1 Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan
2 Department of Agriculture, Junior College, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan
3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence
Kenji Ueda
ueda{at}brs.nihon-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Evidence is accumulating that the onset of morphological differentiation and secondary metabolite formation in streptomycetes is induced by autoregulatory substances which have a -butyrolactone structure. A-factor (2-isocapryloyl-3R-hydroxymethyl-
-butyrolactone) of Streptomyces griseus, the best-studied
-butyrolactone autoregulator, induces the onset of both aerial growth and secondary metabolism in this organism (Horinouchi et al., 1984
). The studies of Horinouchi, Beppu and coworkers have revealed the details of the A-factor signalling cascade, which involves multiple regulatory gene functions (Horinouchi, 1999
, 2002
; Horinouchi & Beppu, 1992
). On the other hand, Nihira, Yamada and colleagues have characterized the role and function of virginiae butanolides in Streptomyces virginiae (Yamada, 1999
). They have also identified the plethora of
-butyrolactones in a variety of Streptomyces species, including Streptomyces coelicolor A3(2), the model organism whose genome has been completely sequenced (Bentley et al., 2002
; Takano et al., 2000
). It is generally understood that the action of
-butyrolactone autoregulators is specific to the producer organism because of the specificity of the cognate receptor proteins.
In contrast to the idea of autoregulation, we have previously shown that interspecific stimulation of morphogenesis and/or secondary metabolism takes place among various Streptomyces species and related organisms (Ueda et al., 2000). Cross-feeding experiments on solid media demonstrated the response of an array of colonies of one strain to a concentration gradient of a substance diffusing from a colony of another strain. Comprehensive cross-feeding tests among 76 Streptomyces strains showed that more than 20 % of the strains showed response(s) to putative metabolite(s) excreted by another strain and exhibited precocious colony development and/or enhanced secondary metabolite formation. These stimulatory events between different species may include those involving the function of a specific metabolite. In this study, we focused on the stimulation of the growth and development of Streptomyces tanashiensis; this stimulation is caused by a substance, revealed to be a type of siderophore, secreted by S. griseus.
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METHODS |
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Isolation of the stimulant produced by S. griseus.
In order to isolate the substance that stimulates the development of S. tanashiensis, S. griseus st-21-2 was cultured in Bennett's/glucose liquid medium. The seed culture was grown at 28 °C for 5 days; shaking was carried out at 135 r.p.m. in a 500 ml baffled Erlenmeyer flask in a working volume of 100 ml. After removing the mycelia by centrifugation and filtration, 100 ml of the culture supernatant was first extracted three times with 100 ml ethyl acetate, and 20 g cation-exchange resin (Amberlite IRC50; Organo) was added to the resultant water phase. Following incubation for 1 h at room temperature with gentle stirring, the resin was removed by filtration. The unabsorbed filtrate thus obtained was then applied onto a Dowex 1x2 anion-exchange column (2·5x7·5 cm; Organo). Following washing with 300 ml distilled water (DW), the activity was eluted by 100 ml 0·2 M NaCl. The recovered fraction was concentrated by evaporation and lyophilization to dryness. The sample was dissolved in 3 ml DW and applied to a Sephadex G-15 gel filtration column (2·5x47 cm; Amersham Biosciences). The column was developed with DW (0·6 ml min1) and fractions of 10 ml were collected. The activity, which was recovered in fractions 1315, was concentrated by evaporation and lyophilization to dryness. The sample was then dissolved in 3 ml DW and applied to a reverse-phase column (ODS-7515-12A, 2·5x14 cm, Amersham). After washing with water, the column was developed with a stepwise gradient of methanol in water (water : methanol 10 : 00 : 10). The activity was recovered by elution with water : methanol (6 : 44 : 6). The fractions were combined and lyophilized to dryness, dissolved in 1 ml water and applied onto a reverse-phase HPLC column (RESOURCE RPC, 5·0x20 cm; Amersham). Following washing with water, the column was developed with a gradient of 1090 % acetonitrile in 0·05 % formic acid at 1 ml min1 and fractions of 1 ml were collected, monitoring UV absorption spectra at 218 nm. The active fractions were combined, lyophilized, dissolved in 1 ml water and applied onto a reverse-phase HPLC column operated under the same conditions as above, except that the elution was performed at 0·8 ml min1. The HPLC performed previously afforded the stimulatory activity as a single peak at a concentration of 25 % acetonitrile. The activity of the purified substance as a siderophore was examined by the CAS assay according to Schwyn & Neilands (1987), which visualizes ferric-sequestering activity by the formation of a clear halo on the CAS assay plate.
Structural analysis.
The 1H NMR spectrum was recorded on a JEOL JMN-500 spectrometer at 500 MHz. The FAB-MS spectrum was obtained on a JEOL JMS-700T spectrometer using xenon as the fast atom. Spectral data of the active substance were as follows: FAB-MS (positive, glycerol matrix) m/z 601 (M+H)+, 623 (M+Na)+; 1H NMR (500 MHz, DMSO-d6): 1·151·24 (m, 6H), 1·331·40 (m, 6H), 1·451·52 (m, 6H), 2·27 (t, J=7 Hz, 6H), 2·58 (t, J=7 Hz, 6H), 2·973·02 (m, 6H), 3·46 (t, J=7 Hz, 6H), 7·77 (br. s, 3H, NH), 9·60 (br. s, 3H, N-OH).
Gene disruption.
The desABCD mutant of S. coelicolor A3(2) was generated by the standard homologous recombination technique, which replaced the wild-type allele with a mutated construct on a disruption plasmid. The disruption plasmid was constructed as follows. The two DNA fragments, a and b (see Fig. 4a), which correspond to the internal region of desA and desD respectively, were amplified from the chromosomal DNA of S. coelicolor A3(2) by standard PCR. The PCR primers that were used were as follows: 5'-CGACAAGCTTGAGGACGTCTA-3' (corresponding to 30358323085852 of SCO2782) (http://www.sanger.ac.uk/Projects/S_coelicolor) (Bentley et al., 2002
) and 5'-AAGGAGATCTCCCAGGTACT-3' (30370493037068 of SCO2782) for fragment a and 5'-CTTCGAGATCTACGAGTACCT-3' (30394163039436 of SCO2785) and 5'-CAGGGAATTCTTCAGGGTAC-3' (30406503040670) for fragment b. Fragments a and b were then digested with the restriction endonucleases HindIII/BglII and BglII/EcoRI, respectively, and cloned onto pUC18, which was digested with HindIII/EcoRI by three-fragment ligation. In order to generate the disruption plasmid, the plasmid thus formed was cleaved with BglII and ligated to an aphII (kanamycin resistance) cassette (Beck et al., 1982
), which was recovered as a BamHI-digested fragment. The disruption plasmid was introduced into S. coelicolor A3(2) M145 cells by standard transformation. As a result, two kanamycin-resistant colonies were obtained; one colony was confirmed for true recombination by the standard Southern hybridization technique using appropriate probes and described as a desABCD mutant (KY1 strain). The KY1 strain lacks the region containing the C-terminal part of desA, the entire region for desB and desC and the N-terminal portion of desD. The insertion of aphII does not affect the gene expression of the flanking regions, because aphII oriented in the opposite direction to the des operon.
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RESULTS |
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By the FAB-MS and NMR spectral data, the active substance was identified as desferrioxamine E (synonym: nocardamine) (Fig. 2). This was confirmed by comparing the retention time on HPLC and the spectral data of the active substance with those of an authentic sample. Desferrioxamine E is a siderophore that is widely produced by Streptomyces species and related organisms (Berner et al., 1988
). The siderophore activity of the purified substance was confirmed by the CAS assay (Schwyn & Neilands, 1987
) (data not shown).
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Desferrioxamines have several analogous forms, including desferrioxamine B (Fig. 2), which is commercially available. In comparison with desferrioxamine E, the growth-stimulation activity of commercial desferrioxamine B against S. tanashiensis was weak (Fig. 1b
), although it showed distinct stimulation when it was mixed with 0·1 µg FeCl2 prior to application onto the filter disc. The exogenous supply of other siderophores, including 2,3-dihydroxybenzoic acid, ferrozine, ferrichrome, 8-quinolinol, transferrin and nocobactin and free ferric ion, did not affect the phenotype of S. tanashiensis (data not shown).
Fig. 3 shows the effect of exogenous desferrioxamine E on the phenotypes of various actinomycete strains. The supply of the substance induced precocious aerial growth in both S. griseus st-21-2, the strain used as a producer of desferrioxamine E in this study, and S. coelicolor A3(2). In addition, various effects were observed with the strains that were freshly isolated from soil (Fig. 3
): the supply of desferrioxamine E stimulated yellow pigment and antibiotic production (strain no. 17), aerial mycelium formation (no. 70 and no. 507), melanin-like pigment production (no. 82) and growth (no. 326).
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KY1 showed impaired growth, morphological development and pigment production on Bennett's/glucose solid medium (Fig. 4b). The deficiency was partially rescued by an exogenous supply of desferrioxamine E isolated from S. griseus culture broth (Fig. 4b
) and was fully complemented by the introduction of a plasmid that carries the intact des operon (pDES) (Fig. 4c
). The cross-feeding assay showed that S. coelicolor A3(2) wild-type exhibits an activity that stimulates the growth and development of S. tanashiensis, while the KY1 strain was defective in the stimulation activity (Fig. 4c
). The stimulation activity was restored by the introduction of pDES. Purification of desferrioxamine E by the same method as employed for S. griseus showed the presence of the compound in the culture broth of S. coelicolor A3(2) wild-type and its absence in that of KY1 (data not shown).
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DISCUSSION |
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Ferric uptake in Streptomyces species has been poorly characterized, except in Streptomyces pilosus, the organism used for the fermentation production of desferrioxamine B (Müller & Raymond, 1984; Müller et al., 1984
). This organism produces various desferrioxamine analogues, among which desferrioxamine B is the major constituent. Müller et al. (1984)
demonstrated that S. pilosus can take up not only desferrioxamines but also ferrichrome, another major microbial siderophore, although the organism cannot produce the latter type of siderophore. On the other hand, we found that the growth and development of S. tanashiensis are not affected by the exogenous supply of ferrichrome. Evidence suggests that there is diversity in the utilization of siderophores in streptomycetes; however, the major siderophores in the group of bacteria may be desferrioxamines.
The stimulation of S. tanashiensis growth and development by S. griseus was not detected in our previous study (Ueda et al., 2000). This may be attributed to the difference in the length of the incubation period. While the stimulation was assessed after a 4 day culture in the previous screening study, the marked stimulation in S. tanashiensis was observed after prolonged incubation for more than 1 week. The wide occurrence of desferrioxamine in Streptomyces species and its stimulatory effect on the growth and/or development of various streptomycetes and related organisms indicate that the stimulatory event is not specific to the two strains mentioned above but common to the constituents of the bacterial group.
The studies on S. pilosus have shown that desferrioxamines are commonly synthesized from lysine (Müller & Raymond, 1984; Schupp et al., 1987
). Although we were successful in isolating only desferrioxamine E from the S. griseus culture broth in this study, during the purification we also observed the presence of several minor fractions with stimulatory activities for S. tanashiensis development (unpublished result). These fractions may contain desferrioxamine analogues, which are also produced by the S. griseus strain. The lower effect of desferrioxamine B on the growth and development of S. tanashiensis in comparison with that of desferrioxamine E (Fig. 1b
) suggests that the siderophore uptake mechanism in S. tanashiensis has higher affinity for the latter compound than for the former. Müller & Raymond (1984)
previously described that S. pilosus takes up desferrioxamine B more efficiently than desferrioxamine E, together with information on the conformation of ferric-desferrioxamine complexes. The diversity in uptake specificity may help to prevent competition for the acquisition of the environmental ferric ion in this group of bacteria. The precise characterization of the activity spectrum of each desferrioxamine will reveal the various roles of specific siderophores in streptomycete physiology.
The S. coelicolor mutant defective in desferrioxamine biosynthesis (KY1) showed impaired growth and development on Bennett's/glucose solid medium. Since ferric ion is essential for the viability of nearly all life forms, the growth impairment is probably due to iron deficiency. The partial but marked restoration activity of desferrioxamine E (Fig. 4b) indicates that the substance transports iron into the cell of this organism. However, the partial effect implies that another type of desferrioxamine(s) also plays an important role in the physiology of S. coelicolor A3(2). Additionally, the fact that KY1 is still viable suggests that the organism has another ferric uptake system(s). Challis & Ravel (2000)
suggested the occurrence of a peptidic siderophore termed coelichelin in S. coelicolor A3(2), although this has not been biochemically confirmed. Both KY1 and S. tanashiensis grow and develop well on Bennett's medium supplied with maltose (our unpublished observation). Thus, we currently speculate that the siderophore production in Streptomyces species is under complex regulation that links not only to ferric limitation but also to carbohydrate metabolism.
Desferrioxamines have long been known as a fungal growth factor (Prelog, 1963). The uptake of desferrioxamines takes place in the budding yeast Saccharomyces cerevisiae (Lesuisse et al., 2001
; Yun et al., 2000
) as well as pathogenic bacteria such as Salmonella (Kingsley et al., 1999
) and Neisseria species (Schryvers & Stojiljkovic, 1999
), which do not have the ability to produce their own ferric-chelating agent. Salmonella strains have been shown to have receptors for siderophores that they do not synthesize (Kingsley et al., 1999
). Since our attempts to amplify des genes from S. tanashiensis genomic DNA by PCR have failed, we assume that the S. tanashiensis strain does not carry the desferrioxamine biosynthesis gene cluster (unpublished observation). Microbes defective in siderophore production may survive in the natural environment by utilizing the siderophores produced by other organisms.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 April 2005;
revised 26 May 2005;
accepted 20 June 2005.
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