JOURNAL OF BACTERIOLOGY, May 1992, p. 2958-29670021-9193/92/092958-10$02.00/0Copyright X 1992, American Society for Microbiology

Vol. 174, No. 9

abaA, a New Pleiotropic Regulatory Locus for AntibioticProduction in Streptomyces coelicolor

M. A. FERN NDEZ-MORENO,"2 A. J. MARTIN-TRIANA,"2 E. MARTINEZ,"2 J NIEMI,3tH. M. KIESER,3 D. A. HOPWOOD,3 AND F. MALPARTIDAl.2*

Centro Nacional de Biotecnologia, Serrano 115, 28006-Madrid, 1 and Facultad de Medicina de la UAM,Departamento de Bioquimica, Arzobispo Morcillo 4, 28029-Madrid, 2 Spain, and John Innes

Institute, John Innes Centre, Norvich NR4 7UH, United Kingdom3

Received 15 November 1991/Accepted 23 February 1992

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans andStreptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosomecloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of thecloned genes revealed that these genes were different from other known S. coelicolor genes concerned withactinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed fiveputative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in onedirection) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed thatORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans.Insertion of a +C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype inwhich the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but notmethylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigorof sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaAconstitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulatesantibiotic production. DNA sequences that hybridize with the cloned DNA are present in several differentStreptomyces species.

Over the last few years, information about the control ofthe two special aspects of Streptomyces differentiation, i.e.,morphological (sporulation) and biochemical (secondary me-tabolite production), has increased rapidly (7-9, 23). Studiesof individual antibiotics have revealed that the genes con-trolling different metabolic steps in a particular biosyntheticpathway are typically located in clusters which also includeregulatory genes specific for that pathway (41, 53). In mostof the examples analyzed, the regulatory genes serve asactivators of the biosynthetic genes, as in the cases ofactinorhodin (19), bialaphos (2), streptomycin (15, 45), andundecylprodigiosin (39, 44), while for methylenomycin, arepressor has been implicated (11).

In addition to this "local" regulation of a particularbiosynthetic pathway, a second level of control connects theregulation of pathways for several secondary metabolites inthe same organism. This level has been revealed in the moststudied strain, Streptomyces coelicolor A3(2), which pro-duces four different antibiotics. These are actinorhodin (56),undecylprodigiosin (50), methylenomycin (57), and the cal-cium-dependent antibiotic (CDA) (31). There is genetic andphysical evidence for clusters of biosynthetic and regulatorygenes for the first three compounds (11, 18, 19, 37, 39, 50).Four genes have been implicated in the pleiotropic control ofmore than one of these pathways: mutations in absA (1) andabsB (6) abolish production of all four antibiotics, while afsBmutations (20) block actinorhodin and undecylprodigiosinproduction (as well as that of A factor) but only reduce the

* Corresponding author.t Present address: State Technological Research Centre, Turku,

Finland.

production of methylenomycin and CDA. The cloned afsRgene (formerly called afsB [29]) suppresses the effects ofafsB mutations (30, 54).A third level of control is manifested by other genes,

called bld. Mutations in these abolish the production ofaerial mycelium and antibiotic. Most characterized amongthis class of genes is bld,4, whose product is a tRNA specificfor translation of the UUA leucine codon (33, 34). Recently,one of the targets for the b1dA gene product was identified asa TTA codon within the actII regulatory region of theactinorhodin cluster: translation of this codon by the bldAproduct would give rise to a transcriptional activator (theactII-open reading frame 4 [ORF4] product) for the acti-norhodin biosynthetic genes (19). Control of the undecylpro-digiosin pathway may share some features with that ofactinorhodin, as suggested by similarities between the redD(44) and the actIl-ORF4 gene products (19). However, thecontrol exerted by bidA seems to be different for the twopathways: redD contains no TTA codon, suggesting theinvolvement of at least one further gene in control of the redgene set.

Clearly, an organism like S. coelicolor, with good geneticsfor in vivo and in vitro experiments and cloned genes forboth aspects of the differentiated state (aerial mycelium andspores on the one hand, and each of several antibioticpathways on the other), is a suitable subject for furtherstudies to build up an understanding of the regulatorynetwork involved in differentiation. Adding to the knowl-edge of this network, we here describe a new locus of the S.coelicolor chromosome involved in global control of second-ary metabolism which, like the abs and afs genes, appears toact independently of sporulation.

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FIG. 1. Restriction maps of the antibiotic-activating sequence (pIJ2344) and its derived subclones. Only relevant restriction sites areshown. Chromosomal DNAs are represented by open bars, plasmid sequences are represented by discontinuous lines, and vectors are givenin brackets.

MATERIALS AND METHODS

Bacterial strains. The Escherichia coli strains used were

JM101 (58) and XL1-blue (5). The S. coelicolor strains wereM145 (prototrophic, SCP1- SCP2-) (25), M138 (argAlproAl cysD18 SCP1+ SCP2-) (25), JF1 [argA1 guaAl act-177(II) redD42 SCP1- SCP2-1 (17), J1501 (hisAl uraAlstrAlpgl SCPI- SCP2-) (13), J1703 (hisAl uraAl strAl pglbldA16 SCPlNF SCP2-) (33), and the actIl mutant strains(49). The Streptomyces lividans strains were DL87/Gyls(selCI gyIB) (produced by lysogenization of DL87 [36] witha 4C31 derivative carrying an internal segment of the gyl-ABX operon) and TK21 (SLP2- SLP3-) (27).

Plasmids and bacteriophages. The E. coli plasmids werepIJ2921 (30a) and pUC18 and pUC19 (58). E. coli M13derivative phages mpl8 and mpl9 (58) were used for DNAsequencing. The Streptomyces plasmid vectors were thelow-copy-number SCP2* derivatives pIJ940 and pIJ941 (35)and the high-copy-number pIJ101 derivatives pIJ486 andpIJ487 (55). The Streptomyces phage vector was the 4C31derivative PM1 (38). Plasmid clones carrying various S.coelicolor genes were pIJ2303 (pIJ922 carrying the entire act

cluster [37]), pIJ2355 (pIJ922 carrying the whole red cluster[39]), and pIJ43-AP3 (pIJ43 carrying the afsR gene [29]).Media, culture conditions, and microbiological procedures.

For Streptomyces spp., agar media and transformation andtransfection were as described in reference 25. Thiostrepton(a gift from S. J. Lucania, E. R. Squibb and Sons, Princeton,N.J.) was used at a concentration of 50 ,ug/ml in agar mediumand 10 ,ug/ml in broth cultures. Hygromycin (Sigma; catalogno. H2638) was used at 200 ,ug/ml in solid media and 50,ug/ml in liquid media. E. coli strains were grown on L agaror L broth (40).DNA sequencing. DNA sequencing was carried out by the

dideoxy-chain termination method (51); we used the 7-deaza-dGTP reagent kit from U.S. Biochemical Corp. (cat-alog no. 70750) according to the manufacturer's recommen-dations. Exonuclease III (ExoIII) digestions were made onplasmids pMF108 and pMF109 (Fig. 1) previously digestedwith KpnI and BamHI. Suitable clones from each set ofExoIlI digestions (22) were chosen to cover most of the2.7-kb DNA fragment, and remaining gaps were sequencedby directed subcloning in the M13 mpl8 and mpl9 deriva-

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AGTCCGCG* A

TTGTCGTACCGCTGCCACCAGCCCGGTTCGCCCGCTCGTTGCAACAGCTTGAGCAGGACCGAGGCCTCGTACGGATCGGTGTCGTAGTGCTCCAGCAGCG101 .-- -- --- -- ---- --------+----- 200

AACAGCATGGCGACGGTGGTCGGGCCAAGCGGGCGAGCAACGTTGTCGAACTCGTCCTGGCTCCGGAGCATGCCTAGCCACAGCATCACGAGGTCGTCGCS T TG S GGA R NAR EN C CS S C SR PR T RI P T TT S W CR

CCCGGACGTCGGTCTCCGAGGGAGCCTGCGGCCCTTGCCCGACTCGATGCGCGAGAGCTTCGCGGCGCTGAAGCCGAGCGCGCGTGGCCTGGTCCTGG201 .--- - -- -- -- - --- -- - -- 300

GGGCCTGCAGCCAGAGGCTCCCTCGGACGCCGGGAACGGGCTGAGCTACGCGCTCTCGAAGCGCCGCGACTTCGGCTCGCGCGCACGCCGGACCAGGACCG ST P RR P LRR G KG SE IR SL KAA SF G LA RAA QD Q

GAGAGGCCGGCATCCTCGCGGAAGCCCGCCAGCTGCACGCCGACGAGCATCTTCAGCAGGGTCGGAGCCGGCTCGGCCCGATCCAGATAGGGTTCGAGAC301 .-- - - -------------------+ 400

S L GA D ERF GA LQ V G VL M KLLT PA PE AR D LY P E L

ORVN

401 .-- - - -- -- --- -----------++. --- 500CCCTCTACGCTACGCTGCGCCGCCTGTAGGACTGAgMTCGR S I R H S A A S M rbs

501 ------ ------..---------- 600GATGTGGTCCACCAGCTTGAGCGGCAGGAAGCGTGGGCGGTCCTTG*V L HDF E GD KA GA L F

GCCGCCACCTCGGCCGGGGTGTAGACGAGCGCGGGGCCGTCGGGGTCCCGGGAGTTGCGCATCGCGATGCCTCCGTCGACGAGGGCGACCTCGACGCAGT601 .--- ----.- --- ---------------------------+--- 700

A AV E A P T Y V LA P GD P DR S NR MA I G G DV L AV E V C

ORFD

701 .--- -- -- ---- ----------++----- 800ACGGGAGCCGCAACGACACGGCTGAGAAGAAGGTCGTCCGCAGGTTGTTCGACCGGACGTGAGGCAACGCGTGACCACCGTGGCGCCcg&QgcGN G EA N SHR SKK W CAD LL SA QV G NRV PP M rbs

801 .---------------------. --------------------------------+----- 900

rbs 0R31k

901 .-- -- -- ------------ -----------+----+--- 1000M P S PAMH P T L R S P G

rbsCGAACCCGGGTCGGAGGCAGGCGAGGCGACCCGCTCCXI&QQCTCCCGTACCCGGTGCGGGCCACCGCCGGCCGGGCCGTCCCGCCGTCGTCCGCCCCG

1001 .------- .-----------------------------------------.-. --+----- 1100E PG 5 E AG E A TR SG G LPY PV RA TA GR A VP P S SA P

1101 - -----.----------------+ 1200Y PG 5 P A L GGRP TA AV LR VA C SGE G FA RAR V FTR D

GGTGGCCGGCGGGCCGGAGATCGGGCTCGGACTCGCCCTGGGCTCCGGCCGGCTGAAGCTGACCGTCTCCGATCCCGGGGACGACGCGCCCCGGCTCAAC1301 --------------.. ---------------------------------+-- -------- 1400

V A GG P EI G L G LA LG S GR LK L TV S D PG D DA PR L N

1401 .--- .--- -- .--- --- --- -- --- - 1500P S DG S ALR EH GR G LC I V DA LAE EW G WT P RP P AG K

rbs A....T 0313 -

AGACGGTCTGGGCCACGTTGTCGACCCGCCCCCTCACCTGACCCGACTGCCGCgQ&&WGCCCCACACCATGCGCAGCGCTCCGACACCCGAGCCGAGCA1501 - ------+. -------- ----- ----- ----- --------------------------+ 1600

T V WA T L S T R P L T * M RS A PT P EP SI

1601 -----------------------------.-+-------------------- --+----- 1700RR T DR QT P P GS PR TT T LL AG LD G VPW S D I Q DS T

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abaA IN S. COELICOLOR 2961

GGGCTCGGCGGCGGCCATTCCGCGGCTGCTGCGCAAGGTCGCCCGGGGCGACGCCGAAACCGCCCGCGCCGCTCTCGGCGACCTGCGCAGGCGCATCTGC1701 + . + . + . + . + - + . + - + - + . + 1800

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

CAGTACGGCTTCGTCGTCGAGCAGGCGACCGCCGCGACCGTGCCCTTCCTGTGGGAGCTGGCGCAGCGGCCCCAGGTGAGCTGCCGGGCGCAGATCATCC1801 +.+- +- +.+- +.+-+-+.+ 1900

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

PvuIIAGCTGCTCAAGAACATCGCCGACGCCCGGCAGTGGGAGACCACCGCCACCGCCTACCCCAAGCTGCTCAACCACCGGGAGAACCCGGTGGCCTGGGAGCG

1901- + +- +- +- +- + +- +- +- + 2000L L K N I A D A R Q W E T T A T A Y P K L L N H R E N P V A W E R

CGCGGCGCGGCAGGCCGTCCGCGCCCGACGGGACGGACTGGAGCGGCTGCTG;GCGGACGACGACACCGAGATCATGCGCGCCACCAGCGAACTCGCCCGC2001 + . + . + . + - + - + - + - + - + . + 2100

A A R Q A V R A R R D G L E R L L A D D D T E I M R A T S E L A R

ACCCTCGGGGACTGAGACCGGCACCGCCTGGACCGGGACCCGCGCCCTCCACGGGGGAGAAGGCrGCGGGTCCCTCCCAAGGCGCGGAACGGTGTGGCGTG2101- + +- +- +- +- +- +- +- +- + 2200

T L GD *

rbs ORFCCTCCCGGCCGACAGGGTAGGAAAGCGCGGGAAGCAAGCGCTTGCCCCACTCGGTCACCAGGAGCCACGCCGATGGCGTCGTCGATGGAAAAGCCGCTC

2201 +.+- +- +.+- +.+-+.+-+ 2300M A S S M E K P L

GATCACCGCTACCGGGGCGAACACCCGATACGCACGCTCGTCTACCTGTTCCGCGCCGACCGCCGCCGACTGGCCGGCGCGGTCGCCGTCTTCACCGTCA2301 +- +- +- +- +- +- +- +- +- + 2400

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

AGCACAGCCCGATCTGGCTGCTGCCCCTCGTCACCGCCGCCATCGTCGACACCGTGGTCCAGCACGGTCCGATCACCGACCTGTGGACCAGCACCGGGCT2401 + . +-+ . +-+-+-+ . + - + - + 2500

H S P I W L L P L V T A A I V D T V V Q H G P I T D L W T S T G L

CATCATGTTCATCCTGGTGGTCAACTACCCGCTGCACCTGCTCTACGTCCGCCTCCTGTACGGCAGCGTGCGCCGCATGGGCACCGCCCTGCGGTCCGCG2501- +- +- +- +- +- +- +- +- +- + 2600

I M F I L V V N Y P L H L L Y V R L L Y G S V R R M G T A L R S A

Sr)hI Pi-tICTGTGCACGCGCATGCAGCAGCTCTCCATCGGCTACCACTCGCGGGTCAGCGCGGGCGTGCTGCAG

2601 +.+- + +- +- +- 2666L C T R M Q Q L S I G Y H S R V S A G V L Q

FIG. 2. Nucleotide sequence of the 2.7-kb PstI fragment from pIJ2344 carrying the antibiotic-activating sequence and its deducedtranslation products. Relevant restriction sites are indicated. The putative ribosomal binding sites are shown by underlined sequences. Thededuced directions of transcription for ORFs are indicated by arrows and named accordingly; only those which fit the standard codon usagefor Streptomyces genes are considered (Fig. 3).

tives. The fragments from the Exolll digests were finallycloned either in M13mpl8 or in M13mpl9 for sequencing. Ofthe DNA, 93.3% was sequenced on both strands.Computer analysis of sequences. The DNA sequence was

analyzed for open reading frames by using CODONPREF-ERENCE (University of Wisconsin Genetics ComputerGroup [UWGCG] [14]) with a Streptomyces codon usagetable for 64 genes (2a). Amino acid sequences were analyzedby using various programs from the UWGCG package(version 6.2, 1990). Comparisons of sequences were madeagainst the EMBL nucleic acid data base (daily update,August 1991) and the Swissprot data base (release 18.0, May1991), using BESTFIT, FASTA, TFASTA, COMPARE, andDOTPLOT.Gene disruption. For gene disruption experiments, we

used insert-directed recombination (10) with the 4C31-att-derivative PM1 as vector and S. coelicolor J1501 as host.DNA and RNA manipulations. For isolation, cloning, and

manipulation of nucleic acids, methods used were thosedescribed in reference 25 for Streptomyces spp. and refer-ence 40 for E. coli. For high-resolution S1 mapping, themethod of Murray (43) was used. The growth medium was

SY liquid medium (46) inoculated with pregerminatedspores, and RNA was extracted after 2 days.

Antibiotic production assays. Production of the pigmentedantibiotics actinorhodin and undecylprodigiosin was de-tected by visual inspection; CDA production was tested asdescribed in reference 28; and methylenomycin activity waschecked by growth inhibition of a methylenomycin-sensitiveS. coelicolor strain against the disrupted S. coelicolor J1501mutant after introduction of the SCP1 plasmid by conjuga-tion with SCP1+ strain M138 and recovery of transconju-gants on minimal medium supplemented with histidine,uracil, and streptomycin. The presence of SCP1 in the strainwas confirmed by the strain's resistance to methylenomycin.

Pulsed-field gel electrophoresis. Analysis of genomic DNAof S. coelicolor digested with AseI and location of the abaAgene by Southern blotting were carried out as describedelsewhere (30b).

Nucleotide sequence accession number. The nucleotidesequence presented in this article has been submitted toGenBank. The accession number is X60316.

RESULTS

Cloning and characterization of the DNA that stimulatesproduction in S. lividans. Fragments of S. coelicolor M145chromosomal DNA were obtained by partial digestion with

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A

B

S3

3'

500 1000 1500 2000 25003,

S5

.v I *1 I

500 1000 1500 2000 2500FIG. 3. CODONPREFERENCE of the 2.7-kb PstI fragment containing the antibiotic-activating sequence. The numbering is as in Fig. 2.

A codon usage table was constructed from 64 different Streptomyces genes (see Materials and Methods) and used with the UWGCG programto scan the DNA sequence. The deduced ORFs are indicated by arrows showing directions of transcription as deduced from the DNAsequence. Their translated products are indicated in Fig. 2 under the DNA sequence.

MboI and ligated into the BamHI site of the high-copy-number plasmid pIJ486. The ligation mixture was introducedby transformation into the S. lividans derivative DL87/Gyls(the choice of this particular host is not relevant for thepresent study), with selection for thiostrepton resistance.Among the transformants, an intensely blue colony wasisolated, and its phenotype was shown to be due to thecloned fragment by retransformation of S. lividans TK21.Analysis of plasmid DNA (named pU2344) from this colonyrevealed a 20-kb insert in the cloning site of the vector (Fig.1). The phenotype associated with pIJ2344 raised the possi-

bility that the cloned DNA might contain either the actllregion (19) or the afsR gene (30). However, its pattern ofrestriction sites was clearly different from either of those.

Transformation of S. lividans TK21 protoplasts with plas-mids produced by ligating a BglII digest of p0J2344 withpIJ486 yielded several blue colonies. Four such transfor-mants analyzed carried the 4.3-kb BglII fragment of pIJ2344.Since both orientations were represented, it was likely thatthis DNA included promoter sequences. One of these re-

combinants was named pIJ2343 and used for further charac-terization.

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A more-detailed restriction map was obtained for pIJ2343(Fig. 1). Location of the activating sequences within asmaller fragment was achieved by digesting pIJ2343 withPstI to give fragments of 1.8, 2.7, and 4.2 kb (the last onecarrying the thiostrepton resistance gene and plasmid repli-cation functions); ligating the DNA; and using it to transformS. lividans TK21. Only clones carrying the 2.7-kb PstIfragment gave a blue-activating phenotype (pMF105 andpMF106 represented the two orientations; Fig. 1). Further-more, deletion of the internal ApaI fragment gave a nonac-tivating phenotype. pMF105 and pMF106 failed to comple-ment actII mutations or to confer an Act+ phenotype on S.coelicolor J1703 (a b1dA4 mutant), suggesting a mode ofoperation different from that of the actII-ORF4 gene, whichcauses actinorhodin production when it is cloned at highcopy number but not at low copy number, in a b1dA mutant(19, 48).

Southern blot analysis. Southern blotting using pMF110(see Fig. 5) as a probe showed no relatedness of the DNA tothe act cluster (in pIJ2303), afsR (in pIJ43-AP3), or the redcluster (in pIJ2355). Southern blots of PstI-digested chromo-somal DNAs from several Streptomyces species [S. coeli-color A3(2) (J1501), S. lividans TK21, S. antibioticus ATCC11891, S. ambofaciens ATCC 15154,S. violaceoruber Tu22,and S. glaucescens ETH 22794) probed with pMF109 (Fig. 1)revealed a single hybridizing band in some chromosomaldigests. Bands of similar sizes and intensities were observedin S. coelicolor, S. lividans, S. antibioticus, and S. violace-oruber, and a smaller band much less intense than the otherswas observed in S. ambofaciens. No hybridizing band wasobserved in S. glaucescens. These results clearly indicatedthe existence of similar genes in several different Streptomy-ces species and confirmed that the sequenced DNA wascolinear with the original S. coelicolor chromosome and didnot result from rearrangement during the ligation of MboIfragments.DNA sequence of the antibiotic-activating 2.7-kb PstI frag-

ment. The DNA sequence of the 2.7-kb PstI fragment (Fig.2) was analyzed for ORFs by using the program CODON-PREFERENCE (UWGCG). Five possible ORFs were de-duced (Fig. 3) and named ORFA, -B, -C, -D, and -E. Thetwo smallest (ORFD and ORFE) are divergently arrangedwith respect to the three largest ones (ORFA, ORFB, andORFC), and ORFC seems to be incomplete in the sequencedfragment. Other putative ORFs were discarded as codingregions because their codon usages did not fit the Strepto-myces pattern. (This included a potential ORF antisense ofORFA.)The most likely start codons for each ORF, as deduced by

considerations of codon usage (4) and the presence of goodputative ribosome-binding sites at appropriate distancesfrom a potential start codon (3), are indicated in Fig. 2. (ForORFA, a GTG codon 31 codons downstream of the mostlikely start of the gene is also preceded by a good potentialribosome-binding site.) The Mrs of the products of the ORFswould be 11,896 for ORFE, 7,869 for ORFD, 19,586 forORFA, and 20,053 for ORFB.Comparison of the deduced amino acid sequences of the

ORFs with sequences in available data bases failed to showany significant homologies with other known proteins. How-ever, the ORFA product showed two repeats of smalldomains (domains A and E, B and D, Fig. 4). The A and Edomains showed the basic motif AxxxGxxxxxV, reported tobe conserved in some DNA binding proteins, in their aminoacid sequences (52). In some such proteins, this sequenceforms a helix-turn-helix motif. However, the calculated

- !enen IV

ORFA product I _ LX

ORFA domains A B

dom A PGEPGSEAGEATRSGGLPYPVRATA:1:1:: I II I...I ::

dom E AGGPEIGLGLALGSGRLKLTVGDPG

C D E

domB RAVPPSSAPYPG:11111 1. 1:

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FIG. 4. Physical organization of ORFA. Above is a schematicmap of the ORFA gene product. The translated sequence is num-bered starting from the first start codon (Fig. 2). The limits of therepeat domains (rectangles) are indicated by numbers. The aminoacid sequences are given below, with boldface letters correspondingto the amino acid sequence motif conserved in some DNA bindingproteins. dom, domain.

scores obtained with the Dodd-Egan matrix (16) for theORFA product are too low (-782 and -1,233, with standarddeviations of -3.48 and -5.01, respectively, for the A and Edomains), making it unlikely that a helix-turn-helix motifexists at these positions (Fig. 4).

Identification of the antibiotic-activating sequences. Figure5 shows the results of transforming S. lividans TK21 withvarious fragments derived from the sequenced PstI frag-ment. Those fragments carrying both complete ORFB and137 bp of DNA downstream of it caused blue pigmentproduction. Since pMF128 with ORFB pointing away fromthe terminator of the vector gave blue colonies, while theopposite orientation was never obtained when blue colonieswere sought, transcription of ORFB when cloned alonemight come from a vector promoter. Consequently, thenatural promoter sequences must lie upstream of the 5' endor within the coding region of ORFA. When the SalIfragment (nucleotides [nt] 675 to 925 upstream of the 5' endof ORFA) was placed in its natural orientation upstream ofORFB to generate pMF129 and pMF130 (opposite orienta-tions in pIJ486), blue S. lividans transformants were ob-tained for both constructions. This result suggests the pos-sibility of a polycistronic mRNA for at least ORFA andORFB.

Transcriptional organization of the activating gene(s). Todetermine the transcription start point of ORFA, total S.coelicolor RNA was hybridized with the 503-bp fragment(BglII-NotI, nt 620 to 1123) from pMF108.3 (a pMF108ExoIII deletion, harboring nt 620 to 2666) previously labelled(5') at the NotI site with [-y-32P]ATP. After S1 digestion, twoprotected fragments of 215 and 213 bp were seen (Fig. 6A),implicating nt 908 and 910 (Fig. 2) as transcription startpoints of ORFA (and perhaps also of ORFB). Full-lengthprotection was observed when a NaeI-XmaI fragment (nt1490 to 1742) labeled at the 5' end of the XmaI site was usedas probe (Fig. 6B), with no other start site indicating theabsence of the promoter in the intergenic region ORFA-ORFB.Sequences upstream of the ORFA transcription start point

(Fig. 7) can be aligned with a group of Streptomyces pro-moters (24) and with other consensus bacterial promoters(21). Furthermore, inverted repeat sequences located aroundthe -35 region resemble those of some bacterial operators(32, 47), which might be the target of a transcriptionalregulator.

Phenotypic characterization of the activating sequences in S.coelicolor. In order to explore a possible role for the ORFBproduct in actinorhodin production in S. coelicolor, ORFBwas disrupted by using a clone carrying the 392-bp SacII-DdeI fragment (nt 1720 to 2112) (previously cloned as blunt

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G CWS:CRfl ~ ~~~~GCA T_ ~~~~~~~~~~~CG

C GG CC GA TG, r

the phage had been inserted through the IS110 sequencesrather than through the cloned fragment. Analogous at-tempts to inactivate ORFA and ORFC were unsuccessful,because all lysogens were formed by the insertion of IS110rather than by homologous recombination between the

251 cloned fragment and the corresponding chromosomal gene.* 242 Map locations of the activating sequences. The cloned DNA217 was mapped on the S. coelicolor chromosome by probing a195 Southern blot of an AseI digest of M145 DNA, separated by

pulsed-field gel electrophoresis, with 32P-labeled pIJ2344* DNA. The probe labeled the sixth largest AseI fragment

(AseI-F), which has been located on the combined genetic-physical map of the S. coelicolor chromosome (30b) at aposition corresponding to approximately 2 o'clock on thestandard genetic map (26). This map location is differentfrom those of absA, at about 10 o'clock (1); absB and afsB,at about 5 o'clock (20); and afsR, at about 7 o'clock (1).Thus, the newly studied DNA represents a new pleiotropiclocus of S. coelicolor which we propose to name, provision-ally, abaA (antibiotic biosynthesis activator).

OR FA

5'3

FIG. 6. High-resolution Si mapping experiments. (A) Transcrip-tion initiation point for the ORFA-ORFB transcript. The start pointis indicated by asterisks within the DNA sequence as derived fromMaxam and Gilbert reactions (42) of the protected fragments. (B)Full-length protection of the NaeI-XmaI fragment (Fig. 2), indicat-ing the absence of promoters initiating within the intergenic regionbetween ORFA and ORFB.

ended in the HincII site of pIJ2921 and rescued with BglII) inthe BglII site of PM1 to lysogenize S. coelicolor J1501. Asubstantial proportion of the lysogens selected on thiostrep-ton and hygromycin were brown-orange, indicating an Act-Red- phenotype. Southern blot analysis of the two of themshowed that ORFB had indeed been interrupted by insertionof the phage through the cloned fragment. The lysogens werefound to be significantly reduced in their abilities to produceCDA but not methylenomycin (tested after introducing theSCP1 plasmid into one of the lysogens). Thus, disruption ofORFB abolished actinorhodin production, almost totallyabolished undecylprodigiosin production, significantly re-duced CDA production, but left methylenomycin productionessentially unchanged. The remaining lysogens were blue,and restriction analysis of the phages liberated from five ofthem showed the presence of IS110 (12); we assumed that

TTrTTTCGCGCAATTrCTCGTGCAATIGC&CGCGAGCGCTCTAAGCGTGG&TAATAGCCGTGGCGTCAA

TTGACa 17 bp TAtAaT a

TTGaca 17 bp tAGgaT b

FIG. 7. DNA sequence upstream of the transcription start site ofthe abaA gene. abaA mRNA is initiated at either of the Gs indicated.Below the DNA sequence are indicated the consensus sequences ofthe -10 and -35 regions of bacterial promoters (a) and Streptomy-ces "vegetative" promoters (b). Putative palindromic sequences areindicated by lines above the sequence; the symmetry axis is indi-cated by a colon (:).

DISCUSSIONBy selecting a clone from S. coelicolor that stimulated

actinorhodin production in S. lividans, we have identified anew S. coelicolor gene (abaA) which participates in thecontrol of antibiotic expression in this species. Insertion ofthe jC31 prophage into the locus does not significantly affectmethylenomycin production but completely abolishes acti-norhodin production and strongly reduces CDA and prodi-giosin production. These differences would make a distinc-tion between this new locus and absA and absB, whoseactions do not differentiate between expression of the fourantibiotics. Perhaps abaA is involved in the complex net-work of regulation at a point closer to the actinorhodin genecluster than to the clusters for the other antibiotics. Thephenotypes of afsB mutants seem to be more similar to thatof the insertional mutant described here, but a different maplocation clearly distinguishes the genes.The DNA sequence of the new locus has revealed at least

two putative ORFs (A and B) whose transcription seems todepend on the same promoter sequences. ORFB seems to beimplicated in the control of actinorhodin, prodigiosin, andCDA production, but the failure to obtain insertions intoORFA leaves open the possibility that ORFA is also in-volved in the control of antibiotic production. Because nosignificant homologies between the ORFA or ORFB prod-ucts and other proteins whose sequences are available in thedata bases were found, the sequences themselves give noclue as to the mode of action. However, the isolation of theabaA locus and its disruption should help in analyzing thenetwork of controls of secondary metabolite production in S.coelicolor.

ACKNOWLEDGMENTSWe thank Judy Ward for the preparation ofMboI-cut S. coelicolor

DNA and the cloning vector used in the isolation of pIJ2344; S. J.Lucania, Squibb Institute for Medical Research, Princeton, N.J., forthe gift of thiostrepton; T. Beppu for the gift of pIJ43-AP3; andK. F. Chater for helpful comments on the manuscript.M.A.F.-M. acknowledges a fellowship from the Spanish PFPI

(Ministerio de Educaci6n). This research was supported in part bypersonal grants (to F.M.) from the Spanish CICYT (PBT86-0025 andB1090-0760), EEC (BAP 0393-E), and Smith, Kline & FrenchS.A.E.; by a grant-in-aid to the John Innes Institute from theAgricultural and Food Research Council and the John Innes Foun-dation; and by NATO Collaborative Research Grant 870118.

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