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1 Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain
2 Instituto de Biotecnología (INBIOTEC), Parque Científico de León, Av. Real 1, 24006 León, Spain
3 Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Correspondence
P. Liras
paloma.liras{at}unileon.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the sequence of the 5.4 kb DNA fragment cloned in this work is AM408890.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and pppGpp (Haseltine et al., 1972). In Escherichia coli and other enterobacteria the intracellular level of (p)ppGpp is controlled by RelA, a (p)ppGpp synthetase encoded by relA, and by the (p)ppGpp 3'-pyrophosphohydrolase activity encoded by spoT (Sy, 1977). RelA is ribosome-associated and is activated, presumably by conformational change, when uncharged tRNAs bind to the A site of the ribosome (Cashel et al., 1996). Subsequent (p)ppGpp synthesis reduces the level of transcription of many genes that are required for rapid growth, while enhancing that of many others associated with stationary phase and other physiological stresses. Indeed, in E. coli ppGpp is now firmly considered to be a global regulator of gene expression rather than simply a modulator of ribosome biosynthesis (Braeken et al., 2006). Its mode of action has been studied extensively in E. coli, and involves reorienting gene transcription via binding to RNA polymerase (Magnusson et al., 2005).
In actinomycetes, the accumulation of (p)ppGpp after amino acid starvation was first demonstrated in Streptomyces hygroscopicus (Riesenberg et al., 1984). The isolation of thiopeptin-resistant mutants of several Streptomyces species, many of which were shown to be deficient in (p)ppGpp synthesis, subsequently revealed a general and positive correlation between (p)ppGpp synthesis, antibiotic production and morphological differentiation (Ochi, 1986, 1990). While in enterobacteria, relA and spoT encode two related proteins with different functions, actinomycetes and other Gram-positive bacteria possess a single bifunctional RelA/SpoT protein (Martínez-Costa et al., 1996; Wendrich & Marahiel, 1997). In Streptomyces coelicolor A3(2), the relA/spoT gene (hereafter named relA; Chakraburtty et al., 1996) encodes a 94 200 Da protein which conferred (p)ppGpp hydrolysis activity on an E. coli relA spoT double mutant (Martínez-Costa et al., 1998), thus behaving in a similar way to the bifunctional RelA/SpoT homologue of Streptococcus equisimilis (Mechold et al., 1996). relA-null mutants of S. coelicolor are impaired in the stationary-phase production of two antibiotics, actinorhodin (Martínez-Costa et al., 1996) and undecylprodigiosin, under conditions of nitrogen limitation (Chakraburtty & Bibb, 1997).
Streptomyces clavuligerus is used for the production of the β-lactamase inhibitor clavulanic acid and consequently is of considerable industrial interest (Liras & Rodríguez-García, 2000); it also produces the β-lactam antibiotic cephamycin C (Liras, 1999). Prior to this study, and in contrast to other streptomycetes, the role of ppGpp and related highly phosphorylated guanosine nucleotides in the control of secondary metabolism in S. clavuligerus was unclear. Bascarán et al. (1991) showed that a stringent response followed amino acid starvation in S. clavuligerus and resulted in increased ppGpp levels. While some mutants impaired in ppGpp synthesis produced higher levels of cephamycin C than the wild-type strain, suggesting that ppGpp is not essential for antibiotic biosynthesis in S. clavuligerus, other mutants produced reduced cephamycin C levels. The effect of these mutations on clavulanic acid biosynthesis was not studied. More recently we studied the effect of a well-characterized rplK (relC) mutation on clavulanic acid and cephamycin C production, finding that the mutation resulted in a reduction in (p)ppGpp synthesis and lower antibiotic production than in the parental strain (Gomez-Escribano et al., 2006). While Jones et al. (1996, 1997) reported a burst of ppGpp synthesis prior to clavulanic acid production, they concluded that ppGpp was not required for transcription of the clavaminate synthase (cas) gene involved in clavulanic acid biosynthesis. It was thus necessary to clarify the role of ppGpp in clavulanic acid biosynthesis using relA-null mutants unable to synthesize ppGpp. We therefore constructed two different relA-null mutants and show here that both surprisingly overproduce clavulanic acid and cephamycin C. This is in contrast to the findings of Jin et al. (2004), who reported that the production of both compounds required a functional relA gene.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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were used for cloning. E. coli Ess22-35 and Klebsiella pneumoniae ATCC 29665 were used to assay for cephamycin C and clavulanic acid production, respectively (Liras & Martín, 2005). S. clavuligerus ATCC 27064 was used as the parental strain. S. clavuligerus relC (Gomez-Escribano et al., 2006) was used for comparison purposes in S1 nuclease protection experiments. TSB complex medium or SA defined medium were used for studies of antibiotic production (Paradkar & Jensen, 1998; Lorenzana et al., 2004). All S. clavuligerus cultures were carried out in triplicate in baffled flasks. Amino acid shift-down was carried out using defined MF medium (Bascarán et al., 1991). Growth of the cultures was determined by dry cell weight or by total DNA content, measured by the diphenylamine method (Burton, 1968). ME agar medium (Sánchez & Braña, 1996), as well as mannitol soya flour (SFM) medium (Kieser et al. 2000), were used to assess morphological differentiation and sporulation.
DNA manipulations.
Nucleic acid purification, DNA manipulation, E. coli and Streptomyces transformation and E. coli–S. clavuligerus conjugation were performed following standard methods (Sambrook et al., 1989; Kieser et al., 2000). Nucleic acid hybridizations were performed using the protocol given in the DIG-System kit (Roche) and colorimetric detection was achieved using nitro blue tetrazolium (NBT) and 5-bromo-1-chloro-3-indolyl phosphate (BCIP). PCR was performed using a Biometra TGradient Thermocycler and the conditions of Kieser et al. (2000). dTNP mixtures were prepared from individual nucleotides (Promega) using a ratio of 15A : 15T : 35G : 35C to improve the amplification efficiency with high-G+C Streptomyces DNA. The oligonucleotides in Table 1 were used for subcloning, detection of the relA-null mutants and to obtain probes for S1 nuclease mapping. DNA sequencing was carried out using double-stranded DNA and the PCR method of Mullis & Faloona (1987). Nucleotide sequences were obtained on an ABI Prism Sequencer 310 (Perkin Elmer), and analysed using the following computer programs: Geneplot from DNASTAR, FASTA3 (EBI), CLUSTAL_X for multiple alignments (Higgins & Sharp, 1989) and the databases Swall SWISS-PROTein, EMall (EMBL) and GenBank (USA). The DNA sequence of the 5.4 kb DNA fragment cloned in this work can be found in the EMBL database under accession number AM408890.
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) and inserted into EcoRI/HindIII-digested pULGE211 to give pULGE212. Finally the neo gene, encoding kanamycin resistance, was isolated with KpnI from plasmid pTC192K (Rodríguez-García et al., 2006) and inserted into the single KpnI site internal to relA1.9 in pULGE212 to yield pULGE220. This plasmid was introduced into S. clavuligerus by conjugation and exconjugants were selected for kanamycin resistance and apramycin sensitivity. One, S. clavuligerus relA : : neo, gave the pattern expected for a relA-disrupted mutant when hybridized with neo or relA1.9 probes (Fig. 1, lanes 3 and 4).
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) was replaced by a neomycin/kanamycin-resistance cassette. The resulting plasmid, with neo in the same orientation as relA, was called pULGE240. The conjugation cassette with the apramycin-resistance marker from pIJ733 was inserted in the NotI site of pULGE240 to yield pULGE244. This plasmid was introduced into S. clavuligerus ATCC 27064 by conjugation and kanamycin-resistant exconjugants were screened for apramycin sensitivity, characteristic of double-crossover recombinants. DNA from kanamycin-resistant, apramycin-sensitive colonies was hybridized with relA1.9 and neo probes. Lack of hybridization with the relA probe and hybridization with the neo probe (Fig. 1
, lane 5), as well as the size of the hybridizing fragments, confirmed the construction of a relA-deleted mutant.
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), which integrates site-specifically into the phage 
C31 attachment site of S. clavuligerus, was digested with BamHI and the 4.6 kb DNA fragment isolated. The amplified fragment carrying relA and its own promoter was ligated to the 4.6 kb BamHI DNA fragment to yield pULGE331, in which relA is in the opposite orientation to aac(3)IV. This plasmid was used to complement the relA-null mutants. Religated BamHI-digested pMS17 (pMS17B) was used as vector control. The incomplete relA gene relARI was amplified from genomic DNA using oligonucleotides relA-O5 and relA-O6, confirmed by sequencing and inserted into XbaI/EcoRV-digested pMS17 to give pULGE261, in which relARI is expressed from the Streptomyces promoter tcp830 (Rodríguez-García et al., 2005).
RNA extraction and purification.
Streptomyces RNA extraction and purification were performed using the RNeasy kit (Qiagen) following the protocol at http://www.surrey.ac.uk/SBMS/Fgenomics. Phenol was obtained from BDH or Appligen-Oncor, and lysozyme from Sigma.
S1 endonuclease mapping.
High-resolution S1 nuclease mapping was performed with sodium trichloroacetate buffer as described by Kieser et al. (2000) but using 1x S1 digestion buffer in step 1 of the protocol. The oligonucleotides used to amplify the probes for high-resolution mapping were labelled with [
-32P]ATP and polynucleotide kinase. Thirty micrograms of RNA was used in each reaction. A bldG probe was included as an internal control in every reaction; that for claR (shown in Fig. 7c) was repeated without the bldG probe to allow visualization of the claR-tsp2 protected fragment, which differs from that of the bldG-tsp1 protected segment by only one nucleotide (Bignell et al., 2005). Reactions for each strain were performed concomitantly, loaded on the same gel and exposed on the same film, allowing quantitative comparison of different time points and different strains for each probe.
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) using 1 litre flasks with stainless steel springs for dispersed growth and containing 200 ml MF medium supplemented with 1 % (w/v) Casamino acids (MFA). Flasks were inoculated with mycelium from a 24 h TSB culture to a final OD600 of 0.25. During rapid growth (15–17 h after inoculation), 50 ml of each culture was quickly filtered (15 s maximum) through Whatman no. 1 filters (8.5 cm diameter) using a vacuum pump, washed with fresh MF medium and the mycelium resuspended in the same volume of MF or MFA (as control) medium.
Quantification of nucleotides.
Nucleotides were extracted as described by Ochi (1986). Samples of the cultures taken from 0 to 60 min after shift-down were quickly filtered as above and the filters submerged upside-down in ice-cooled 1 M formic acid in a Petri dish. The extraction was kept at 4 °C for 60 min; the formic acid extract was then isolated by centrifugation, filtered through 0.45 µm pore cellulose acetate filters, frozen in liquid nitrogen and freeze-dried. The dry samples were kept at –80 °C until analysed. Separation and quantification of the nucleotides were achieved using a 4.6x250 mm 10 µm particle size Partisil SAX column on an Agilent 1100 HPLC system fitted with a photodiode array detector. The mobile phases (A) KH2PO4 7 mM adjusted to pH 4.0 with phosphoric acid, and (B) KH2PO4 0.5 M containing Na2SO4 0.5 M adjusted to pH 5.4 with NaOH, were used with the following gradient: time 0 min, 0 % B; 3 min, 20 % B; 15 min, 70 % B; 25 min, 75 % B; 35–45 min, 100 % B. Under these conditions retention times were: ATP, 13.6 min; GTP, 15.4 min; ppGpp, 25.2 min; pppGpp, 37.5 min. Nucleotide standards were obtained from Sigma. ppGpp was kindly supplied by K. Ochi, National Food Research Institute, Tsukuba, Ibaraki, Japan. ppGpp and pppGpp were quantified using the absorption coefficient for GTP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and indicates that it encodes amino acids 140–771 of RelA. This fragment was then used to disrupt relA in S. clavuligerus (Fig. 1a, lanes 3 and 4). Since there are no NotI sites in either the relA1.9 fragment or the inserted neo gene, the regions flanking relA were cloned by marker rescue using NotI-digested total DNA from S. clavuligerus relA : : neo. A kanamycin-resistant E. coli transformant was found to carry a plasmid (pULGE221) with an insert of about 26 kb in which the neo gene was located between an 8 kb DNA fragment carrying the 5'-end of relA and its upstream region and a 16.6 kb DNA fragment carrying the 3'-end of relA and its downstream region. The complete sequence of relA was obtained by sequencing fragments of pULGE221. While the sequence of the relA protein-coding region was identical to that published by Jin et al. (2004), there were marked differences in the upstream region (see below). Upstream of relA, and separated by 176 nt, was a gene (apt) encoding a protein 84.2 % identical to the adenine phosphoribosyltransferase of S. coelicolor (SCO1514), an enzyme involved in purine nucleotide biosynthesis (Fig. 2). Downstream of relA, in the opposite orientation and separated by 105 nt, was an ORF encoding a putative peroxidase with 52.2 % identity to Bpro DRAFT_3308 from Polaromonas sp. The ends of several fragments obtained by BamHI and NcoI digestion were also sequenced (Fig. 2). Analysis of the sequences revealed an ORF (alaS) encoding an alanyl-tRNA synthetase homologous to SCO1501, an ORF (ppiB) encoding a putative peptidyl-prolyl cis/trans isomerase homologous to SCO1510, and an ORF (hisS) encoding a histidyl-tRNA synthetase homologous to SCO1508. The arrangement of the S. clavuligerus genes is similar to that found in S. coelicolor (Fig. 2) and in S. avermitilis (data not shown).
Interestingly, the sequence of the intergenic region of S. clavuligerus between relA and apt is markedly different from that published by Jin et al. (2004). We found an intergenic region of 176 nt between apt and relA, whereas only 29 nt were reported by Jin et al. (2004). A tandem duplication containing the probable relA ribosome-binding site in the sequence of Jin et al. (2004) was not present in our intergenic region. The nucleotide sequence found in our work is identical to that obtained independently by DSM, Delft, The Netherlands (M. van den Berg, personal communication) for the same wild-type strain. Thus the strain used by Jin et al. (2004) appears to have undergone deletion and rearrangement in the relA promoter region.
Construction of a relA-deleted mutant of S. clavuligerus
The S. clavuligerus relA : : neo insertion mutant described above contains the whole relA gene in two fragments separated by neo. Since internal fragments of relA might still encode a functional ppGpp synthetase (Martínez-Costa et al., 1998), we proceeded to construct a relA-deletion mutant of S. clavuligerus. The 
relA mutant was confirmed by hybridization with the relA1.9 and neo probes (Fig. 1a, lane 5) and was named S. clavuligerus relA : : neo. This mutant carries only 104 nt of the 5'-end of relA.
Characteristics of the S. clavuligerus relA-null mutants and of complemented transformants
Morphological differentiation.
The relA-null mutants did not sporulate on ME agar (Fig. 3), the medium commonly used for S. clavuligerus sporulation, nor on SFM medium. In addition, the mutants failed to produce aerial mycelium and the brown pigment characteristic of S. clavuligerus when grown on ME agar (Fig. 3b, cultures 3 and 5) or on SFM medium (data not shown)
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relA : : neo and S. clavuligerus relA : : neo. Exconjugants carrying the vector pMS17B were used as controls. Morphological differentiation was restored in S. clavuligerus relA : : neo(pULGE331) and S. clavuligerus relA : : neo(pULGE331), but not in derivatives containing pMS17B (data not shown). The integrative plasmid pULGE261, which carries an incomplete relARI gene encoding a truncated RelA protein (amino acids 228–495) that is predicted to be able to form ppGpp in a ribosome-independent manner (Martínez-Costa et al., 1998), was also introduced into the relA-null mutants by conjugation. Both S. clavuligerus relA : : neo(pULGE261) and S. clavuligerus relA : : neo(pULGE261) expressing the relARI DNA fragment in trans recovered the ability to form aerial mycelium, spores and pigment (Fig. 3, cultures 4 and 6). Integration of the vector (pMS17) alone lacking the truncated relA failed to complement the two mutations.
Physiological differentiation.
The ability of S. clavuligerus relA : : neo and S. clavuligerus relA : : neo to produce cephamycin C and clavulanic acid was assessed in TSB and SA liquid media. Both mutants gave lower biomass yields (expressed as mg per mg DNA) in TSB at 36 h, and in SA at 72 h. The sequential pattern of production of cephamycin C and clavulanic acid in both relA mutants was similar to that of the wild-type strain. However, the yield of clavulanic acid and cephamycin C, expressed as µg per mg DNA, was consistently much higher in the relA-null mutants (Fig. 4). The yield of clavulanic acid from S. clavuligerus relA : : neo grown in TSB medium was 3- to 4-fold higher than that from the wild-type strain, and cephamycin C production was 2.6-fold higher. In SA medium, the increases were even higher: 4-fold for clavulanic acid and 6-fold for cephamycin C at 72 h. The S. clavuligerus relA : : neo mutant showed a similar pattern of antibiotic production and higher yields of clavulanic acid and cephamycin C than the parental strain (Fig. 4).
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Nucleotide levels in S. clavuligerus ATCC 27064 and in the relA : : neo mutant
S. clavuligerus wild-type and relA : : neo were grown in SA medium and their ATP, GTP and polyphosphorylated nucleotide levels determined (Ochi, 1986). Samples were taken from the same cultures to measure clavulanic acid and cephamycin C production, and for S1 nuclease analysis of the expression of antibiotic biosynthesis genes (see later). Both cultures exhibited a similar increase in ATP levels (about 0.2 nmol per mg cell dry weight) as growth proceeded, with that in the mutant showing a delayed and more gradual rise (Fig. 5). A marked decrease in GTP level (about threefold) occurred in the wild-type strain during growth, but then levelled out upon entry into stationary phase; in contrast, the GTP content of the S. clavuligerus relA : : neo mutant remained fairly steady throughout the fermentation. As previously described (Gomez Escribano et al., 2006), ppGpp and (p)ppGpp peaked at 48 of growth in the wild-type. As expected, no polyphosphorylated guanine nucleotides were detected in the relA-deletion mutant.
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), and of ceaS2, encoding the carboxyethylarginine synthase for clavulanic acid biosynthesis, was assessed by high-resolution S1 nuclease protection analysis. In parallel, transcription of the regulatory genes ccaR (Pérez-Llarena et al., 1997) and bldG (Bignell et al., 2005) (both involved in the regulation of cephamycin C and clavulanic acid production), and of claR (Pérez-Redondo et al., 1998, Paradkar & Jensen, 1998) (involved in regulating clavulanic acid biosynthesis), was determined, as was that of relA.
While the transcription profiles of relA, bldG, claR and ccaR were broadly similar in both strains (although transcription of ccaR appeared somewhat higher and persisted for longer in the relA-deletion mutant), transcription of cefD and particularly of ceaS2 (encoding the enzyme catalysing the first step in clavulanic acid biosynthesis) was much higher in the relA-deletion mutant. This was especially noticeable after 48 h, when expression dropped markedly in the wild-type strain. This difference in expression of cefD and ceaS2 correlates well with the overproduction of both cephamycin C and clavulanic acid in the relA-deletion mutant (Fig. 6a, b).
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relA : : neo, the pattern of transcription of this gene could also be studied by S1 nuclease protection analysis. Transcription initiation of relA occurred at a thymine located 43 nt upstream of the triplet TTG proposed by Jin et al. (2004) as start codon (Fig. 7a). This thymine lies in the segment of the intergenic region that appears to be deleted in the strain used by Jin et al. (2004).
Nutritional shift-down switches expression of claR from tsp1 to tsp2
The behaviour of S. clavuligerus ATCC 27064 and of S. clavuligerus relA : : neo after amino acid shift-down was determined by transferring cells from MFA medium to MF medium, lacking amino acids. Nucleotide contents after shift-down and transcription of the same set of antibiotic biosynthesis genes were analysed. As expected, (p)ppGpp production was higher in the wild-type strain 15 min after amino acid shift-down, concomitant with a reduction in the GTP level and an increase in ATP content. ppGpp and pppGpp were never detected in S. clavuligerus relA : : neo, which showed a strong rise in both ATP and GTP levels after shift-down (Fig. 8).
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), corresponding to a cytosine located 155 nt upstream of the ATG start codon (Paradkar & Jensen, 1998), changed in the relA mutant 30 min after shift-down to be transcribed predominantly from tsp2, an adenine located 107 nt upstream of the ATG start codon, a tsp not previously described (Figs 7c, d and 8). This adenine is located 5 nt downstream of a putative TACAGT Pribnow box. tsp2 was used poorly by the wild-type and the S. clavuligerus relCPALG mutant (data not shown), in both SA and MF cultures, but gave a strong signal in S. clavuligerus relA : : neo 30–60 min after shift-down. Thus this switch of tsp was observed only in the relA mutant, suggesting that it is dependent on the absence of RelA. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). S. clavuligerus relA-null mutants are unable to form aerial mycelium and to sporulate (Jin et al., 2004). In batch cultures in liquid SA medium, S. clavuligerus relA : : neo maintains a steady intracellular GTP content, which is twice that of the wild-type strain upon entry into stationary phase (Fig. 5); after amino acid shift-down, the GTP content in the relA-null mutant increases threefold, in contrast to that in the wild-type, which remains relatively stable (Fig. 8). Thus our data are in agreement with the hypothesis of Ochi (1986) linking a decrease in GTP content with the initiation of sporulation in Streptomyces.
Ochi (1986) also proposed that (p)ppGpp synthesis is required for antibiotic production in streptomycetes. In support of this, a relA-null mutant of S. antibioticus was unable to produce actinomycin (Hoyt & Jones, 1999) and a S. coelicolor M600-derived relA-null mutant was impaired in antibiotic production under conditions of nitrogen, but not phosphate, limitation (Chakraburtty & Bibb, 1997). However, a S. coelicolor J1501-derived relA-null mutant was impaired in actinorhodin but not in undecylprodigiosin or calcium-dependent antibiotic production (Martínez-Costa et al., 1996), suggesting that the requirement for RelA is dependent upon background genotype and the growth medium used.
The work described here is believed to be the first report of a Streptomyces relA-null mutant that overproduces antibiotics. S. clavuligerus relA-null mutants do not synthesize detectable amounts of (p)ppGpp, yet they overproduce both clavulanic acid and cephamycin C when compared to the wild-type strain. This is also reflected in increased levels of transcription of antibiotic biosynthesis genes. Antibiotic production and morphological differentiation were restored to levels similar to those observed in the wild-type strain by complementation with a full-length relA or a truncated relARI gene. The antibiotic phenotype of the S. clavuligerus relA-null mutants (Fig. 4) contrasts with the results of Jin & et al. (2004). These authors constructed a S. clavuligerus relA-null mutant unable to synthesize (p)ppGpp that was impaired in both sporulation and antibiotic production. In addition, they constructed a null mutant of a relA-homologous gene, rshA. The rshA-deleted mutant exhibited reduced (p)ppGpp synthesis (about 67 % ppGpp and 42 % pppGpp compared to wild-type), and was as severely impaired in antibiotic production as the relA mutant, but showed almost normal sporulation. The authors concluded that just a slight decrease in (p)ppGpp can severely affect antibiotic production in S. clavuligerus without affecting morphological differentiation. The role of rshA is not clear since relA-null mutants completely lack (p)ppGpp formation (Jin et al., 2004; this work) and an rshA-deleted mutant of S. coelicolor is unaffected in antibiotic production (Sun et al., 2001).
The differing behaviour of our S. clavuligerus relA-null mutants and that published by Jin et al. (2004) appears to reflect differences in the parental strains used and potentially the growth media adopted in the respective studies. Sequencing of the region between apt and relA, and the 3'-end of apt (data not shown), revealed marked differences between the two strains. We found 147 additional nucleotides between apt and relA, and located the relA tsp at a thymine 43 nt upstream of the translation start codon, in a region not present in the strain used by Jin et al. (2004). The sequence we determined is identical to that obtained independently by DSM (Delft, The Netherlands) for S. clavuligerus ATCC 27064 (M. van den Berg, personal communication), and it appears that the strain used by Jin et al. (2004), also described as ATCC 27064, has undergone a relA promoter deletion (note that we cannot exclude that the two isolates may differ further through the occurrence of additional DNA rearrangements and mutations acquired during separate subculturing). In addition, the culture media and growth conditions used in the two studies are different. While we used SA medium in baffled flasks (Paradkar & Jensen, 1998; Lorenzana et al., 2004), Jin et al. (2004) used a different medium in a jar fermenter, conditions in which the antibiotic production occurred after entry into stationary phase in their wild-type strain. Either or both of these differences may be responsible for the contrasting patterns of antibiotic production and the markedly different phenotypes of the relA-null mutants. However, our results clearly indicate that (p)ppGpp is not required for antibiotic biosynthesis in our strain of S. clavuligerus, as previously implied from the isolation of thiostrepton-resistant mutants that were proficient in cephamycin C production (Bascarán et al., 1991).
Consistent with our mutant analysis, we observed a peak in (p)ppGpp synthesis in SA-grown cultures of S. clavuligerus ATCC 27064 at the beginning of stationary phase that coincided with a clear decrease in the abundance of antibiotic biosynthesis gene transcripts. This growth-dependent negative regulation of antibiotic biosynthesis gene expression does not occur in the relA mutant (see Figs 5 and 6), suggesting a negative role for (p)ppGpp in the expression of such genes in wild-type S. clavuligerus. While this may seem to disagree with earlier work in other streptomycetes, a striking difference is that in S. clavuligerus expression of the cephamycin C and clavulanic acid biosynthesis genes is growth-associated (Figs 4 and 6); i.e. it occurs during rapid growth and declines upon entry into stationary phase. Consequently, a priori, expression of the biosynthesis genes for both of these compounds would not be expected to be (p)ppGpp-dependent. Nevertheless, this is the first report of the negative regulation of secondary metabolite biosynthesis by (p)ppGpp. It will be interesting to see whether the expression of other secondary metabolic gene clusters that are transcribed during rapid growth are also negatively regulated by (p)ppGpp, and whether their levels of production increase in a relA-null mutant.
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ACKNOWLEDGEMENTS |
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Edited by: L. Heide
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Received 22 July 2007;
revised 16 November 2007;
accepted 29 November 2007.
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, S. clavuligerus ATCC 27064; 
, S. clavuligerus relA : : neo; 
, S. clavuligerus 
, 
, 
, ppGpp; 
, 
, pppGpp. CDW, cell dry weight. The insets show the location of the peaks of ppGpp (3) and pppGpp (4) in extracts of the wild-type strain as analysed by HPLC upon maximal synthesis after amino acid deprivation, and their absence in the similarly treated 
