Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku,Tokyo 113-8657, Japan1
Author for correspondence: Sueharu Horinouchi. Tel: +81 3 5841 5123. Fax: +81 3 5841 8021. e-mail: asuhori{at}hongo.ecc.u-tokyo.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Streptomyces coelicolor A3(2), amfC, aerial mycelium formation, antibiotic production, polyketide spore pigment
The GenBank accession number for the amfC promoter sequence reported in this paper is D63677.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These observations prompted us to determine the role of amfC in S. coelicolor A3(2), the most intensively studied streptomycete. Our results show that amfC plays the same role in aerial mycelium formation as in S. griseus. In addition, we found that disruption of amfC severely reduced actinorhodin production. Overexpression of amfC abolished spore pigment formation almost completely. This paper describes the phenotypes of amfC disruptants and strains containing extra copies of amfC. It also describes transcription in these strains of whiE, responsible for spore pigment formation, and actII-ORF4, responsible for actinorhodin production.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant DNA studies.
Restriction enzymes, T4 DNA ligase and other DNA-modifying enzymes were purchased from Takara Shuzo. [-32P]dCTP (110 TBq mmol-1) for DNA labelling with the Takara BcaBest DNA labelling system and [
-32P]ATP (220 TBq mmol-1) for end-labelling at 5' ends with T4 polynucleotide kinase were purchased from Amersham. DNA was manipulated in Streptomyces as described by Hopwood et al. (1985) and in E. coli as described by Maniatis et al. (1982)
.
amfC was cloned in pKU209 and pIJ486 as follows: an 897 bp EcoT14IAccIII fragment containing amfC and its promoter (see below) was flush-ended with the Klenow fragment and inserted into the HincII site of pUC19. The recombinant pUC19 plasmid was digested with HindIII and flush-ended with the Klenow fragment. An EcoRI linker was then attached to the ends and the amfC sequence, excised as an EcoRI fragment, was inserted into the EcoRI sites of pIJ486 and pKU209 to give pIJ486-amfC and pKU209-amfC, respectively.
Gene disruption.
To disrupt the chromosomal amfC in S. coelicolor A3(2), we used single-stranded M13 phage DNA as described by Hillemann et al. (1991) . For construction of an M13 recombinant plasmid containing a neomycin phosphotransferase (neo) gene, a 1·32 kb SmaIHindIII fragment from Tn5 (Beck et al., 1982
) was first cloned in pUC19. A 307 bp SmaIPmaCI fragment encoding the internal part (Arg37Val140) of AmfC was then inserted into the SmaI site. Single-stranded DNA was prepared from E. coli JM109 and introduced into S. coelicolor A3(2) strains M130 and M145 by transformation. After neomycin (20 µg ml-1)-resistant transformants had been selected, they were screened for true disruptants by Southern hybridization of their BamHI-digested chromosomal DNA with the neo sequence and the 307 bp SmaIPmaCI fragment as probes.
Electron microscopy.
Spores and hyphae of S. coelicolor A3(2) strains grown at 28 °C for 7 d on R2YE agar were examined by scanning electron microscopy (Takamatsu et al., 1976 ). To prepare specimens, agar blocks were fixed with 2% osmium tetroxide for 40 h and then dehydrated by air-drying. Each specimen was sputter-coated with platinum/gold and examined with a Hitachi S4000 scanning electron microscope.
Assay of actinorhodin and undecylprodigiosin.
Actinorhodin was detected visually by its blue colour. For quantification, actinorhodin production in R2YE liquid medium was estimated from the A610 of the culture broth at pH 12, as described by Onaka et al. (1998) . Undecylprodigiosin in mycelium was measured (Onaka et al., 1998
) by extracting the pigments with methanol from mycelium grown at 28 °C for 79 d on a cellophane sheet laid on the surface of R2YE agar medium. After concentration of the extract by evaporation, the orange pigment at Rf 0·35 was separated out by thin-layer (Whatman KC18F reverse-phase plate) chromatography with 100% methanol as the solvent and eluted with the same solvent. The amount was calculated from the A533 of the eluate. Undecylprodigiosin shows absorption maxima at 533 and 468 nm at acidic and alkaline pH, respectively.
S1 nuclease mapping.
RNA was isolated from mycelium grown at 30 °C for 27 d on cellophane placed on the surface of R2YE or SFM agar, as described previously (Horinouchi et al., 1987 ). 32P-labelled probes were prepared by PCR with primers I and II and a template of strain M145 chromosomal DNA. When the 5' end of primer II was 32P-labelled with T4 polynucleotide kinase, the PCR product could be used as the probe. To determine the transcriptional start point of amfC, primer I (5'-TAGGGTGTGGAGGGGCGGGGCCGT-3'; corresponding to -155 to -132, taking the A residue of the ATG start codon of amfC as +1) and primer II (5'-ACCACCCAGGCCGGCCGGACCGCT-3'; corresponding to +96 to +73) were used. For actII-ORF4, primer I (5'-AATTTTTGATCAATAGGAGAGATCGCTTG-3'; corresponding to -108 to -82, taking the A residue of the ATG start codon of actII-ORF4 as +1; Fernándes-Moreno et al., 1991
) and primer II (5'-CGAGCAGCCGGCCGCCGGTGCGGATC-3'; corresponding to +358 to +333) were used. For whiE, primer I (5'-GCTTCACCCGCTTAACCCTCC-3'; corresponding to -147 to -127, taking the A residue of the ATG start codon of whiE as +1; Kelemen et al., 1998
) and primer II (5'-GTCGCTACGACGGGAGAGACC-3'; corresponding to +26 to +6) were used. For hrdB, primer I (5'-GGCCGCAAGGTACGAGTTGATGACCTTGTTTATCC-3'; corresponding to -279 to -245, taking the first G residue of the GTG start codon of hrdB as +1; Buttner et al., 1990
) and primer II (5'-AGGCCCGACGCACGTCATCGCCGGCGATCTGCCCC-3'; corresponding to +87 to +121) were used. Marker 10 (pBR322/MspI digest; Nippon Gene) was used to provide size markers. For high-resolution S1 mapping, protected DNA fragments were analysed on DNA sequencing gels by the method of Maxam & Gilbert (1980)
.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(ii) Sparse aerial mycelium formation by amfC disruptants. Both mutants M130amfC and M145
amfC formed very few spores on R2YE medium. Fig. 1a
shows delayed and infrequent sporulation by M130
amfC. The parental strains M130 and M145 began to form aerial hyphae at 3 d after inoculation, whereas aerial hyphae of strains M130
amfC and M145
amfC appeared at 5 d. Spores in the sporulating area of these mutants were less abundant, although the shape and size of spores of the mutants were indistiguishable from those of the parental strain (Fig. 1c
). The delayed and reduced sporulation of M130
amfC and M145
amfC was also observed on media containing maltose, mannitol or glycerol in place of glucose, in contrast to the situation with many bld mutants whose aerial mycelium formation depends on the carbon source (Merrick, 1976
; Chater, 1984). The effect of the amfC disruption on aerial mycelium formation was also seen on R2, SFM and TSB media. Introduction of amfC on a low-copy-number plasmid, pKU209 (plasmid pKU209-amfC) into these mutants restored the defect (data not shown), although a difference in spore pigment formation between the parental strain and these amfC disruptants was noted (see below). These observations indicated an important, but not essential, role for amfC in aerial mycelium formation.
|
|
|
(v) Lack of spore pigment formation by S. coelicolor A3(2) harbouring extra copies of amfC. To investigate possible effects of overexpression, we introduced amfC on a high-copy-number plasmid, pIJ486 (pIJ486-amfC), into strains M130 and M145. The transformants grew normally and, on R2YE, R2, TSB and SFM solid media, their colonies became white (indicative of aerial hyphae formation) at the same time as the parental strains. The colonies also developed spores at the same time as the parental strains. However, they remained white even after 7 d, whereas colonies of the parents turned grey due to synthesis of the polyketide spore pigment (Davis & Chater, 1990 ). This effect of amfC on spore pigment formation was also observed with pKU209-amfC whose copy number is reported to be 12 per genome (Kakinuma et al., 1989
) (Fig. 4
). The white area in the colony of pIJ486-amfC- or pKU209-amfC-harbouring strains was full of spores with the same shape and size as those of the strains lacking the plasmids (Fig. 1c
). In addition, coils of spore chains of strains M130 and M145 harbouring pKU209-amfC were indistiguishable from those of strains that did not harbour the plasmids. The spores with no pigment remained white after prolonged incubation. Thus, even one or two extra copies of amfC abolished spore pigment formation in strains M130 and M145. Since spore pigment formation by strains M130
amfC and M145
amfC harbouring pKU209-amfC was unstable, a very slight increase in the amount of AmfC seemed to suppress pigment formation.
|
The same effect of amfC overexpression on spore pigment formation was observed in S. lividans; the spores of S. lividans TK21 and TK24 harbouring pIJ486-amfC or pKU209-amfC remained white even after prolonged incubation. We also observed an inhibitory effect of pIJ486-amfC on spore pigment formation in Streptomyces lavendulae IFO 12789 and Streptomyces lipmanii IFO 12791. The deep-brown and red-brown colours, respectively, of these strains were completely abolished; spores of the strains harbouring pKU209-amfC remained white. However, pIJ486-amfC did not have any effect on the spores of Streptomyces alboniger IFO 12738 or Streptomyces ambofaciens IFO 12651. The chemical structures of these spore pigments are not known.
Transcriptional analysis of amfC
Transcription of amfC was examined by high-resolution S1 nuclease mapping with RNA prepared from mycelium grown on R2YE agar medium (Fig. 5a). The amfC mRNA was detected throughout growth, as was hrdB mRNA encoding one of the major
factors (Buttner et al., 1990
; Shiina et al., 1991
); mRNAs were investigated in substrate mycelium obtained after 2 d growth and in a mixture of aerial mycelium and spores obtained after 6 d. The transcriptional start point was determined to be the G residue 29 nucleotides upstream of the ATG start codon. In front of the transcriptional start point, GTCACA and CACGAT with a 19 bp spacer are present (Fig. 5b
). These sequences show similarity to those (TTGACA for -35 and TATAAT for -10 with a 17 bp spacer) of other prokaryotic promoters, as well as to one type (TTGACA for -35 and TAGGAT for -10 with a 18 bp spacer) of Streptomyces promoter believed to be active during vegetative growth (Hopwood et al., 1986
).
|
|
In strain M145amfC, the whiEP1 mRNA was detected at 3 and 4 d. Strain M145
amfC began to form aerial hyphae at this time on SFM agar medium. This was consistent with our observation that the sparce spores of this mutant were grey.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The amfC mutants of S. coelicolor A3(2) M130 produced much less actinorhodin than the parent. In addition, undecylprodigiosin production was delayed. The difference in the effect of amfC mutations on these antibiotics may reflect differences in the regulatory cascades leading to transcription of the respective pathway-specific regulatory genes (Bibb, 1996 ). Because of the difference in regulation, the timing of production of these antibiotics is also different. The influence of amfC on secondary metabolism in S. coelicolor A3(2) is in contrast to the situation in S. griseus, where amfC mutations did not cause any detectable effect on A-factor or streptomycin production (Kudo et al., 1995
). In the absence of amfC, almost no transcription of the pathway-specific transcriptional regulator actII-ORF4 occurs, and thus actinorhodin biosynthetic genes are not transcribed. This means that amfC is required for a particular signal transduction pathway to start transcription of the pathway-specific regulatory gene. In S. griseus, the corresponding signal transduction pathway leading to streptomycin biosynthesis may not require the amfC product. It is also possible that amfC affects streptomycin production, but to an undetectable level.
amfC mutations did not affect the yield of actinorhodin produced by S. coelicolor A3(2) M145. Ochi & Hosoya (1998 ) identified a mutation, sre-1 (suppression of relC effect), to which they ascribed the overproduction of actinorhodin by strain M145. The sre-1 mutation phenotypically suppresses or overcomes the defect in amfC mutants that affects actinorhodin and undecylprodigiosin production by strain M145. The sre-1 mutation, which is perhaps in the ribosomal protein S12 (Shima et al., 1996
), exerts its effect at the translational level. Since amfC exerts its influence on secondary metabolism at the transcriptional level, sre-1 seems to suppress the amfC mutation in an independent way.
Extra copies of amfC in S. coelicolor A3(2) M130 and M145 caused no detectable effects on the morphology of aerial mycelium or spore chains, suggesting that the whi genes (whiA, -B, -G, -H, -I and -J) required for sporulation septum formation in the regulatory cascade of aerial hyphae development function normally (Kelemen et al., 1998 ; Ryding et al., 1998
). However, the absence of spore pigmentation in these strains due to decreased transcription of whiE suggests that an increase in the amount of AmfC disturbs a regulatory signal to commence transcription of the whiE promoters in the hierarchy of gene expression controlling aerial mycelium development and spore maturation. Because overexpression of amfC phenotypically separates morphogenesis of spore chains from spore pigment formation, genetic studies using amfC will elucidate the molecular basis for the steps of spore maturation.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beck, E., Ludwig, G., Auerswald, A., Reiss, B. & Schaller, H. (1982). Nucleotide sequence and exact localisation of the neomycin phosphotransferase gene from transposon Tn5. Gene 19, 327-336.[Medline]
Bibb, M. J. (1996). The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology 142, 1335-1344.[Medline]
Buttner, M. J., Chater, K. F. & Bibb, M. J. (1990). Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). J Bacteriol 172, 3367-3378.[Medline]
Bystrykh, L. V., Fernándes-Moreno, M. A., Herrema, J. K., Malpartida, F., Hopwood, D. A. & Dijkhuizen, L. (1996). Production of actinorhodin-related blue pigments by Streptomyces coelicolor A3(2). J Bacteriol 178, 2238-2244.[Abstract]
Chater, K. F. (1984). Morphological and physiological differentiation in Streptomyces. In Microbial Development, pp. 89-115. Edited by R. Losick & L. Shapiro. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Chater, K. F. (1989). Sporulation in Streptomyces. In Regulation of Procaryotic Development: Structural and Functional Analysis of Bacterial Sporulation and Germination, pp. 277-299. Edited by I. Smith, R. A. Slepecky & P. Setlow. Washington, DC: American Society for Microbiology.
Davis, N. K. & Chater, K. F. (1990). Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics. Mol Microbiol 4, 1679-1691.[Medline]
Fernándes-Moreno, M. A., Caballero, J. L., Hopwood, D. A. & Malpartida, F. (1991). The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces. Cell 66, 769-780.[Medline]
Floriano, B. & Bibb, M. (1996). afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol Microbiol 21, 385-396.[Medline]
Fujii, T., Gramajo, H. C., Takano, E. & Bibb, M. J. (1996). redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing hrdD. J Bacteriol 178, 3402-3405.[Abstract]
Hara, O., Horinouchi, S., Uozumi, T. & Beppu, T. (1983). Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus. J Gen Microbiol 129, 2939-2944.[Medline]
Hillemann, D., Pühler, A. & Wohlleben, W. (1991). Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors. Nucleic Acids Res 19, 727-731.[Abstract]
Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
Hopwood, D. A., Bibb, M. J., Chater, K. F., Janssen, G. R., Malpartida, F. & Smith, C. P. (1986). Regulation of gene expression in antibiotic-producing Streptomyces. In Regulation of Gene Expression 25 Years On, pp. 251-276. Edited by I. R. Booth & C. F. Higgins. Cambridge: Cambridge University Press.
Horinouchi, S. (1996). Streptomyces genes involved in aerial mycelium formation. FEMS Microbiol Lett 141, 1-9.
Horinouchi, S. & Beppu, T. (1992). Autoregulatory factors and communication in actinomycetes. Annu Rev Microbiol 46, 377-398.[Medline]
Horinouchi, S. & Beppu, T. (1994). A-factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus. Mol Microbiol 12, 859-864.[Medline]
Horinouchi, S., Furuya, K., Nishiyama, M., Suzuki, H. & Beppu, T. (1987). Nucleotide sequence of the streptothricin acetyltransferase gene from Streptomyces lavendulae and its expression in heterologous hosts. J Bacteriol 169, 1929-1937.[Medline]
Kakinuma, S., Takada, Y., Ikeda, H., Tanaka, H. & Omura, S. (1991). Cloning of large DNA fragments, which hybridize with actinorhodin biosynthesis genes, from kalafungin and nanaomycin A methyl ester and identification of genes for kalafungin biosynthesis of the kalafungin producer. J Antibiot 44, 995-1005.[Medline]
Kelemen, G. H., Brian, P., Flärdh, K., Chamberlin, L., Chater, K. F. & Buttner, M. J. (1998). Developmental regulation of transcription of whiE, a locus specifying the polyketide spore pigment in Streptomyces coelicolor A3(2). J Bacteriol 180, 2515-2521.
Kudo, N., Kimura, M., Beppu, T. & Horinouchi, S. (1995). Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus. J Bacteriol 177, 6401-6410.[Abstract]
Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Maxam, A. M. & Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65, 499-560.[Medline]
Merrick, M. J. (1976). A morphological and genetic mapping study of bald colony mutants of Streptomyces coelicolor. J Gen Microbiol 96, 299-315.[Medline]
Ochi, K. & Hosoya, Y. (1998). Genetic mapping and characterization of novel mutations which suppress the effect of a relC mutation on antibiotic production in Streptomyces coelicolor A3(2). J Antibiot 51, 592-595.[Medline]
Onaka, H., Nakagawa, T. & Horinouchi, S. (1998). Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis. Mol Microbiol 28, 743-753.[Medline]
Ryding, N. J., Kelemen, G. H., Whatling, C. A., Flärdh, K., Buttner, M. J. & Chater, K. F. (1998). A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2). Mol Microbiol 29, 343-357.[Medline]
Shima, J., Hesketh, A., Okamoto, S., Kawamoto, S. & Ochi, K. (1996). Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). J Bacteriol 178, 7276-7284.[Abstract]
Sollner-Webb, B. & Reeder, R. H. (1979). The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis. Cell 18, 485-499.[Medline]
Takamatsu, S., Kunoh, H. & Ishizaki, H. (1976). Scanning electron microscopy observations on the perithecia of several powdery mildew fungi. I. Erysiphe and Sphaerotheca. Trans Mycol Soc Jpn 17, 409-417.
Ueda, K., Miyake, K., Horinouchi, S. & Beppu, T. (1993). A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators. J Bacteriol 175, 2006-2016.[Abstract]
Ueda, K., Hsheh, C.-W., Tosaki, T., Shinkawa, H., Beppu, T. & Horinouchi, S. (1998). Characterization of an A-factor-responsive repressor for amfR essential for onset of aerial mycelium formation in Streptomyces griseus. J Bacteriol 180, 5085-5093.
Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J. & Bibb, M. J. (1986). Construction and characterization of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen Genet 203, 468-478.[Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Received 23 February 1999;
revised 4 June 1999;
accepted 15 June 1999.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |