Involvement of amfC in physiological and morphological development in Streptomyces coelicolor A3(2)

Tohru Yonekawa1, Yasuo Ohnishi1 and Sueharu Horinouchi1

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
amfC plays a regulatory role in aerial mycelium formation in Streptomyces griseus and is distributed widely among Streptomyces species. Disruption of the chromosomal amfC gene in Streptomyces coelicolor A3(2) severely reduced formation of aerial hyphae, indicating that amfC is important in morphological development. In addition, the disruption caused S. coelicolor A3(2) M130 to produce much less actinorhodin, and to produce undecylprodigiosin at a later stage of growth, indicating that amfC also regulates secondary metabolism. S1 nuclease mapping showed that transcription of actII-ORF4, the pathway-specific transcriptional activator in the act gene cluster, was greatly reduced in the amfC disruptants. The defect in secondary metabolite formation was suppressed or overcome by a mutation in sre-1. Consequently, an amfC-disrupted strain derived from S. coelicolor A3(2) M145, an actinorhodin-overproducing strain due to the sre-1 mutation, still produced a large amount of actinorhodin. Extra copies of amfC in strains M130 and M145 did not change spore-chain morphology or secondary metabolite formation. However, the spores in these strains remained white even after prolonged incubation. Since only spore pigmentation was affected, all known whi genes, except whiE, responsible for the polyketide spore pigment formation, were assumed to function normally. S1 nuclease mapping showed that transcription of whiEP1, one of the promoters in the whiE locus, was reduced in S. coelicolor A3(2) containing extra copies of amfC. Introducing amfC into several other Streptomyces species, such as Streptomyces lividans, Streptomyces lavendulae and Streptomyces lipmanii, also abolished spore pigment formation. An increase in the amount of AmfC appeared to disturb the maturation of spores.

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Gram-positive soil bacteria of the genus Streptomyces are characterized by complex morphological differentiation resembling that of filamentous fungi, and by the ability to produce a wide variety of secondary metabolites, including antibiotics (Chater, 1984 , 1989 ). In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-{gamma}-butyrolactone) acts at an extremely low concentration as a switch for streptomycin production, streptomycin resistance, yellow pigment production and aerial mycelium formation (Horinouchi & Beppu, 1992 , 1994 ). Because of the essential role of A-factor in physiological and morphological development, A-factor-deficient mutants of S. griseus cannot form streptomycin or aerial mycelium (Horinouchi, 1996 ). Screening a DNA library of chromosomal genes from the wild-type strain for DNA fragments that restored aerial mycelium formation in the A-factor-deficient mutant strain HH1 has led to the isolation of an operon and two genes: amfR–amfA–amfB (Ueda et al., 1993 , 1998 ), orf1590 (Babcock & Kendrick, 1990 ; Ueda et al., 1993 ) and amfC (Kudo et al., 1995 ). amfC encodes a 218 aa protein that does not restore A-factor or streptomycin production, indicating that this gene acts in aerial mycelium formation independently of its secondary metabolic function (Kudo et al., 1995 ). Disruption of the chromosomal amfC in wild-type S. griseus severely reduced the abundance of spores due to infrequent sporulation. Nucleotide sequences homologous to amfC are distributed in all 12 Streptomyces species tested (Kudo et al., 1995 ), which suggests a common role of amfC. An AmfC homologue (222 aa) in Streptomyces coelicolor A3(2) shows 60% identity in amino acid sequence to AmfC in S. griseus.

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
S. griseus IFO 13350 (Hara et al., 1983 ) was obtained from the Institute of Fermentation, Osaka, Japan (IFO). S. coelicolor A3(2) strains M130 (hisA1 uraA1 SCP1- SCP2-) and M145 (sre-1 SCP1- SCP2-) and S. lividans strains TK21 and TK24 were obtained from D. A. Hopwood, John Innes Research Centre, Norwich, UK (Hopwood et al., 1985 ). A pUC19 derivative containing the whole amfC sequence in a 2·7 kb BamHI fragment (Kudo et al., 1995 ) was used as a source of amfC from S. coelicolor A3(2) M130. Other Streptomyces strains were obtained from IFO. Plasmids pIJ486, with a copy number of 40–100 per genome (Ward et al., 1986 ), and pKU209, with a copy number of 1–2 (Kakinuma et al., 1991 ), were used as Streptomyces cloning vectors. DNA was manipulated in Escherichia coli JM109 with pUC19 and M13mp19 (Yanisch-Perron et al., 1985 ). R2YE, R2 and MM agar media for S. coelicolor A3(2) were described by Hopwood et al. (1985) . SFM and TSB media were described by Floriano & Bibb (1996 ) and Onaka et al. (1998) , respectively. Histidine (50 mg l-1) and uracil (7·5 mg l-1) were added when necessary.

Recombinant DNA studies.
Restriction enzymes, T4 DNA ligase and other DNA-modifying enzymes were purchased from Takara Shuzo. [{alpha}-32P]dCTP (110 TBq mmol-1) for DNA labelling with the Takara BcaBest DNA labelling system and [{gamma}-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 EcoT14I–AccIII 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 SmaI–HindIII fragment from Tn5 (Beck et al., 1982 ) was first cloned in pUC19. A 307 bp SmaI–PmaCI fragment encoding the internal part (Arg37–Val140) 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 SmaI–PmaCI 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 7–9 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 2–7 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phenotypes of amfC mutants derived from S. coelicolor A3(2)
(i) Construction of amfC disruptants from strains M130 and M145. To examine the function of amfC, we disrupted the chromosomal amfC gene of S. coelicolor A3(2) M130 by using the neomycin resistance (neo) gene as a selection marker and the amfC sequence truncated at both ends to provide homology for integration of a non-replicating circular molecule by a single cross-over and, as a result of which, to disrupt the gene. True disruptants were selected by Southern hybridization with the neo and truncated amfC sequences as probes (data not shown). This procedure yielded four amfC disruptants (M130{Delta}amfC). We also generated eight amfC disruptants (M145{Delta}amfC) from a prototrophic strain, M145. The mutation did not cause any defects in growth, as determined by measuring cell mass in liquid culture or the diameters of colonies growing on solid medium.

(ii) Sparse aerial mycelium formation by amfC disruptants. Both mutants M130{Delta}amfC and M145{Delta}amfC formed very few spores on R2YE medium. Fig. 1a shows delayed and infrequent sporulation by M130{Delta}amfC. The parental strains M130 and M145 began to form aerial hyphae at 3 d after inoculation, whereas aerial hyphae of strains M130{Delta}amfC and M145{Delta}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{Delta}amfC and M145{Delta}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.



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Fig. 1. Phenotypes of S. coelicolor A3(2) strains M130 and M145 containing the disrupted amfC gene. (a) The parental strain M130 (as a control) and mutant M130{Delta}amfC were inoculated on R2YE agar and grown for the indicated time (in days) at 30 °C. Mutant M130{Delta}amfC formed sparse aerial hyphae at 5 d, whereas the parent formed aerial hyphae at 3 d. The blue colour in the centre of the colony of the parent at 5 d is due to actinorhodin produced. Actinorhodin production by the parent was apparent at 4 d, whereas almost no pigment was produced by mutant M130{Delta}amfC. (b) S. coelicolor A3(2) strains M145 and M145{Delta}amfC produced almost the same amount of actinorhodin on R2YE agar. The photograph was taken after 7 d growth at 30 °C. Strain M145 forms abundant grey spores, whereas the mycelium of mutant M145{Delta}amfC looks blue because of scarce aerial hyphae and spore formation. (c) Scanning electron micrographs of S. coelicolor A3(2) strains M130, M130{Delta}amfC and M130 harbouring pKU209-amfC. Strains M130 and M130 harbouring pKU209-amfC form coils of spore chains. The micrograph of mutant M130{Delta}amfC is of a relatively abundantly sporulating area in the colony.

 
(iii) Effects of amfC mutation on secondary metabolism in strain M130. Strain M130{Delta}amfC produced almost no blue pigments on R2YE medium (Fig. 1a) as well as R2YE medium containing maltose, mannitol or glycerol in place of glucose. The effect of the amfC disruption on actinorhodin production could also be detected on R2 and TSB media. On SFM medium, both the parental strain and M130{Delta}amfC produced little actinorhodin. For better quantification (Fig. 2), we examined production of the blue pigment in R2YE liquid cultures by measuring the A610, reported to reflect mainly the amount of actinorhodin (Bystrykh et al., 1996 ). The amount of the pigment produced by M130{Delta}amfC in liquid culture was also reduced. Introduction of pKU209-amfC restored pigment production to the same level as in the parental strain, both on solid and in liquid media. Thus, it was evident that amfC influenced actinorhodin production in strain M130.



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Fig. 2. Actinorhodin production by S. coelicolor A3(2) strains M130 ({circ}), M130{Delta}amfC ({bullet}) and M130{Delta}amfC harbouring pKU209-amfC ({blacktriangleup}). Each plot is the mean of values obtained from three independent cultures in R2YE medium.

 
We examined production of undecylprodigiosin, another pigmented antibiotic produced in the mycelium of S. coelicolor A3(2), by macroscopic observation of the lower surfaces of mycelial mats. This indicated that M130{Delta}amfC produced undecylprodigiosin at a much later stage of growth than the parent. Measurements of undecylprodigiosin extracted from mycelium grown on solid medium, purified by TLC and analysed on a scanning spectrophotometer (Fig. 3)gave results consistent with the macroscopic observation. At 7 d after inoculation, when the amount produced by the parental strain reached a maximum, M130{Delta}amfC produced almost no undecylprodigiosin. However, at 9 d after inoculation, M130{Delta}amfC began to produce undecylprodigiosin, indicating that the amfC mutation delayed but did not prevent production. All of these observations suggested that amfC mutations in strain M130 influenced secondary metabolism as well as morphogenesis, in contrast to the situation in S. griseus where amfC mutations caused no detectable effects on streptomycin or A-factor production (Kudo et al., 1995 ).



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Fig. 3. Effects of amfC disruption on undecylprodigiosin production. Undecylprodigiosin produced in mycelium on R2YE agar by S. coelicolor A3(2) strains M130 (—) and M130{Delta}amfC (---) was measured as described in Methods. The spectra shown are those that are close to the average profiles obtained from repeated experiments.

 
(iv) The amfC mutation had no detectable effect on secondary metabolism in strain M145. The prototrophic strain M145 produces much more actinorhodin and undecylprodigiosin than strain M130. Comparison of actinorhodin production by strains M145 and M145{Delta}amfC did not show any detectable difference (Fig. 1b). The two strains produced almost the same amount of blue pigments in R2YE liquid culture (data not shown), as well as on solid medium. The parental and M145{Delta}amfC strains also produced undecylprodigiosin in the same time frame and in the same yield. As discussed below, we assume that strain M145 overproduces actinorhodin and undecylprodigiosin due to the sre-1 mutation (Ochi & Hosoya, 1998 ), and this mutation overcomes the defect in amfC. Since mutant M130{Delta}amfC showed the same defects on media exogenously supplemented with histidine at 50 µg ml-1 and uracil at 7·5 µg ml-1, the hisA1 or uraA1 mutation did not interfere with the defects in physiological and morphological development caused by disruption of amfC.

(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 1–2 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{Delta}amfC and M145{Delta}amfC harbouring pKU209-amfC was unstable, a very slight increase in the amount of AmfC seemed to suppress pigment formation.



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Fig. 4. Lack of spore pigment formation by S. coelicolor A3(2) strains M145 and M130 harbouring pKU209-amfC. Strain M145 formed spores at 3–4 d after inoculation on SFM agar and the spores turned grey at 7 d, whereas the spores of strain M145 harbouring pKU209-amfC remained white even at 7 d. In the perimeter of the patch of strain M145 harbouring pKU209-amfC, grey spores appeared at 7 d. The spores of strain M130 harbouring pKU209-amfC also remained white 7 d after inoculation, whereas those of strain M130 turned grey.

 
On the other hand, strains M130 and M145 harbouring pIJ486-amfC or pKU209-amfC produced almost the same amounts of actinorhodin and undecylprodigiosin as the plasmid-free strains when examined as described above (data not shown). These observations suggested that overexpression of amfC caused no detectable effects on production of the pigmented polyketide-derived antibiotics actinorhodin and undecylprodigiosin, but reduced synthesis of the polyketide spore pigment.

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 {sigma} 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 ).



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Fig. 5. Transcriptional analysis of amfC in S. coelicolor A3(2) M130. (a) High-resolution S1 mapping of amfC mRNA prepared from cells grown on cellophane on the surface of R2YE medium for the time indicated (in days). S1-protected fragments were analysed in parallel with the sequence ladders (lanes G+A and T+C). The 32P-labelled probe alone (lane P) was included as a control. The arrowhead indicates the position of the S1-protected fragment. The 5' terminus of the mRNA was assigned to the indicated position because the fragments generated by the chemical sequencing reactions migrate 1·5 nt further than the corresponding fragments generated by S1 nuclease digestion of the DNA–RNA hybrids (half a residue from the presence of the 3'-terminal phosphate group and one residue from the elimination of the 3'-terminal nucleotide) (Sollner-Webb & Reeder, 1979 ). The time course of hrdB mRNA abundance was determined using the same mRNA preparations. (b) Nucleotide sequence of the amfC promoter region. The transcriptional start point is shown by an arrow. Probable -35 (GTCACA) and -10 (CACGAT) sequences, separated by a 19 nucleotide spacer, are underlined.

 
Transcriptional analysis of actII-ORF4 in amfC disruptants
actII-ORF4 is a pathway-specific transcriptional activator for the whole cluster of actinorhodin biosynthetic genes (Fernándes-Moreno et al., 1991 ; Fujii et al., 1996 ). Since the amfC-disrupted strains produced greatly reduced amounts of actinorhodin, we examined transcription of actII-ORF4 by S1 nuclease mapping with RNA prepared from mycelium grown on R2YE agar medium (Fig. 6a). In strain M130, actII-ORF4 mRNA with the expected size of 390 bp was detected after 2 d when cells grew as substrate mycelium, and it was still detected after 3 d when aerial hyphae appeared and after 4 d when cells grew as a mixture of aerial hyphae and spores. This is perhaps due to the mRNA preparation procedure; the harvested cell material used for RNA extraction was not fractionated and thus the late samples were mixtures of vegetative and aerial mycelium and spores. On the other hand, no actII-ORF4 mRNA was detected in M130{Delta}amfC, although hrdB mRNA was detected throughout growth. We concluded that the amfC disruption inhibited actII-ORF4 transcription, which resulted in severe reduction in actinorhodin yield.



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Fig. 6. Transcription of actII-ORF4 in S. coelicolor A3(2) strains M130 and M130{Delta}amfC and of whiE in S. coelicolor A3(2) M145, M145{Delta}amfC and M145 harbouring pKU209-amfC. (a) RNA was prepared from cells grown on cellophane on the surface of R2YE agar at 30 °C for the time indicated (in days). Under these conditions, strain M130 began to form aerial mycelium (Am) at 3 d after inoculation, whereas mutant M130{Delta}amfC began to form aerial hyphae at 4 d. In strain M130, actII-ORF4 mRNA of 390 bp was produced just before aerial mycelium formation. In mutant M130{Delta}amfC, only a very faint signal at this position was seen. The 32P-labelled probe (lane P) was included as a control. hrdB mRNA was produced throughout growth in both strains M130 and M130{Delta}amfC. (b) RNA was prepared from cells grown on cellophane on the surface of SFM agar at 30 °C for the time indicated (in days). Under these conditions, strains M145 and M145 harbouring pKU209 formed aerial mycelium (Am) at 2 d after inoculation and spores (Sp) at 3 d, whereas mutant M145{Delta}amfC formed less abundant aerial hyphae and spores at 3 and 4 d, respectively. In strains M145 and M145{Delta}amfC, whiE mRNA of 116 bp was produced at the onset of aerial mycelium formation. In strain M145 harbouring pKU209-amfC, however, little whiE mRNA was produced. In all three strains, hrdB mRNA was produced throughout growth.

 
Transcriptional analysis of whiE in strain M145 containing extra copies of amfC
The whiE gene cluster specifies the polyketide spore pigment in S. coelicolor A3(2), and two divergently oriented promoters, whiEP1 and whiEP2, are responsible for transcription of the gene cluster (Kelemen et al., 1998 ). The whiEP1 promoter transcribes most of the grey pigment production genes encoding ketosynthase, chain length factor, acyl carrier protein, aromatase and cyclase. Although both strains M130 and M145 containing extra copies of amfC did not form the spore pigment, we analysed transcription of whiEP1 in strain M145 harbouring pKU209-amfC because M145 formed spores more abundantly than M130. We also examined whiEP1 transcription in M145{Delta}amfC for comparison. RNA was prepared from mycelium grown on SFM solid medium because M145 formed more spores on this medium than on any other medium tested. In the parental strain M145, the whiEP1 mRNA with the expected size of 116 bp was detected in a large amount after 2 d of growth when aerial hyphae began to appear and decreased thereafter (Fig. 6b). Although the mRNA was still detected at 3 d when sporulation began, this is perhaps due to the mRNA preparation procedure, as described above. The whiEP1 mRNA pattern in strain M145 was in agreement with that reported by Kelemen et al. (1998 ). In strain M145 harbouring pKU209-amfC, a small amount of the whiEP1 mRNA appeared at 4 d. We assume that this mRNA derived from mycelium in the perimeter of the mycelium patch where the grey spore pigment was formed (Fig. 4). Thus, lack of pigment formation due to extra copies of amfC resulted from inhibition of whiE transcription.

In strain M145{Delta}amfC, the whiEP1 mRNA was detected at 3 and 4 d. Strain M145{Delta}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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Disruption of amfC in S. coelicolor A3(2) severely reduced the formation of aerial mycelium, irrespective of the carbon source in the medium. Many bld mutants (Merrick, 1976 ; Chater, 1989 ) show defects in aerial mycelium formation and antibiotic production, depending on the carbon source. amfC disruptants of S. griseus also showed sparse sporulation and amfC from S. coelicolor A3(2) restored its sporulation to normal (Kudo et al., 1995 ). Introducing amfC from S. griseus into S. coelicolor A3(2) and S. lividans abolished spore pigment formation (data not shown), consistent with the idea that amfC in both strains is functionally the same. Since amfC appears to be distributed widely among Streptomyces spp. (Kudo et al., 1995 ), we assume that it plays a common, but not essential, regulatory role in aerial mycelium formation in this genus. Lack of spore pigment formation by several other Streptomyces species harbouring pIJ486-amfC also supports this idea.

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
 
We are grateful to K. F. Chater and M. J. Bibb for providing us with the DNA fragments containing whiE and actII-ORF4 and the data on whiE transcription before publication. This work was supported, in part, by the Nissan Science Foundation, by the Research for the Future Program of JSPS and by the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan (BDP-99-VI-2-3).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 23 February 1999; revised 4 June 1999; accepted 15 June 1999.



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