JOURNAL OF BACTERIOLOGY,0021-9193/97/$04.0010

Sept. 1997, p. 5854–5861 Vol. 179, No. 18

Copyright © 1997, American Society for Microbiology

The ppGpp Synthetase Gene (relA) of Streptomyces coelicolor A3(2)Plays a Conditional Role in Antibiotic Production

and Morphological DifferentiationREKHA CHAKRABURTTY AND MERVYN BIBB*

Department of Genetics, John Innes Centre, Norwich Research Park, Colney,Norwich NR4 7UH, United Kingdom

Received 24 March 1997/Accepted 2 July 1997

Deletion of most of the coding region of the ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2)resulted in loss of ppGpp synthesis, both upon entry into stationary phase under conditions of nitrogenlimitation and following amino acid starvation during exponential growth, but had no effect on growth rate. TherelA mutant, which showed continued rRNA synthesis upon amino acid depletion (the relaxed response), failedto produce the antibiotics undecylprodigiosin (Red) and actinorhodin (Act) under conditions of nitrogenlimitation. The latter appears to reflect diminished transcription of pathway-specific regulatory genes for Redand Act production, redD and actII-ORF4, respectively. In addition to the changes in secondary metabolism,the relA mutant showed a marked delay in the onset and extent of morphological differentiation, resulting ina conspicuously altered colony morphology.

Streptomycetes are gram-positive mycelial soil bacteria thatproduce a wide variety of secondary metabolites, many withimportant applications as antibiotics or other useful com-pounds in human medicine and in agriculture (6, 40). Second-ary metabolism is generally confined to stationary phase inliquid culture and usually coincides with the onset of morpho-logical differentiation in surface-grown cultures. This latterprocess involves the growth of aerial hyphae from the filamen-tous substrate mycelium and subsequent septation of the aerialhyphae to yield chains of unigenomic spores (14). While thephysiological signals and underlying regulatory mechanismsthat regulate antibiotic production remain to be fully eluci-dated (12), most of the published data are consistent with arole for a low growth rate, or the cessation of growth, indetermining the onset of secondary metabolism (13).

In Escherichia coli, the highly phosphorylated guanine nu-cleotides ppGpp and pppGpp have been implicated in growthrate control of gene expression (26, 28, 50, 51; see also refer-ences 1a, 15a, and 20a) and in the regulation of stationary-phase gene expression (22). In streptomycetes, several studies(reviewed in references 11 and 56) have noted a correlationbetween ppGpp synthesis and the onset of antibiotic produc-tion, leading to the suggestion that ppGpp plays a central rolein triggering antibiotic biosynthesis (47). In our own work, wehave examined the relationship between ppGpp synthesis andthe production of undecylprodigiosin (Red) and actinorhodin(Act) in Streptomyces coelicolor A3(2) (54, 56, 58); in general,we have noted a positive correlation between the appearanceof ppGpp and transcription of pathway-specific activator genesfor each antibiotic (redD for Red and actII-ORF4 for Act).Although such correlations are intriguing, they do not establisha signalling role for ppGpp in triggering antibiotic biosynthesis.Moreover, the reduction in antibiotic production frequentlyobserved in relC mutants of several Streptomyces species (42,43, 45–47) could, in principle, reflect impaired protein synthe-sis rather than reduced levels of ppGpp (relC mutants aredefective in ribosomal protein L11, which is required for acti-

vation of the ribosome-bound ppGpp synthetase, and grow atabout half the maximal rate of the wild-type strains).

In E. coli, (p)ppGpp is synthesized from ATP and GTPpredominantly by the ribosome-bound RelA, which is acti-vated when an uncharged tRNA binds to the acceptor site oftranslating ribosomes (9). To address the role of ppGpp indetermining the onset of antibiotic production in a more directmanner, we isolated the ppGpp synthetase gene (relA) ofS. coelicolor, the genetically best-characterized streptomycete(30), and used the cloned fragment to construct relA insertionand deletion mutants (11). Detailed analysis of the insertionmutant, which appeared to be unaffected in antibiotic produc-tion, indicated that it was still capable of ppGpp synthesis,potentially reflecting residual RelA activity (11). In contrast,the relA deletion mutant failed to make Act and Red, but ina medium-dependent manner. In similar studies, Martınez-Costa et al. (37) made a relA deletion mutant of a different S.coelicolor strain, J1501; while Act production was lost, therewas no apparent effect on Red synthesis. To address the basisof this conditional phenotype and assess the effect of a nullmutation in relA on the expression of the pathway-specificregulatory genes for Act and Red, we carried out a detailedphenotypic analysis of our relA deletion mutant and concludedthat relA is required for antibiotic production under conditionsof nitrogen limitation.

MATERIALS AND METHODS

Bacterial strains, culture conditions, and microbiological procedures. E. coliK-12 strains DH5a (25) and ET12567 (35) were used for routine subcloning andwere grown and transformed by standard procedures (49). The S. coelicolorA3(2) strains used were M600 (SCP12 SCP22; 6a); M145 (SCP12 SCP22; 29)and its Pgl2 derivative J1929 (4), and J1501 (hisA1 uraA1 strA1 Pgl2 SCP12

SCP22; 15). Transformation was done as described previously (29). SFM me-dium (20) was used to make spore suspensions. SMMS (a solidified version ofSMM; 20, 58), R5 (29), and R2 (29) were used to assess antibiotic production.RNA was isolated from liquid cultures grown in SMM (58). Calcium-dependentantibiotic (CDA) production was determined by the method of S. Podmore andC. P. Smith (48a).

Isolation and manipulation of DNA and RNA. Plasmid DNA was isolatedfrom E. coli derivatives, and plasmid and total DNAs were isolated from S.coelicolor strains as previously described (references 11 and 29, respectively).Southern analyses (53) were performed by using 32P-labelled probes made by

* Corresponding author. Phone: 44 1603 452571. Fax: 44 1603456844. E-mail: bibb@bbsrc.ac.uk.

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random oligonucleotide priming (49). RNA was isolated as described earlier(29).

Construction of relA deletion mutants. A 3.4-kb BamHI fragment containingthe first 500 nucleotides of the relA coding region and a 4.5-kb NotI fragmentcontaining the last 491 nucleotides of relA were cloned, in stepwise fashion andin the same relative orientation as in the chromosome, in the BamHI and NotIsites of the polylinker of pIJ2406, yielding pIJ6072. pIJ2406, a pBluescript SK1derivative that is unable to replicate autonomously in streptomycetes, containstsr, which confers resistance to thiostrepton (20). A 1.7-kb BglII fragment con-taining a hygromycin (Hyg) resistance gene (hyg; 63) was blunt ended by usingthe Klenow fragment of DNA polymerase I and cloned in pIJ6072, which hadbeen digested with XbaI and similarly blunt ended, yielding pIJ6073; the XbaIsite of pIJ6072 lies between the BamHI and NotI sites used to clone the frag-ments containing the 59 and 39 segments of relA, respectively. Samples (5 mg) ofpIJ6073 DNA in 9 ml of water were denatured by adding 2 ml of NaOH,incubated at 37°C for 10 min, and transferred to ice, and the solution wasneutralized by adding 2 ml of 1 M HCl. The resulting single-stranded DNA wasused to transform protoplasts of M600, M145, J1929, and J1501 (48). Transfor-mants were selected on R5 supplemented with 200 mg of Hyg ml21 and subse-quently replicated to R5 containing 5 mg of thiostrepton ml21. Putative recom-binants, in which a double-crossover recombination event might have replacedthe internal segment of relA with hyg, were identified as thiostrepton-sensitive,Hyg-resistant colonies and purified by single-colony isolation. Southern analysisof PstI- and PvuII-digested total DNA isolated from each strain, by using a 1.7-kbXbaI-PvuII fragment from pIJ6054 (10) that contained the 59 half of relA labelledwith 32P by random oligonucleotide priming as a probe, confirmed replacementof the chromosomal copy of relA with the mutant allele. A 2.8-kb PvuII fragmentand PstI fragments of 4 and 6 kb in the relA1 strains were replaced with 5.9-kbPvuII and 5-kb PstI fragments, respectively, in the mutants, in agreement with therestriction maps of the relA region and pIJ2406 (see Fig. 1).

Complementation of relA null mutants. A 3.7-kb XbaI-EcoRI fragment con-taining the entire relA coding sequence and promoter region was isolated frompIJ6055 (10) and cloned in pSET152 (7), which had been digested with XbaI andEcoRI, yielding pIJ6074. pIJ6074 was introduced by transformation into thenonmethylating E. coli ET12567 strain, which contained the RP4 derivativepUB307 (5). Conjugal transfer of DNA from E. coli to S. coelicolor was per-formed by using a modification (19a) of the method of Mazodier et al. (38) asdescribed in reference 32. Exconjugants were purified by single-colony isolation,and plasmid integration into the chromosome was confirmed by Southern anal-ysis of total DNA.

Amino acid depletion and nucleotide pool assays. Amino acid depletion andnucleotide pool assays were conducted essentially as described in reference 54.

S1 nuclease protection assays. A 570-bp AccI-BanI fragment from pIJ2820(54) that contains the 16S rRNA promoter region was used as a probe to detectrrnD transcripts; the fragment was uniquely labelled at the 59 end of the AccI sitelocated immediately upstream of the 16S rRNA coding region with [g-32P]ATPand T4 polynucleotide kinase. A 465-bp XhoI-AseI fragment from pIJ2334 (36)containing the actII-ORF4 promoter region was used as a probe to detectactII-ORF4 transcripts; the fragment was uniquely labelled with [g-32P]ATP atthe 59 end of the XhoI site that lies 356 bp downstream of the actII-ORF4translational start site. A 1.3-kb ClaI-NdeI fragment from pIJ6013 (58) thatcontains the redD promoter region was used as a probe to detect redD transcripts;

the fragment was uniquely labelled at the 59 end of the ClaI site that lies 29 bpdownstream of the redD translational start codon. A 498-bp SacI-SalI fragmentfrom pIJ6068 (11), uniquely labelled at the 59 end of the SalI site that lies 136nucleotides downstream of the relA translational start site, was used as a probeto detect relA transcripts. All of the S1 nuclease protection experiments werecarried out at least twice by using RNA isolated from independently growncultures, and the results presented were shown to be reproducible.

RESULTS

A relA deletion mutant of S. coelicolor M600 lacks detectableppGpp synthesis under conditions of nitrogen limitation.Since it appeared likely that the relA insertion mutant of pro-totrophic S. coelicolor J1929 reported previously (11) was not anull mutant, a relA deletion mutant was made by gene replace-ment. A mutant relA allele was constructed in which most ofthe relA coding region (corresponding to amino acid residues167 to 683 of 847) was replaced by a hygromycin resistancegene (hyg). This allele was used to replace the chromosomalcopy of relA in S. coelicolor A3(2) strain M600, a prototrophic,plasmid-free derivative of the wild-type strain, to give M570(Fig. 1).

To assess the effect of the relA mutation on ppGpp synthesisin S. coelicolor, M600 and M570 were grown in SMM liquidmedium until mid-exponential phase (optical density at 450 nm[OD450], 0.5) and subjected to amino acid depletion by rapidlytransferring the cultures to SMM lacking amino acids. Nucle-otides were extracted with 1 M formic acid 0, 7.5, and 15 minafter this nutritional shiftdown and analyzed by high-perfor-mance liquid chromatography (HPLC). The levels of ppGpp inM600 7.5 and 15 min after amino acid depletion (223 and 347pmol mg21 [dry weight], respectively) were comparable to ear-lier estimates for relA1 S. coelicolor M145 (200 pmol mg21

[dry weight]; 54) and J1929::KC301 (292 and 542 pmol mg21

[dry weight] in duplicate experiments) and fell in the rangeobserved for other Streptomyces species (56); in contrast, therewas no detectable ppGpp in M570. While the level of GTP inrelA1 M600 dropped markedly on shiftdown, as previouslyobserved in M145 (54) and J1929::KC301 (11), there was no

FIG. 1. Construction of a relA deletion mutant (M570) from S. coelicolorM600. Coding regions are indicated by broad arrows. The shaded segmentindicates the extent of the relA deletion and its replacement with a hygromycinresistance gene (hyg); apt encodes a homolog of adenosine phosphoribosyltrans-ferase (11). The probe used for Southern analysis to confirm the deletion eventand the fragment cloned in pIJ6074 for the complementation analysis are shownabove the map (the XbaI and EcoRI sites are derived from cloning vectors). Thelocations of two of the relA transcriptional start sites are shown by open circles,and the direction of transcription is shown by the associated arrow.

FIG. 2. HPLC analysis of ppGpp and nucleoside triphosphate levels afteramino acid depletion of M600 (relA1) and M570 (DrelA). The results shown areaverage values from two independent experiments. Nucleoside triphosphate lev-els were standardized to those observed immediately before amino acid deple-tion in each culture (100%); ppGpp levels were standardized to the levelsobserved 7.5 min after shiftdown in the M600 cultures. Average nucleotide con-centrations (in picomoles per milligram [dry weight]) for M600 immediatelybefore shiftdown were as follows: ATP, 6,635; CTP, 1,012; UTP, 1,225; GTP,1,400; ppGpp, 2.1. Those for M570 were as follows: ATP, 3,513; CTP, 823; UTP,1,349; GTP, 1,480; ppGpp, ,1.0.

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reduction in GTP in M570 (Fig. 2). Growth was essentiallyarrested following nutritional shiftdown; no significant in-crease in OD450 was observed for either M600 or M570.

Growth of S. coelicolor in SMM is limited by the availabilityof nitrogen (57). When relA1 strains of S. coelicolor are grownin this medium, ppGpp synthesis occurs during the transitionbetween exponential growth and stationary phase (11, 54). Inthe present experiments, a ppGpp level of 15 pmol mg21

(dry weight) was observed in M600 (consistent with the 16pmol mg21 [dry weight] observed in M145 [54] and the 14.3pmol mg21 [dry weight] observed in J1929 [11]) but no ppGppwas detected in M570 (Fig. 3).

The relA null mutant shows relaxed control of stable RNAsynthesis. The hallmark of the stringent response in E. coli isa drastic reduction in stable RNA (tRNA and rRNA) synthesisupon amino acid starvation (21). Mutants defective in thestringent response (relaxed mutants) continue to synthesizestable RNA under these conditions (9). The effect of aminoacid starvation on stable RNA synthesis in M570 was studiedby using rrnD, one of the six rRNA gene sets of S. coelicolor(2). rrnD is transcribed from four promoters (p1 to p4), all ofwhich are subject to negative stringent control (54).

RNA was isolated immediately before (0 h) and 1 h afterexponentially growing cultures of M600 and M570 (OD450;0.5) were subjected to amino acid depletion. S1 nuclease pro-tection assays of rrnD transcripts revealed six RNA-protectedfragments (Fig. 4), as described previously (3). Four of thesecorrespond to transcript initiation at the rrnD promoters p1(516 nucleotides), p2 (466 nucleotides), p3 (319 nucleotides),and p4 (239 nucleotides). Two additional bands were presentthat are not observed in in vitro transcription experiments (3);one represents the premature form of the 16S rRNA andcorresponds to a processing site (170 nucleotides), and theother is a protected fragment of unknown origin (S5, 201nucleotides) that probably represents partial hybridization ofthe probe to transcripts originating from one or more of theother rRNA gene sets. In exponentially growing cultures ofM600 and M570, p3 and p4 were the strongest promoters whiletranscription from p2 was virtually undetectable. Following nu-tritional shiftdown at an OD450 of 0.5, marked reductions inthe levels of the four primary transcripts were observed within1 h in M600 (Fig. 4), consistent with previous observations(54). However, amino acid depletion caused only a small re-

duction in rrnD transcript levels in M570, confirming the re-laxed phenotype with respect to rRNA synthesis.

The relA null mutant is conditionally defective in antibioticproduction and morphological differentiation. Unlike that ofthe relA disruptant J1929::KC936 (11), the growth rate ofM570 in SMM was not impaired, and in this respect, M570resembles E. coli relA null mutants (19). Both M600 and M570grew with a doubling time of about 2 h, the cultures enteringstationary phase 18 to 20 h after inoculation, at an OD450 ofapproximately 2.0 (Fig. 3). In M600, Red production occurredimmediately upon entry into stationary phase and Act synthe-sis was detected 7 to 10 h later. Neither pigmented antibioticwas detected in M570. Moreover, while Act (but not Red)production occurred in M600 after a mid-exponential-phase(OD450, 0.5) culture was subjected to amino acid starvation,consistent with earlier observations using S. coelicolor M145(54) and J1929::KC301 (11), neither pigmented antibiotic wasproduced by similarly treated cultures of M570.

When grown on SMMS, a solidified version of SMM, M570failed to produce either Red or Act (Fig. 5). No perceptibleeffect on antibiotic production was observed on R2 medium, inwhich growth is likely to be phosphate limited (see below). Onthe rich R5 medium (R2 supplemented with 0.5% [wt/vol]yeast extract), production of blue diffusible pigment (presum-ably the lactone form of Act, g-actinorhodin; 8) was abolished,production of the purple mycelial form of Act was markedlyreduced, and Red production was apparently unaffected. Todetermine whether relA is required for production of the (cal-cium-dependent antibiotic (CDA) made by S. coelicolor, M600and M570 were monitored for CDA synthesis on Oxoid Nu-

FIG. 3. Growth and production of antibiotics and ppGpp in S. coelicolorM600 (relA1) and M570 (DrelA) grown in SMM. The filled and open squaresshow the intracellular levels of ppGpp determined by HPLC in M600 and M570,respectively. tD, doubling time during exponential growth. The arrows indicatethe points of detection of Act and Red in the mycelium of M600.

FIG. 4. Transcription of rrnD after amino acid starvation of S. coelicolorM600 (relA1) and M570 (DrelA). RNA was isolated immediately before (0 h) and1 h after exponentially growing (SMM) cultures of M600 and M570 were sub-jected to amino acid depletion at an OD450 of 0.5 and used for S1 nucleaseprotection analysis with a uniquely end-labelled, rrnD-specific probe. P1, P2, P3,and P4 correspond to transcripts originating at the four rrnD promoters; P.S.represents a processing site, and S5 represents a protected fragment of unknownorigin. SM, 32P-end-labelled, HaeIII-digested fX174 size markers. Probe, loca-tion of the full-length probe. nt, nucleotides.

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trient Agar, SMMS, R2, and R5. The mutation had no discern-ible effect on CDA production.

To determine the basis of the conditional phenotype of theDrelA mutant, the level of phosphate in SMMS and SMM wasreduced from the usual 1.2 mM to 0.2 mM, resulting in phos-phate-limited, rather than nitrogen-limited, growth (the cul-ture entered stationary phase at an OD450 of 1.2, rather thanthe 1.8 to 2.0 observed in SMM). Production of Red and Actwas restored in M570 apparently to wild-type levels, beginningduring transition and stationary phase, respectively, in an iden-tical manner to M600. Comparison of the compositions of R2and SMM suggests that growth on R2 is likely to be phosphatelimited. Moreover, since R2 agar medium contains a high levelof CaCl2 (20 mM) a significant proportion of the 0.36 mMphosphate initially present is likely to be sequestered in theform of highly insoluble Ca3(PO4)2. Consistent with the notionthat relA is required for sensing of nitrogen starvation as a

signal for antibiotic production, increasing the amount of phos-phate in R2 from 0.36 to 1 mM severely reduced Act produc-tion in M570 but had no effect on M600. Correspondingly,increasing the CaCl2 concentration in SMMS from the usual 9mM to the 20 mM present in R2, potentially resulting in phos-phate limitation, restored Act and Red production in M570almost to wild-type levels.

The relA null mutant was also defective in morphologicaldifferentiation on SMMS but not on R2 or R5. Although M570produced a characteristic white aerial mycelium, the grey poly-ketide pigment associated with mature spores (16) was absent(Fig. 6). Phase-contrast microscopy revealed a marked reduc-tion in the number of spores formed. In addition, gross colonymorphology was altered and the diameter of individual colo-nies of M570 was significantly greater than that of the congenicrelA1 strain; vegetative growth of the M570 substrate myce-lium continued radially after that of the M600 colonies hadceased and aerial mycelium had begun to form, presumably inresponse to nitrogen limitation.

Complementation of the relA mutation. To confirm that themutant phenotypes were due to the absence of a functionalrelA rather than to a mutation elsewhere in the genome, awild-type copy of relA was reintroduced into M570 by using theconjugative vector pSET152 (7), which integrates into thechromosome of S. coelicolor by site-specific recombination atthe bacteriophage fC31 attachment site, attB (34). Conjugaltransfer of DNA from E. coli to Streptomyces requires oriT andthe tra gene functions of RP4 supplied in trans by the E. colidonor strain (38). A 3.7-kb XbaI-EcoRI fragment containingthe entire relA coding region and upstream promoter se-quences was cloned in pSET152. The resulting plasmid,pIJ6074, was transferred to S. coelicolor by conjugation via theE. coli donor ET12567 containing the RP4 derivative pUB307.Exconjugants were purified by single-colony isolation. Plasmidintegration into the chromosome was confirmed by Southernanalysis of total DNA. Surprisingly, as well as the expectedhybridizing fragments, additional bands were detected in threeof the four exconjugants analyzed; these appeared to haveresulted from integration of pIJ6074 by homologous recombi-nation on both sides of the mutant relA locus (deduced fromrestriction maps of the relA region of the chromosome and ofpIJ6074). Antibiotic production and normal morphological dif-ferentiation were restored on SMMS in all four of the excon-jugants. Moreover, ppGpp production following amino aciddepletion of an exponentially growing SMM culture of M574,which contained a single copy of pIJ6074 integrated at attB,was restored to wild-type levels (215 pmol mg21 [dry weight]).

relA is required for transcription of redD, the pathway-spe-cific regulatory gene for Red production, under conditions ofnitrogen starvation. Previous S1 nuclease protection experi-ments using RNA isolated from S. coelicolor M145 had shownthat the level of the redD transcript, which was barely detect-able during exponential growth, increased dramatically uponentry into stationary phase. Nutritional shiftdown at an OD450of 0.5 had no immediate stimulatory effect on redD transcrip-tion (58). Transcription of redD was assessed both during nor-mal growth and following amino acid depletion in M600 andM570. Transcription of redD was undetectable during expo-nential growth of M600 but increased upon entry into station-ary phase (Fig. 7A); Red production soon followed, consistentwith earlier data for M145 (58). Neither transcription of redDnor Red production was detected in M570 (Fig. 7A). Followingnutritional shiftdown at an OD450 of 0.5, neither transcriptionof redD nor Red production was detectable in M600 or M570(data not shown), consistent with previous observations onM145 (58). However, amino acid depletion of M600 at an

FIG. 5. Effect of deletion of relA on antibiotic production in S. coelicolor.Antibiotic production by M600 (relA1) (bottom culture) and M570 (DrelA) (topculture) on SMMS, R2, and R5 agar media after 5 days of incubation at 30°C.

FIG. 6. Effect of deletion of relA on colony development in S. coelicolor. Amixed plating of M600 (relA1; smaller grey colonies) and M570 (DrelA; larger,white, Hyg-resistant colonies) spores after 7 days of incubation on SMMS at 30°Cis shown.

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OD450 of 0.6 resulted in redD transcription within 1 h (Fig. 7B)and Red production in 4 h, again similar to earlier reports forM145 (55); neither occurred in an identically treated culture ofM570 (Fig. 7B).

Transcription of the pathway-specific regulatory gene foractinorhodin production, actII-ORF4, is markedly reduced inthe relA mutant. Earlier S1 nuclease protection experimentshad shown a marked increase in the level of the actII-ORF4transcript during the transition of strain M145 from exponen-tial growth to stationary phase (23). In M600, a low level of theactII-ORF4 transcript was detected during exponential growth,which increased sharply upon entry into stationary phase (Fig.7C); Act production followed 7 to 10 h later. In M570, tran-scription of actII-ORF4 was also observed upon entry intostationary phase, but at a lower level than in M600 (Fig. 7C),and Act was not produced, even on prolonged incubation.

Similar results were obtained when nutritional shiftdownwas applied to an exponentially growing culture (OD450, 0.5) ofM600. Transcription of actII-ORF4 was detected within 1 h ofamino acid starvation (Fig. 7D), consistent with previous ob-servations for M145 (55); Act production was observed 7 to10 h later. No stimulation of actII-ORF4 transcription wasobserved following identical treatment of M570 (Fig. 7D), andno Act was produced.

Transcription of relAp1 is not subject to positive stringentcontrol. S. coelicolor relA is transcribed from two promoters,relAp1 and relAp2, and also by transcription from a promoterlocated upstream of, or within, the adjacent apt gene (11).Transcription from relAp1 peaked during transition phase andfollowing nutritional shiftdown, suggesting that relAp1 is sub-ject to positive stringent control mediated by ppGpp (11). Toaddress this possibility, transcription of relA was assessed fol-lowing amino acid starvation of M570. The same pattern wasobserved for both M600 and M570: transcription from relAp1increased considerably after nutritional shiftdown, while therelAp2 transcript persisted at a relatively constant level (Fig. 8),as previously observed (11).

DISCUSSION

In contrast to its congenic parental strain (M600), the relAdeletion mutant M570 produced no detectable ppGpp on entryinto stationary phase upon nitrogen limitation, nor followingamino acid starvation during exponential growth. This con-trasts with the relA disruptant J1929::KC936 (11), in whichelevated basal levels of ppGpp were observed which did notincrease upon nitrogen limitation. This suggests that the pro-phage insertion in J1929::KC936 did not produce a null relAallele. Indeed, insertions in, and partial deletions of, relA inE. coli can result in the retention of ppGpp synthetase activity(51). The ability of E. coli RelA to bind to ribosomes has beenattributed to the carboxyl-terminal domain of the protein (39),and a C-terminally truncated RelA containing the N-terminal455 (of a total of 743) amino acids, retained ribosome-inde-pendent ppGpp synthetase activity. However, a truncated de-rivative that contained the 331 N-terminal amino acids was

FIG. 7. Effect of deletion of relA on transcription of redD and actII-ORF4.(A and C) Transcription of redD (A) and actII-ORF4 (C) during growth ofS. coelicolor M600 (relA1) and M570 (DrelA) in SMM. RNA was isolated duringthe exponential (EX), transition (TR), and stationary (ST) phases of growth and

subjected to S1 nuclease protection analysis using uniquely end-labelled redD-and actII-ORF4 specific probes. RNA-protected fragments of 225 and 384 nu-cleotides (nt) were observed for redD and actII-ORF4, respectively, consistentwith previously published results (17, 54, 58). SM, 32P end-labelled, HaeIII-digested fX174 size markers. (B and D) Transcription of redD (B) and actII-ORF4 (D) after amino acid depletion at OD450s of 0.6 and 0.5, respectively, ofM600 (relA1) and M570 (DrelA). RNA was isolated at the times indicated afterthe strains were subjected to amino acid depletion and was then used for S1nuclease protection analysis. See above for further details.

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inactive (51). It is therefore likely that the N-terminal segmentof S. coelicolor RelA that remains after disruption in J1929::KC936 (and which corresponds to the first 380 amino acids ofE. coli RelA) possesses ribosome-independent ppGpp synthe-tase activity.

Previous studies of ppGpp and antibiotic production inS. coelicolor used M145 as the model strain (54, 58), and in ourearlier analysis of relA (11), we disrupted the gene in J1929, aphage-sensitive (Pgl2) derivative of M145 (4). In an indepen-dent study (37), a relA mutant was created in the auxotrophic,plasmid-free strain J1501, whose genome is known to haveundergone several deletions (33). Thus, for comparison, themutant allele constructed in this study was also used to replacethe chromosomal copy of relA in S. coelicolor M145, J1929, andJ1501 (data not shown). The conditional defects in Act andRed production observed in the M600 derivative M570 werealso observed in the relA mutants of these latter strains. Suchmedium-dependent phenotypes are frequently associated withpleiotropic regulatory mutants of S. coelicolor that are defec-tive in antibiotic production (13). While this might indicate theabsence of null mutant alleles, it is more likely to reflect theexistence of a complex network of regulatory signals and mech-anisms that can act independently to activate expression ofantibiotic biosynthetic pathways. The different compositions ofSMMS and R2 and attempts to identify critical factors bysupplementation and subtraction of medium components in-dicate an essential role for ppGpp in the activation of antibi-otic production in response to nitrogen limitation. Presumably,some other relA-independent mechanism is active under theconditions of phosphate limitation predicted for R2. It is un-clear whether this mechanism involves ppGpp. If there is relA-independent ppGpp synthesis, it might arise from the novelppGpp synthetase/polynucleotide phosphorylase activity re-cently identified in S. coelicolor (31) or from the syntheticactivity of a thus far unidentified homolog of SpoT, which inE. coli encodes a 39-pyrophosphohydrolase activity usually in-volved in ppGpp degradation but which appears to possess

synthetic activity under conditions of carbon starvation (27,62).

The principal limitation to Act and Red production appearsto be the concentration of the pathway-specific activator pro-teins ActII-ORF4 and RedD. Threshold concentrations ofActII-ORF4 and RedD are required for transcription of theircognate biosynthetic structural genes and the subsequent pro-duction of the antibiotics, and premature expression of eitherregulatory gene leads to precocious overproduction of the cor-responding antibiotic (23, 58). Deletion of relA, to give M570,abolished Act production in liquid culture under conditions ofnitrogen limitation but only reduced the level of actII-ORF4transcription upon entry into stationary phase. In comparablestudies, Martınez-Costa et al. (37) made a relA deletion mutantof S. coelicolor J1501. While the residual level of actII-ORF4transcription in their mutant was markedly lower than thatobserved in M570, deletion of relA in J1501 abolished tran-scription of the biosynthetic structural genes actI-ORF1 andactVI-ORF1. Consequently, we believe that the level of ActII-ORF4 in M570 is insufficient to activate Act production. Al-ternatively, the relA mutation may have affected the expressionof bldA, which encodes the only tRNA of S. coelicolor that canefficiently translate the rare leucine codon, UUA, one of whichis present in the actII-ORF4 transcript (18). However, sinceearlier studies failed to reveal any limitation on the translationof UUA codons during exponential growth in SMM (23), whenppGpp is undetectable, it seems unlikely that relA is requiredfor bldA activity. Transcription of actII-ORF4 and of redD wasstimulated following nutritional shiftdown of M600 at OD450sof 0.5 and 0.6, respectively. Although this may have been anindirect effect of nutritional shiftdown, rather than of specificinduction by ppGpp, no such stimulatory effect was observed inthe relA null mutant, consistent with a direct role for ppGpp inantibiotic production.

Transcription of redD in transition phase cultures of M600correlates with the appearance of ppGpp, and both are absentfrom comparable cultures of M570. However, while amino aciddepletion of M600 at an OD450 of 0.6 resulted in relA-depen-dent activation of redD transcription, no such effect was ob-served at an OD450 of 0.5; similar observations have been madefor M145 (55), indicating that the apparent ability of ppGpp toelicit redD expression is influenced by other physiological fac-tors. Recent results (59) have demonstrated a role for redZ(24) in the activation of the Red biosynthetic pathway, withRedZ apparently functioning as a transcriptional activator ofredD; no homolog of redZ occurs in the act biosynthetic genecluster. While ppGpp might have been required for expressionof redZ, S1 nuclease protection experiments failed to revealany deleterious effect of the relA deletion on redZ transcription(60). The reason for the growth stage-dependent effect of nu-tritional shiftdown on redD transcription remains to be deter-mined, but these results indicate that there are physiologicalfactors other than nitrogen limitation that influence the ex-pression of redD.

The M600 relA null mutant is altered in colony developmentunder conditions of nitrogen limitation, showing delayed aerialmycelium formation, suggesting a role for relA in morpholog-ical differentiation. The isolation and characterization of re-laxed mutants of several Streptomyces spp. led Ochi (42–45) topropose that morphological differentiation results from a de-crease in the GTP pool, whereas secondary metabolism is dueto elevated levels of ppGpp. Addition of the GTP synthetaseinhibitor decoynine to an exponentially growing culture ofS. coelicolor M145 resulted in reductions in growth rate andintracellular GTP levels very similar to those observed afteramino acid depletion, but there was no associated ppGpp, Act,

FIG. 8. Transcription from relAp1 and relAp2 after amino acid starvation ofS. coelicolor M600 (relA1) and M570 (DrelA). RNA was isolated at the timesindicated after the strains were subjected to amino acid depletion and was thenused for S1 nuclease protection analysis. FLP, location of the fully protectedsegment of the probe that is homologous to relA.

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or Red synthesis (61). These results are consistent with animportant role for elevated levels of ppGpp (rather than areduction in GTP levels) in triggering the onset of antibioticproduction. In S. griseus, the reduction in GTP levels duringthe stringent response is due partly to inhibition of IMP dehy-drogenase (an enzyme required for GMP, and hence GTP,synthesis) by ppGpp (42, 44), suggesting an indirect role forppGpp in morphological differentiation. This would be consis-tent with the delayed appearance of aerial mycelium in relC(42–44) and relA (this study) mutants. In these relaxed strains,the eventual decline in GTP levels presumably reflects thecurtailment of purine nucleotide synthesis upon glycine deple-tion (41), which is likely to occur more slowly than the inhibi-tion of IMP dehydrogenase by ppGpp. However, increasingthe intracellular level of ppGpp in Myxococcus xanthus by in-ducing the expression of an introduced copy of E. coli relA wassufficient to activate the developmental program in the absenceof a decline in the GTP level (52). Furthermore, lowering theGTP pool by adding specific inhibitors of GTP synthesis didnot induce development.

The diameters of M570 colonies on SMMS were significantlylarger than those of M600, whereas colonies of the relA dis-ruptant J1929::KC936, which showed elevated levels of ppGppduring exponential growth, were much smaller than those ofthe congenic relA1 strain (11). Vegetative growth of the sub-strate mycelium of M570 on SMMS appears to continue longerthan in M600 and perhaps reflects the inability of the relA nullmutants to sense nitrogen limitation. Such continued growthmay result in the depletion of resources normally used forefficient erection of aerial hyphae and sporulation. The phe-notype with respect to morphological differentiation was es-sentially the same when relA was deleted in M145 and J1929.However, the J1501 relA deletion mutant showed delayed aer-ial mycelium formation on all of the media tested, although nosignificant difference in colony size or gross colony morphologywas apparent, consistent with previous results (37). The reasonfor the difference in phenotype observed with the J1501 relAmutant and the other strains is not clear; perhaps ppGppmetabolism in the auxotrophic J1501 differs from that in thewild-type strain. Other differences between J1501 and the pro-totrophic plasmid-free strains exist; J1501 produces consider-ably less Act and Red than does either M600 or M145 on mostmedia and differs from M145 in the regulation of C sourceutilization (1).

The levels of the relAp1 transcript following nutritional shift-down were the same in the mutant and the relA1 strain; hence,transcription from relAp1 is not directly related to the levelof ppGpp, as suggested by earlier results (11). The apparentpositive stringent control of transcription from relAp1 and itsgrowth phase dependence remain to be explained.

ACKNOWLEDGMENTS

We thank Mark Buttner, Keith Chater, David Hopwood, MarkPaget, and Eriko Takano for comments on the manuscript and ColinSmith for the gift of ET12567 containing pUB307.

This work was supported by a grant to the John Innes Centrefrom the Biotechnology and Biological Sciences Research Council(BBSRC) and by a BBSRC studentship awarded to R.C.

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