A Gene Cluster Involved in Aerial Mycelium Formation in

Vol. 175, No. 7JOURNAL OF BACTERIOLOGY, Apr. 1993, p. 2006-20160021-9193/93/072006-11$02.00/0Copyright X 1993, American Society for Microbiology

A Gene Cluster Involved in Aerial Mycelium Formation inStreptomyces griseus Encodes Proteins Similar to theResponse Regulators of Two-Component Regulatory

Systems and Membrane TranslocatorsKENJI UEDA, KATSUHIDE MIYAKE,t SUEHARU HORINOUCHI,* AND TERUHIKO BEPPU

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

Received 10 December 1992/Accepted 29 January 1993

Mutants of Streptomyces griseus deficient in A-factor production are sporulation negative, since A-factor is anessential hormonal regulator for the induction of morphological and physiological differentiation in thisbacterium. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficientmutant strain, S. griseus HH1, was cloned from this mutant strain. Subcloning experiments and nucleotidesequencing showed that two open reading frames, ORF1 with 656 amino acids and ORF2 with 201 amino acids,were required in order to induce sporulation. The amino acid sequence of ORF1 significantly resembled that ofthe Escherichia cofl HlyB protein, a member of a family of bacterial membrane proteins engaged in ATP-dependent secretion mechanisms. Conserved features of this surface translocator family, such as the transmem-brane structure predicted by their hydropathy profiles and the amino acid sequence forming an ATP-bindingfold, were also conserved in ORF1. The ORF1 gene appeared to constitute a transcriptional unit with anadditional upstream gene encoding ORF3, which was greatly similar to ORF1 in size and amino acid sequence.The other protein, ORF2, showed significant end-to-end homology with the E. coli uhpA product, a regulatoryprotein for the uptake of sugar phosphates. Like UhpA as a response regulator of a bacterial two-componentregulatory system, ORF2 contained a helix-turn-helix DNA-binding domain at its COOH-terminal portion andan Asp residue (Asp-54) probably to be phosphorylated at its NH2-terminal portion. An amino acid replacementfrom Asp-54 to Asn resulted in the loss of the ability of ORF2 to induce sporulation in strain HH1.

The gram-positive bacterial genus Streptomyces showscharacteristic morphological differentiation resembling thatof filamentous fungi (8, 9). During vegetative growth, Strep-tomyces spp. form a branched, multinucleoid substratemycelium. In response to nutrient limitation, the aerialmycelium is produced; its growth is supported by the can-nibalization of the vegetative mycelium. After septa havebeen formed at regular intervals along the hyphae, longchains of uninucleoid spores are formed. Two mutant classesdefective in these differentiation processes have been ob-tained from Streptomyces coelicolor; bld mutants defectivein the formation of aerial mycelium and whi mutants defec-tive in the formation of spores. These mutants have beensuccessfully used as hosts for cloning the genes complement-ing the respective defect.Our approach to studying morphological differentiation in

Streptomyces spp. has focused on a microbial hormone,A-factor (2-isocapryloyl -3R-hydroxymethyl -y -butyrolac-tone), which is essential for aerial mycelium formation andstreptomycin production in Streptomyces griseus (19, 26,27). An A-factor-deficient mutant S. griseus strain has a Bldphenotype, and exogenous supplementation of A-factor at aconcentration of 1 nM to this strain restores aerial myceliumformation, sporulation, and streptomycin production. Thisindicates that A-factor triggers the expression of a certaingene(s) required for aerial mycelium formation and second-

* Corresponding author.t Present address: Department of Biotechnology, Faculty of

Engineering, Nagoya University, Chikusa-ku, Nagoya 464-01, Ja-pan.

ary metabolite production. This is also supported by theobservation that the A-factor-deficient mutant strain formsconidia without aerial mycelium production on some agarmedia (42). It seemed possible that the overexpression of theputative A-factor-controlled genes in the absence of theA-factor signal might lead to sporulation and/or streptomy-cin production in an A-factor-deficient host. Such geneswere expected to include some that encode regulatoryproteins that mediate the transfer of the A-factor signal tothe downstream genes responsible for aerial mycelium for-mation and streptomycin production.Such cloning experiments brought forth two DNA frag-

ments which induced aerial mycelium formation and sporu-lation exclusively; streptomycin production was not re-stored. This article deals with one of the two cloned DNAfragments, since the other was previously characterized byBabcock and Kendrick (1, 2). The region essential for theinduction of sporulation was found to contain two openreading frames; one resembles response regulators of pro-karyotic two-component regulatory systems responsible foradaptive responses, and the other resembles proteins of afamily of membrane translocators including hemolysin B andthe mammalian P glycoprotein.

MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. S. gri-

seus IFO 13350 was obtained from the Institute of Fermen-tation, Osaka, Japan. This strain was formerly classified asStreptomyces bikiniensis. An A-factor-deficient mutantstrain, S. griseus HH1 (25), was derived from strain IFO13350 by incubation at 370C. This mutant is defective in

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SPORULATION GENES IN S. GRISEUS 2007

aerial mycelium formation and streptomycin productionbecause of its A-factor deficiency. Plasmids pIJ487 (carryingthiostrepton resistance) with a copy number of 40 to 100 pergenome (46) and pIJ922 (carrying thiostrepton resistance)with a copy number of 1 per genome (22) were used ascloning vectors. Each of these plasmids presumably had thesame copy number in S. griseus as in S. coelicolor. Forsite-directed mutagenesis, Eschenchia coli JM109 [A(lac-pro) thi-1 endAl gyrA96 hsdR17 rel4l recAl/F' traD36proAB lacIq lacZAM15] (50) and CJ236 (dut-i ung-1 thi-1rel4I/pCJ105) (34) and phage vector M13mp19 (50) wereused. DNA was manipulated in E. coli JM109 by cloning onpUC19 (50). S. griseus strains were grown in YMPG mediumcontaining the following, in grams per liter: yeast extract(Difco Laboratories), 2; meat extract (Wako Pure Chemi-cals), 2; Bacto Peptone (Difco), 4; NaCl, 5; MgSO4. 7H20,2; glucose (pH 7.2), 10. Growth conditions for E. coli strainswere as described by Maniatis et al. (31).

General recombinant DNA techniques. Restriction endonu-cleases, T4 DNA ligase, T4 polynucleotide kinase, reversetranscriptase, and DNA polymerase were purchased fromTakara Shuzo, Co., Ltd., or Boehringer-Mannheim GmbH.[ao-32P]dCTP at 400 Ci/mmol for nucleotide sequencing bythe M13-dideoxynucleotide method (39) with M13mp18 andM13mp19 (50) and at 3,000 Ci/mmol for the Amershammultiprime DNA labeling system was purchased from Am-ersham International. Thiostrepton was a gift from AsahiChemical Industry, Shizuoka, Japan. DNA manipulations inE. coli were as described by Maniatis et al. (31), and those inStreptomyces organisms were as described by Hopwood etal. (22).

Subcloning experiments. The originally cloned 9-kilobasepair (kb) Sau3AI fragment on pSPO1 was at the BamHI siteof pIJ487. pSH1 was constructed by the insertion of the3.5-kb BamHI fragment (see Fig. 2) into the BamHI site ofpIJ487. pSH2 was constructed by deleting the 3.5-kb BamHIfragment on pSPO1. For construction of pSH5, the PmaCIsite in the cloned fragment was first changed into an EcoRIsite by use of an 8-mer EcoRI linker and then a 5.2-kbEcoRI-BamHI fragment was inserted in the multilinker ofpUC19. The fragment was recovered as an EcoRI-HindIIIfragment and inserted between the EcoRI and HindIlI sitesof pIJ487. For construction of pSH6 and pSH7, the SmaI siteof pUC19 was first changed into an NcoI site by use of an8-mer NcoI linker and the respective BamHI-NcoI frag-ments (see Fig. 2) were cloned between theBamHI and NcoIsites of the mutated pUC19. The fragments were recoveredas BamHI-EcoRI fragments and inserted into pIJ487. pSH9was constructed by cloning a 4.9-kb XbaI fragment intopIJ487 after each of the two NcoI sites had been changedinto an XbaI site by the use of an 8-mer XbaI linker. Forconstruction of pSH10, the 3.3-kb BamHI-SalI fragment wasfirst cloned into the polylinker of pUC19 and then thefragment was recovered as a BamHI-HindIII fragment. TheBamiHI-HindIII fragment was then inserted in pIJ487. Forconstruction of pSL1, the XbaI-XhoI fragment containingthe whole cloned region was recovered from pSPO1 andinserted between the XbaI and XhoI sites of pIJ922.

Site-directed mutagenesis. For generation of an amino acidreplacement of residue 54 (GAC, Asp) of the ORF2 proteinwith Asn (AAT), a nucleotide 26 bp in length (5'-CACCGACCTCAATTGCCCGGGCGCAC-3'; italic letters indi-cate the codon to be replaced; see nucleotides 4708 to 4733in Fig. 3) was used. As the target DNA, a 1,639-bp NcoI-BamHI fragment (see nucleotides 3789 to 5428 in Fig. 3)containing this region was subcloned into the polylinker of

the above-mentioned mutated M13mp19 containing an NcoIsite instead of the SmaI site. The phage DNA was propa-gated once in E. coli CJ236 to prepare uracil-containingsingle-stranded DNA (28). The oligonucleotide was annealedwith the single-stranded DNA, and the complementarystrand was synthesized with DNA polymerase and ligase. E.coli JM109 was then transfected with the reaction mixture(51). The mutation thus generated was checked by nucle-otide sequencing, and the mutated DNA fragment wastransferred to the original Streptomyces plasmid, pSPO1,after DNA manipulations with pUC19 in E. coli.

Scanning electron microscopy. The spores and hyphae of S.grtseus strains were observed by scanning electron micros-copy (43) following growth for 10 days on nutrient agarmedium. For preparation of specimens, agar blocks werefixed in 1% osmium tetroxide for 12 h and then dehydratedby freeze-drying. Each specimen was sputter coated withplatinum-gold and examined with a Hitachi S4000 scanningelectron microscope.

Nucleotide sequence accession number. The nucleotidesequence of the genes reported has been submitted to theDDBJ, EMBL, and GenBank nucleotide sequence databases under accession number D13614.

RESULTS

Cloning of two DNA fragments conferring the ability tosporulate on an A-factor-deficient mutant strain. We con-structed a bank of Sau3AI-partially digested chromosomalfragments of the A-factor-deficient S. griseus HH1 mutant,with a multicopy plasmid, pIJ487, as the cloning vector. Wethen transformed this strain with the resulting genomiclibrary. Our expectation was that an increase in the copynumber of a regulatory gene involved in sporulation wouldresult in an increase in the amount of gene product therebysuppressing the sporulation-negative phenotype of strainHH1 caused by A-factor deficiency. As expected, twosporulating colonies were found among about 2,000 thio-strepton-resistant transformants grown on YMPG agar me-dium. One of the two colonies was found to harbor a plasmidcontaining a 3-kb insert at the BamHI site of pIJ487. Restric-tion mapping and partial nucleotide sequencing revealed thatthe 3-kb insert contained a region identical to that cloned andcharacterized by Babcock and Kendrick (1, 2), as a genecomplementing a Bld phenotype of S. griseus. The othercolony harbored a plasmid, named pSPO1, containing a 9-kbinsert on pIJ487. Because the 9-kb insert did not overlapwith the 3-kb insert, we further studied the 9-kb insert onpIJ487. Restoration of the Bld phenotype of the A-factor-deficient mutant, HH1, by introduction of pSPO1 into thisstrain is shown in Fig. 1. On agar medium, the transformantappeared to form spores less abundantly than the wild-typestrain, S. griseus IFO 13350. In addition, electron micros-copy revealed that the major axis of each spore of thetransformants containing the pIJ487-derived plasmids wasslightly longer than that of the wild-type strain (Fig. 1,photographs 1 and 3). The average axis of spores of thewild-type strain was 2.0 pum, whereas that of the transfor-mants containing the pIJ487-derived plasmids was 3 to 4 pum.The spores of the transformant containing pSL1 were almostthe same size as those of the wild-type strain.

Subcloning experiments. The restriction map of the 9-kbfragment is shown in Fig. 2. Subsequent subcloning experi-ments with pIJ487 as the vector showed that the 3.3-kbBamHI fragment on pSH1 was capable of restoring sporu-lation in strain HH1. In this region, two open reading frames,

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FIG. 1. Scanning electron micrographs of S. grneus strains grown on nutrient agar medium. The wild-type strain, S. griseus IFO 13350,forming chains of spores (photograph 1) and the A-factor-deficient mutant strain, HH1, growing as a form of substrate mycelium (photograph2) are shown as controls. Introduction of pSPO1 (photograph 3) and pSL1 (photograph 4) into strain HH1 restores the aerial myceliumformation and, accordingly, sporulation.

ORFi and ORF2, were encoded, as described below. BothORFi and ORF2 were required in order to induce sporula-tion, since neither pSH6 nor pSH10 containing the region forORFi or ORF2, respectively, caused sporulation in strainHH1. To exclude a possibility that the failure of pSH6 tocause sporulation is due to the lack of the promoter, weconfirmed that the ORF2 region in the same fragmentinserted downstream of the neo promoter in pIJ385 (22) alsofailed to induce sporulation in strain HH1. During thesesubcloning experiments, we noticed that the 5.2-kb PmaCI-BamHI fragment on pSH5 caused the same extent of sporu-lation as did the originally cloned 9-kb fragment but that the3.3-kb BamHI fragment on pSH1 caused slightly less abun-dant sporulation. This implied that ORF3 (see below) en-coded by the region upstream from ORFi enhanced sporu-lation induced by the cooperation of ORFi and ORF2.To determine the effect of the copy number of the cloned

genes on sporulation, we constructed pSL1, in which theoriginally cloned 9-kb fragment was cloned on pIJ922 with acopy number of 1 per genome, and introduced it into strainHH1. The copy number of pIJ922 in S. griseus was roughlyjudged to be similar to that in S. coelicolor, as estimated byagarose gel electrophoresis of cleared lysates. Contrary toour expectation, however, the transformant harboring pSL1sporulated more abundantly than that harboring pSPO1 butstill not as much as the wild-type strain. This observationsuggested that some region at a high copy number exerted a

negative effect on the induction of sporulation. We thenintroduced all the plasmids shown in Fig. 2 into the wild-typestrain, IFO 13350, and found that the plasmids containing theORF2 region, such as pSH1, pSH5, and pSH6, exerted aninhibitory effect on sporulation. Although these plasmidsalso appeared to inhibit the growth of substrate myceliumslightly, sporulation at a low level was observed at almostthe same time as in the case of the wild-type strain contain-ing pIJ487. These data, together with the observation thatthe wild-type strain harboring pSL1 sporulated normally,suggested that an excess of the ORF2 protein exerted anegative effect on sporulation in the wild-type strain in someunknown way.The additional phenotype controlled by A-factor in S.

griseus is streptomycin production. None of the above-mentioned plasmids restored streptomycin production instrain HH1 when the transformants harboring each of theplasmids were bioassayed with Bacillus subtilis ATCC 6633as the indicator. Introduction of these plasmids into thewild-type strain, IFO 13350, did not significantly disturbstreptomycin production. We therefore concluded that thecloned genes were associated only with sporulation and notwith streptomycin production.

Nucleotide sequence of the cloned fragment. On the basis ofthe data from the subcloning experiments, we determinedthe nucleotide sequence of the 5.4-kb SmaI-BamHI fragmentincluding the 3.3-kb BamHI fragment essentially required for

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(kb)2.5 3.8 5.7 7.4 8.8 9.0

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pSPOl pIJ487 +

pSLI pIJ922 ++

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ORF2 ORF4

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(Base number)FIG. 2. Restriction map and subcloning of the cloned DNA fragment and FRAME analysis of the nucleotide sequence. Plasmid pSPO1

contains the originally cloned 9-kb Sau3AI fragment at the BamHI site of pIJ487. The other plasmids were constructed as described inMaterials and Methods. The nucleotide sequence of the 5,428-bp SmaI-BamHI fragment was analyzed by the FRAME analysis (3), using a

sliding window of 80 codons.

induction of sporulation in strain HH1 and the adjacentSmaI-BamHI fragment exerting a stimulatory effect on thisinduction. The whole nucleotide sequence in a total of 5,428bp is shown in Fig. 3. Computer-aided FRAME analysis (3)predicted three large open reading frames, ORF1 with 656amino acids, ORF2 with 201 amino acids, and ORF3 with629 amino acids, in addition to a truncated open readingframe named ORF4 (Fig. 2). The putative translationalinitiation codons for ORF1, ORF2, and ORF3 are precededby potential ribosome-binding sequences (12), GGAGG or

GGAG, GAGGG, and AAAGGG, respectively, all of whichare present at an appropriate position with respect to theinitiation codon. The COOH termini of ORF1 and ORF2 are

overlapped by 48 amino acids. This suggests the presence oftranscriptional and translational regulation for the expres-

sion of these genes. As described above in relation to thesubcloning experiments, it was apparent that ORFi andORF2 were both required for induction of sporulation. ORF3was assumed to enhance the sporulation induced by ORFiplus ORF2. An additional small open reading frame, ORF6with 43 amino acids, which was preceded by a possibleribosome-binding sequence, AAAGGA, was also predicted.ORF6 was not required for induction of sporulation, sincepSH5 without this open reading frame restored sporulationin strain HH1.

Sequence similarity of ORF2 to response regulators ofprokaryotic two-component regulatory systems. A computer-aided search revealed that ORF2 resembled the E. coli uhpAgene product (11, 48), a transcriptional activator for the genecluster responsible for the sugar-phosphate transport system

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SmaICCCGGGCGAGCAGTTGATGAGCCTGTCGATGGACCTGGCCACCGGGACCGCCGGCGCCCTrCCTCGCCCTGGGCGCCGCGCTCGGCGGCACCACCGGACTCCCTTCCTGGCCGCCGCCCC

GGCGGCGCGCCCCGGCCGCGCGAGCGGCCCCACGGACCGGCCCCACCAGGGGTCGTAGCAACACACCGTTCAGTACCACTACGTCCCCGAAAGGACATCATCATGGCGCTTCTCGACCTTORF6- M A L L D L

Sia ICAGGCGATGGACACCCCGGCCGAGGACTCCTTCGGCGAGCTCCGCACGGGCAGCCAGGTCTCGCTGCTGGTCTGTGAGTACAGCTCGCTCAGCGTGGTCCTCTCCACCCCGTCACCCCCGQ A M D T P A E D S F C E L R T C S Q V S L L V C E Y S S L S V V L C T P$

PuaCI S aIGGCGCCCCMCGTCGGTGTTCCGCGGGCCGGCCGGCCCACGTCCACCCACAAAGGGCCACCCCGTCCGGCTGCACCCACGCCCGGGCACGGACTCCGGCACCCGGCGGCTCGCCCGGGCAGC

ORF3MM R L H P R P G T E S GT R R L A R Q HSall

ACCCGGCCGCCCTCCTCCTCGGGCTCCTCCTCTGCTCCACCGGCGGGGCGCTCGCCGCCCTCGCCCTGCCCGCCGCCCTCGGCCACACCGTCGACCTGCTCATCGCCGGCGGCCCCGTGCP A A L L L G L L L C S T G G A L A A L A L P A A L G H T V D L L I A G G P V P

CCTGGCCGGGCCTGCTGCTCTGCGCGGCACTGATCCTCGCCGAGACCGCGTTCGACGCGACGGCCGCCGTGACCGCCACGACCACCACCGCCCGGCTCACCGCCTCGCTGCGCACCCGCAW P G L L L C A A L I L A E T A F D A T A A V T G T T T T A R L T A S L R T R T

Sma ICCGCCGCCCGGGTCCTGGCCGCCGAACCCCGCCGCGCCCTGGCCCTCCCCACCGGCGACCTCACCGCCCGGCTCACCGCCCGCACCGCCGACGCCGCCGCCGCTCCCGTCACCGCCGCCG

A A R V L A A E P R R A L L P T G D L T A R L T A R T A D A A A A P V T A A G

GAGCCGTCTCCGGCGTCCTGCTCCCGCTCGGCGCGATCGCCGGCCTCCTCCTCATCGACGTCTGGACGGCCGCCGCCCTCCTTCTCGGCGCCCCCCTCCTCGTCGCGCTGCTGCGCCCCTA V S G V L L P L G A I A V 6L L I D V W T A A A L L L A P L TV A L L R A F

TCACCCGCAGGACCGCCCACGCCGGGGCCGACTACCACCCCGCCCAGTCGCTCATCCCCCACCGGCTCACCGACCCCCTCCACGGCCCCGACACCATCCGCCCCGCCCCGCACCCGAGCCCT R R T A D A G A D Y Q R A Q S L I A H R L T E A L D G A D T I R A A R T C A R

GCCAGCACCCCCGCCTCCTGGMACCCCTGACQCCGCTCGCCGMA~CcGGCAGGCCCACCTGGTCGGTGTACGCGCGGGCGGTCGGCCCCAGCGGGCTGCTGCTGCCCCTCCTCATGCTGCE H R R V L E P L T A L A E H G R R T W S V Y G R A V G R S G L L L P L L M L L

aiH . ...

TGGTCCTCGCGGTCGCCCGCCTCCCGTCCGCGCGCGGTGCCATCGGTGTCGGCGACCTGCTCCCCCCCTCCCCCTACCCCAGCCTCCCCGTCCGCATCGGCCCCCTCACCCGCGCGCTCCV L A V A G L R L G A G A I G V G D L V A A S R Y A S L A V G I G A L T G A 1. C

GCGCCCTCGCCCCCAGCCCAGCCGCCGCCCCCGAGCCTCGACCCCCTCCTGGCCCTTCCCCCGCTGCCCCACCCCCGCCTCGGCCCCGCCCCCCCACCCACCCGGCACCTCCAACTCCCGCGA L A R S R A A A R S 1, D P L L A 1, P IA1, P 11 R G L G P A P D A P G H L E L R D

ACCTCGGCGTCGMACAGGACGGCGGCCGGCTGCTGACCGCGCTCCATCTGACCGTCCCCGGCGGCACCTCCCTGGCGGTCCTCGGGCCCTCCCGCTCCCGTAAGTCCGTGCTGGCCCCGGV G V E Q D G G R L L T G V D L T V P G G T S L A V V G R S G S G K S V L A A V

Si.nai.

TCCCCCGGCGGCTGCTCGACCCGGACACCGGMAGCCTGCTGCTGGACGGCCTCCCCGATGGACGGCATGCMACCCGACCGGCTGCCCCCGCCAGCTCGCCTACGCCTTCCCCCGCCCCCGCCCA G R L L D P D T G S V L L D G V P M D G M E P D R L R R E V A Y A F A R P A L

TCCCCGGCACCACCCTCCAGGACACGATCCCCTACGGGCCCTGGACCCCCTCGCCCCAGGCCCTACGGGACGCCGCCCGCCCGGCCCCCGCCGACGGGTTCCTCGCCCTGCTCCCGTACGP G T T V E D T I A Y G P W T A S P E A V R D A A R A A R A D G F L A L L P Y G

GCTACGCCACCCCGCTCGCCGACGCCCCGCTCTCCGGCGGCGAACGCCMACGCCTCGGCCTGGCACCCGCGTTCGCCCACCCCGGCCCTCTGATGATCCTCCACCACCCGCTGTCCAGCCY A T P L A D A P L S G G E R Q R L G L A R A F A H P G R L M I L D D A L S S L

TCGACACCGTGACCGAGCACCACGTCCGGCGCGCGCTCGACGCCCGGGCGGGGCACTGCACCCGCATCCTCGTCCCCCACCGGCTGTCCTCGGCCCCGCGGGCGGACCGGGTCGCCTGGCD T V T E H H V R R A L D A R A G H C T R I V V A H R L S S A A R A D R V A W L

Bamfl ITCGAGGACGGCCGGATCCGTGCGACGGGCCCCCACGAGGMACTCTCGGCGGACCCGCACTACCGGGCGGTCTTCCCCACGCAGGCCACCCGCCCCGAGGGCCCCGCGCCCCGCCGCMACC

E D G R I R A T G R H E E L W A D P D Y R A V F R T E A T R A E G P A P A G N P

CCGCGCCCGCCGGGMATCCCCCGCCCCCCGCGMATCCCCCCCCCGCCCCACCAGGAGCACCCCCCCAGMACCCCCCGCCATCCCACCACCCAGCCCCCGCCGCAGGCTCCGCGCCCACCGAA P A G N P A P A G N P A P A D E E H P P R T P R H P T T Q R P P Q A P R P P K

MATCGCGGCGCCGCGCAGCCGTACGACCGTGACGCCCCCCGCCGCAGCCGAGCGCCGGTGMCGGCCGGCCGAGCCCCCGGGCGCCACCAGCAGGCCCCCGGCGCACGCCCCCGCCAGGCCS R R R R A V R R ORF1Ib M K A G R A P G R D E Q A P G A R P R Q A

GGGCAGCCGCACGACGCACGCCCCGCGCGCGACGGCMAGCGGCACGGCGCGCGCGGCCGGCGCGACACCTCGCTGCGGCGGGTCGGCCGGGAGGCCCGGCCCTTCCTGCCGCGCCGGACCG Q R H D A R P A R D G K R H G A R G R R D T S L R R Y G R E A R P F L P A R T

G A I L R L A G W S L L E F A Q T F L G G F G V A R A L D D G F L A G R P T T G

ClTCCTCTGGCITGGCCGTCGCCGCCCGCCGCCGTGCTGCCGTCCGTGCCCGCCGCCCGCGGAGTGTTCGGCCGGCTCGCGGACCTGGTGGAGCCACTGCGCGACGGACTGGTCCGGCGCGCCL L W L A V A A A A V L P S V P A A R G V F G R L A D L V E P L R D G L V R R A

GTGTCCCGGGCGCTCTCCGGGGCGCTCGACCGGGCCGGCGACGTCAGCACCCGGTCCCTGTCGCAGGTCACCCATCAGAGCGAGATCGCCCGCGACGGCTGGGCCGGACTCGTCCTGACCV S R A 1, S C A L D R A G D V S T R S L S Q V T H Q S E I A R D G W A G L V L T

120

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FIG. 3. Nucleotide sequence of the cloned DNA fragment. The nucleotide sequence of the 5,428-bp SmaI-BamHI fragment and deducedamino acid sequences in single-letter code are presented. The possible ribosome-binding sequence for each open reading frame is underlined.A TTA codon (Leu-141) in ORF2 is also underlined. Asterisks denote translational termination codons.

(Fig. 4). The UhpA protein is f member of response regula'tors of the prokaryotic two-component regulatory systems(6, 40, 41). Members of this family of the response regulatorshave an NH2-terminal domain of similar structure containingan Asp residue which is phosphorylated by sensory histidine

* protein-kinases. Because of significant end-to-end sequence- similarity of ORF2 to UhpA as well as other members of theresponse regulators, alignment of these proteins allowed usto predict that Asp-54 of ORF2 was the site of phosphory-lation. In addition to this Asp residue, an Asp and a Lys

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CTGCGCTCGTTCGTGTTCACGGCGCCGCGGGCCGCTCACCGGTCTGCTGGCCCTCTCCCCCGCCCTCCTCCTGATCGTCCTGCCCCCGCTGCTCCTCGGGCCGCCCCTGTTCCTCGCCACCL R S F V F T A A G A V T G L L A L S P A L L L I V L P P L L L G A A L F L A T

CTGCCCGATCGCCGCCCGCCAGCCGCACTACCTCGCCGCCCCACGAGCCGTACCCCGCCCACGOCGGCCGGCTCCCCGCCTCCCTACCCCACATCGCCGCCGCCCGCGGCCGCCGAGCGGL P P M1 A A R Q R D Y L A A D E A Y A A H A C R L A A S V R D I A A A G A A E R

ACGGTGACCGAGTCCCGGACCCTCGCCGAGGAGCAGCTGGCCGCCTCCCGCTCGCTGGCCCGCTGGTCGCCGTCCGGGTCCTGGCCCTCGCCGTCTGGCCGGMCCGCGCTCCTGT V T E S R T L A E E Q L A A S R S L A R 1 S G V R V L A L A V C C R F P P L L

CTGCTC"CTG=CCCCCCCGTCTGCTCGGAAC&GGCTCACGCCGGGCGCGCTGGTGGCGGGCGCTGACCTACCTCACCCAGGCGCTCGCGCCCGCCGTGCAGGCGCTGATGACCATGCTCL L L C A P W L L R N G L T P C A L V G A L T Y L T QA L A P A V Q A L 11 T M L

GGCACCoGCCGGGo GCCGTGGT CCTGGAC CGGTTCACcGACG~C~ccCGccCCGCCGCCCGAooCccGACCcGccc GGGAGGCAGCCCCGCCGCACoCGGGAACCCCoGGTCG T A G G R L V V V L D R F T D A P P P P A D D P P P A R E A A P P H R G T P V

GCcG~TCCGA GGTCAGTTCGuTACGGACCGGGCGCCCAACCCGT CCTGGACOGGCTCTCCCTGACGATOGAGGocGG MAGCACTCGCGGTCGTCGGCCC AGCGGCAGCCGGGA E L H E V S F A Y G P G A Q P V L D R L S L T I E A G E H L A V V G P S G S G

AAATOCACCCTCGCCGCCCCTCCTCGCOGCGAOCGAGCGGCCCACOCGGGGGAGOCGTGOGCTGGOGGGCGCGGCCCGTCOCCCOCGCCGACGCCACCGCCGTACCCGTCCTCCTGCCCCAGK S T L A A V L A G T E R P T G G A V R 1 R G R P V R P A D A T A V R V L L P Q

NcolCACGOCTACGTnGTCAGc MCTcrGCGGGACAACCTCCGCTACCACCGcCCCGGTGCCoCTGACOGGGAGATCGCGGCCATGGCGGACACGCTCGGCCTGGACCTCTcCCCGCTCGGCH A Y V F S G S L R D N L R Y H R P G A R D R E I A A M1 A D T L G L D L S R L G

TCCCPGGACGCGDCCGTCGACCLGACGGCTCTCCCAGGGCADCGGCAGCTGATCAAGC CCGGGCCTACCT SRCDCCGCCCICGuATACTCAGAGACCAGCCGGS L D A A V E P D R L S Q G E R Q L I A L G R A Y L A A P P L L I L D E A T S R

CTCGATCOGGCCGCoCAGACCCGCGTChGcAcGCCCTTCMCc~ACTGCCcGGGCCGCTCGToCTCGTGCCoCACCGGCTCAGCTOcGcGCCCGCCGCCGGACGGACCCTGGTGATGGACL D P A A E T R V E H A F G A L P G A L V V V A H R L S S A R R A G R T L V M D

GGCCCGCGCACCCCnAGTCGCACCCACCGTGCTCCTGGACTCCTGCGCCCTGTACCGGGAC~lCTGCTGGGCTIAGCGCGGACGGAAGGGGCGAGGGGAGCGGAAGAOCGGAGCGG P R T Q C G T H G E L L D S C A L Y R D L A G L 1 E R G R K G R G E R K T G G

* I W G A E K A R R I A D LCTTGCaCGTCCC'CCGCCTCGCGCCTTCCOCCATCCTCGCTOCCTOCTCCCCTOGTCACCCCCGCCOCTCIC I£GaSGTCTAGCTC&GGOCMAGGMAGCGGGCAGACTAGCGCAGGTCGGGAACGGCAGGGGGGAGCAGGCGGAAGGGGGTAGGAGCAGGGAGCAGGGGAGCAGTGGGGGCCGGGGAGGAGCGGCGTCAGATCCAGCCCCCTTCCTTCGCCCGTCTGATCGCGTCCAGOCE R Q G G G A R K G V G A G R R G A V G A G R S G V R S S P L P S P V *

R N R A G S K R I A A A L Y N R V T G P T L H L R G A I G A V T D G G E A M S LCTAACGCTCGGGGCCTGAAGGCATAoCGoCGoCCGTCTATCAACGCCTGTCAaGGcGGCCCGTCCACGTCGGCCGGOOGCTAGGGCCGGTGCCACAGCGGGGGGAGCCGGTAGCTCrCGT

V S L E R P S L P M D A V Q L L S F A L S E D I H R G G D A L K H M V E S L D DGTGAGTCAAGGGCGCCGCTATrCCCGTACAGCCGGTGGACCTCCTCCGACTTCCGGTCGCTGAGCAGCTACAC&GCAGGGGGCAGGCGGTCAAACACGTACTGGAGCCTGTCCAGCAGCT

Sall.V P R Y K D I Y G L A G A E Y A R R L G S P R D S R T L V A L P C P G G H P A TGG;=; MTGcAACACTACATCGGCTCcGCGGGGCGGAGCATGMCG~GGCGTCTGGCGAGCCGGCCAGOGACGCGCACTCCTGCCGCTCGCCTGTGOCAGGCGGCACGCCACGCCAGT

V T K V E D L V D L A G P C D L D T L L V D P S P G S L A A R I A G H E A T R LGCCAAAG~cTGGAGCGCTC~CGoCGGcTTCACGGGCCTGCAGCTCCAA CTCCGTGTAGoCTGA AGCGGGCGAGTCGCGCCGCGCATAGCGGGCACAAMGCCGoCACGCCTCGA

E I G R G T G L L S A L S A R 11 L P V T H V L L V T T Id -ORF2GATACOGGCGGGCGCACGGCTCCTCGCTGCGGTCGCTGCGCGOCGTGTTGCOGTGCCACACCTGGTCGTOGTGCCATCAGTAGTCAGAC&GAGTCGAAAAGAGAAGGCCGGGCGGGCTG

CGCCTTCCGCTGCCOGACATGCGTAGGCCAMTCCCATAGGTCAACTCGCGTACCTGTAGCGCATGMMTrl~GOCaXGCTTATGGCGAGGGCACCa;CGCTTCCCCCGCAAGCC

TACAMAGCCCTCGCGTGTAGCGAGTGCCTGGGTAGGCAGAGCGGTGAGGGACCCGTGGCCGTCCCGOCGTCGCOCGCAGCTGlCACCTAAGCCOGGCGCTAGGGCGTCCTGCOGGCSall

$N L L A R A A H A K R R V N E A T T P E I G D L V A A Y E E Id E A A G R L FCAGTCAGTCGTCCCGR CGCVCGCACLGGARFGCGGCCTGCDGAGOPPCAGCAGCCAAGCTACDGPAGCTPCTGCGGCGTATGAGGAGGTAGAGG TPGCGTVC

D G R Q V L L Q A R E F R L R A P H S P L L L S L E V A W L A V D T R E P R A AAGTGGCGCGACCTGGTCCTCGA(CGCCAGCTTCGCCT CGGACCGGCCTACGCTCCCGTCGTCGTCGCrGTOGAGGTGGCGGGTGTCCOGGTGTAGGCACGOGAGCCCGGCGCGGCGG

.~~~Nco ..

A W A R V N N L 1(-ORF4)GCG MTCCGTHTAGG

.BamHIFIG. 3-Continued.

(Asp-9 and Lys-102 of UhpA) tend to be highly conserved inthe response regulators. One of them (Lys-104) is alsoconserved in ORF2.

In addition to the sequence similarity of the NH2-terminaldomain, ORF2 contains a helix-turn-helix motif at its COOHterminus. The amino acid sequences forming this motif ofORF2 and UhpA show great similarity, as is found for otherresponse regulators (Fig. 4). Evidence suggests that thesehelix-turn-helix motifs in the response regulators are DNA-binding domains (40, 41); thus, it is most likely that theconserved amino acid sequence forming a helix-turn-helixmotif at the COOH terminus of ORF2 serves as a DNA-binding domain recognizing a specific DNA sequence.

Amino acid replacement at Asp-54 of ORF2. As describedabove, Asp-54 of ORF2 was predicted to be phosphorylated.Since phosphorylation of the response regulators of thetwo-component regulatory system is essential for their reg-ulatory activity, we generated an amino acid replacement atAsp-54 by site-directed mutagenesis to examine the ability ofthe mutated ORF2 to suppress A-factor deficiency. In agree-ment with the prediction, pSPO1 containing a mutated orf2gene encoding a protein with an Asn residue in place ofAsp-54 failed to induce sporulation in strain HH1. Thesedata indicated the importance of Asp-54 of ORF2 and are

consistent with the idea that this residue is a target for

phosphorylation.

3000

3120

3240

3360

3480

3600

3720

3840

3940

4080

4200

4320

4440

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*

UhpA * MITVALIDDHLIVRSGFAQLLGLEPDLQV-VAEFGSGREALAGLPGRGVQVCICD-ISMPD-ISGLELLSQLPKGMATI-MLSVHDSPALVEQALNAGA 96$*** ***RA $ *E * **S$ * * ** * *** 99

ORF2 > MdTTVLLVHTVPLWRASLASLLGTGRGIELRTAEHGAIRAALSGPSPDVLLTDL-DCPGALDVLDEVKTVTAPHGGP CPLAVLTRSDRPSGLRRAYEAGA 99

S* RGFLSKRCSPDEL IAAVHTVATGGCYLTPDI A IKLASGRQDPLTKRERQVAEKLAQGM

> LGY I DKYRPVDDLSE VMHKLADGGRH I DESLAFSLLQVADMPLSPRELSVLSMAEGGD

ComA 154 .T...CLI. QEV. K.-GerE 12 .TK. .RE.FEKLVQD-FixJ 142 .SE. .RQ.. .AVVA.-

ANLMEKLGVSNDVELARRMFD-GW 196*

AAAIRKSGARNRLDAIRRAKEAGWI 201

SIFN-.LNVGS.TE.N. MQ-.L.VKG. SQ.NVMA-. MK. KSLPHL

FIG. 4. Alignment of amino acid sequences between ORF2 and UhpA. Identical amino acids are marked by asterisks. The Asp residueto be phosphorylated is shown by a vertical arrowhead. Two Asp and a Lys residue which tend to be conserved in this family are markedby solid circles. Probable helix-turn-helix DNA-binding motifs are boxed and are aligned with those of other response regulators; theseinclude ComA, which is responsible for the development of competence in B. subtilis (47); GerE, which is responsible for sporulation in B.subtilis (21); and FixJ, which is responsible for nitrogen fixation in Rhizobium meliloti (10). Dots indicate amino acids identical to those ofORF2. Dashes indicate gaps introduced for alignment.

Sequence similarity of ORF1 and ORF3 to membranetranslocators. We noticed significant similarity in the aminoacid sequences, especially in the COOH-terminal portions,of ORF1 and ORF3. In their COOH-terminal portions, thereare two amino acid sequences resembling the consensus

sequence for ATP binding (45). One (amino acids 416 to 428in ORF1 and 368 to 380 in ORF3) contains the A-typeconserved sequence G-(X)4-G-K-(X)5-I/V, which is found in,for example, adenylate kinase, E. coli ATPase at and Lsubunits, E. coli RecA protein, and the P glycoprotein (Fig.5). The Lys residue in this consensus sequence is highlyconserved and is thought to interact with the al-phosphate(45). The other (amino acids 525 to 537 in ORF1 and 482 to495 in ORF3) contains a sequence similar to the B-typeconserved sequence R/K-(X)2 3-G-(X)3-L-(hydrophobic)4-D,which is found in, for example, adenylate kinase, E. coliATPase at and IB subunits, phosphofructokinase, and the Pglycoprotein. Most of the ATP-requiring enzymes containboth the A- and B-type conserved sequences in a molecule.As shown in Fig. 5, the COOH-terminal portions of ORF1

and ORF3, containing the ATP-binding folds, were found tobe similar in amino acid sequence to that of the E. coli HlyBprotein, a membrane protein essential for translocation ofhemolysin (HlyA). HlyB, a member of a family of membraneproteins engaged in ATP-dependent secretion mechanisms(5, 13), is thought to form a transmembrane complex totransport HlyA into the medium (20). The proteins belongingto this family in a wide variety of organisms are concernedwith the transport of many different compounds specific foreach transport protein. These proteins therefore containhydrophobic membrane-spanning sequences, in most casesat their NH2-terminal portions. The hydropathy profiles ofORF1 and ORF3, which were determined by the method ofKyte and Doolittle (29), are quite similar to that of HlyB aswell as many other membrane translocators (data notshown), although no significant similarity in amino acidsequence of the membrane-spanning regions is found amongthese proteins. The topological organizations of ORF1 andORF3 are therefore likely to be similar to those of othermembrane translocators. P glycoprotein and HlyB have sixpotential NH2-terminal transmembrane domains, as deducedby hydropathy analysis. In the case of HlyB, two moleculesof HlyB and an accessory protein, HlyD, form a porethrough which HlyA is pumped out by the energy of ATPhydrolysis.

DISCUSSIONThe DNA fragment cloned as a suppressor for A-factor

deficiency contained two open reading frames, one resem-

bling response regulators of the prokaryotic two-componentregulatory systems and the other encoding a protein resem-

bling membrane translocators. Since A-factor acts as a

switch for aerial mycelium formation, these two proteins areconcluded to be involved in aerial mycelium formation. Wepropose to designate the genes coding for ORF1, ORF2, andORF3 as amfA (aerial mycelium formation), amfR, andamfB, respectively. According to our model of the A-factor-regulatory network (23, 24), the A-factor signal is firstaccepted by the A-factor receptor protein, which acts as arepressor-type regulator (35, 36). The expression of a still-unknown gene that has been repressed by the A-factorreceptor protein during the early growth phase is dere-pressed on binding of A-factor to the receptor protein, andthe gene product then causes expression of the other genesnecessary for aerial mycelium formation and streptomycinproduction via multiple regulatory steps. In this model, thereis a branch point of the signal relay, from which theregulatory cascade common to development and secondarymetabolism is divided into two parts, one for the develop-mental process and the other for secondary metabolism. Oneof the proteins on the signal relay for streptomycin produc-tion after the branch point is an A-factor-responsive proteinthat binds to and probably activates the expression of strR,a regulatory gene in the streptomycin biosynthetic gene

cluster (44). The amfgenes in the present study can be listedon the cascade for the developmental process downstreamfrom the branch point.ORF2, or AmfR, is most likely a member of response

regulators of the two-component regulatory systems in-volved in signal transduction as suggested by the similarityin amino acid sequence of both the signal receiver domain atits NH2-terminal portion and the DNA-binding domain at itsCOOH-terminal portion. In addition, the complete loss ofthe ability to induce aerial mycelium formation after Asp-54was replaced with Asn supports the idea that the activity ofAmfR is modulated by phosphorylation of this Asp residue,as are the other response regulators (40, 41). Overexpressionof AmfR belonging to this family may disturb the physiolog-ical conditions and make the host cell sick. This may explainwhy amfR on a high-copy-number plasmid exerts a negativeeffect on sporulation in the wild-type strain. The role of

AVKE I AAELGLSPKTVHVHR* ** * * $ **

TVAG IAGRLHLTPGTVRNYL

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MKAGRAPGRDEQAPGARPRQAG*-LYQGH II L ITSRSSVTGKLAKFDFTWIF IPA I IKYRR I FIETLVVSVFLQLFAL ITPLFFQVVMSDKVLVHRGFSTLNV ITVALSVVVVFE I ILSGLRTY-- IFAHSTSR IDVELGAKLFRH

*l * * $ * 8+to QRHDARPARDGKRHGARGRRDTSLRRVGREARPFLPARTGA I LRLAGVSLLEFAQTFLGGFGVARAL-DDGFLAGRPTTGLLWLAVAAAAVLPSVPAARGVFGRLADLVE-PLRDGLVRR

* LLALPISYFESRRVGDTVARVRELDQIRNFLTGQALTSVLDLLFSLIFFAV.-----IWYYSPKLTLVILFSLPCYAAWSVFISPILRRRLDDKFSRNADNQSFLVESVTAINTIKAMAVS

tx AVSRALS-GALDRAGDVSTRSLSQVTHQSE I ARDGWAGLVLTLRSFVFTAAGAVTGLLALSPALLL I VLPPLLLGAALFLATLPPMAARQRDYLAADEAYAAHAGRLAASVRD I AAAGAA

* PQMTNIWDIQLAGYVAAGFKVTVLATIGQQGIQLIQKTVMI INLWLGAHLVISGDLSIGQLI-AFNMLAGQIVAPVIRLAQIWQDF-QQVGISVTRLGDVLNSPT-ESYHGKLTLPEING** *88* * * G* * * $ * * *

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ORF3Bovine ATPaseE.coli ATPaseAdenylate kinasetdrlSte6

359 PG.TS148 AK.GKIGLF160 GR.QRELII

5 KKSKIIF..417 KS. QTV. L.383 S.. QFTFI.

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.... AGRVFIMELIAL. ID. I

QCEKIVQTVQLMQR.SNL. LR

*KILAGAHDFISELREGYNTIVGEQGAGLSGGQRQRIAIA R ALVNNPK ILIF DEATSALDYESEHVIMRNMHKICKGRTVI I IAHRLSTVKNADRI IVMEKGKIVEQGKHKELLSEPE* ** ***** *

tF AMADTLGLDLS-RLG-SLDAAVEPDRLSQGERQLIALG R AYLAAPP LLILI DEATSRLDPAAETRVEHAFGAL--PGALVVVAHRLSSARRAGRTLVMD-GPRTQCGTHGELL-DSC

ORF 3Bovine ATPaseE.coli ATPaseAdenylate kinaseMdrlSte6

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FIG. 5. Alignment of amino acid sequences of ORF1 and HlyB. Identical amino acids are marked by asterisks. The amino acids which tendto be highly conserved in the ATP-binding folds of the membrane translocators are boxed. The probable ATP-binding folds are aligned withthose of ATP-requiring enzymes and membrane translocators; these include ORF3, bovine and E. coli ATPase subunits a and (45),adenylate kinase (45), Mdrl (P glycoprotein) (14), and Ste6 (33). The Lys residue in the A-type consensus sequence, which is thought tointeract with the a-phosphate of ATP, is indicated by a solid circle. Identical amino acids in the ATP-binding motifs are shown by dots. Dashesindicate gaps introduced for alignment.

AmfR in aerial mycelium formation may be analogous to thatof the phosphorelay for the initiation of sporulation in B.subtilis (7, 15). The initiation of sporulation in B. subtilisrequires a phosphorelay including KinA, SpoOF, SpoOB, andSpoOA. Among the components in this phosphorelay, SpoOAand SpoOF are members of the response regulators, althoughSpoOF contains only the conserved NH2-terminal domain. Itis conceivable that, in the absence of the A-factor signal, theoverall phosphorelay including AmfR is inactive and that anincrease in the amount of a component in the cascade leadsto an increase in the amount of the final component in aphosphorylated (active) form, which then results in switch-ing on aerial mycelium formation. However, no data areavailable to show the presence of extra components, inaddition to AmfR and its putative kinase, that are engaged inthe putative phosphorelay in Streptomyces spp. Also unclearis how many pathways including a phosphorelay are in-volved, either dependently or independently, in the initiationof aerial mycelium formation.ORFi and ORF3 (or AmfA and AmfB, respectively)

appear to be members belonging to the HlyB subfamily ofhighly conserved polypeptides involved in the transport ofmany different compounds, specific for each transport pro-tein (5, 13). The HlyB subfamily has a highly conserved

domain containing an ATP-binding site in most cases at theCOOH-terminal portion and a large transmembrane domainat the NH2-terminal portion. This family in most cases drivesthe secretion of a variety of compounds. In Streptomycesspp., the DrrA protein responsible for self-resistance todaunorubicin in S. peucetius is known as a member of thisfamily (18). Involvement of a protein of the HlyB subfamilyin aerial mycelial formation in S. griseus reminds us, again,of the SpoOK transport system necessary for the initiation ofsporulation in B. subtilis (37, 38). The SpoOK transportsystem, which is homologous to the oligopeptide permeasesystem of Salmonella typhimurium includes a substrate-binding protein localized to the outside of the cell, twomembrane-integrated proteins probably facilitating thetransport of the substrate into the cell, and two ATPases thatcouple transport to ATP hydrolysis (37, 38). The assembly ofthese proteins constitutes a structure similar in topology tothe functional domains of the membrane translocators of theHlyB subfamily. We therefore speculate that the AmfAIAmfB system is homologous to the B. subtilis SpoOK sys-tem.The SpoOK system is hypothesized to have a role in

sensing extracellular pheromone-like factors and to activatethe above-described phosphorelay through activation of

HlyB * IDSCHKIDYGLYALEILAQYHNVSVNPEEIKHRFDTDGTGLGLTSWLLAAKSLELKVKQVKKTIDRLNFIFLPALVWREDGRHFILTKISKEVNRYLIFDLEQRNPRVLEQSEFEAORFI to

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some phosphokinases, including KinA (15, 38). The com-pound transported by the AmfA/AmfB system remains un-known. However, it has been shown that some proteins andoligopeptides are required for aerial mycelium formation inStreptomyces spp. (9, 17) and sporulation in B. subtilis (16).For instance, SapB is a small protein involved in aerialmycelium formation which may enable hyphae on the sur-face of colonies to break surface tension and thereby grow asaerial mycelium into the air, or alternatively, may serve asan intracellular pheromone-like signal that stimulates theexpression of genes required for aerial mycelium formationin the recipient cells (49). The AmfA/AmfB system mayfacilitate the transport of one or more of these proteins andoligopeptides. An extracellular 34-kDa protein, factor C,which stimulates aerial mycelium and spore formation in S.griseus (4), is also a candidate.

The close location and direction of amfA and amfBsuggest that they constitute a single transcriptional unit,although transcriptional analysis has not yet been done.Some of the proteins belonging to the HlyB subfamily, suchas the mammalian P glycoprotein (13) and Ste6 from Sac-charomyces cerevisiae involved in the export of the yeastmating factor a (33), form a tandem duplication giving rise totwo homologous halves, each with homology to H1yB. Fromthese observations, we assume that AmfA and AmfB as-semble to form a heterodimer rather than a homodimer.However, the finding that AmfB is not absolutely requiredfor the induction of aerial mycelium formation in strain HH1is puzzling. It is possible that the homodimer of AmfAformed in the absence of a sufficient amount of AmfB canalso function but at a lower efficiency. This assumption isa possible explanation for the enhancing effect of AmfBon the aerial mycelium formation induced by AmfA andAmfR. Whether the combination of AmfB and AmfR in-duces aerial mycelium formation in strain HH1 and, if so,whether AmfA exerts a similar enhancing effect remain to beexamined.We would like to point out that a TTA codon for Leu,

which is confined to a very small number of Streptomycesgenes engaged in the regulation of morphogenesis and sec-ondary metabolism, is present at Leu-141 in the AmfRcoding sequence. In S. coelicolor, bldA specifying this tRNAcontrols both aerial mycelium formation and secondarymetabolite formation at the translational level (9, 30). Thissuggests that the expression ofamfR is regulated by the bldAgene, which appears to control morphogenesis in S. griseus(32) as in S. coelicolor. If the 1TA codon in AmfR actuallyrequires the bldA gene product for its translation, then AmfRmust function at a stage later than bldA. Interestingly, anadditional gene involved in aerial mycelium formation,which was cloned during our present study and by Babcockand Kendrick (1), was also reported to contain a 1TA codon(2).Our attempts to disrupt the S. griseus chromosomal loci

by means of gene replacement in order to determine thephenotypes of null mutations of amfR and amfA failed. Wethen sought to make such mutants in S. coelicolor, withwhich these techniques have been established (22). How-ever, Southern hybridization at normal stringency with theamfA/amfBl or amfR gene sequence, as a first step to clonethe corresponding genes from S. coelicolor, did not reveal asequence homologous to these genes in S. coelicolor (datanot shown). Of 14 actinomycete strains examined, includingS. coelicolor, Streptomyces lividans, Streptomyces fradiae,Streptomyces albus, and Streptomyces viridochromogenes,only Actinomyces citreofluorescens IFO 12853 and Actino-

myces fluorescens IFO 12861 were shown to contain se-quences strongly hybridizing with the probes. These obser-vations suggest that the nucleotide sequences of amJfR andamfA/amfB are not highly conserved among Streptomycesspp., although we assume that the genes with functionssimilar to those of amfiR and amfA/amfB are also involved inaerial mycelium formation in general in other Streptomycesspp. It is also possible that other species do not use a systemlike the amfR-amfA/am/B system and that there are diverseperipheral routes to the activation of the core developmentalgenes. In relation to this, A-factor itself exerts its regulatoryfunction only in limited species, although there is a possibil-ity that -y-butyrolactones structurally similar to A-factorserve as hormonal regulators in general in a given Strepto-myces sp. (23).We also cloned an additional gene which suppressed the

Bld phenotype because of A-factor deficiency, which wasfound to be identical to that cloned with a high-copy-numberplasmid pIJ702 by Babcock and Kendrick (1), who screenedfor genetic complementation of the Bld phenotype of a S.griseus mutant. They observed that this gene encodes a55.5-kDa protein whose COOH-terminal portion shows 35%identity to that of the COOH-terminal portion of the E. coliNusA protein (2). However, they observed that it failed tosuppress the Bld phenotype in an A-factor-deficient S.grseus mutant strain. A possible explanation for this dis-crepancy is that the mutant strain they used has a geneticbackground different from that of the mutant we used. Theexistence of the two different suppressors of the Bld pheno-type suggests that expression of one suppressor at a certainlevel can bypass a repressed expression of the other. It ispossible that there are multiple signal transfer systemsinvolved in aerial mycelium formation, acting either inde-pendently or downstream of the other systems. Such aredundancy of signal transfer systems may be one possibleexplanation of the general absence of bid mutants that areunaffected in secondary metabolism. Further study willreveal the role of this additional suppressor gene and itsrelationship with the amfR-amfAlam/B genes.

ACKNOWLEDGMENTS

This work was supported in part by the Waksman Foundation ofJapan and by the Ministry of Agriculture, Forestry and Fisheries ofJapan (grant BMP93-III-2-1). K. Ueda and K. Miyake were sup-ported by the Japan Society for the Promotion of Science.

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3. Bibb, M. J., P. R. Findlay, and M. W. Johnson. 1984. Therelationship between base composition and codon usage inbacterial genes and its use for the simple and reliable identifi-cation of protein-coding sequences. Gene 30:157-166.

4. Bir6, S., I. Bekesi, S. Vitfilis, and G. Szab6. 1980. A substanceaffecting differentiation in Streptomyces griseus. Purificationand properties. Eur. J. Biochem. 103:359-363.

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A Gene Cluster Involved in Aerial Mycelium Formation in