Vol. 173, No. 7JOURNAL OF BACTERIOLOGY, Apr. 1991, p. 2311-23180021-9193/91/072311-08$02.00/0Copyright © 1991, American Society for Microbiology

Phosphorylation of the AfsR Product, a Global RegulatoryProtein for Secondary-Metabolite Formation in

Streptomyces coelicolor A3(2)SOON-KWANG HONG, MORIKAZU KITO,t TERUHIKO BEPPU, AND SUEHARU HORINOUCHI*

Department ofAgricultural Chemistry, Faculty of Agriculture, The University of Tokyo,Bunkyo-ku, Tokyo 113, Japan

Received 29 October 1990/Accepted 31 January 1991

The AfsR protein is essential for the biosynthesis at the wild-type level of A-factor, actinorhodin, andundecylprodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans. Because overexpression of theafsR gene caused some deleterious effect on these strains, a multicopy plasmid carrying the whole afsR gene wasintroduced into Streptomyces griseus, from which a crude cell lysate was prepared as a protein source. The AfsRprotein was purified to homogeneity from the cytoplasmic fraction through several steps of chromatography,including affinity column chromatography with ATP-agarose and use of anti-AfsR antibody for its detection.The molecular weight of AfsR was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis andby gel filtration to be 105,300, which is in good agreement with that deduced from the nucleotide sequence ofafsR. The purified AfsR protein was found to be phosphorylated through the transfer of the y-phosphate groupof ATP in the presence of the cell extracts of S. coelicolor A3(2) and S. lividans. This phosphorylation proceededvery rapidly, and no competition was observed with CTP, GTP, UTP, or cyclic AMP. In the cell extract of S.griseus, no activity phosphorylating the AfsR protein was detected, suggesting that this activity is not generallypresent in Streptomyces spp. but is specific to certain species. It is conceivable that the extent of phosphorylationof the AfsR protein modulates its regulatory activity which, in turn, regulates expression of some target gene(s)involved in the secondary-metabolite formation in S. coelicolor A3(2).

The afsR gene is a pleiotropic and essential regulatorygene for secondary metabolism in Streptomyces coelicolorA3(2) and a related species, Streptomyces lividans (11, 17,33). Introduction of the gene cloned on a plasmid into S.lividans caused marked production of the pigmented antibi-otics actinorhodin and undecylprodigiosin (16). The induc-tion of actinorhodin production was found to occur throughtranscriptional stimulation of the genes involved in theantibiotic biosynthesis (20). Nucleotide sequencing of theafsR gene revealed that it codes for a 993-amino-acid proteinwith a molecular weight of 105,600 (18). The AfsR proteincontains A- and B-type ATP-binding consensus sequences atits NH2-terminal portion and two DNA-binding consensussequences with a helix-turn-helix motif at its COOH-termi-nal portion. A mutation of either of the two ATP-bindingconsensus sequences, which was generated by site-directedmutagenesis, resulted in the loss of the ability of afsR tocause production of pigments in S. lividans. In addition,disruption of the chromosomal afsR gene by use of thecloned afsR gene and phage 4C31KC515 resulted in asignificant loss of pigment production in S. coelicolor A3(2),which indicates that afsR has an obligatory role in normalantibiotic synthesis (18).

Historically, the afsR gene was identified because it com-plemented an afsB mutation on the chromosome (16, 17).Although the cloned afsR gene was assumed to coincide withthe afsB gene, subsequent experiments have shown that theafsR product is a bypass function with regard to afsBcomplementation (33). More recently, the AfsR protein hasbeen shown to be important in its own right, since disruption

* Corresponding author.t Present address: Applied Research Laboratories, Ajinomoto

Co., Kawasaki-ku, Kanagawa 210, Japan.

of its function results in a loss of pigment production (18).Each of the NH2- and COOH-terminal halves could partiallyconfer pigment production on S. lividans.The above observations prompted us to examine the

possible ability of the AfsR product to interact with ATP,which might be associated with its regulatory function. Forthis purpose, we purified the AfsR protein to homogeneityand determined its characteristics. In this paper, we describethe purification of the AfsR protein and the phosphorylationof it by a phosphokinase activity present in S. coelicolorA3(2) and S. lividans.

MATERIALS AND METHODS

Materials. Restriction endonucleases, DNA-modifying en-zymes, were purchased from Takara Shuzo Co., Ltd., andwere used according to the recommendation of the supplier.[_y-32P]ATP (5,000 Ci/mmol), [a-32P]ATP (3,000 Ci/mmol),and an oligonucleotide-directed mutagenesis kit were ob-tained from Amersham Corp. ATP, CTP, GTP, UTP, cyclicAMP, and ATP-agarose (AGATP type 4) were purchasedfrom Pharmacia, Inc., and cellulose-nitrate membranes werefrom Schleicher & Schuell Corp. The nonionic detergentThesit, which has the structural formula dodecylpoly(ethyl-ene glycol ether)n, was supplied by Boehringer GmbH.Thiostrepton was a gift from Asahi Chemical Industry,Shizuoka, Japan.

Strains and plasmids. The bacterial strains and plasmidsused are listed in Table 1.Recombinant DNA work. Preparation of plasmid DNA,

recombinant DNA work, and protoplast transformationwere performed as previously described (14). For expressionof the AfsR protein at a higher level, plasmid pIJ702-C81 wasconstructed (see Fig. 1). Plasmid pIJ702 (25), a high-copy-

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2312 HONG ET AL.

TABLE 1. Bacterial strains and plasmids

Designation Relevant characteristics Reference

Streptomyces strainsS. lividans TK21 15S. coelicolor A3(2) hisAl uraAl strAl Scp-1- 5M130 Scp-2-; A-factor producer

S. griseus HH1 A-factor-deficient mutant 19derived from S. griseusIFO 13350

Escherichia coli F- hsdS20 recA131 ara-14HB101 proA2 lacYl galK2 rpsL20

supE44

PlasmidspIJ41 Neomycin and thiostrepton 4

resistance; copy number,3-4/chromosome

pIJ702 Thiostrepton resistance; 25melanin'; copy number,40-300/chromosome

pIJ41-AP3 Thiostrepton resistance; 17contains the afsR gene

pIJ702-C81 Thiostrepton resistance; This studycontains the afsR gene

pCA1 Ampicillin resistance; 18contains a prochymosin-afsR fused gene

number plasmid (40 to 300 copies per chromosome) inStreptomyces spp., was digested with PstI and BglII restric-tion enzymes, and the larger fragment (5.3 kb) was purifiedby agarose gel electrophoresis. For preparation of a DNAfragment containing the promoter and whole coding regionof the afsR gene by cleavage with restriction enzymes ofpIJ41-AP3, a SmaI site at nucleotide position 1 reported inreference 18 was changed into a PstI site by attaching ahexamer PstI linker. Then, a 3.8-kb PstI-BclI fragmentcontaining the whole afsR gene was ligated with PstI-plus-BglII-digested pIJ702. The ligation mixture was introducedby transformation into protoplasts of Streptomyces griseusHH1. Transformants were selected on medium containing 40,g of thiostrepton per ml.Media. The liquid medium for S. griseus HH1 had a pH of

7.2 and contained, in grams per liter, yeast extract (DifcoLaboratories), 2; meat extract (Kyokuto), 2; Bacto-Peptone(Difco), 4; NaCl, 5; MgSO4 7H20, 2; and glucose, 10.R2YE medium (14) containing 0.2% asparagine instead ofproline was used for regeneration of protoplasts. For theliquid medium of S. coelicolor A3(2) M130, R2YE mediumwithout agar was used. YMPG medium (pH 7.2) (containing,in grams per liter, yeast extract, 4; malt extract [Difco], 10;MgCl2 .6H20, 2; Bacto-Peptone, 1; and glucose, 10) wasused for the cultivation of S. lividans TK21.

Preparation of anti-AfsR protein antibody. E. coli HB101harboring plasmid pCA1 (18) was used to produce theprochymosin-AfsR fused protein (1,323 amino acids; Ala-1to Leu-361 of prochymosin and Ala-43 to Arg-993 of AfsR).The strain was grown in M9 medium supplemented with 2%Casamino Acids and 0.5% glucose to an optical density at600 nm of about 0.5, and 3-,-indolylacrylic acid was addedto a final concentration of 15 pug/ml. After incubation for 15h with shaking at 37°C, cells were harvested, washed with 50mM Tris - Cl (pH 7.5) buffer, and disrupted by sonication.The fused protein overproduced in the form of inclusion

bodies was collected by low-speed centrifugation at 5,000 xg for 10 min. The precipitate was suspended in the samebuffer, and the proteins were applied onto sodium dodecylsulfate (SDS)-polyacrylamide (7% acrylamide) gels for elec-trophoresis. The 145-kDa protein corresponding to the pro-chymosin-AfsR fused protein was electroeluted from the gel(22), and 500 pLg of the protein was injected into two rabbits(New Zealand White rabbits, male; body weight, 2 kg). Therabbits were booster injected 1 week later with a further 500ptg of the protein and were bled 4 weeks after the lastinjection. The immunoglobulin G fraction was purified fromthe antiserum by protein A-affinity column chromatography(26). The antibodies specific to prochymosin in the immuno-globulin G fraction were removed by immunoprecipitation(3) with prochymosin, and the supernatant which did notshow any positive reaction against prochymosin was used asthe anti-AfsR antibody.

Purification of the AfsR protein. Throughout the purifica-tion procedure, the AfsR protein was identified by SDS-polyacrylamide gel electrophoresis and Western immunoblotanalysis (6) with the anti-AfsR protein antibody. All of theoperations were performed at 4°C, except fast protein liquidchromatography (FPLC) operations were performed at roomtemperature. The amount of protein was measured by themethod of Lowry et al. (29).

(i) Preparation of crude extract. A 3-day-old culture of S.griseus HH1 containing plasmid pIJ702-C81 in the presenceof 20 ,ug of thiostrepton per ml was diluted 1:10 into freshliquid medium and grown at 30°C for 3 days on a reciprocalshaker. Mycelia (wet weight, 210 g) obtained from 10 liters ofculture were washed with buffer A (10 mM Tris Cl [pH6.3], 1 mM EDTA, 1 mM ,B-mercaptoethanol) and suspendedin 1.5 liters of buffer A. They were disrupted by sonication.Cell debris was removed by centrifugation for 1 h at 20,000x g in a high-speed centrifuge. Proteins in the supernatantfluid that were precipitated with 33%- to 55%-saturatedammonium sulfate were collected by centrifugation at 20,000x g for 1 h. The precipitate was dissolved in 500 ml of bufferA and dialyzed against 20 liters of the same buffer overnightwith three changes of the buffer.

(ii) DEAE-Toyo Pearl column chromatography. After re-moval of precipitates of the dialyzed sample by centrifuga-tion, the supernatant was diluted with buffer A to a finalvolume of 2 liters. This sample solution was applied to aDEAE-Toyo Pearl column (Toyo Soda Manufacturing Co.,Ltd., Tokyo, Japan; 4 by 40 cm) previously equilibrated withbuffer A. The column was washed with the same buffer andwashed again with buffer A containing 0.1 M NaCl until nomore protein was eluted. Buffer A containing 0.2 M NaClwas then applied to elute the AfsR protein. The fractionscontaining the AfsR protein were pooled and dialyzedagainst 5 liters of buffer B (10 mM Tris Cl [pH 7.0], 5 mMEDTA, 5 mM ,B-mercaptoethanol) overnight with twochanges of the buffer.

(iii) Affinity chromatography with an ATP-agarose column.After centrifugation to remove a small amount of the precip-itates in the dialysate, the supernatant was applied to anATP-agarose column (1.5 by 10 cm) (28) previously equili-brated with buffer B. After the column had been washed withthe same buffer, proteins were eluted with a linear NaClgradient from 0 to 1 M in buffer B containing 0.01% Thesit,a nonionic detergent, to prevent the aggregation betweenprotein molecules. The fractions from 0.3 to 0.45 M NaClgradient portions containing the AfsR protein were pooled.

(iv) Phenyl-superose column chromatography. The samplethus obtained was brought to 20% saturation of ammonium

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PHOSPHORYLATION OF THE AfsR PROTEIN 2313

sulfate. After filtration through a 0.22-,um-pore-size mem-brane filter, it was applied to a phenyl-superose column (0.5by 5 cm) previously equilibrated with buffer B containing20% ammonium sulfate. The AfsR protein was eluted with alinear ammonium sulfate gradient from 20 to 0% in the samebuffer containing 0.01% Thesit in a Pharmacia FPLC system.

(v) Gel permeation chromatography. For further purifica-tion, superose-6 and superose-12 were connected in theFPLC system and gel permeation chromatography of theAfsR protein was performed twice in succession, once inbuffer C (10 mM Tris Cl [pH 7.4], 1 mM EDTA, 1 mMP-mercaptoethanol) containing 0.01% Thesit and once inbuffer C only to remove the nonionic detergent. The AfsRprotein purified in this way was stored in 50% glycerol at-800C.Preparation of crude extracts as a source of phosphokinases.

S. coelicolor A3(2) M130, S. lividans TK21 and S. griseusHH1 were cultivated at 30°C for 3 days on a reciprocalshaker, and the mycelia were harvested. After the myceliahad been washed with buffer C, they were suspended in thesame buffer and disrupted by sonication. Cell debris wasremoved by centrifugation at 35,000 x g for 30 min, and thesupernatants were used as a mediator for phosphorylation ofthe AfsR protein.

Phosphorylation protocol. The purified AfsR protein wasphosphorylated with the crude extracts of S. coeli-olorA3(2) M130, S. lividans TK21, or S. griseus HH1 as aphosphokinase preparation in a mixture containing 10 mMTris * Cl (pH 7.4), 4 mM MgCl2 - 6H20, 3 mM P-mercapto-ethanol, 2 mM MnCl2, 1 mM EDTA, and 10 ,uCi of[_y-32P]ATP, mainly according to the conditions described byCherry et al. (8). After 5 min of incubation at room temper-ature, the reaction was terminated by the addition, withboiling for 2 min, of the SDS sample buffer for electropho-resis to give a 0.1% concentration of SDS. Phosphorylatedproteins were separated by 0.1% SDS-10% polyacrylamidegel electrophoresis and detected by autoradiography at-80°C with a Du Pont Cronex intensifying screen.Polyacrylamide gel electrophoresis. An SDS-polyacryl-

amide gel (10% acrylamide) system (27) was used to examinethe purity of the AfsR preparation and to determine itsmolecular weight. A polyacrylamide gel system (10% acryl-amide) without SDS was also used (10).

RESULTS

Purification of the AfsR protein. For overexpression of theAfsR protein, we constructed pIJ702-C81 containing thewhole afsR gene on the multicopy plasmid vector pIJ702(Fig. 1). As previously reported (18), neither S. coelicolorA3(2) nor S. lividans allowed the replication of a multicopyplasmid carrying the afsR gene, probably because the pres-ence of afsR at a high copy number causes production ofactinorhodin and undecylprodigiosin to such an extent thatthese pigmented antibiotics led to the death of the host cells.The afsR gene, even on pIJ41, whose copy number is 3 to 4per chromosome (14), caused a decrease in the growth rateof S. lividans. We therefore introduced pIJ702-C81 into S.griseus HH1, which does not contain the biosynthetic genesfor these antibiotics; from this a crude extract as the startingmaterial for purification of the AfsR protein was prepared. Inaddition, S. griseus HH1 was expected to produce noprotein homologous to the AfsR protein, because it containsno DNA sequence homologous to the afsR gene (21). Al-though the growth of S. griseus HH1 containing pIJ702-C81

Ai

plJ702-C81 #

PstlI afsR cii/BgIII

I IfsR 1T D A-idn

ATP-binding DNA-bindingsequence sequence

FIG. 1. Construction of plasmid pIJ702-C81 carrying the afsRgene. The single line of the circle represents the sequence of plasmidpIJ702 containing the thiostrepton resistance gene (tsr) and a mela-nin production gene (mel), and the open bar represents the DNAfragment containing the S. coelicolor A3(2) afsR gene. Just up-stream of the coding sequence, there are two transcriptional startpoints (18), as shown by an arrow. The AfsR protein of 993 aminoacids contains two ATP-binding consensus sequences at its NH2-terminal portion and two DNA-binding consensus sequences at itsCOOH-terminal portion.

was considerably reduced, the addition of thiostrepton to themedium allowed the plasmid to be maintained.Western blot hybridization with the anti-AfsR protein

antibody was employed for detection of the AfsR protein. Aprotein band giving positive hybridization in the Westernblot was detected in the supernatant of the sonic extract ofmycelium, i.e., in the soluble fraction but not the membranefraction (Fig. 2C, lane 2). Starting with this crude lysate, wepurified the AfsR protein to homogeneity by ammoniumsulfate fractionation and several steps of column chromatog-raphy. Among these steps, the affinity column chromatogra-phy with ATP-agarose was very useful for its purification.The final preparation gave a single band on an SDS-poly-acrylamide gel even by silver-staining (Fig. 2B). The appar-ent molecular weight of the AfsR protein was estimated to be105,300 from its mobility on SDS-polyacrylamide gel elec-trophoresis. This value was in good agreement with that (M,105,600) deduced from the nucleotide sequence of afsR (18).The molecular weight of the native AfsR protein estimatedon the basis of both SDS-polyacrylamide gel electrophoresiswith a nondenaturing buffer (0.06 M Tris * Cl, pH 6.8, 10%glycerol) without a boiling treatment (9) and superose-6 gelfiltration was exactly the same as that determined on thedenaturing SDS-polyacrylamide gel. These data show that

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2314 HONG ET AL.

M 1 2 34 5 6 7 M 7 M 1 2 3

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A BFIG. 2. Purification of the AfsR protein. (A) SDS

gel used for detection of the AfsR protein and detepurity; (B) silver staining of the purified AfsR protein; (hybridization performed with the anti-AfsR antibodytein is indicated by the arrow. Lanes 1, Total-cell lysat4HH1 harboring pI702 as a control (10 ,ug of prsupernatant of sonic extract of total-cell lysate prgriseus HH1 harboring pLJ702-C81 (10 ,ug of proteinammonium sulfate fractionation (5 ,ug of protein);DEAE-cellulose chromatography (2.5 ,ug of proteinATP-agarose chromatography (1.5 ,ug of protein); lane,superose chromatography (1 ,ug of protein); lanes 7, asuperose-12 chromatography (1 ,ug of protein). The fclar mass markers were used in lanes M: phosphorylbovine serum albumin (67 kDa), ovalbumin (43 kDa),drase (30 kDa), and soybean trypsin inhibitor (20.1 kI

4 5 6 7 the AfsR protein thus purified exists as a monomer in thecytoplasmic fraction.

Phosphorylation of the AfsR protein. The amino acidsequence of AfsR protein deduced from its nucleotide se-

- - quence indicated that it contains A- and B-type ATP-bindingconsensus sequences at its NH2-terminal portion (18). Byanalogy with two-component regulatory systems (32), wehad expected that the AfsR protein might be autophospho-rylated and that it could regulate secondary metabolismdepending on the extent of phosphorylation or dephospho-rylation of AfsR. The purified AfsR protein was thereforeassayed for autophosphorylation by incubating it with[_y-32P]ATP for 5 min in the presence of 4 mM MgCl2.Contrary to our expectation, however, no phosphorylationoccurred in this assay system (Fig. 3, lane 1).We next examined phosphorylation of AfsR protein with

-' the cell lysate of S. coelicolor A3(2) M130, from which the0-polyacrylamide afsR gene was derived. Reaction conditions were basedrmrnation of its mainly on those of Cherry et al. (8). A protein with an(C) Western blot apparent molecular weight of 105,300, in addition to several1.The AfsR pro-efrom S. griseus other proteins, was 32P labeled when the purified AfsRotein); lanes 2, protein was incubated with the cell lysate in the presence of*epared from S. [y-32P]ATP (Fig. 3). When [ot-32P]ATP instead of [_y-32P]ATP); lanes 3, after was used, no labeling occurred, as described below. Thelanes 4, after degree of 32p labeling of this protein was apparently propor-

); lanes 5, after tional to the amount of the AfsR protein added. In addition,s 6, after phenyl- when the AfsR protein was not added to the assay mixture,ifter superose-6- no detectable labeling could be observed under these condi-lase b (94kDa)m tions (lane 6). However, a very long exposure of the gel, carbonic anhy- showed a faint signal in lane 6 at the position correspondingDa). to the AfsR protein (data not shown). All of these data

.---:?, -- Z~- CC 0 0~- :_ - 2: 007 -L;me

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A BFIG. 3. Phosphorylation of the AfsR protein mediated by the crude extract from S. coelicolor A3(2) M130. Reaction mixtures containing

[-y-32P]ATP and various amounts of the purified AfsR protein and the cell extract were incubated for 5 min at room temperature. The mixtureswere then separated by SDS-polyacrylamide gel electrophoresis, and the autoradiogram (A) and Western blot (B) were obtained. The two32P-labeled bands indicated by arrowheads are apparently degraded products of the 32P-labeled AfsR protein. The phosphorylated AfsRprotein is indicated by the arrow. Molecular masses (in kilodaltons) are indicated on the left.

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PHOSPHORYLATION OF THE AfsR PROTEIN 2315

clearly show that the AfsR protein is phosphorylated when itis incubated with ATP and the cell extract of S. coelicolorA3(2) cells. The experiments with a lesser amount of the celllysate, as described below, showed that this phosphorylationwas very rapid and that the dephosphorylation was alsorapid; this is an explanation for the lack of a comparableincrease of 32p signals of corresponding proteins with eitheradded AfsR or various amounts of cell extract.

Concerning the additional 32P-labeled proteins, two dis-tinct bands, indicated by arrowheads in Fig. 3, are appar-ently degraded products of the AfsR protein, since thereaction mixture without exogenous AfsR (lane 6) does notyield these bands. This is also supported by the Western blothybridization which shows that these are immunoreactivewith the anti-AfsR antibody. On the other hand, other32P-labeled bands observed are assumed to be proteins thatare 32p labeled by this reaction system. Because no 32plabeling of these proteins occurs with [a-32P]ATP, as de-scribed below, this implies that the incubation of the cellextract with ATP under these conditions yields phosphory-lated proteins in addition to the AfsR protein, indicating thepresence of a protein phosphokinase(s). It is not clear atpresent whether the phosphokinase activity responsible forthe phosphorylation of AfsR also phosphorylates some ofthese proteins.The above assay also suggested that the content of the

AfsR protein in S. coelicolor A3(2) cells was too low to bedetected by this assay system, although the AfsR proteinthat had already been phosphorylated could not be detectedby this assay. Consistent with this, an immunoblot assaywith the anti-AfsR antibody failed to show any proteinsreactive with the antibody, even in concentrated cell lysatesprepared from 3- or 4-day-old culture, probably because ofthe small number of the AfsR molecules.

Effects of various nucleotides on phosphorylation of theAfsR protein. As mentioned above, incubation of the AfsRprotein with the cell lysate of S. coelicolor A3(2) in thepresence of [ct-32P]ATP instead of [_y-32P]ATP did not yield a32P-labeled AfsR protein (Fig. 4, lane 3). The 32p labeling ofthe AfsR protein with [_-32P]ATP was competitively inhib-ited by an excess of unlabeled ATP (Fig. 4, lane 4). Theseresults show that the y-phosphate group of ATP is trans-ferred to the AfsR protein. We next examined the effects ofvarious nucleotides in the reaction mixture on phosphoryla-tion. Unlike ATP, an excess of CTP, GTP, UTP, or cyclicAMP caused no significant effect on the phosphorylation ofthe AfsR protein with [y-32P]ATP (Fig. 4, lanes 5 to 8).

Kinetics of phosphorylation of the AfsR protein. The timecourse of phosphorylation of the AfsR protein in the abovesystem was examined. The reaction occurred very rapidly,and most of the AfsR molecules in the reaction mixtureappeared to be phosphorylated within 20 s (Fig. 5A). Theamount of phosphorylated AfsR protein continued to in-crease for 2.5 min. After 5 min, it gradually decreased,presumably because the reaction reached equilibrium andthen phosphatase and protease in the cell lysate degraded thephosphorylated AfsR protein.Because of the unexpectedly rapid phosphorylation, we

used a lesser amount of the cell lysate in the reaction mixturein order to confirm that the reaction was linear with time(Fig. SB). The amount of 32P-labeled AfsR gradually in-creased with the incubation period, indicating clearly thatthe phosphorylation is linear with time.

Presence of a kinase activity specific to AfsR in S. lividansbut not in S. griseus. A phosphokinase activity similar to thatin S. coelicolor A3(2) was expected to exist in S. lividans,

1 234 5 6 7 8

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FIG. 4. Autoradiogram showing the effects of various nucleo-tides on phosphorylation of the AfsR protein. The standard reactionmixture (lane 2) in a final volume of 20 ,ul containing 10 ,uCi of[y-32P]ATP, 2.2 pmol of the purified AfsR protein, and the cellextract (10 ,ug of protein) of S. coelicolor A3(2) was incubated for 5min at room temperature. The cell extract was omitted from thestandard mixture as a control (lane 1). [_y-32P]ATP was replaced by[a-32P]ATP (lane 3). Lanes 4 to 8 show the phosphorylation con-taining 20 nmol (each) of ATP (lane 4), CTP (lane 5), GTP (lane 6),UTP (lane 7), and cyclic AMP (lane 8). Molecular masses (inkilodaltons) are indicated on the left.

since introduction of the afsR gene in this strain causedproduction of actinorhodin and undecylprodigiosin in verylarge amounts. Examination for a phosphokinase capable ofphosphorylating the AfsR protein clearly showed the pres-ence of such an activity in the cell lysate of S. lividans TK21(Fig. 6, lanes 2 to 9). When the reaction mixture (lane 7)containing the cell lysate of S. lividans and [_y-32P]ATP withno exogenous supply of the purified AfsR protein wasanalyzed similarly by means of a prolonged exposure, a faint

1 2 3 4 5 6

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1 2 3 4 5 6

BFIG. 5. Autoradiogram showing phosphorylation of the AfsR

protein depending on reaction time. Each lane contained 2.2 pmol ofthe AfsR protein in 20 ,ul of the reaction mixture. The reactionmixture was incubated for 0 s (lanes 1), 20 s (lanes 2), 1 min (lanes3), 2.5 min (lanes 4), 5 min (lanes 5), and 20 min (lanes 6) at roomtemperature and immediately subjected to gel electrophoresis. (A)Crude extract (10 ,ug of protein) from S. coelicolor A3(2) M130 and10 ,uCi of [-y-32P]ATP; (B) crude extract (4 ,g of protein) and 20 ,uCiof [y-32P]ATP. Molecular masses (in kilodaltons) are indicated onthe left.

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2316 HONG ETAL.J.BCEOL

-. N enCw u N.O ,- C OrO - N rl"tA UNr O"D - -4 N m A UgN O :l- o OyO - N r.iJrc %nO rl-Lane ----- - - - .."

Puriried AfVsR (pmnole) N~ N NN N~N~N~N~ N~ -~ N" NN N N N N '4 N N N N Ny Nm - NmNNN N N N 0 NC N N N N NO0 - NN ' O

S. Iividanscrude extract (jug)

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FIG. 6. Phosphorylation of the AfsR protein by crude extracts from S. lividans TK21 and S. griseus HH1: SDS-polyacrylamide gel of the

phosphorylating reaction mixtures (A) and its autoradiogram (B). Lanes 2 to 9 contain the crude extract of S. lividans HH21, and lanes 10

to 17 contain the crude extract of S. griseus HH1. The reaction was continued fo'r min at room temperature. The AfsR protein band isindicated by the arrow. Molecular mas'ses (in kilodaltons) are indicated on the left.

32P-labeled signal at the position corresponding to AfsR was

observed. This implies that in S. lividans the AfsR protein

which can be phosphorylated is produced in a very small

amount, as in the case of S. coelicolor A3(2). The other

proteins of S. lividans that were 32P labeled in this system

appeared to be the same as those in S. coelicolor A3(2). A

distinct band of a higher molecular weight than AfsR that is

seen in all of the lanes appears to be missing in lane 7 in this

photograph, but a weak 32P signal corresponding to this

position is seen on the X-ray ffilm. These data reflect theobservations that these two strains are very similar to each

other in many aspects.

On the other hand, a similar experiment with the cell

lysate of S. griseus HH1 showed the absence of a phosphoki-

nase activity specific to the AfsR protein (Fig. 6, lanes 10 to

17). The number and sizes of S. griseus proteins that were

32P labeled are different from those of S. coelicolor A3(2) and

S. lividans proteins.

DISCUSSION

Despite the ability of both COOH- and NH2-terminal

portions to complement the afsB mutation, the large size of

the AfsR protein predicted by the nucleotide sequence has

been confirmed by this study. The present study also clearly

demonstrates that the AfsR protein accepts the y~-phosphateof ATP when it is incubated with the cell extracts of S.

coelicolor A3(2) and S. lividans. It was suggested that the

phosphorylated A.fsR protein exerts its positive function,

probably by enhancing the transcription of its target gene(s)

(20). The phosphokinase activity specific for AfsR appears

not to be present in general in Streptomyces spp. These

features of the AfsR protein, together with the presence of a.

specific phosphorylating activity, remind us of so-called

two-component regulatory systems such as NtrB-NtrC (31,

34), EnvZ-OmpR (2, 24), PhoB-PhoR (30) and CheA-CheY

(12, 13). The two-component modulator-effector pairs are

very likely prevalent in a variety of procaryotes. The genet-

ical and biochemical features so far observed for the AfsR

protein seem to conform with those of a transcriptional

regulator, called an effector, of the two-component systems,

although no significant similarity in amino acid sequence

between AfsR and other components of these systems (23) is

found. If so, the protein kinase activity for AfsR which we

have detected in this study is then specified by a protein, a

modulator, which is autophosphorylated and is able to

transfer a phosphate group from itself to the AfsR protein.

Further extensive studies on the AfsR protein. and the

putative protein kinase are apparently required to reveal the

mechanisms of regulation exerted by the pair of AfsR and

the A&fR-phosphorylating activity. We are now trying topurify the kinase by following the AfsR-phosphorylating

activity.S. lividans growing without actinorhodin production pro-

duces a very small amount of the AfsR counterpart that can

be phosphorylated (data not shown), just like the case of S.

coelicolor A3(2). Why does S. lividans produce actinorhodin

only under unusual cultural conditions, for example, on

media containing sucrose at a high concentration? Thedifference in the profile of actinorhodin production between

these two strains is presumably ascribed to some other

downstream machinery to which the positive regulatory

LA 0 UN1 0 UN 0 0 0-. v- N N UN% UN UNA

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PHOSPHORYLATION OF THE AfsR PROTEIN 2317

signal of the phosphorylated AfsR is transferred. We spec-ulate as a model that in the case of actinorhodin, thephosphorylated AfsR protein binds the regulatory region ofactll controlling the expression of the biosynthetic clusterand stimulates its transcription. According to this specula-tion, the actll regulatory region may possess some differ-ence from that of S. coelicolor A3(2); as a result, a largeramount of AfsR is required for full expression of actll thanin the case of S. coelicolor A3(2). However, this speculationstill cannot answer the above question.

In addition to afsR, afsB (20) and absA (1) also globallycontrol the secondary metabolite formation in S. coelicolorA3(2). Both afsB and absA mutations are circumventedwhen afsR is introduced on a plasmid. Interestingly, amulticopy plasmid, pIJ702-AP22, containing the DNA se-quence coding for only a COOH-terminal region of the AfsRprotein is capable of complementing, to some extent, thedeficiency in actinorhodin production in afsB and absAmutants (7). We also showed that only an NH2-terminalregion of AfsR still possessed the ability to confer actinorho-din production on S. lividans (18). The experiments with themutated AfsR protein suggest that the truncated AfsR pro-teins are not phosphorylated (data not shown). We cannotfind any explanation for these interesting phenomena, but itis clear that all three of these genes so far identified arefunctioning for the secondary metabolism in S. coelicolorA3(2) and that at least in the case of AfsR, the extent ofphosphorylation is profoundly associated with the regula-tion.The AfsR gene can be maintained at a high copy number in

S. griseus, although it affects the growth rate significantly.No enhancement of streptomycin production by the pres-ence of afsR was observed. It is clear that the AfsR proteinis not phosphorylated in S. griseus, and this is a plausibleexplanation for the maintenance of a multicopy of afsR. Thereduction of the growth rate may be related to some un-known mechanism by which even an NH2-terminal portionand a COOH-terminal portion still possess a regulatoryfunction to some extent.

ACKNOWLEDGMENTS

This work was supported in part by a research grant from theMinistry of Education, Science and Culture of Japan and by a grantfrom the Kato Memorial Foundation for Bioscience Researches.

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