A novel extracytoplasmic phenol oxidase of Streptomyces: its possible involvement in the onset of morphogenesis

Kohki Endo1, Kuniaki Hosono1, Teruhiko Beppu1 and Kenji Ueda1

Department of Applied Biological Sciences, Nihon University, 1866 Kameino, Fujisawa, 252-8510, Japan1

Author for correspondence: Kenji Ueda. Tel: +81 466 84 3936. Fax: +81 466 84 3935. e-mail: ueda{at}brs.nihon-u.ac.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Exogenous addition of copper stimulates cellular differentiation in Streptomyces spp. Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in Streptomyces griseus. Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized. EpoA activity, examined by an in-gel stain procedure with N,N'-dimethyl-p-phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium. The enzyme activity was abolished and markedly reduced in the mutants forA-factor biosynthesis and amfR, respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in S. griseus. EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined. A homologous sequence identified in the genomic database of Streptomyces coelicolorA3(2) was used as a probe to clone the complete epoA gene of S. griseus. The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme. EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment. Introduction of epoA on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant. Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in S. griseus and several other species. Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.

Keywords: laccase, EpoA, melanin, copper, morphological differentiation, Streptomyces griseus

Abbreviations: BCDA, bathocuproinedisulfonic acid; DOPA, dihydroxyphenylalanine; EpoA, extracytoplasmic phenol oxidase; PMP, p-methoxyphenol

The DDBJ accession number for the sequence reported in this paper is AB056583.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The filamentous, soil-living, Gram-positive bacterial genus Streptomyces performs complex morphological differentiation, resembling that of filamentous fungi (Chater, 1993 ). Early in the life-cycle, the organism grows as a branching, multinucleate substrate mycelium. In response to nutritional limitation and various environmental signals, the substrate mycelium produces aerial hyphae, which culminate into spore chains via septum formation at regular intervals. Streptomyces is also characterized by its ability to produce a wide variety of secondary metabolites, including antibiotics, that have important applications in pharmaceutical industries (Miyadoh, 1993 ). It is known that the initial regulatory steps for the morphological and physiological development of this organism are controlled by common regulatory elements. For example, several bld mutants of Streptomyces coelicolor A3(2) are defective in both aerial mycelium formation and antibiotic production (Chater, 1993 ). In Streptomyces griseus, an autoregulatory substance, A-factor (2-isocapryloyl-3R-hydroxymethyl-{gamma}-butyrolactone), acts as a switch for both morphological development and secondary metabolism (Horinouchi, 1999 ).

Our previous attempt to explore novel regulatory mechanisms for cellular differentiation and antibiotic production in Streptomyces revealed that exogenous copper remarkably stimulates those phenotypes in various Streptomyces spp. (Ueda et al., 1997 ). We noticed the phenomenon by the phenotype of a bald mutant of Streptomyces tanashiensis, which was restored to wild-type morphology and antibiotic productivity by supplementing with 1 µM or less of copper. The potent and specific effect suggested the presence of an intrinsic copper-dependent regulatory mechanism for the onset of morphogenesis and antibiosis in Streptomyces.

A coincidental event caused by exogenous copper in various Streptomyces spp. is melanin production. The initial reaction of microbial melanin biosynthesis is catalysed by phenol oxidases, copper-containing enzymes, including tyrosinase, which oxidizes tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to DOPA quinone. Subsequent oxidation and autopolymerization of DOPA quinone result in the formation of a pigment termed DOPA melanin (Bell & Wheeler, 1986 ). Another enzyme involved in melanin synthesis is laccase, which also catalyses the oxidation of DOPA to produce the melanin pigment (Thurston, 1994 ). Recently, we cloned and characterized a gene encoding tyrosinase widespread among Streptomyces, melC1C2, in S. griseus and found that overexpression of the operon stimulated aerial mycelium formation of this organism, which implied that the enzyme has some role in the onset of morphogenesis (Endo, 2001 ). Involvement of phenol-oxidizing enzymes in cellular development has also been suggested by studies of fungal differentiation (reviewed by Bell & Wheeler, 1986 ; Thurston, 1994 ). A melC2 mutant of S. griseus, however, performed normal cellular differentiation and melanin production, which suggested the presence of another phenol oxidase in this organism. Here we report identification and characterization of a novel phenol oxidase in S. griseus. The enzyme is a laccase-like secreted enzyme whose activity is closely linked to cellular differentiation of this organism. The results strongly suggest that a substance(s) synthesized by the enzyme has an activity to stimulate morphogenesis in Streptomyces.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and media.
The wild-type strain S. griseus IFO 13350 and an A-factor-deficient mutant strain, HH1, were described previously (Horinouchi et al., 1984 ). An amfR-deficient mutant has also been described (Ueda et al., 1998 ). S. coelicolor A3(2) M145 was obtained from D. A. Hopwood (John Innes Centre, Norwich, UK). Streptomyces olivaceus NRRL B-1125 and Streptomyces aureus IAM0092 were obtained from the culture collection of the Laboratory of Fermentation and Microbiology, University of Tokyo, Japan. An environmental actinomycete strain was isolated from soil collected at Fujisawa-shi, Kanagawa, Japan. Bacillus subtilis ATCC 6633 was used for the bioassay of streptomycin. Escherichia coli JM109 [{Delta}(lac-pro) thi-1 endA1 gyrA96 hsdR17 relA1 recA1/F' traD36 proAB lacIq lacZ{Delta}M15] and pUC19 (Yanisch-Perron et al., 1985 ) were used for general DNA manipulation. Media and growth conditions for E. coli were as described by Maniatis et al. (1982) . Plasmid pIJ922 (carrying thiostrepton resistance) has a copy number of 1 to 2 (Hopwood et al., 1985 ). pSL1 used for genetic complementation of an amfR mutant was described previously (Ueda et al., 1993 ). S. griseus strains were grown in Bennett’s/glucose [containing (g l-1): yeast extract (Difco), 1; meat extract (Kyokuto), 1; NZ amine (Wako), 2; glucose (Kokusan), 10; pH 7·2], YMP/glucose [containing (g l-1): yeast extract (Difco), 2; meat extract (Kyokuto), 2; Bacto Peptone (Difco), 4; NaCl, 5; MgSO4.7H2O, 2; glucose (Kokusan), 10; pH 7·2] and nutrient broth (Difco). For Bennett’s/maltose and YMP/maltose, glucose was replaced with maltose. Agar (Kokusan) was supplied at 1·5% for solid media. For the selection of transformants, ampicillin and kanamycin (Wako) at a final concentration of 50 µg ml-1 were used for E. coli. For S. griseus, thiostrepton (Sigma) and neomycin (Wako) were added at a final concentration of 20 µg ml-1. CuSO4 (Kokusan) was supplemented at 10 or 100 µM, and bathocuproinedisulfonic acid (BCDA; Sigma) was added at 200 µM. Synthetic A-factor is a kind gift from Y. Yamada (Fukuyama University, Hiroshima, Japan).

Filter disc assays.
As for the experiments shown in Figs 1 and 7, filter discs (8 mm diam., thick; Advantec) were soaked with 50 µl 100 mM CuSO4, 2 mg A-factor ml-1, 0·8 M p-methoxyphenol (PMP; Kokusan), 1 mg synthetic melanin ml-1 (Sigma), 10 mM DOPA and its resultant product with extracytoplasmic phenol oxidase (EpoA). All chemicals were dissolved in deionized water except for DOPA (see below). All solutions sterilized by filtration through a 0·22 µm membrane filter (Millipore) were placed onto solid medium inoculated with S. griseus and other Streptomyces spp. Cells were inoculated with sterile toothpicks either to form single colonies in a line at an equal distance or to form a confluent lawn. Plates were incubated for 2–5 days at 28 °C.



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Fig. 1. (a) Filter disc assay for stimulation or inhibition of morphogenesis in S. griseus. The filter discs, soaked with 50 µl 100 mM CuSO4 and 0·8 M PMP, were placed onto Bennett’s/glucose solid medium inoculated with wild-type S. griseus. Patches were photographed after 5 (copper) and 2·5 days (PMP) growth. The disc soaked with 50 µl 2 mg synthetic A-factor ml-1 was placed onto YMP/glucose solid medium supplied with 100 µM CuSO4 and inoculated with S. griseus HH1. The photograph was taken after 3 days growth. (b) Inhibition of morphogenesis and melanogenesis of S. griseus by BCDA. Wild-type S. griseus was inoculated on YMP/glucose solid medium with or without BCDA. Each colony was photographed from the top (left) and bottom (right) after 5 d growth.

 
Detection of EpoA activity by in-gel stain.
To detect EpoA activity, an in-gel activity stain method was used. Since EpoA was active even in the presence of SDS, all in-gel stain assays were done in SDS-polyacrylamide gel. The procedure for SDS-PAGE followed the standard protocol and the samples were boiled for 2 min prior to application onto the gel. The concentration of polyacrylamide was 12·5% in separating gels and 4% in stacking gels. For detection of enzyme activities, gels were washed with deionized water for 30 min and stained for 10 min at 37 °C with a solution containing 10 mM N,N'-dimethyl-p-phenylenediamine sulfate and 10 mM 1-naphthol in sodium citrate buffer (containing 37 mM citric acid, 126 mM Na2HPO4, pH 6·0). The activity of EpoA was detected by the indigo-coloured precipitate.

Proteins for the assay were prepared as follows. Various S. griseus strains were cultured on solid media, whose surfaces were covered with sterilized cellophane sheets that enabled efficient collection of the mycelium. Cells were washed twice with buffer A containing 10 mM Tris/HCl (pH 7·0), 1 mM EDTA and 10% glycerol, finally resuspended in 10 ml buffer A and disrupted by sonication (model XL2020; Misonix). The disrupted mycelium was then centrifuged at 10000 g for 30 min at 4 °C and the resultant supernatant containing 10 µg proteins was subjected to the in-gel stain. For the restoration of EpoA activity by A-factor, a filter disc soaked with 0·1 µg synthetic A-factor was placed onto a Bennett’s/maltose agar plate supplemented with 10 µM CuSO4 and inoculated with HH1 strain to produce a confluent lawn. The plate was incubated for 3 days and the sporulating colonies in response to the exogenous A-factor were collected and processed as above. For the detection of EpoA activity in the culture supernatant and the cell-free extract, wild-type S. griseus was cultured in 50 ml Bennett’s/maltose liquid medium with 10 µM CuSO4 and cells were harvested by centrifugation after 3 days growth. A sample (30 µl) of the resultant supernatant containing 3 µg proteins was assayed as a culture supernatant fraction. The resultant cellular precipitates were processed by the same procedure as the plate culture described above and the resultant 3 µg proteins was assayed as a cell-free extract fraction.

Purification of EpoA

S. griseus IFO 13350 was used as a source of EpoA. The in-gel stain with SDS-polyacrylamide gels was used to detect enzyme activities, monitor protein purification and estimate molecular sizes. For monitoring protein purification, gels were stained with Coomassie brilliant blue R-250 or a silver stain kit (Pharmacia). During purification, protein concentrations were measured with a Bio-Rad protein assay kit with bovine serum albumin as standard.

(i) Preparation of cell extract.
Mycelia of S. griseus IFO 13350 were harvested from the culture on 100 Petri dishes (85 mm diam.) of Bennett’s/maltose solid medium with 10 µM CuSO4. Each Petri dish contained 25 ml medium and the cells were grown for 4 days at 28 °C. Cells were washed twice with buffer A, resuspended in 200 ml buffer A and disrupted by sonication. The supernatant obtained by centrifugation of the disrupted mycelium at 10000 g for 30 min at 4 °C was used as a cell extract. Proteins (3·0 g) were recovered by this step.

(ii) DEAE-Toyopearl column chromatography.
The cell extract was applied to a DEAE-Toyopearl column (42x320 mm; Tosoh) previously equilibrated with buffer A. After the column had been washed with 0·5 l buffer A, proteins were eluted with a linear gradient of KCl from 0 to 0·25 M in a total volume of 0·5 l buffer A at a flow rate of 2 ml min-1. Fractions (90 ml) containing 75 mg proteins with oxidase activity were recovered.

(iii) Copper-affinity chromatography.
The sample was then applied to a HiTrap chelating column equilibrated with buffer B, containing 10 mM Tris/HCl (pH 7·0) and 10% glycerol, with a fast protein liquid chromatograph (Pharmacia), according to the protocol of the manufacturer. Prior to application to the column, samples were dialysed against buffer B and 0·5 M NaCl was added to prevent non-specific proteins from binding to the column by ionic exchange. Proteins were eluted with buffer B and 0·05 M EDTA in a total volume of 50 ml at a flow rate of 1 ml min-1. The active fraction containing 40 mg protein was recovered.

(iv) Gel filtration chromatography.
The sample was dialysed overnight against buffer A and then applied to a Sephadex 200 HR 10/30 column equilibrated with buffer A with a fast protein liquid chromatograph, according to the protocol of the manufacturer. Proteins were eluted with buffer A in a total volume of 25 ml at a flow rate of 0·25 ml min-1. The active fraction containing 3 mg protein was recovered.

(v) Non-denaturing PAGE and protein elution.
The proteins obtained above were concentrated to 2 ml by membrane filtration (Centricon with molecular cut off 3000 Da; Amicon) and subjected to non-denaturing PAGE (containing 10 and 2·5% polyacrylamide in the separating and stacking gels, respectively). The gel pieces containing EpoA were sliced, homogenized with a glass homogenizer and resuspended in 20 ml buffer A. The suspension was gently stirred at 4 °C overnight and centrifuged at 10000 g for 20 min to remove gel particles. The eluates were collected and concentrated by membrane filtration to yield 1 ml of the purified protein solution. The amount of EpoA recovered was 0·3 mg.

Reaction of EpoA with several substrates.
The purified EpoA protein was reacted with L-tyrosine, L-DOPA, catechol and PMP. Each substrate (10 mM), dissolved in sodium citrate buffer, was mixed with 40 µg purified EpoA in a total volume of 1 ml and incubated overnight at 30 °C. For the reaction with DOPA, the absorbance of the reaction mixture was scanned every 1 h during 5 h incubation. The reaction was terminated by boiling for 10 min. The reactant (50 µl) after 5 h incubation or a diluted solution of it in citrate buffer was subjected to the filter disc assay described above. To estimate the molecular mass of the active substance, the reaction mixture was dialysed against an appropriate volume of sodium citrate buffer with dialysis membranes of various molecular cut-off sizes (Spectrum Laboratories).

Amino acid sequence determination.
For determination of the N-terminal amino acid sequence of the EpoA protein, the purified enzyme was electrophoresed in an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Immobilon Transfer, 0·45 µm pore size; Millipore). The membrane was stained with 0·1% amide black in methanol/acetic acid (40:10, v/v) and destained with 2-propanol/acetic acid (10:10, v/v). The membrane pieces containing the protein were cut out and washed with distilled water. The amino acid sequence was determined with an Applied Biosystems model 494HT pulsed liquid sequencer. All reagents for sequencing were purchased from Applied Biosystems.

General recombinant DNA studies.
Restriction enzymes and other DNA-modifying enzymes were purchased from Takara Shuzo. [{alpha}-32P]dCTP (110 TBq mmol-1) for DNA labelling with a Takara BcaBest DNA Labelling System was purchased from Amersham Pharmacia Biotech. DNA manipulation, Southern hybridization and colony hybridization were as described by Maniatis et al. (1982) and genetic manipulation in Streptomyces was as described by Hopwood et al. (1985) . The nucleotide sequence was determined by the dideoxy chain-termination method with a Thermo Sequenase Cycle Sequencing kit (Amersham) on an automated DNA sequencer (LiCor model 4200).

Cloning of a DNA fragment containing epoA of S. griseus.
To clone the epoA gene of S. griseus, the homologous gene of S. coelicolor was used as probe. Using the primers EPC1 and EPC2 (Table 1), a 1·8 kb DNA fragment containing the epoA homologue was amplified from the chromosomal DNA of S. coelicolor A3(2) by PCR. The amplicon was then used as a probe for Southern hybridization against the S. griseus chromosome. The 4·2 kb BamHI fragment that hybridized to the S. coelicolor probe was cloned into the BamHI site of pUC19 to generate pUC-A (see Fig. 5) by using standard DNA manipulation techniques, including colony hybridization. Nucleotide sequencing of the fragment revealed the presence of the complete ORF for epoA at the terminal region of the 4·2 kb BamHI fragment. The 3·0 kb BglII fragment containing the upstream region from epoA was also cloned by following a standard gene-walking protocol, using a 0·9 kb BamHI–BglII fragment as probe. The BglII fragment was inserted into the BglII site of pUC-Bgl to generate pUC-B. pUC-Bgl was constructed by attaching an 8-mer BglII linker at the HincII site of pUC19. Finally, the nucleotide sequence of the 4·6 kb BamHI region was determined, including part of the fragment cloned into pUC-B. To construct pEPO1 (see Fig. 5), the epoA-containing 1·4 kb fragment was amplified from S. griseus IFO 13350 chromosomal DNA by PCR using primers EPG1 and EPG2 (Table 1), recovered as an XhoI-digested fragment and cloned into the XhoI site of pIJ922.


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Table 1. Oligonucleotide primers used in this study

 
Gene disruption.
epoA was disrupted by a standard homologous recombination technique using pUC-C (see Fig. 5). The mutagenized construct on pUC-C was prepared as follows. Fragment a was excised from pUC-A as a BamHI/BglII-digested fragment. For fragment b, the 1 kb region was amplified by PCR using primers EPG3 and EPG4 (Table 1) and recovered as a BglII/HindIII-digested fragment. Thus prepared fragments a and b were inserted between the BamHI and HindIII sites of pUC19 by three-fragment ligation. The resultant plasmid was digested with BglII and ligated to an aphII (Beck et al., 1982 ) cassette prepared as a BamHI-digested fragment by appropriate PCR to generate pUC-C. The mutagenized epoA on pUC-C lacked the C-terminal portion containing the putative site for copper-binding. The mutagenized construct was then recovered from pUC-C as an EcoRI/HindIII-digested fragment and the linear DNA was introduced into S. griseus IFO 13350 by a standard transformation procedure. The mutants were screened among the resultant kanamycin-resistant colonies carrying the mutagenized construct via a double-crossover event. A subsequent check by Southern hybridization with appropriate probes confirmed the true disruptant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Stimulatory effect of copper in S. griseus
The effect of exogenous copper was assessed by a filter disc assay in S. griseus (Fig. 1a, top). In response to exogenous copper diffusing from the filter disc, wild-type S. griseus formed abundant spores and produced marked diffusible melanin. Streptomycin productivity was also stimulated (not shown). The essential requirement for copper in cellular differentiation and melanin production of S. griseus was shown by the addition of BCDA, a specific chelator for copper ions (Zak, 1958 ). Addition of BCDA at 200 µM did not affect cellular growth, but strongly inhibited aerial mycelium formation and melanin production (Fig. 1b). A similar effect of BCDA on cellular differentiation in S. coelicolor A3(2) was reported by Keijer et al. (2001) .

The diffusible melanin produced by the wild-type was visible by adding 100 µM CuSO4 in YMP/glucose agar medium. The culture conditions revealed that S. griseus HH1, an A-factor-deficient strain (Horinouchi et al., 1984 ), was unable to produce the pigment and the deficiency was restored by an exogenous supply of synthetic A-factor (Fig. 1a, middle). This result indicates that melanogenesis is controlled by A-factor in S. griseus.

The initial reaction of microbial melanin biosynthesis is mediated by phenol oxidases, copper-containing enzymes such as tyrosinase and laccase (Bell & Wheeler, 1986 ). To assess possible involvement of phenol oxidase in morphogenesis, the effect of PMP, an inhibitor of melanin production in Streptomyces bikiniensis (Tomita et al., 1990 ), was examined (Fig. 1a, bottom). The result showed that the inhibitor specifically blocked aerial mycelium formation in S. griseus without affecting vegetative growth. The colony next to the filter disc normally formed aerial mycelium, showing that an optimum concentration of PMP is required to exert its inhibitory effect on morphogenesis in S. griseus, as observed with inhibitors of eukaryotic protein kinases (Hong et al., 1993 ). A notable feature was that the colonies at the inhibitory range produced a marked reddish pigment, different from the melanin normally synthesized by the organism. The pigment was observed in a previous study and was assumed to be an oxidized product of PMP synthesized by the activities of phenol oxidases in S. griseus (Endo et al., 2001 ).

Identification of a copper- and A-factor-dependent phenol oxidase activity
Our previous study suggested the presence of a phenol oxidase different to MelC2 (Endo et al., 2001 ), and the above results suggested the presence of a phenol-oxidizing enzyme that depends on copper and A-factor for its activity. Such an enzyme activity was identified in the cell extract of S. griseus IFO 13350 by the in-gel stain procedure using N,N'-dimethyl-p-phenylenediamine sulfate as a substrate. The enzyme showed marked activity even in SDS-PAGE, which allowed us to estimate its apparent molecular mass of approximately 100 kDa. The enzyme was produced by a melC2 mutant (Endo et al., 2001 ), which indicated that the oxidase is not identical to the MelC2 tyrosinase. The time-course experiment revealed that the enzyme activity depends on copper and the carbon-source supplied in the medium (Fig. 2a); the mycelium grown on Bennett’s/maltose solid medium showed strong activities, whereas the activity was significantly reduced when the cells were grown on Bennett’s/glucose medium. The activities were markedly enhanced by the addition of 10 µM CuSO4. The activity profile of the enzyme was parallel to the abundance of sporulating colonies, i.e. the best conditions for sporulation were Bennett’s/maltose with copper and the second was Bennett’s/maltose without copper or Bennett’s/glucose with copper. On Bennett’s/glucose without copper, the organism sporulated poorly until the late growth phase. Supplying 1% galactose resulted in a similar reduction of enzyme activity as observed with glucose, while supplying 1% mannitol or no sugar caused a similar effect to maltose (data not shown). The oxidase was named EpoA, according to the properties described below.



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Fig. 2. In-gel activity stain of EpoA. (a) Time-course (in days) of EpoA activity in wild-type S. griseus grown on Bennett’s/maltose (Mal) or Bennett’s/glucose (Glc) solid media with or without 10 µM CuSO4. (b) EpoA activity in various strains of S. griseus grown on Bennett’s/maltose solid medium with 10 µM CuSO4 (left panel). The activities in cell extract (CE) and culture supernatant (sup) of the wild-type strain cultured in Bennett’s/maltose liquid medium with 10 µM CuSO4 are also shown (right panel).

 
To assess the relationship of EpoA to morphogenesis further, two bald mutants of S. griseus were examined for enzyme activity. Strain HH1, an A-factor-deficient mutant, produced no EpoA activity (Fig. 2b). Exogenous supplementation of A-factor restored activity to the wild-type level in this mutant, which confirmed that the lack of EpoA activity is due to A-factor-deficiency. The other bald mutant depleted in the amfR gene, encoding an essential transcriptional regulator for the onset of morphogenesis (Ueda et al., 1993 , 1998 ), produced a markedly reduced EpoA activity. Introduction of pSL1 carrying an intact amfR gene into the mutant restored the wild-type activity.

Purification and N-terminal sequence of EpoA
EpoA was purified from the cell extract of wild-type S. griseus. Mycelium harvested from solid culture was used as the starting material for the purification, since these culture conditions enabled a higher yield of the enzyme than liquid culture. Starting with the culture in one hundred 25 ml Bennett’s/maltose agar plates supplemented with 10 µM CuSO4, the enzyme was purified to homogeneity by DEAE column chromatography, copper-affinity chromatography, gel filtration chromatography and elution from a non-denaturing gel (Fig. 3). The fractionation by DEAE was very effective and the enzyme was eluted at a very low concentration of KCl (approx. 50 mM), eliminating most of the contaminated proteins.



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Fig. 3. Monitoring of EpoA purification. SDS-polyacrylamide gels of silver stain (left panel) and in-gel activity stain (right panel) are shown. Lanes: 1 and 4, the active fraction after DEAE-column chromatography; 2 and 5, the active fraction after elution from a non-denaturing gel; 3, the denatured fraction of the eluate from a non-denaturing gel.

 
We determined the N-terminal amino acid sequence of the purified EpoA by Edman degradation after the transfer of the protein to a polyvinylidene difluoride membrane. The determined sequence was: N-Ala-Glu-Asn-Pro-Pro-Arg-Thr-Ala-Pro-Ala-Gly-Gly-Val-Val-Arg-Arg-Leu-Lys-Met-Tyr-Ala-Glu-Lys-Leu-Pro-Asn-Gly-C (27 aa).

The purified EpoA protein was checked for its oxidizing activity against several substrates. While overnight incubation with tyrosine, PMP or catechol failed to produce pigment, reaction with DOPA resulted in the formation of a melanin-like pigment. The polymerization profile of DOPA during the 5 h of incubation with purified EpoA is shown in Fig. 4. The colour of the pigment produced by EpoA appeared to be identical to that of the melanin produced by S. griseus, which suggested that EpoA is one of the enzymes that mediates melanin biosynthesis in this organism.



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Fig. 4. Polymerization of DOPA by purified EpoA, monitored by scanning changes in absorbance from 320 to 520 nm.

 
Molecular cloning of epoA
A homology search in the genomic database of S. coelicolor (http://www.sanger.ac.uk/Projects/S_coelicolor/) revealed that the gene product of the 22nd ORF on cosmid SC4C6 (accession no. T35030) carried a sequence homologous to the N-terminal sequence of EpoA. The homologue consisted of 343 aa with a molecular mass of 37 kDa, and possessed a feature characteristic of copper oxidase.

The high sequence similarity and the copper oxidase feature prompted us to use the S. coelicolor sequence as a probe to clone epoA of S. griseus. A 1·8 kb fragment containing the epoA homologue of S. coelicolor was amplified by PCR, and used as probe to clone the epoA-containing fragments of S. griseus (see Methods). Nucleotide sequencing and analysis by the FRAME program (Bibb et al., 1984 ) revealed the presence of two complete (epoA and orf1) and two truncated (orf2 and orf3) ORFs in the 4·6 kb region (Fig. 5a). The gene organization in the epoA region of S. griseus was different from that of S. coelicolorA3(2) and there was no strong similarity among the proteins encoded by the ORFs flanking the epoA gene of the two strains.



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Fig. 5. Restriction map of the epoA-containing fragment with the positions and directions of ORFs (a), and sequence alignment of EpoA with its homologue in S. coelicolor (b). (a) The extents and directions of ORFs predicted by using the FRAME program (Bibb et al., 1984 ) are indicated by arrows. The positions of the fragments cloned by colony hybridization and the region used in the knockout experiment are also shown. pEPO1 carries a 1·4 kb fragment on a low-copy-number plasmid, pIJ922 (Hopwood et al., 1985 ). The gene organization in the epoA region of S. coelicolor A3(2) is also shown. (b) The amino acid sequence of EpoA of S. griseus (EpoAg) was aligned with the homologous sequence of S. coelicolor A3(2) (EpoAc) by using the CLUSTAL W program (Thompson et al., 1994 ). Asterisks show the conserved amino acid residues between EpoAg and EpoAc. The putative copper-binding sites are highlighted with the corresponding sequences of three microbial laccases: NCR, Neurospora crassa (accession no. A28523); CHI, Coriolus hirsutus (A35883); MME, Marinomonas mediterranea (AF184209). The amino acid residues conserved among these oxidases are boxed. The amino acid residues for the two types of potential copper-binding sites are indicated by open (Type I) and closed (Type III) triangles. The residues corresponding to the N-terminal amino acid sequence determined by Edman degradation are underlined and the putative cleavage site for the signal peptidase is indicated by an arrow.

 
The EpoA product of S. griseus consisted of 348 aa with a molecular mass of 37 kDa, with 71% identity to that of S. coelicolor (Fig. 5b). A protein motif search in the PROSITE database (Hofmann et al., 1999 ) revealed that EpoA proteins carry the multicopper oxidases signatures (PROSITE nos PS00079 and 00080), which are known to be contained in laccase. The 5 aa residues involved in copper-binding were conserved in EpoA (His287, Cys288, His 289, His293 and Met298; Fig. 5b). A homology search in the protein databases revealed that EpoA shows distinct, though not end-to-end, similarity to various laccases identified mostly in fungi.

The determined N-terminal amino acid sequence of the purified EpoA of S. griseus corresponded to the internal portion, Ala35–Gly61 (Fig. 5b), which suggested that the N-terminal polypeptide of 34 aa preceding the determined sequence functions as a signal peptide. The Ser (S. griseus) and Ala (S. coelicolor) residues at 33–34 aa were assumed to be the cleavage sites for processing during secretion. The property of EpoA as a secreted enzyme was confirmed by the activity detected both in the cell-free extract and culture supernatant of wild-type S. griseus grown in liquid medium (Fig. 2b, right panel). The EpoA activity in the cell extract was detected at the position of relatively higher molecular mass than that of the enzyme in the culture supernatant.

The apparent molecular mass of active EpoA by SDS-PAGE (ca 100 kDa) was three times higher than that deduced from the primary sequence (34 kDa in its mature form). After boiling for 20 min in the loading buffer for SDS-PAGE, the purified EpoA migrated to a single band at the position of the deduced molecular mass (Fig. 3, lane 3). The denatured protein showed no oxidase activity. These results suggest that the active EpoA consists of a homotrimeric structure.

Effects of inactivation and introduction of epoA in S. griseus
To assess the role of epoA, a null epoA mutant was generated by homologous recombination (see Methods). The resultant epoA mutant lost the oxidase activity in the in-gel stain. However, it did not show marked phenotypic changes in comparison to the wild-type except for a slight decrease in the apparent amount of melanin pigment produced (not shown). Introduction of an intact epoA on a low-copy-number plasmid (pEPO1; see Fig. 5a) restored both the oxidase activity and the wild-type melanin productivity in the epoA mutant, indicating that the reduced melanin productivity was due to the inactivation of epoA.

pEPO1 was also introduced into S. griseus HH1, an A-factor-deficient mutant, which was shown to lack EpoA activity in the above experiment. Introduction of the plasmid restored a low level of extracellular EpoA activity (Fig. 2b). Furthermore, the introduction of pEPO1 suppressed the deficiency in morphogenesis and melanogenesis in HH1; the transformant partially restored both aerial mycelium formation (Fig. 6) and melanin production (not shown) on Bennett’s/glucose solid medium. These phenotypes were abolished by adding BCDA. The restoration of aerial mycelium was also observed on Bennett’s/maltose medium (Fig. 6). A similar experiment showed that the introduction of pEPO1 caused precocious aerial mycelium formation in the wild-type strain of S. griseus.



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Fig. 6. Stimulation of aerial mycelium formation by the introduction of an epoA-containing plasmid. Strain HH1 harbouring pEPO1 or pIJ922 (negative control) was inoculated on Bennett’s solid medium (+10 µM CuSO4) supplied with glucose or maltose. Colonies on Bennett’s/glucose supplemented with 200 µM BCDA are also shown. Patches were photographed after 4 days growth.

 
Effect of in vitro reaction product of EpoA
The above result indicated that EpoA has an in vivo activity inducing aerial mycelium formation in S. griseus. We speculated that the activity might possibly be mediated by melanin or a related substance synthesized by the extracytoplasmic activity of EpoA. To assess this possibility, we examined the exogenous effect of the in vitro reaction product of EpoA with DOPA on morphogenesis and found that adding the reactant caused precocious aerial mycelium formation in wild-type S. griseus in a dose-dependent manner (Fig. 7). Neither the same amount of DOPA similarly treated without EpoA nor purified EpoA, as used for the reaction, exhibited the stimulatory effect. The absence of stimulation of EpoA itself would be due to a low level of DOPA produced by the organism during the early growth phase. A notable feature was that the original reaction product without dilution (Fig. 7a, left end) formed a stimulation zone in a ring, implying that the effective concentration of the putative stimulant is in a rather narrow range. A similar effect was observed with commercial synthetic melanin prepared by incubating L-tyrosine with a fungal tyrosinase. We also observed that the reaction product of EpoA caused precocious aerial mycelium formation in several other species, including a strain freshly isolated from soil (Fig. 7b). Ultrafiltration of the EpoA reaction product by dialysis membranes with various molecular cut-off sizes clearly showed that the molecular mass of the stimulatory substance is between 500 and 1000 Da. The activity of fungal melanin also separated into the same molecular size fraction. The active fractions were slightly yellow, but almost colourless.



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Fig. 7. Exogenous effect of the substance synthesized by EpoA in S. griseus (a) and other species (b). (a) The original reaction mixture was sequentially diluted and applied onto the discs to examine the dose effect. Each 50 µl of 10 mM DOPA and 1 mg fungal melanin ml-1 solution was also assayed. Patches were photographed after 1 day growth on YMP/glucose agar with 10 µM CuSO4. (b) The effect of the EpoA reaction product was examined in various species. Patches were photographed after growth for 2 (strains from culture collection) and 1 day (environmental strain). Each disc contained 50 µl reaction mixture without dilution. All strains were grown on Bennett’s/maltose agar.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present study, we have identified a novel phenol oxidase in S. griseus. The activity of the enzyme was correlated with the stimulatory effect of copper on morphogenesis in this organism. The A-factor-deficient mutant HH1 lacked EpoA activity and the introduction of epoA on a plasmid into the mutant partially restored both the enzyme activity and the ability to form aerial mycelium. We previously observed a similar effect of the meC1C2 genes of this organism, in which introduction of the genes on a plasmid caused precocious aerial mycelium formation in S. griseus and S. lividans (Endo et al., 2001 ). These results support the notion that phenol oxidases play some role in morphological differentiation of Streptomyces, which is also suggested by the inhibitory effect of PMP (Fig. 1).

EpoA probably comprises a homotrimer that is stably maintained even during migration in SDS-PAGE. A recombinant EpoA protein expressed in E. coli showed oxidase activity at the same position in SDS-PAGE, which confirmed the homocomplex structure (unpublished data). A similar SDS-resistant property was reported by Edens et al. (1999) for a laccase of Gaeumannomyces graminis, which is assumed to consist of a stable homodimer of its glycosylated form.

EpoA contained the multicopper oxidase signatures that are common to the laccase family in its C-terminal region. The substrate selectivity, oxidizing not tyrosine but DOPA, also implied that EpoA is a laccase-type phenol oxidase. Although it has been predicted that not only fungi, but also bacteria could produce laccase activity (Alexandre & Zhulin, 2000 ), there has been no report of the identification of the prokaryotic enzyme, except for a multifunctional oxidase identified in a marine bacterium (Solano et al., 2000 ) and CotA of B. subtilis, which was recently shown to have a laccase activity (Hullo et al., 2001 ). The presence of a homologue of EpoA in S. coelicolor A3(2), a phylogenetically diverged organism from S. griseus, implies wide distribution of the enzyme among Streptomyces. Future studies should reveal the precise biochemical properties of this novel enzyme.

The N-terminal amino acid sequence of the purified EpoA indicated that EpoA is an extracytoplasmic enzyme. We assume that the majority of the enzyme is localized in the cell wall, which explains the efficient collection of the enzyme from the mycelia grown on solid medium. The possible interaction of the enzyme with cell-wall components may be strong, and thus it requires sonication to extract the enzyme. It is known that several fungal laccases are also located in the cell wall (Palmieri et al.; 2000 ; Xudong et al., 2001 ). The relatively large molecular mass of the enzyme detected in the cell extract from liquid culture (Fig. 2b) implies that the mature EpoA is released into the medium and the cells mostly contain the unsecreted form in the submerged culture. These results may reflect the differences between the cell-wall structures of S. griseus cultured in solid and liquid media.

We discovered that a substance(s) synthesized by EpoA from DOPA stimulates morphogenesis in Streptomyces spp. A similar effect observed with commercial melanin, a mixture of substances generated by oxidation of tyrosine with a fungal tyrosinase, suggests that the stimulant is generally produced by these phenol oxidases. Our studies have elucidated that S. griseus possesses at least one tyrosinase (MelC2) and one laccase-type oxidase (EpoA), both of which probably catalyse oxidation of DOPA to produce DOPA melanin. Furthermore, we assume the presence of another tyrosine-oxidizing enzyme in this organism since a melC2 mutant produced melanin pigment at the wild-type level (Endo et al., 2001 ). EpoA could not be an alternative to MelC2 because the enzyme is not capable of oxidizing tyrosine, the probable starting material available for melanin production in the medium. The partial restoration of melanin pigment production and aerial mycelium formation in strain HH1 by the introduction of pEPO1 (Fig. 6) could be explained by weak activity of MelC2 and/or another possible enzyme in the mutant supplying a low level of DOPA to EpoA-mediated oxidation.

The result of ultrafiltration of the EpoA reaction products demonstrated that the active compound is not a melanin pigment, but a colourless substance with a relatively small molecular size. It is probable that the stimulant is synthesized by oxidation of DOPA via the formation of quinone structures. Recently, it was reported that quinones act as a direct signal for transcriptional control in E. coli (Georgellis et al., 2001 ). In parasitic plants, it is known that quinones and phenolics induce development of pathogenic structures and one of those compounds has been shown to globally induce transcription for both defensive responses and morphogenesis (Matvienko et al., 2001 ). The possible stimulative molecule generated by EpoA could play a similar role in the morphological development of Streptomyces.


   ACKNOWLEDGEMENTS
 
We thank B. Keijser, Y. Yamada and S. Horinouchi for helpful discussions. This study was supported by Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, the Research for the Future Program of the Japan Society for the Promotion of Science, and the High-tech Research Center Project of the Ministry of Education, Science, Sports and Culture, Japan.


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Received 19 November 2001; revised 14 January 2002; accepted 6 February 2002.