptibility experiments were performed using the media with varying compositions.
In this report, a novel effect of scandium on secondary metabolism has been found. Given that rare earths are distributed ubiquitously throughout the world, it is conceivable that microorganisms have acquired the ability to respond to low levels of these elements over the course of their long evolutionary history, possibly as a means of adapting their physiology to the prevailing conditions. In this context, it is noteworthy that lanthanum (La) was recently shown to stimulate the growth of some plants under water-limiting conditions (Peverrill et al., 1997). The compelling effect of low levels of scandium on antibiotic production implies that scandium functions in situ as a factor that induces or stimulates the production of secondary metabolites, which can include pigments, mycotoxins, phytotoxins, and antibiotics. Although the mechanism of action remains to be clarified, it is possible that scandium (and also other rare earths) acts on the ribosome, eventually leading to modulation of ribosomal function, because addition of scandium immediately reduced the level of ppGpp, which is synthesized on the ribosome (Fig. 3). ‘Rare earth microbiology’ may thus offer new insights into entirely unknown regulatory events that occur in all organisms, possibly including humans.
This work was supported by grants to K.O. from the Organized Research Combination System and the Effective Promotion of Joint Research of Special Coordination Funds and by a grant to K.K. from Scientific Research on Priority Area (440) (the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government).