A Single Target Is Sufficient To Account for the Biological Effects of the A-Factor Receptor Protein of Streptomyces griseus

In the model of the A-factor (2-isocapryloyl-3R-hydroxymethyl- ${gamma}$ -butyrolactone)regulatory cascade in Streptomyces griseus, A-factor binds ArpA,the A-factor receptor protein, that has bound to the adpA promoterand dissociates it from the DNA, thus inducing the transcriptionof adpA. AdpA switches on the transcription of a number of genesrequired for secondary metabolism and morphological differentiation,forming an AdpA regulon. Consistent with this model, arpA nullmutants produced streptomycin and a yellow pigment in largeramounts and formed aerial hyphae from an earlier growth stagethan the wild-type strain. On the other hand, mutant MK2, expressinga mutant ArpA (Trp119Ala), neither produced secondary metabolitesnor formed aerial hyphae, because this A-factor-insensitivemutant ArpA always bound to and repressed the adpA promoterdue to the amino acid replacement of Trp-119 with Ala. Introductionof adpA under the control of a foreign promoter into mutantMK2 restored all of the phenotypes that we could observe, whichsuggests that the only significant target of ArpA is adpA. Incontrast to other ${gamma}$ -butyrolactone regulatory systems, disruptionof arpA had no effect on A-factor production, indicating thatArpA does not regulate A-factor biosynthesis. Instead, A-factorproduction was found to be repressed by AdpA in a two-step regulatoryfeedback loop.

INTRODUCTION

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Introduction

The transcriptional factors ArpA and AdpA which we have longstudied are key members of the A-factor (2-isocapryloyl-3R-hydroxymethyl- ${gamma}$ -butyrolactone)regulatory cascade controlling the initiation of secondary metabolismand morphological differentiation in Streptomyces griseus (2,5-7). A-factor acts as a switch for these processes, and anA-factor-deficient mutant neither produces any of the secondarymetabolites nor forms aerial mycelium. During the early stagesof growth, when the concentration of A-factor is still low,the A-factor-specific receptor, ArpA, binds to the promoterregion of adpA and represses its transcription. When the concentrationof A-factor reaches a critical level at or near the decisionpoint, A-factor binds to the DNA-bound ArpA and releases itfrom the DNA, thus activating the transcription of adpA. Thedecision point is at the middle of the exponential growth phase(5), and the transcription of adpA therefore starts just beforeor at the decision point. A number of genes required for secondarymetabolism and morphological development are then switched onby the transcriptional activator, AdpA. These include strR,encoding the pathway-specific transcriptional activator forstreptomycin biosynthetic genes (19); adsA, encoding an extracytoplasmicfunction sigma factor essential for aerial mycelium formation(31); amfR, encoding a transcriptional factor for the amf operonessential for aerial mycelium formation (33); sgmA, encodinga zinc-containing metalloendopeptidase important for aerialmycelium formation (9); and ssgA, encoding a small acidic proteinessential for septum formation in aerial hyphae (32). We havealso identified several proteases as targets of AdpA (our unpublisheddata).

The above model for the early steps of the A-factor regulatorycascade has been formulated by the following observations. S.griseus mutant HH1 is unable to form aerial hyphae or streptomycinbecause it cannot synthesize A-factor. Suppressor mutants KM7and HO1, which form aerial hyphae and produce streptomycin evenin the absence of A-factor, lack the A-factor receptor activity(15, 23). Consistent with the repressor-like behavior of ArpAin vivo, in vitro analysis showed that it binds a 22-bp palindromewith the sequence 5'-GG(T/C)CGGT(A/T)(T/C)G(T/G)-3' as one halfof the palindrome, and upon binding A-factor it immediatelydissociates from the DNA (22). At this stage of the research,we postulated that ArpA bound specific genes, named Xs, andrepressed their transcription in an early stage of growth. Apartfrom these studies, we purified an A-factor-dependent protein(AdpA) that can bind the upstream activation sequence of strRand identified adpA as one of the gene Xs, since ArpA boundthe promoter of adpA (19). Disruption of adpA caused S. griseusto be unable to form aerial hyphae or produce secondary metabolitessuch as streptomycin and a yellow pigment. The yellow pigmenthas been determined to be a phenoxazinone derivative and itwas named grixazone (our unpublished data).

Because the arpA mutants examined so far were derived from mutantHH1, which contains large deletions (14), including afsA, whichis essential for A-factor biosynthesis (1), , we generated anarpA null mutation, starting with S. griseus IFO13350, whichhas the wild-type genetic background. One of the purposes ofconstructing this mutant was to examine A-factor production,since orthologous ${gamma}$ -butyrolactone receptor mutations, includingBarA, which serves as a receptor for virginiae butanolides inStreptomyces virginiae (11, 16, 20), FarA for IM-2 in Streptomyceslavendulae (12, 13, 30), and ScbR for SCB1 in Streptomyces coelicolorA3(2) (26), affect the biosynthesis of the respective ${gamma}$ -butyrolactonesin some unknown way. We also generated an arpA mutant (MK2)that contained a site-directed mutation at position 119 of ArpA;the tryptophan residue at this position is essential for A-factorbinding (25). The Trp residue corresponding to Trp119 of ArpAis conserved in all the ${gamma}$ -butyrolactone receptors so far reportedand is involved in the formation of a ligand-binding pocket,as determined by X-ray crystallography of CprB (17), an ArpAhomologue in S. coelicolor A3(2). Site-directed mutagenesisof ArpA has shown that the Trp119Ala mutant lacks the A-factorbinding activity but not the DNA-binding activity (25). Characterizationof the arpA null mutant and MK2 clearly showed the functionof ArpA as a repressor of morphogenesis and secondary metaboliteproduction.

Concerning this model of the A-factor regulatory cascade, thequestion of whether ArpA targets additional genes other thanadpA was raised. To answer this question, we expressed adpAunder a foreign promoter in mutant MK2. If ArpA has additionaltargets, expression of adpA under a constitutive promoter wouldnot be enough to restore the phenotypes of MK2, because theadditional target gene(s), which might also be essential formorphogenesis and secondary metabolite production, must be repressedby the mutant Trp119Ala ArpA. In this short communication wediscuss that the target of ArpA must be adpA for all the phenotypesthat we can observe and that, unlike in other ${gamma}$ -butyrolactoneregulatory systems, the null arpA mutation does not affect A-factorbiosynthesis. To fully understand the regulatory mechanism bywhich various ${gamma}$ -butyrolactones switch on the expression of thegenes important for secondary metabolism and morphogenesis invarious Streptomyces spp., it is important to determine thedetailed phenotypes resulting from mutations of arpA and adpAin the A-factor regulatory cascade and to determine clear differencesbetween the A-factor regulatory system in S. griseus and other ${gamma}$ -butyrolactone regulatory systems in various Streptomyces strains.

Phenotypes of an arpA null mutant.

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Phenotypes of an arpa...

The S. griseus mutant KM7, encoding an ArpA protein that isdefective for A-factor binding, developed normally and producedstreptomycin and grixazone in the absence of A-factor. Thissuggested that the A-factor receptor protein (ArpA) served asa repressor for these phenotypes (15). Consistent with thisidea, mutant HO1, containing a mutant ArpA (Pro115Ser) withA-factor-binding ability but without DNA-binding ability, showedthe same phenotype as mutant KM7 (23). Both KM7 and HO1 werederived from S. griseus mutant HH1, which had large deletions,including afsA, probably encoding an enzyme for the biosynthesisof A-factor (1). We therefore generated a null arpA mutant byin-frame deletion in the wild-type genetic background, startingwith S. griseus IFO13350, as follows. The 4.4-kb SalI fragmentcontaining the whole arpA gene was cloned previously in pUC19(pARP-Sal) (21). By Southern hybridization and colony hybridization,the 2.8-kb BamHI fragment containing the region downstream ofarpA was cloned in pUC19 (pARP-Bam). The 0.3-kb BamHI-EcoRIfragment (corresponding to the sequence upstream from the Gln-4codon of arpA) was amplified with primers 5'-GGGTTCGGTTCGCCGGGACC-3'and 5'-CGgaattcCTGCTTCGCCATTTCCTGCC-3' (the lowercase lettersindicate an EcoRI site), and the absence of PCR errors was confirmedby nucleotide sequencing. The 1.3-kb BamHI-SalI fragment containingarpA in pARP-Sal was replaced with this amplified fragment,and the resultant 3.4-kb SalI-EcoRI fragment was ligated withthe 2.8-kb EcoRI-BamHI fragment from pARP-Bam in pUC19. A 1.9-kbBamHI-HindIII fragment carrying the kanamycin resistance genefrom Tn5 was inserted in the multicloning site.

The plasmid prepared from Escherichia coli JM110 was introducedby transformation into S. griseus IFO13350, and kanamycin-resistanttransformants were isolated. Kanamycin-sensitive colonies, derivedafter a second crossover in one of the transformants, were candidatesfor arpA-disrupted strains. Southern hybridization with the4.4-kb SalI fragment in pARP-Sal and the kanamycin resistancegene as ³²P-labeled probes against the chromosomal DNA digestedwith EcoRI plus SalI identified true arpA disruptants ( ${Delta}$ arpA).Mutant ${Delta}$ arpA strains constructed in this way contained a deletionfrom Ala-5 to Gly-245 of ArpA (Fig. 1A).

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FIG. 1. Phenotypes of an arpA null mutant, the ${Delta}$ arpA mutant, of S. griseus. (A) Schematic representation of the structures of arpA on the chromosome of S. griseus IFO13350 (wild type, wt) and an arpA deletion mutant ( ${Delta}$ arpA). ArpA contains a helix-turn-helix (HTH) DNA-binding motif near the N terminus. The ${Delta}$ arpA mutant contains an in-frame deletion from Ala-5 to Gly-245. (B) Effects of the ${Delta}$ arpA mutation on morphological differentiation. Both strains were inoculated with a toothpick and grown on YMPD agar medium (19) at 28°C for the indicated periods. (C) Effects of the ${Delta}$ arpA mutation on streptomycin production. Strains were inoculated with a toothpick and grown on Bennett agar medium (10) without glucose at 28°C for the indicated periods. Spores of Bacillus subtilis were then overlaid, and the plates were incubated at 37°C for 1 day. Streptomycin production was detected by the formation of a growth inhibition zone. (D) Morphological differentiation of the ${Delta}$ arpA mutant harboring pARPL at the normal time. The ${Delta}$ arpA mutant harboring pKU209, grown for 36 h in the same way as in B, prematurely forms aerial mycelium, whereas the mutant harboring pARPL remains as substrate hyphae. (E) Streptomycin production by the ${Delta}$ arpA mutant harboring pARPL in the normal yield at the normal time. Strains were grown for 3 days, and streptomycin production was assayed in the same way as in C.

The ${Delta}$ arpA mutant produced A-factor with the same kinetics andin the same amount as the wild-type strain (see below). However,it formed aerial mycelium and spores earlier than the wild type;earlier sporulation of the mutant slightly reduced the diameterof its colony on agar medium (Fig. 1B). The ${Delta}$ arpA mutant producedstreptomycin earlier than the wild type and accumulated it ina larger amount (Fig. 1C). Consistent with this, adpA was transcribedmore actively at all time points in the ${Delta}$ arpA mutant than inthe wild-type strain (Fig. 2A). Introduction of pARPL containingthe whole arpA gene on low-copy-number plasmid pKU209 into the ${Delta}$ arpA mutant repressed the early aerial mycelium formation at36 h (Fig. 1D) and the overproduction of streptomycin (Fig.1E). These phenotypes of the ${Delta}$ arpA mutant were the same as thoseof mutants KM7 and HO1, showing that elimination of the repressorfunction of ArpA led to uncontrolled switch-on of the A-factorregulatory cascade irrespective of the presence of afsA.

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FIG. 2. Transcription of adpA in ${Delta}$ arpA (A) and MK2 (Trp119Ala) (B) mutants. RNA was prepared from mycelium grown in YMPD liquid medium (A) and on YMPD agar medium (B). We used the liquid medium for RNA preparation in A because we could hardly prepare enough RNA from the mycelium grown on cellophane on the surface of agar medium for S1 mapping at 6 h. S1 mapping was performed with the ³²P-labeled probe corresponding to nucleotide positions -274 to + 150 with respect to the transcriptional start point of adpA, as described previously (19). hrdB, encoding a principal ${sigma}$ factor of RNA polymerase, was used to check the purity and amounts of RNA used.

Phenotypes of a mutant containing the Trp119Ala mutation.

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Phenotypes of a mutant...

Site-directed mutagenesis of ArpA showed that Trp-119 is essentialfor A-factor binding but nonessential for DNA binding. The Trp119Alamutant is an A-factor-insensitive mutant that binds to its targetDNA normally in both the presence and absence of A-factor (25).If the in vitro A-factor-insensitive feature of the Trp119Alamutant is also the case in vivo, S. griseus mutant MK2 containingthe Trp119Ala mutation would fail to develop aerial hyphae andto produce secondary metabolites because of constant bindingto and hence repression of the target promoters.

We introduced the mutated arpA gene containing the Trp119Alamutation into S. griseus. For generation of the S. griseus mutant,we used the mutated arpA gene containing the Trp119Ala mutationand introduced it into S. griseus IFO13350, essentially in thesame way as for generating the ${Delta}$ arpA mutant. A single crossoverby homologous recombination yielded mutant MK1 and the secondcrossover yielded mutant MK2 (Fig. 3A). As expected, mutantMK2 remained bald (Fig. 3B, left) and failed to produce streptomycin(Fig. 3C, left) or grixazone (Fig. 3D, left). Neither additionof A-factor nor introduction of pADP10L containing the entireadpA gene and its promoter on low-copy-number plasmid pKU209(19) corrected these defects of mutant MK2, whereas pADP20L,containing the adpA sequence under the control of the E. colilac promoter on pKU209 (19), restored the full wild-type phenotypes(Fig. 3B, C, and D, right). The spores of mutant MK2 harboringpADP20L were the same in shape, length, and diameter as thoseof the wild-type strain (Fig. 3E).

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FIG. 3. Phenotypes of mutants MK1 and MK2. (A) Schematic representation of the arpA region on the chromosomes of mutants MK1 and MK2. Mutant MK1 contains both the intact arpA and the mutated arpA (Trp119Ala) in addition to the kanamycin resistance gene. (B) Effects of the arpA W119A mutation on morphological differentiation. S. griseus IFO13350 (wild type, wt), MK1, and MK2 were grown on YMPD agar medium at 28°C for 3 days. Neither MK1 nor MK2 formed aerial hyphae or spores. Introduction of pADP20L containing the adpA-coding sequence under the control of the E. coli lac promoter resulted in restoration of sporulation in MK2, whereas pADP10L, containing the intact adpA, did not restore the defect in aerial hypha formation of MK2. (C) Effects of the Trp119Ala mutation on streptomycin production. Strains were grown on Bennett agar medium without glucose at 28°C for 3 days, as described in the legend toFig. 1. (D) Effects of the Trp119Ala mutation on grixazone production. Strains were grown on phosphate-depleted standard minimal medium at 28°C for 3 days, as described previously (31). Grixazone is produced only on phosphate-depleted medium. (E) Scanning electron micrographs of spores formed by S. griseus IFO13350 and mutant MK2 harboring pADP20L. Both strains were grown on R2YE agar medium (10) at 28°C for 3 days. Bar, 2 µm.

The E. coli lac promoter is known to be expressed in Streptomycesspp. (19, 29), and we confirmed constant transcription of thelac promoter by S1 mapping (data not shown). In mutant MK2,no transcription of adpA was detected by S1 nuclease mapping(Fig. 2B). The failure of pADP10L to complement the defectstherefore resulted from the constant repression of the adpApromoter by the Trp119Ala form of ArpA. Since the presence ofAdpA corrects all the defects arising from the constant bindingof ArpA (Trp119Ala) to the target promoters, the adpA promoteris the only critical target of ArpA.

Consistent with this idea, we failed to isolate other significanttargets for ArpA during repeated attempts by a gel mobilityshift-PCR method (8) and Southern hybridization. The gel mobilityshift-PCR method was used successfully for isolation of a numberof DNA fragments to which AdpA binds (8, 31). Although we isolatedtwo additional ArpA-binding sites (ABS2 and ABS3) other thanthe adpA promoter by this method, both were found to have nobiological functions. ABS2, with the sequence 5'-CGATACCTGCCGTTCGGTATTT-3',was located between two divergent genes, each encoding a proteinwith no similarity to known proteins, and ABS3, with the sequence5'-CAGAACCGGGCGGTCGGTCGGC-3', was located between convergentgenes, each encoding a product with no similarity to known proteins.Replacement of ABS2 on the chromosome with a mutant site unableto bind ArpA had no effects on the transcription of the twodivergent genes or on detectable phenotypes. Concerning ABS3,we examined the presence of promoters near or at ABS3 by S1mapping but found no promoter activity. We suppose that thesetwo ArpA-binding sites, showing similarity to the ArpA-bindingconsensus sequence 5'-(A/C)C(A/G)(T/A)ACCG(A/G)CCGG(T/C)CGGT(A/T)(T/C)G(T/G)-3',have no significant biological functions. Repeated Southernhybridization with the 22-bp ArpA-binding consensus sequenceas a ³²P-labeled probe and the chromosomal DNA of S. griseusdigested with various restriction enzymes as targets also gaveABS3.

Mutant MK1, obtained by a single crossover, contained both theintact arpA gene and the mutated arpA (Trp119Ala) gene (Fig.3A). Because this mutant showed the same phenotypes as mutantMK2, Trp119Ala is a dominant-negative mutation.

A-factor production by arpA and adpA mutants.

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A-factor production by arpa...

Disruption of the arpA homologues barA in S. virginiae (16)and scbR in S. coelicolor A3(2) (26) resulted in complete lossof production of the respective ${gamma}$ -butyrolactones. In addition,a farA mutant of S. lavendulae produced a greatly reduced amountof the ${gamma}$ -butyrolactone (13). The S. griseus ${Delta}$ arpA mutant, however,produced the same amount of A-factor in the same amount of time,as determined by the streptomycin cosynthesis method (4) (Fig.4), which is a vivid contrast to the above ${gamma}$ -butyrolactone regulatorysystems. Mutant MK2 (Trp119Ala) unexpectedly produced A-factorin a larger amount than the wild type and ${Delta}$ arpA mutant (Fig.4). Furthermore, the ${Delta}$ adpA mutant produced a much larger amountof A-factor. Since AdpA is absent in the ${Delta}$ adpA mutant and theAdpA activity in mutant MK2 probably remains to a very smallextent, we assume that AdpA acts as a repressor of A-factorproduction in some unknown way. The ${Delta}$ arpA mutant, in which AdpAmust be present in a larger amount than in the wild-type strain,produced almost the same amount of A-factor as the wild type.We assume that the amount of AdpA in the wild-type strain issufficient for full repression of A-factor production.

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FIG. 4. A-factor production by the wild-type (wt) S. griseus strain and ${Delta}$ arpA, MK2, and ${Delta}$ adpA mutants. (A) Growth was monitored by measuring the wet weight of mycelium grown in YMPD liquid medium. (B) The A-factor concentration was measured by the streptomycin cosynthesis method, as described previously (4). The values are the averages of results for two independent samples.

Similarity and differences between the A-factor regulatory cascade and other ${gamma}$ -butyrolactone regulatory systems.

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Similarity and Differences...

${gamma}$ -Butyrolactone regulatory systems have been reported in severalStreptomyces spp. There are four such systems in which a receptorand its cognate ${gamma}$ -butyrolactone are known; these are ArpA andA-factor in S. griseus, BarA and virginiae butanolides in S.virginiae (20), FarA and IM-2 in S. lavendulae (30), and ScbRand SCB1 in S. coelicolor A3(2) (26). The phenotypes of MK1and MK2, in combination with adpA, enable us to compare thefunction of the A-factor receptor with that of other receptors.Of the four receptors, only arpA acts as a switch for aerialmycelium formation, and the others exert no detectable effectson morphogenesis. Concerning secondary metabolism, arpA actsas a repressor of the production of streptomycin, grixazone,and other secondary metabolites. Similarly, barA acts as a repressorof virginiamycin production in S. virginiae (16), and farA actsas a repressor of production of a blue pigment and a nucleosidicantibiotic in S. lavendulae (13). In S. coelicolor A3(2), however,an ScbR-SCB1 complex is required for actinorhodin production;SCB1 induces actinorhodin production, and an scbR disruptantdoes not produce actinorhodin (26, 27).

A vivid contrast between the A-factor regulatory system andthe other ${gamma}$ -butyrolactone regulatory systems is that arpA appearsnot to affect A-factor production, but barA, farA, and scbRare required for production of the respective ${gamma}$ -butyrolactones(13, 16, 26). In S. griseus, A-factor production is controlleddirectly or indirectly by adpA, which we suppose is the uniquetarget of ArpA. A-factor at a critical concentration dissociatesArpA from the adpA promoter and induces the production of AdpA,which in turn serves as a negative regulator of A-factor production,thus preventing overproduction of A-factor. We speculate that,in the case of BarA (11), FarA (12), and ScbR (26), these receptorscontrol the production of the respective ${gamma}$ -butyrolactones asa positive factor by a different mechanism, autorepression oftheir own promoters. In contrast, ArpA does not bind its ownpromoter (data not shown).

In S. griseus, adpA serves as a global transcriptional activatorgene for a number of genes required for both secondary metabolismand morphological differentiation. Because of this linear cascade,starting with A-factor to ArpA to AdpA and then to a numberof genes in S. griseus, both secondary metabolism and morphologicaldifferentiation are switched on by A-factor. The targets ofAdpA include the pathway-specific transcriptional activator(strR) in the streptomycin biosynthetic gene cluster (19). Inthe regulation of antibiotic production by other ${gamma}$ -butyrolactoneregulatory systems, a receptor (TylP) itself appears to bindto and control the promoter of the pathway-specific transcriptionalactivator (tylS) in the tylosin biosynthetic gene cluster inStreptomyces fradiae (24). Similarly, a ${gamma}$ -butyrolactone receptor(SpbR) also appears to control the pathway-specific regulator(papR1) in the pristinamycin biosynthetic gene cluster in Streptomycespristinaespiralis (3). Furthermore, in some gene clusters forantibiotic biosynthesis, ${gamma}$ -butyrolactone receptor homologueswere found; MmfR for methylenomycin biosynthesis in S. coelicolorA3(2) (accession no. AJ276673), JadR₂ for jadomycin B biosynthesisin Streptomyces venezuelae (34), and AAM78022.1 for neocarzinostatinbiosynthesis in Streptomyces carzinostaticus subsp. neocarzinostaticus(accession no. AY117439).

BarA is also probably encoded in the virginiamycin biosyntheticgene cluster. This means that BarA and these probable receptorsdirectly control the expression of the individual gene clusters.We assume that the presence of AdpA between ArpA and the pathway-specificregulators, including StrR in the linear A-factor regulatorycascade, and the diverse regulatory functions of AdpA make A-factorthe key master switch for production of most, if not all, secondarymetabolites and aerial mycelium formation in S. griseus. Inother Streptomyces spp., the strict signal relay from ArpA toAdpA may be lost; in S. coelicolor A3(2), for example, a probableadpA orthologue (bldH) that is involved both in morphologicaldifferentiation and in secondary metabolism is not under thecontrol of ${gamma}$ -butyrolactones (18, 28).

ACKNOWLEDGMENTS

J. Kato was supported by the Japan Society for the Promotionof Science. This work was supported by the Asahi Glass Foundationand by the Bio Design Program of the Ministry of Agriculture,Forestry, and Fisheries of Japan (BDP-03-VI-2-7).

FOOTNOTES

* Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81 3 5841 5123. Fax: 81 3 5841 8021. E-mail: asuhori{at}mail.ecc.u-tokyo.ac.jp.

REFERENCES

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