Induction of Holomycin Production and Complex Metabolic Changes by the argR Mutation in Streptomyces clavuligerus NP1 Hua Yin,a Sihai Xiang,a Jianting Zheng,a Keqiang Fan,a Tingting Yu,a Xu Yang,a Yanfeng Peng,a Haibin Wang,b Deqin Feng,a Yuanming Luo,a Hua Bai,b and Keqian Yanga State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of China,a and Hisun Pharmaceutical Company Ltd., Taizhou, People’s Republic of Chinab
In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetylCoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in S. clavuligerus.
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treptomyces clavuligerus, an industrially important producer of clavulanic acid (CA) (48), also produces several other secondary metabolites with interesting pharmacological activities, among them the -lactam compounds cephamycin C (42) and 5S clavams (6, 19, 47), the pyrrothine structure antibiotic holomycin (26), and the glucosamine-containing antibiotic tunicamycin (26). CA is a -lactamase inhibitor widely used to treat bacterial infections (5, 48). It has a typical -lactam structure characterized by a bicyclic nucleus comprising a four-member -lactam ring fused to a five-member oxazolidine ring (21, 48), but despite its structural similarity to the conventional -lactam antibiotics, such as penicillins and cephalosporins, which are synthesized by a nonribosomal peptide synthetase mechanism, CA utilizes a carboxyethylarginine synthase (CeaS)-catalyzed reaction for the initial condensation of precursors: glyceraldehyde-3-phosphate (G3P) and arginine (14, 27, 51, 62). CA and 5S clavams share an early biosynthetic pathway that gives rise to the common intermediate clavaminic acid (13, 22). Earlier genetic studies on the genes involved in CA and 5S clavam biosynthesis revealed one of the most complicated examples of gene organization governing antibiotic biosynthesis, showing that the genetic information needed for the production of CA and the 5S clavams is distributed among three distinct gene clusters located in unlinked regions of the S. clavuligerus genome (54, 60). These clusters have been designated the CA gene cluster, the clavam gene cluster, and the paralog gene cluster; both the CA and paralog gene clusters (23, 61) are involved in CA biosynthesis, whereas the clavam cluster contains most of genes responsible for clavam biosynthesis. The CA cluster is located right beside the cephamycin C gene cluster on the chromosome, and they form the so-called supercluster (64). Both the CA supercluster and the clavam cluster lie on the chromosome, whereas the paralog cluster is located on the pSCL4 plasmid (37). The complexity of CA-clavam biosynthesis is also reflected at the
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level of regulation, and at least 6 regulatory genes (ccaR [45, 52], claR [43], cvm7P [59], and orf21 to orf23 [25, 55]) were identified among the three gene clusters. Intricate cross-regulation between the arginine and CA (12, 51), cephamycin C and CA (44, 45), and CA and holomycin (11) pathways were also reported. In the case of arginine-CA cross-regulation, the oat genes (homologous to the arginine biosynthetic gene argJ, encoding ornithine acetyltransferase) were found in both the CA (oat2) and the paralog (oat1) clusters, and the oat2 mutant showed interesting changes in CA production levels, depending on the arginine concentrations, indicating it plays a role in controlling the flux between arginine and CA (12). Metabolic engineering of the precursor supply has been successfully applied to increase CA production. For example, the glycolytic pathway has been targeted to overcome G3P limitation; the disruption of gap1, encoding one of the glyceraldehyde-3-phosphate dehydrogenases, increased the flux of G3P to CA biosynthesis, and as a result, the mutant showed 2-fold improvement in CA production (33). G3P could also be supplied by the glycerol utilization pathway. Thus, amplification of the glycerol-utilizing gene cluster (glpF1K1D1) and subsequent fermentation in a high-glycerol medium led to 7.5-fold-increased CA production (1). In wild-type S. clavuligerus ATCC 27064, oversupply of arginine and ornithine extracellularly showed stimulatory effects on CA production (51, 53); however, the effect of ornithine was more
Received 26 November 2011 Accepted 11 February 2012 Published ahead of print 17 February 2012 Address correspondence to Keqian Yang,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.07699-11
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TABLE 1 Bacterial strains and plasmids used Strain or plasmid Plasmids pKC1139 pGH112 pSET152 pIMEP
pDAG pIMEP-argR pKHps Strains E. coli DH5␣ ET12567(pUZ8002) S. clavuligerus NP1 CZR CZR-argROE HPS
High-copy-number, temperature-sensitive E. coli-Streptomyces shuttle vector High-copy-number E. coli-Streptomyces shuttle vector with apr, tsr, and oriT(RK2) E. coli-Streptomyces shuttle vector capable of integration into 31 attB site in streptomycetes Contains ermE*p and a ribosomal binding site sequence upstream of the multiple cloning site of pSET152; constructed in our laboratory and effectively used for gene overexpression in Streptomyces argR disruption plasmid based on pGH112 argR overexpression plasmid based on pIMEP hlmE disruption plasmid based on pKC1139
3 39 28 2, 63
General cloning host for plasmid manipulation Donor strain for conjugation between E. coli and streptomycetes Mutant partially blocked in cephamycin synthesis argR disruption mutant of NP1 argR overexpression mutant of CZR hlmE disruption mutant of CZR
Invitrogen 28 46, 67 This work This work This work
consistent than that of arginine (9), probably because arginine was converted into ornithine and urea by the induced arginase in S. clavuligerus, and high levels of urea could induce stress responses. To increase the arginine supply in vivo, one strategy is to inactivate the key repressor gene of arginine biosynthesis, argR. argR disruption in wild-type S. clavuligerus ATCC 27064 resulted in derepression of the transcription of arginine biosynthetic genes and oat2 (12, 49, 50) and lower production of CA (P. Liras et al., personal communication). However, little is known about the effects of argR mutation on the overall cellular metabolism. Since the arginine supply is important for CA yield, characterization of the general cellular responses to argR mutation will help in understanding the regulation of CA biosynthesis in relation to the arginine supply and the regulatory connections between primary and secondary metabolism. For these reasons, we reexamined the arginine supply problem by disruption of argR in S. clavuligerus NP1. NP1 is a cephamycin C mutant of wild-type S. clavuligerus isolated by B. Mahro and A. L. Demain after nitrosoguanidine (NTG) mutagenesis (46, 67). It is partially blocked in cephamycin C biosynthesis but is normal in growth and differentiation. Indeed, the argR mutant of NP1 (designated CZR) displayed dramatic growth and metabolic phenotypes compared with NP1. The most striking phenotype is the overproduction of holomycin. Holomycin is a dithiolopyrrolone antibiotic with RNA synthesis-inhibiting activity (18, 26, 65) and is known for its activity against rifamycin-resistant bacteria (41). It is synthesized from one acetyl-coenzyme A (acetyl-CoA) and two cysteines (11, 31). The gene cluster responsible for holomycin biosynthesis was recently identified and characterized. In the cluster, a standalone nonribosomal peptide synthetase (SSCG_03488; hlmE) was postulated to catalyze the condensation of two cysteines to form the L-Cys–L-Cys dipeptide intermediate (31). Holomycin is not produced by the wild-type strain under the reported growth conditions (40), but it was produced by some CA mutants: de la Fuente et al. reported that several CA mutants (ceaS2, bls2, and oat2) blocked in an early stage of CA biosynthesis before the formation of clavaminic acid could not produce holomycin, whereas mutants blocked in the second half of biosynthesis, after clavaminic
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This work This work This work
acid formation, overproduced holomycin (11); they proposed a cross-regulation mechanism between the CA and holomycin pathways mediated by an intermediate of CA biosynthesis. As a starting point to understand the complex phenotypic changes and the intricate regulatory relationships in CZR, we set out to characterize the gene expression changes by comparative proteomic analysis and found significant restructuring of primary and secondary metabolism. MATERIALS AND METHODS Strains and culture conditions. The bacterial strains and plasmids used are listed in Table 1. S. clavuligerus NP1, CZR, CZR-argROE, and HPS were grown on YD agar (58). To evaluate CA production, the strains were grown in seed and fermentation media. The seed medium contains 20 g/liter defatted soy flour, 10 g/liter dextrin, 5.0 g/liter soybean oil, 0.6 g/liter KH2PO4, and 10.5 g/liter 3-(N-morpholino)propanesulfonic acid (MOPS) adjusted to pH 7.1. The fermentation medium contains 40 g/liter defatted soy flour, 10 g/liter dextrin, 23 g/liter soybean oil, 1.2 g/liter KH2PO4, 10.5 g/liter MOPS, and 10 ml/liter trace elements adjusted to pH 6.8. The trace elements contain 10 g/liter CaCl2 · H2O, 10 g/liter MgCl2 · 6H2O, 10 g/liter NaCl, 0.05 g/liter FeCl3, 0.05 g/liter ZnCl2, 0.05 g/liter CuCl2, and 0.05 g/liter MnSO4. Before inoculation, the strains were incubated at 28°C for 3 to 5 days on YD agar. Where appropriate, media were supplemented with apramycin (Am) (50 g/ml) or thiostrepton (Tsr) (20 g/ml). A piece of bacterial lawn (1 cm2) was cut from the YD plate and inoculated into 50 ml seed culture in a 250-ml flask, the flasks were placed on a rotary shaker (New Brunswick Scientific) and incubated at 28°C with shaking at 250 rpm for 44 h, and then 3 ml (about 6 ml CZR) seed cultures were inoculated in 30 ml fermentation medium in 250-ml flasks and incubated at 28°C with shaking at 250 rpm. Construction of argR disruption mutants. To construct the argR disruption plasmid, a 1.3-kb upstream region was amplified with primers 5=-GATTCTAGAACTCGATCCTGCTGGAG-3= (the XbaI restriction site is underlined) and 5=-GTAAAGCTTTGCGCCTCGGTCATCGTC-3= (the HindIII restriction site is underlined); a 1.6-kb downstream region was amplified with primers 5=-CATGGATCCGACACCCTGATGCTGA TC-3= (the BamHI restriction site is underlined) and 5=-CTAGAATTCC GTTGTTGCTGCTCACTG-3= (the EcoRI restriction site is underlined). The apramycin resistance gene (apr) was amplified with primers 5=-CTT AAGCTTGGCCTAACTACGGCTACAC-3= (the HindIII restriction site is underlined) and 5=-CATGGATCCGTCATCTCGTTCTCCGCTC-3=
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FIG 1 (A and B) Schemes for the disruption of argR (A) and hlmE (B). The KpnI sites and the probes used in Southern hybridization are indicated. (C and D) Southern blot results. (C) Hybridization bands in the argR mutant (CZR) of S. clavuligerus. Genomic DNA samples (5 g) from NP1 and CZR were digested with KpnI and separated on a 0.8% agarose gel. Lane 1, probe; lane 2, marker; lane 3, NP1; lane 4, CZR. (D) Hybridization bands of the hlmE mutant (HPS) of CZR. Genomic DNA (8 g) from CZR and HPS was digested with KpnI and separated on a 0.8% agarose gel. Lane 1, marker; lane 2, HPS; lane 3, CZR; lane 4, probe.
(the BamHI restriction site is underlined) from pKC1139 (3). These fragments were digested by appropriate enzymes and inserted between the EcoRI and HindIII sites of pGH112 (39) to obtain pDAG. The plasmid was verified by restriction digestion and sequencing. To generate an argR disruption mutant, pDAG was first used to transform E. coli ET12567(pUZ8002) and conjugated into S. clavuligerus NP1 according to the method of Kieser et al. (28). Tsr-sensitive Am-resistant colonies were isolated and first examined by PCR using primers 5=-ACCGGAGAATGA GACGAC-3= and 5=-AGCCGCCAGTCGAAGTGCAC-3=. Colonies that gave the expected 2.9-kb PCR product were candidate argR disruption mutants and were named CZR. Southern blotting was performed to further confirm the disruption of argR by using the DIG DNA labeling and detection kit (Roche) according to the manufacturer’s protocol. Briefly, genomic DNAs were completely digested by KpnI and resolved on agarose gels. A 0.6-kb probe template was amplified by PCR with primers 5=-AC CCTGATGCTGATCAGCAG-3= and 5=-AGCGGGCGTTGGCCTTGAT C-3=. The scheme of disruption of argR is shown in Fig. 1A. Construction of hlmE disruption mutants. To construct the holomycin disruption mutant, the structural gene hlmE (SSCG_03488) of the holomycin biosynthesis gene cluster, encoding a standalone nonribosomal peptide synthetase (31), was targeted for disruption. A 1.3-kb region upstream was amplified with primers 5=-CATGGATCCACGGTGA GGGCGCCGTTCTC-3= (the BamHI restriction site is underlined) and 5=-CTAGAATTCATGGAAGACCTTGTTGCCGAG-3= (the EcoRI restriction site is underlined). A 1.2-kb region downstream of hlmE was
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amplified with primers 5=-GTAAAGCTTCGCCCACGAGTTCGTCCCA C-3= (the HindIII restriction site is underlined) and 5=-GATTCTAGAGC ATGCGGACGATCAGCCTG-3= (the XbaI restriction site is underlined); the thiostrepton resistance gene (tsr) was amplified with primers 5=-GAT TCTAGACTGGCGTAATAGCGAAG-3= (the Xbal restriction site is underlined) and 5=-CATGGATCCACAAGCAGGAAGGAAGCGTC-3= (the BamHI restriction site is underlined) from pGH112 (39). These fragments were digested by appropriate enzymes and inserted between the EcoRI and HindIII sites of pKC1139 (3) to obtain pKHps. The plasmid was verified by restriction digestion and sequencing. pKHps was used to transform into E. coli ET12567(pUZ8002) and conjugated into CZR according to the method of Kieser et al. (28). The colonies were first screened by PCR using primers 5=-ACGGGTATCGCTCCGTGGTC-3= and 5=-TCGGGGT TGTTCGACGCAC-3=. Colonies that gave the expected 3.85-kb PCR product were hlmE mutants and were named HPS. Then, Southern blotting was performed to further confirm the disruption of hlmE by using the DIG DNA labeling and detection kit (Roche) according to the manufacturer’s instructions. Genomic DNAs were completely digested by KpnI. A 0.6-kb probe template was amplified by PCR with primers 5=-TCGGGT CAGTTCACGCAGGAC-3= and 5=-AGCGTCGACCTGCTCTTCGTC3=. The scheme of disruption of hlmE is shown in Fig. 1B. Complementation of the argR disruption mutant CZR. pIMEP (63) in our laboratory was modified from pSET152 by inserting ermE*p (2) with a ribosomal binding site sequence (purine rich; GAACGGA) upstream of the multiple cloning sites. pIMEP-argR was constructed from
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pIMEP by inserting argR and a Tsr resistance gene (tsr) in the multiple cloning sites. A 540-bp argR fragment was amplified with primers 5=-TG ACCGAGGCGCACGCCACCGAC-3= and 5=-AAAAGGATCCTCAGGC CCGGTCGTTCTG-3= (the BamHI restriction site is underlined). The fragments were digested by BamHI and then inserted into EcoRV- and BamHI-digested pIMEP to obtain pIMEP-argR. pIMEP-argR was used to transform into E. coli ET12567(pUZ8002) conjugated into CZR to obtain the overexpression strain CZR-argROE according to the method of Kieser et al. (28). Transconjugants were selected by their Tsr resistance. Purification and Identification of holomycin produced by CZR. Holomycin was purified initially from the culture of CZR grown for 36 h in fermentation medium by high-performance liquid chromatography (HPLC). The purified holomycin was assayed for activity against E. coli and Bacillus subtilis, examined by thin-layer chromatography (TLC), and analyzed by UV spectrum, electrospray ionization-mass spectrometry (ESI-MS), and tandem mass spectrometry (MS-MS). HPLC was run on a reverse-phase column (DikmaDiamonsil-C18; 5 m; 250 by 4.6 mm) with 0% to 40% acetonitrile as the mobile phase; the flow rate was 1 ml/min, and the detection wavelength was 360 nm (11). Under these conditions, holomycin was detected at 18.3 min. The culture supernatants were obtained by centrifugation at 20,000 ⫻ g for 5 min and then were filtered through a 0.2-m filter before injection into HPLC. TLC was applied on a Silica Gel 60 plate and developed in chloroform-methanol (9:1) and benzene-acetone (1:1) (26). The UV spectrum of holomycin in methanol was scanned with a DU800 spectrophotometer. ESI-MS and MS-MS were performed in positive-ionization mode. Detection of CA and holomycin. CZR and NP1 were grown in fermentation medium, and four flasks were withdrawn every 12 h to measure cell dry weight and CA and holomycin levels. The cultures were collected on filter papers and dried at 60°C before the weight measurements. To measure CA production, culture supernatants (50 l) were injected and run on a reverse-phase column (DikmaDiamonsil-C18; 5 mm; 250 by 4.6 mm) with an isocratic buffer system consisting of 0.05 M NaH2PO4 (pH 4.4); dual-wavelength detection was adopted (220 nm and 268 nm for CA); CA was detected at 19.5 min (66). Holomycin production was measured by HPLC (100-l culture supernatants) as described above. Protein extraction and 2DE. The growth conditions of the strains were as described above (see “Strains and culture conditions”), except that the 20 g defatted soy flour in culture medium was replaced by an aqueous extract prepared by filtering a suspension of 20 g defatted soy flour in water after it had been treated in an autoclave at 121°C for 30 min. At 24 h of incubation in fermentation medium, the cell pellets of NP1 and CZR mycelia were harvested from cultures by centrifugation at 10,000 ⫻ g at 4°C for 10 min, washed three times with ice-cold TE buffer (40 mM Tris-HCl, 10 mM EDTA, pH 9.0, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 10 g/ml leupeptin). The cell pellets (1 g) were resuspended in 10 ml TE and sonicated (10-s pulses 24 times with 15-s intervals) on ice. The cell extracts were centrifuged at 200,000 ⫻ g in a 90 Ti rotor (Beckman, Fullerton, CA) at 4°C for 60 min. The pellet was discarded, and 100% (wt/vol) trichloroacetic acid was added to the supernatant to bring the trichloroacetic acid concentration to 17%. After incubation on ice for 4 h, proteins in the samples were collected by centrifugation at 20,000 ⫻ g at 4°C for 30 min and then rinsed four times in ice-cold acetone. Then, the protein samples were dried in a speed vacuum and resuspended in 2 ml lysis buffer (7 M urea, 2 M thiourea, 4% [wt/vol] CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 20 mM dithiothreitol [DTT], 1 mM PMSF, and 10 g/ml leupeptin). After a brief sonication, the protein lysis solution was incubated at 20°C for at least 2 h. The supernatants were collected after centrifugation and stored at ⫺80°C for further use. Protein concentrations were measured with the Quantify Kit (GE Healthcare). The proteins extracted from both NP1 and CZR were subjected to two-dimensional electrophoresis (2DE). Isoelectric focusing (IEF) was performed with an Ettan IPGphor 3 system (GE Healthcare, Uppsala, Sweden) using IPG strips (GE Healthcare). For 18-cm IPG strips, pH 4 to 7, approximately 1 mg protein per sample in 350 l rehy-
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dration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, 10 g/ml leupeptin, and 0.001% bromophenol blue) with a final concentration of 20 mM DTT and 1.0% Pharmalyte (GE Healthcare), pH 4 to 7, was run using the in-gel sample rehydration technique according to the manufacturer’s instructions for IEF. IEF was performed using the following voltage program: 30 V constant for 13 h, step to 200 V for 2 h, step to 500 V within 2 h, step to 1,000 V within 1 h, gradient to 8,000 V within 1 h, and then 8,000 V until a total of 79,000 V · h had been achieved. The temperature was maintained at 20°C for IEF. After completion of the first-dimension IEF, the focused IPG strips were equilibrated twice for 15 min each time in 10 ml of equilibration buffer (375 mM Tris-HCl, pH 8.8, 6 M urea, 20% [vol/vol] glycerol, 2% [wt/vol] SDS) that contained 16 mM DTT during the first equilibration step and 100 mM iodoacetamide during the second equilibration step. The equilibrated IPG strips were then applied onto 15% polyacrylamide gels. A denaturing solution (0.5% agarose, 0.1% SDS, 192 mM glycine, 25 mM Tris-HCl, pH 8.8, 0.001% bromophenol blue) was loaded onto the gel strips. After agarose solidification, electrophoresis was performed in a buffer (pH 8.3) containing 0.3% Tris, 1.44% glycine, and 0.1% SDS at 25°C for 30 min at 10 W/gel, followed by 20 W/gel till the bromophenol blue reached the bottom. The 2D gels were fixed for 2 h in fixing solution containing 50% methanol and 10% glacial acetic acid. Then, they were stained overnight with gentle shaking in staining solution (50% methanol, 10% glacial acetic acid, and 0.1% colloidal Coomassie brilliant blue R250). Finally, the slab gels were destained for about 2 h in fixing solution and then overnight in destaining solution (5% methanol and 7% glacial acetic acid). The 2D-PAGE protein patterns were recorded as digitized images with ImageScannerIII (GE Healthcare). The gels were stored at 4°C in fresh film after scanning. Spot detection, quantification, and analysis were performed using ImageMaster 2D Platinum 6.0 (GE Healthcare). Spots from the 2D images were automatically detected and manually checked to eliminate any artifacts. Spot normalization was performed using relative volumes to quantify and compare the gel spots. Protein spots that showed statistically significant differences of 2-fold or more in their mean spot volumes (with a P value of ⬍0.05) on all gels were excised from the gels manually. In-gel digestion and protein identification. The gel pieces were first washed three times with water and then washed twice with 25 mM NH4HCO3-50% acetonitrile to remove excess Coomassie brilliant blue at room temperature and dried completely by speed vacuum. The gels were rehydrated in 5 l of proteomics sequencing-grade trypsin (12.5 ng/l; Sigma) at 4°C for 45 min, and then the excess trypsin solution was removed. Ten microliters of 25 mM NH4HCO3 was added, followed by incubation at 37°C for 16 h. The peptides were extracted from the gels with 50% acetonitrile and 5% trifluoroacetic acid (TFA), dried in a speed vacuum, and stored at ⫺20°C for MS analysis. The dried tryptic peptide mixtures were dissolved in 2 l of a saturated matrix solution (5 mg/ml ␣-cyano-4-hydroxycinnamic acid [CHCA] in 50% acetonitrile-0.1% TFA), and 1 l of the mixture was spotted on a matrix-assisted laser desorption ionization (MALDI) plate. After air drying, the crystallized spots were analyzed on the Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Framingham, MA). MS calibration was automatically performed with a peptide standard kit (Applied Biosystems) containing des-Arg1bradykinin (m/z 904), angiotensin I (m/z 1,296.6851), Glu1-fibrinopeptide B (m/z 1,570.6774), adrenocorticotropic hormone (ACTH) (peptide 1-17; m/z 2,903.0867), ACTH (peptide 18-39; m/z 2,465.1989), and ACTH (peptide 7-38; m/z 3,657.9294), and MS-MS calibration was performed by the MS-MS fragment peaks of Glul-fibrinopeptide B. All MS mass spectra were recorded in the reflector positive mode using a laser operated at a 200-Hz repetition rate with a wavelength of 355 nm. The accelerated voltage was operated at 2 kV. The MS-MS mass spectra were acquired by the data-dependent acquisition method with the 10 strongest precursors selected from one MS scan. All MS and MS-MS spectra were
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FIG 2 Growth morphology of CZR and NP1 on YD agar. (A and B) CZR and NP1 were incubated and observed at 3 days (A) and 7 days (B). (C and D) Scanning electron microscope observation (magnification, ⫻8,000) of CZR and NP1 after 7 days of incubation on YD. CZR (C) and NP1 (D) showing aerial mycelia of CZR and spores of NP1.
obtained by accumulation of at least 1,000 and 3,000 laser shots, respectively. Neither baseline subtraction nor smoothing was applied to the recorded spectra. MS and MS-MS data were analyzed, and peak lists were generated using GPS Explorer 3.5 (Applied Biosystems). MS peaks were selected between 850 and 3,700 Da and filtered with a signal-to-noise ratio greater than 20. A peak intensity filter was used with no more than 50 peaks per 200 Da. MS-MS peaks were selected based on a signal-to-noise ratio greater than 10 over a mass range of 60 to 20 Da below the precursor mass (36). MS and MS-MS data were analyzed using the MASCOT 2.0 search engine (Matrix Science, London, United Kingdom) to search against the S. clavuligerus protein sequence database downloaded from the Broad Institute database (http://www.broadinstitute.org/). The search parameters were set as follows: trypsin digestion with one missed cleavage; a charge state of ⫹1; monoisotopic; carbamidomethylation of cysteine as a fixed modification; oxidation of methionine as a variable modification; and mass tolerance of precursor ion and fragment ion at 0.2 Da and 0.5 Da, respectively. For all proteins identified, a MASCOT score greater than 100 was accepted as significant (P ⬍ 0.05).
RESULTS
Growth phenotypes of argR disruption mutants. The argR gene in S. clavuligerus NP1 was disrupted by insertion of apr (in the same orientation as argR). The disruption mutants, designated CZR, were isolated initially as Am-resistant, Tsr-sensitive colonies. The disruption was further confirmed by Southern blotting (Fig. 1C). The growth phenotypes of CZR with NP1 on YD agar were very different. As shown in Fig. 2A, after a 3-day incubation, NP1 grew into a lawn of thick white aerial hyphae, whereas CZR grew much less, with sparse aerial hyphae; after a 7-day incubation, NP1 had grown into a thick mycelium mat covered by grayish green spores (Fig. 2B), whereas CZR only produced white aerial hyphae. Under a scanning electron microscope, NP1 (Fig. 2D) produced spores and CZR produced only aerial hyphae (Fig. 2C). These results
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demonstrated that CZR grew more slowly than NP1 and did not sporulate during incubation. Detection and identification of holomycin produced in CZR culture. Unlike NP1, the culture broths of CZR grown in YD or fermentation medium were bright yellow, indicating the production of a new product, probably holomycin. The yellow compound was purified from CZR culture broth by HPLC (Fig. 3A) and had activity against both Gram-positive and Gram-negative bacteria; it was developed on a Silica Gel 60 TLC plate with chloroform-methanol (9:1) or benzene-acetone (1:1) as the solvent system; the Rf values for the two systems were 0.5 and 0.3, respectively (data not shown), similar to the Rfs of holomycin reported previously (26). The UV spectrum of the purified compound in methanol showed peaks at 244, 300, and 388 nm (Fig. 3B), which correspond to the characteristic peaks of the pyrroline ring (11). The mass spectrum of the purified compound gave an m/z peak of 215 (M ⫹ H)⫹ (Fig. 3C). The primary fragment ions of complexes obtained from MS-MS included the peak of 173 (M ⫺ CH2CO ⫹ H)⫹ (11, 26, 31) and the peak of 197 (M ⫺ H2O ⫹ H)⫹ (Fig. 3D). These results were in accordance with reported results and proved the compound was holomycin. The hlmE gene (SSCG_03488) in CZR was disrupted by tsr insertion. The disruption mutants were designated HPS and isolated as Am- and Tsr-resistant colonies. The disruption of hlmE was further confirmed by Southern blotting (Fig. 1D). HPS strains were grown in fermentation medium and assayed for holomycin production by HPLC. The HPS cultures were pale, and no holomycin was detected during fermentation. The loss of holomycin production in HPS strongly supports the notion that the yellow compound produced by CZR was holomycin. Disruption of argR induced holomycin production but depressed CA production. Because CZR grew poorly on solid media
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FIG 3 Identification of holomycin. (A) Holomycin purified by HPLC. Au, absorbance units. (B) The UV spectrum of purified holomycin in methanol, showing peaks at 246, 300, and 388 nm. (C) Identification of holomycin by ESI-MS. The m/z peak of holomycin is 215 (M ⫹ H)⫹. (D) Identification of holomycin by MS-MS. The primary characteristic fragment ions of holomycin obtained from MS-MS in a tandem mass spectrometer included peaks of 173 (M ⫺ CH2CO ⫹ H)⫹ and 197 (M ⫺ H2O ⫹ H)⫹.
and was unable to sporulate, we tried to synchronize the growth of CZR and NP1 in fermentation cultures by vegetative inoculations (Fig. 4A). Similar amounts of CZR and NP1 mycelia were inoculated in fermentation medium, and four independent flasks were taken out of the rotary shaker every 12 h to assay for holomycin and CA production by HPLC to 120 h (Fig. 4B). We used HPLC to quickly detect holomycin in culture supernatants. At 24 h, CZR produced the highest level of holomycin but did not produce CA; CA production was detected at 48 h and gradually rose with time. NP1 did not produce holomycin throughout fermentation, but at 24 h, it already started to produce CA; its CA production levels were generally higher than those of CZR during fermentation. Overall, our results clearly showed that argR mutation induced holomycin production but delayed CA production to a lower level in CZR. argR mutation is the initial cause of the holomycin overproduction phenotype. In order to confirm that the disruption of argR was responsible for the activation of holomycin production and the reduced CA production in CZR and to exclude the possibility that spontaneous mutations in other loci of the S. clavuligerus genome caused the mutant phenotypes, complementation experiments were performed using an ArgR overexpression plasmid. Previous work revealed that argR is transcribed both as argCJBDR polycistronic mRNA (accounting for most of the argR in the cells) and also as monocistronic argR from its own pro-
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moter at a low level constitutively (49). To avoid the complexity of argR expression, argR was expressed using ermE*p (2). Three CZR-argROE strains were selected and cultivated in fermentation medium, and their cultures were sampled every 24 h for the detection of holomycin and CA. While CZR produced significant amounts of holomycin, all cultures of CZR-argROE strains did not produce holomycin (Fig. 5); these clearly demonstrated that production of holomycin was inhibited by argR expression. Similar to NP1, the CZR-argROE strains grew to be denser and more viscous than CZR during fermentation; however, they still did not sporulate on solid medium. Overexpression of argR did not restore the CA production level to that of NP1. This is expected, as a high level of ArgR would repress arginine biosynthesis and, at the same time, activate arginine catabolism. Both effects are detrimental to CA production. 2DE reference map of S. clavuligerus and the proteomes of NP1 and CZR. To simulate the distribution of all predicted proteins on the gel, a theoretical 2DE map of S. clavuligerus ATCC 27064 was generated according to the incomplete genome annotation information (from the Broad Institute) using the software JVirGel 2.0 (http: //www.jvirgel.de/) (data not shown) and other Web tools (http://pro -161-70.ib.unicamp.br/⬃itaraju/tools/pimw/) (data not shown). The theoretical proteome predicted that most proteins have molecular weights between 10,000 and 50,000 and about 60% of the proteins have pI values between 4 and 7. After preliminary
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FIG 5 Holomycin production by CZR and CZR-argROE strains at 24 h of fermentation analyzed by HPLC. The retention time of holomycin is 18.3 min with detection at 360 nm. Three CZR-argROE strains showed no holomycin production during fermentation.
FIG 4 Growth and secondary-metabolite production of S. clavuligerus NP1 and CZR during fermentation. (A) Growth curves of NP1 and CZR measured as dry weight. (B) CA and holomycin production by NP1 and CZR analyzed by HPLC.
experiments using pH 3 to 10 NL IPG strips for IEF, few proteins could be detected in the alkaline region. Therefore, IPG strips of pH 4 to 7 were used for 2DE proteomic analysis, and 15% polyacrylamide gels were used in the second-dimension SDS-PAGE to maximize proteome coverage. More than 1,000 protein spots were reproducibly detected and matched. Representative 2D gel maps are shown in Fig. 6; the pI of the spots ranged from 4.2 to 7, and the mass ranged from 10 to 100 kDa. Using a 2-fold change with a P value of ⬍0.05 as the minimal criteria for differential expression, 37 proteins were identified as significantly changed in expression levels, and they were successfully identified by MS and MASCOT database searching. The protein identification data, including the identifier (ID), Mr, pI, MASCOT score, and sequence coverage ratio are listed in Table 2 containing the proteins overexpressed and underexpressed in CZR relative to NP1. Comparative proteomic analyses between NP1 and CZR. Overexpressed proteins in CZR were identified; they include two arginine biosynthetic enzymes (ArgC and ArgH), indicating that both the argCJBDR and argGH transcripts are upregulated, and they resulted in the synthesis of large amounts of arginine biosynthetic enzymes at 24 h of fermentation. As expected, five proteins from the recently identified holomycin pathway (32), acyl-CoA dehydrogenase domain-containing protein (SSCG_03485; hlmB), glucose-methanol-choline oxidoreductase (SSCG_03487; hlmD), lantibiotic biosynthesis protein (SSCG_03489; hlmF), globin (SSCG_03490; hlmG), and thiore-
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doxin-disulfide reductase (SSCG_03492; hlmH), were detected as overexpressed proteins in CZR. Our results provide strong evidence that the five overexpressed proteins are directly involved in holomycin synthesis. The three genes hlmA, hlmB, and hlmE were also detected as upregulated genes by microarray analysis (data not shown). Four proteins from the CA cluster, CeaS2, Pah2, Bls2, and Oat2, involved in the early stage of CA biosynthesis (38), especially CeaS2 and Oat2, were overexpressed in CZR. However, the proteins CeaS1, OrfA, and OrfB from the paralog cluster and Cvm2 from the clavam cluster were severely downregulated in CZR, which was confirmed by microarray analyses (data not shown). In addition, CefE and CmcI from the cephamycin cluster (34) and TunF, an enzyme involved in the biosynthesis of tunicamycin (8), were overproduced in CZR. These results indicate that argR mutation had different effects on different pathways in CZR. A significant change in the proteomic results was the overexpression of proteins involved in sulfate assimilation and cysteine synthesis. For example, the rhodanese-like protein (SSCG_01126; RhlA [40]) was identified as a highly overproduced protein in CZR. The overexpression of cysteine synthase (SSCG_02363; CysM), thiamine S protein (SSCG_02362), and thioredoxin was also detected. Overexpression of these proteins can enhance the production of O3-acetyl-L-serine and hydrogen sulfide, thus increasing the precursor supply for cysteine synthesis (16, 29, 56). An increased supply of cysteine might be one of the factors leading to holomycin overproduction. Other metabolic changes observed at the proteomic level include the reduced production of the tricarboxylic acid cycle enzyme malate dehydrogenase (MDH) (SSCG_05972) and the increased expression of an acetyl-coenzyme A synthetase (SSCG_04276), which could enhance the synthesis of acetyl-coenzyme A from acetate, ATP, and CoA (57). Also, it is important to note that proteins involved in pyrimidine metabolism showed significant differential expression between NP1 and CZR. The pyrimidine regulatory protein (PyrR; SSCG_03803), which regulates expression of genes involved in de novo pyrimidine biosynthesis in most bacteria, was overproduced in CZR (17). The biosyntheses of arginine and pyrimidine share a common precursor, carbamoylphosphate (CP), which is synthesized by carbamoylphosphate synthase (CPS). It is understandable that pyrimi-
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FIG 6 2DE images of the proteomes of CZR and NP1. Approximately 1,000 g protein was loaded on each IPG strip (pH 4.0 to 7.0; 18 cm). After isoelectric focusing electrophoresis, the strips were run on 15% SDS-PAGE gels in the second-dimension separation. Protein spots showing different intensities in CZR and NP1 are marked with arrows and numbered. L1 and L2 are two landmarks used for matching gels.
dine metabolism is affected by arginine overproduction or argR mutation. DISCUSSION
Disruption of argR in S. clavuligerus NP1 induced phenotypic changes not observed with the same mutation against the wildtype background. Since NP1 was obtained by NTG mutagenesis, it may contain many nonspecific mutations in the genome, and a causal relationship between argR mutation and holomycin production must be carefully examined. Thus, the dramatic phenotypic changes in CZR were characterized at the metabolite, proteome, and transcription levels (data not shown). Although gene expression changes were best represented by genome-wide transcriptome analysis, proteomic results provide more convincing evidence for functional expression. Both gene disruption and complementation experiments showed that argR mutation induced holomycin overproduction. Overexpression of enzymes involved in holomycin, acetyl-CoA, and cysteine biosynthesis in CZR provides convincing evidence in support of the holomycin overproduction phenotype at 24 h of fermentation. A connection between holomycin production and sulfur metabolism was reported previously. Interestingly, holomycin biosynthesis was stimulated by cysteine but inhibited by methionine and ethionine (4, 40). RhlA was recently identified as a highly overexpressed protein in the holomycin-overproducing oppA2::aph mutant of S. clavuligerus (40), and the protein is indicated to be involved in the formation of a cysteine precursor for holomycin. RhlA was also detected as a highly overexpressed protein in CZR. Arginine biosynthesis is tightly regulated in bacteria by a universally conserved repressor, ArgR, which recognizes a palindrome operator sequence, termed the ARG box (10). The hexameric ArgR with bound L-arginine serves as both the master repressor and activator of diverse regulons to coordinate cellular functions (7, 15, 20, 24, 30, 35). In S. clavuligerus, most genes responsible for arginine biosynthesis were found in the argCJBDRGH cluster, except argF (encoding ornithine carbamoyltransferase), which is located in a separate location (49). Normally, ArgR binds to the ARG box in front of
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argC and argG and represses arginine production. Overexpression of argR lowered the biosynthetic ornithine carbamoyltransferase activity but enhanced the catabolic ornithine aminotransferase activity (50). ArgR is implicated in the regulation of CA biosynthesis by directly repressing oat2 expression. However, the role of Oat2 in CA biosynthesis is still not well understood. At high arginine concentrations (above 1 mM), the Oat2 protein or one of its reaction products may negatively affect CA biosynthesis (12). A bioinformatics analysis of the genome sequence of S. clavuligerus (data not shown) detected a limited number of potential ARG box sequences in front of putative target genes. They do not include any sites from the holomycin gene cluster, indicating the genes are not directly regulated by ArgR, so the holomycin overproduction phenotype is probably due to secondary effects from either arginine overproduction or deregulation of certain ArgR targets. In an earlier report, de la Fuente et al. indicated that an intermediate in CA biosynthesis may be the signal to induce holomycin biosynthesis (11). They speculated that the stimulatory effect of arginine on holomycin production could also be due to the accumulation of a CA biosynthetic intermediate in strains that produced holomycin. Both CA and the 5S clavams arise from a common biosynthetic pathway, giving rise to the common intermediate clavaminic acid. Genes encoding enzymes for the early shared part of the pathway include ceaS2, bls2, pah2, and cas2, which are also present in the paralog cluster, where they are designated ceaS1, bls1, pah1, and cas1 (61). Interestingly, argR mutation caused opposite effects on the expression level of CA and the paralog gene clusters: the former was overexpressed, while the latter was downregulated. These may be part of the reason for the observed lower CA production phenotype. The CA production level in S. clavuligerus is influenced by many other factors, among them precursor supplies and the expression levels of the competing biosynthetic pathways, such as the cephamycin C and 5S clavam pathways. Therefore, although CZR could have overproduced arginine and overexpressed CA biosynthetic genes, due to the reduced expression of the paralog gene cluster and the negative effects of overexpressed
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TABLE 2 Proteins identified as differently expressed in CZR relative to NP1
Spot IDa
Protein descriptionb
Gene locusc
Overexpressed proteins 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Argininosuccinate synthetase N-Acetyl-gamma-glutamyl-phosphate reductase Carboxyethylarginine synthase Proclavaminate amidino hydrolase Carboxyethyl-arginine beta-lactam-synthase Glutamate N-acetyltransferase 2 Acyl-CoA dehydrogenase domain-containing protein Glucose-methanol-choline oxidoreductase Lantibiotic biosynthesis protein Globin Thioredoxin-disulfide reductase Cysteine synthase Thiosulfate sulfurtransferase Thioredoxin Thiamine S protein S-Adenosyl-L-homocysteine hydrolase DsbA oxidoreductase Acetyl-CoA synthetase Acyl-CoA dehydrogenase Cephalosporin hydroxylase Deacetoxycephalosporin C synthetase Pyrimidine regulatory protein UDP-glucose 4-epimerase Bromoperoxidase-catalase
SSCG_05695 SSCG_05690 SSCG_00150 SSCG_00152 SSCG_00151 SSCG_00154 SSCG_03485 SSCG_03487 SSCG_03489 SSCG_03490 SSCG_03492 SSCG_02363 SSCG_01126 SSCG_04064 SSCG_02362 SSCG_04409 SSCG_05980 SSCG_04276 SSCG_07331 SSCG_00138 SSCG_00134 SSCG_03803 SSCG_00066 SSCG_01385
Underexpressed proteins 25 26 27 28 29 30 31 32 33 34 35 36 37
Carboxyethyl arginine synthase isoenzyme 1 Conserved hypothetical protein Malate dehydrogenase Serine hydroxymethyltransferase BldKD YjgF family regulator Beta-lactamase domain-containing protein Guanylate kinase Bacterial luciferase domain-containing protein O-Acetylhomoserine aminocarboxypropyltransferase Protease Beta-ketoadipyl CoA thiolase Methylenetetrahydromethanopterin reductase
SSCG_00629 SSCG_06975 SSCG_05972 SSCG_00627 SSCG_02719 SSCG_00626 SSCG_00563 SSCG_03810 SSCG_08125 SSCG_06530 SSCG_05363 SSCG_06569 SSCG_00780
a b c
No. of peptides detected by MS-MS
Gene name
NCBI accession no.
Theoretical Mr
Theoretical pI
MASCOT score
Sequence coverage (%)
argH argC ceaS2 pah2 bls2 oat2 holB holD holF holG holI cysM rhlA
gi:254393199 gi:254393193 gi:254387584 gi:254387586 gi:254387585 gi:254387588 gi:254390828 gi:254390830 gi:254390832 gi:254390833 gi:254390835 gi:254389812 gi:254388565 gi:254391438 gi:254389811 gi:254392040 gi:254393490 gi:254391696 gi:254392359 gi:254387571 gi:254387568 gi:254391342 gi:254387500 gi:254388967
43,536 35,652 61,040 33,494 54,616 41,810 42,720 59,338 21,323 17,779 32,572 33,844 31,710 11,899 10,096 51,809 23,671 71,277 42,153 27,738 34,933 20,734 35,355 54,190
4.88 5.6 5.04 5.69 5.92 4.73 6.16 5.8 5.8 5.46 5.6 5.64 4.68 5.01 4.47 4.94 4.7 5.16 5.52 4.8 5.14 6.32 5.58 5.53
420 309 398 614 264 428 209 573 475 181 330 536 661 346 454 533 340 480 616 469 239 199 222 592
32 54 45 65 42 27 46 72 82 49 70 68 50 78 63 38 62 50 73 81 46 74 56 48
5 5 5 6 4 5 3 6 5 5 5 6 6 4 6 5 5 6 6 4 5 3 3 6
gi:254388064 gi:294813756 gi:254393481 gi:254388063 gi:254390253 gi:254388062 gi:254387999 gi:254391349 gi:294817230 gi:294816384 gi:254392867 gi:294816329 gi:254388217
59,381 16,923 34,622 42,014 38,100 13,498 70,690 11,498 36,947 27,654 20,497 40,945 38,232
4.8 5.26 5.1 5.81 5.56 4.63 4.9 10.4 10.6 6.92 5.11 5.9 5.34
423 305 639 217 242 408 644 306 532 111 230 456 212
50 71 68 38 34 75 56 83 51 27 58 76 27
5 4 4 4 4 4 3 3 5 2 5 6 5
acs cmcI cefE pyrR tunF
ceaS1 cvm2 orfA orfB
Spot ID numbers correspond to the numbers in the 2D images in Fig. 6. The functional descriptions of identified proteins are based on the genome annotation of S. clavuligerus ATCC 27064 of the Broad Institute. Gene loci were obtained from the genome of S. clavuligerus ATCC 27064 at http://www.broadinstitute.org/ and NCBI.
oat2 and cephamycin C pathway genes, the overall outcome is that CA production is reduced. The overexpression of the early enzymes in CA biosynthesis and the reduced expression of enzymes in 5S clavam biosynthesis could lead to the accumulation of an unknown intermediate of CA or 5S clavams. This may be the signal to induce holomycin production in CZR. Thus, CZR is a useful starting point to identify the signal(s) responsible for the induction of holomycin production and to produce a large amount of holomycin. ACKNOWLEDGMENTS This work was supported by Ministry of Science and Technology of China Grant 2009CB118905. We gratefully acknowledge Qian Wang for her assistance in protein identification and data analyses.
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