1 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea
2 Department of Biological Science, Myongji University, Yongin, Kyunggido 449-728, Korea
3 National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
Correspondence
Kye Joon Lee
kjoonlee{at}plaza.snu.ac.kr
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AF421216 and AF421217.
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INTRODUCTION |
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It has also been reported that (p)ppGpp plays an important role during secondary metabolism and morphological differentiation in Streptomyces spp. (Ochi, 1986, 1987
; Strauch et al., 1991
; Bascaran et al., 1991
; Hoyt & Jones, 1999
). However, when the (p)ppGpp synthetase-encoding gene (relA) of Streptomyces coelicolor A3(2) was characterized (Chakraburtty et al., 1996
), a null mutation in the relA gene failed to produce actinorhodin and undecylprodigiosin under conditions of nitrogen limitation (Chakraburtty & Bibb, 1997
). A second RelA homologous protein, RshA, was also identified from the genome sequence of S. coelicolor (Mittenhuber, 2001
). However, no experimental evidence is available to indicate if RshA, like RelA, can act as a bifunctional enzyme, both synthesizing and degrading (p)ppGpp (Sun et al., 2001
). It has been reported that ppGpp was detected at very low levels prior to the accumulation of cephamycin C in S. clavuligerus NRRL3585, and that another form of guanine nucleotide, ppGp, was also simultaneously detected (Jones et al., 1996
). The synthesis of ppGp was not overtly associated with any ppGpp synthetic activity but rather with the ribosome. Nevertheless, ppGp was shown to play a regulatory role in the production of cephamycin C. In the current study, we describe the identification of the relA/spoT homologous genes, relA and rsh, from Streptomyces clavuligerus ATCC 27064. The involvement of the genes in morphological differentiation and the biosynthesis of antibiotics was demonstrated using mutants in which the relA and rsh genes were disrupted.
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METHODS |
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Cloning and characterization of relA/spoT homologous genes.
DNA manipulation in E. coli and Streptomyces spp. was carried out according to the methods reported elsewhere (Sambrook & Russell, 2001; Kieser et al., 2000
). Restriction and modifying enzymes were used according to the manufacturers' recommendations (Poscochem and Boehringer-Mannheim). DNA fragments were purified from agarose gels using the EZNA Gel Extraction Kit (Omega). For the construction of the cosmid library of S. clavuligerus, 100 µg S. clavuligerus chromosomal DNA was partially digested with Sau3AI (Poscochem) to produce a mean fragment size of approximately 50 kb. The reaction was stopped by phenol extraction, and the mixture was applied to a 50 ml sucrose gradient ranging from 10 to 40 % and then ultracentrifuged at 100 000 g for 20 h. Fractions (1 ml) containing DNA fragments of 4060 kb were pooled and concentrated by ethanol precipitation. The size-fractionated DNA (500 ng) was then ligated in a total volume of 20 µl with 1·4 µg BamHI-digested pWE15 that had been treated with calf intestinal alkaline phosphatase (Poscochem) to prevent recircularization. After ligation at 16 °C for 12 h, the ligated DNA was packaged in bacteriophage
particles by using the Giga Pack III Gold kit (Stratagene) and was introduced into E. coli DH5
by transduction. Oligonucleotide primers for PCR were designed based on the conserved sequences of relA/spoT homologous genes from various organisms. The synthetic oligonucleotides relAP1 (5'-CTSCACGACRTSATCGAGGACAC-3'), relAP2 (5'-CTTSGGSVHSGCGATGTAGTCC-3'), spoTP1 (5'-CCCAAGTTCAACSTSTACCAGTCGCTGACG-3') and spoTP2 (5'-CTCSGGSGGSACGGGGGTGCAGCA-3') were used as PCR primers for the amplification of internal segments of the relA/spoT homologue from S. clavuligerus. The PCR-amplified fragments were sequenced to verify their identities and then used as probes to screen a pWE15-based cosmid library containing fragments of S. clavuligerus genomic DNA for cosmids containing relA/spoT homologous genes. The nucleotide sequence of the relA/spoT homologous gene region was determined by dideoxy-sequencing (Sanger method) using an automatic sequencer (ABI3730; Applied Biosystems). The regions encoding the relA/spoT homologous genes were sequenced on both strands and the entire sequence was assembled from sequence information obtained from analysis of overlapping clones using the DNASIS and WINSTAR programs (Hitachi Software). Sequences were compared with the database using the BLAST program (Altschul et al., 1990
) and multiple alignments were done with GENEDOC and CLUSTAL W (Bibb et al., 1984
; Higgins et al., 1992
). ORF analysis and prediction were done with FRAME 2.3.2 (www.nih.go.jp/
jun/cgi-bin/frameplot.pl; Ishikawa & Hotta, 1999
). Phylogenetic analysis of the RelA/SpoT family of proteins was displayed using the PHYLIP program (Felsenstein, 1993
).
Disruption of relA and rsh and phenotypic complementation.
The relA/spoT homologous genes were disrupted by insertion of a hygromycin resistance gene (hyg) and an apramycin resistance gene (apr) in the middle of each gene, respectively. In the case of the relA : : hyg mutant construction, the BglIISmaI fragment (1542 bp) of relA in pSMF4103, a recombinant cosmid containing the relA region, was replaced with the hyg gene (1410 bp) in pJOE829. The DNA fragment (2400 bp) containing relA : : hyg was cloned into pBluescript KS(), creating pSMF384. A BglIIXhoI fragment (3500 bp) from pSMF384 (which includes the hyg gene together with the remainder of relA and downstream sequences) was inserted into BglII/XhoI-digested pIJ702, creating pSMF387 and introduced into S. lividans, reisolated and introduced into S. clavuligerus, creating pSMF387. A disruption mutant defective in the relA gene resulting from replacement of relA by homologous recombination (double crossover) was isolated as described elsewhere (Paradkar & Jensen, 1995). The transformant harbouring pSMF387 was subcultured five times in a rich medium (MYM; Stuttard, 1982
) containing only hygromycin (200 µg ml1) and then transformants resistant to hygromycin but sensitive to thiostrepton (50 µg ml1) were selected as putative relA gene disruption mutants (
relA). The insertion inactivation of relA was confirmed by Southern hybridization (Sambrook & Russell, 2001
) using the 717 bp SacII fragment, containing part of relA and part of hyg, labelled with the ECL direct nucleic acid labelling system (Amersham Pharmacia Biotech) as a probe. For the phenotypic complementation of the disrupted relA gene, a BamHIEcoRI fragment (4500 bp) containing the entire relA sequence in pSMF4013 was cloned into an expression vector (pSET152), creating pSMF389. Plasmid pSMF389 was transformed into the non-methylating strain of E. coli ET12567 and the plasmid reisolated from the transformant of E. coli ET12567 was introduced by transformation to the protoplasts of the relA-disrupted mutant (
relA). Strains showing a hygromycin-sensitive (HygS) and thiostrepton-sensitive (TsrS) phenotype were designated relA-complemented strains (CrelA).
To construct an rsh : : apr mutant, an NcoI fragment (3840 bp) in pSMF41025, a recombinant cosmid containing the rsh (2217 bp) gene was used where the EcoRIApaI fragment (551 bp) was replaced with the apr gene (1920 bp) in pSET152. The NcoI fragment (5210 bp) containing rsh : : apr was cloned into pBluescript KS(), creating pSMF3812. A KpnIXbaI fragment (5800 bp) from pSMF3812 was ligated to KpnI/XbaI-digested pUWL-KS, creating pSMF3813 and then introduced into S. lividans. Plasmid pSMF3813, purified from S. lividans, was then transformed into S. clavuligerus. A transformant harbouring pSMF3813 was subcultured five times using a rich medium (trypticase soy broth, TSB) containing only apramycin (50 µg ml1) and then AprR TsrS colonies were selected as potential disruption mutants (rsh). The insertion inactivation of the rsh gene was confirmed by Southern hybridization using a 1·7 kb NcoIEcoRI fragment, containing part of rsh and part of apr, labelled with the ECL direct nucleic acid labelling system as a probe. For phenotypic complementation of the disrupted rsh, a 3·8 kb BamHIEcoRI fragment, containing the entire rsh sequence and putative promoter region, in pSMF41025 was cloned into pUWL-KS, yielding plasmid pSMF3814. pSMF3814 was introduced by transformation into the non-methylating E. coli strain ET12567 and then reisolated and used to transform protoplasts of the rsh-disrupted mutant (
rsh). Strains showing an AprS TsrS phenotype were designated rsh-complemented strains (Crsh).
Analysis of intracellular (p)ppGpp levels.
The stored spores were inoculated into a rich medium containing 0·4 % (w/v) glucose, 0·4 % yeast extract, 1·0 % malt extract, 0·1 % NZ-amine, 0·2 % NaCl and 0·1 % (v/v) inorganic salts mixture. The inorganic salts mixture consisted of 0·0025 % MgSO4.7H2O, 0·0008 % CuSO4.5H2O, 0·0015 % FeSO4.7H2O, 0·0008 % MnSO4.4H2O, 0·003 % CaCl2.2H2O and 0·002 % ZnSO4.7H2O. Culture grown in the seed culture medium was inoculated into the rich medium and cultured at 30 °C on a rotary shaker (150 r.p.m.). Seed cultures (2·5 ml) were used to inoculate 100 ml chemically defined (CD) medium supplemented with 1 % Casamino acids. The CD medium consisted of 2 % (w/v) glucose, 0·3 % (NH4)2SO4, 0·52 % K2HPO4, 0·24 %, NaH2PO4, 0·05 % NaCl, 0·005 % KCl, 0·005 % MgSO4.7H2O and 0·0001 % ZnSO4.7H2O (pH was adjusted to 7·0 before steam sterilization). Cultures growing in CD medium containing 1 % Casamino acids were harvested during exponential growth phase by filtration (Whatman GF/C filters), washed rapidly with CD medium, resuspended in CD medium without Casamino acids and quickly returned to the shaking incubator. After the downshift, the culture samples were harvested at 0, 5, 10, 15, 30 and 60 min, then filtered. The filter cakes were transferred to 10 ml formic acid, incubated at 4 °C for 1 h and centrifuged for 10 min at 6000 g. The supernatants (formic acid-extractable fractions) were clarified by filtration using a 0·45 µm filter and freeze-dried. The amount of ppGpp, pppGpp and GTP was determined by HPLC using a Partisil-10 SAX (4·6x250 mm) column (Whatman) as described previously (Ochi, 1986).
Antibiotic production and cultural characteristics of mutant strains.
Antibiotic production by the wild-type and mutant strains of S. clavuligerus was assessed in a jar fermenter (KF-5L; Ko-Biotec). One millilitre of spore suspension was inoculated into 100 ml seed culture medium in 500 ml culture flasks and cultured at 30 °C for 3 d on a rotary shaker (150 r.p.m.). Seed culture medium (TSB) consisted of 1·0 % (w/v) maltose, 0·5 % glucose, 1·7 % Bacto tryptone, 0·3 % Bacto soytone, 0·25 % NaCl, 0·25 % K2HPO4 (pH was adjusted to 7·0 before steam sterilization). The seed culture (300 ml) was used to inoculate 3 l production medium in the jar fermenter. Production medium consisted of 1·0 % (w/v) glycerol, 0·6 % arginine, 0·2 % KH2PO4, 0·06 % MgSO4.7H2O, 0·005 % FeSO4.7H2O, 0·005 % CaCl2.2H2O, 0·005 % MnCl2.4H2O, 0·005 % ZnCl2. The culture temperature was maintained at 30 °C and pH was adjusted to 7·0 by automatic addition of 1 M HCl or 1 M NaOH. Agitation was fixed at 300 r.p.m. and aeration was controlled to 1 vol. air (vol. medium)1 min1. To measure cell growth, triplicate samples (10 ml) of cultures were collected on pre-weighed filters (Whatman GF/C) by vacuum filtration. The filter cakes were washed twice with 10 ml distilled water and dried at 80 °C for 24 h and reweighed. The concentration of cephamycin C was assayed using the agar-diffusion method with the indicator organism E. coli ESS, and the concentration of clavulanic acid was quantified by HPLC after derivatization with imidazole (Foulstone & Reading, 1982; Paradkar & Jensen, 1995
).
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RESULTS |
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DISCUSSION |
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The morphological differentiation of S. clavuligerus was also very significantly affected by the disruption of the relA gene, but not greatly by the disruption of the rsh gene. The altered morphological characteristics were completely restored by the complementation of the corresponding disrupted genes. Failure to produce aerial mycelium in the relA mutant accompanied by no decrease in GTP pool size after nutritional downshift (Fig. 4c
) is in agreement with the proposal that morphological differentiation of Streptomyces spp. is directly controlled by intracellular GTP level (Okamoto & Ochi, 1998
). Thus, it was apparent that mycelial morphology and antibiotic production are severely affected by the disruption of the relA gene and both of these phenotypes are presumably associated with the consequent inability of the mutants to accumulate ppGpp and pppGpp after amino acid starvation. These results were very similar to those observed for the relA null mutant of S. coelicolor (Chakraburtty & Bibb, 1997
). However, mutation of the rsh gene gave only minor changes in ppGpp and pppGpp accumulation or mycelial morphology, but nonetheless resulted in a major defect in production of cephamycin C and clavulanic acid. The effects of the rsh gene on physiological differentiation in S. clavuligerus are very different from those observed for S. coelicolor, where deletion of rshA had no effect on the production of the pigmented antibiotics, actinorhodin and undecylprodigiosin (Sun et al., 2001
). It is conceivable that production of cephamycin C and clavulanic acid in S. clavuligerus is more sensitive to the intracellular level of (p)ppGpp than is actinorhodin production in S. coelicolor. Thus, only minor changes in (p)ppGpp production resulted in a major defect in antibiotic production in S. clavuligerus. It seems plausible that the stringent response of S. clavuligerus to starvation for amino acids is governed mainly by RelA and that the (p)ppGpp synthesized immediately after depletion of amino acids triggers the initiation of pathways for both morphological and physiological differentiation in this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 October 2003;
revised 15 December 2003;
accepted 26 January 2004.
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