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Characterization of virginiamycin S biosynthetic genes from Streptomyces virginiae
Gene 286 (2002) 283–290 www.elsevier.com/locate/gene
Characterization of virginiamycin S biosynthetic genes from Streptomyces virginiae Wises Namwat a, Yuji Kamioka a, Hiroshi Kinoshita a, Yasuhiro Yamada b, Takuya Nihira a,* a
b
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Applied Biological Science, Faculty of Engineering, Fukuyama University, 1 Gakuenmachi, Fukuyama, Hiroshima 729-0292, Japan Received 17 September 2001; received in revised form 27 December 2001; accepted 21 January 2002 Received by A.M. Campbell
Abstract Streptomyces virginiae produces g-butyrolactone autoregulators (virginiae butanolide, VB), which control the biosynthesis of virginiamycin M1 and S. A 6.3-kb region downstream of the virginiamycin S (VS)-resistance operon in S. virginiae was sequenced, and four plausible open reading frames (ORFs) (visA, 1,260 bp; visB, 1,656 bp; visC, 888 bp; visD, 1209 bp) were identified. Homology analysis revealed significant similarities with enzymes involved in the biosynthesis of cyclopeptolide antibiotics: VisA (53% identity, 65% similarity) to l-lysine 2-aminotransferase (NikC) of nikkomycin D biosynthesis, VisB (66% identity, 72% similarity) to 3-hydroxypicolinic acid:AMP ligase of pristinamycin I biosynthesis, VisC (48% identity, 59% similarity) to lysine cyclodeaminase of ascomycin biosynthesis, and VisD (43% identity, 56% similarity) to erythromycin C-22 hydroxylase of erythromycin biosynthesis. Northern blotting as well as high-resolution S1 analysis of the ORFs revealed that they were transcribed as two bicistronic transcripts, namely 3.0-kb visB-visA and another 2.7-kb visCvisD transcript, with promoters locating upstream of visB and visC, respectively. Transcription of the two operons was observed only 1 h after the VB production, which was 2 h before the virginiamycin production. Furthermore, prompt induction of the transcription was observed as a result of external VB addition, suggesting that the expression of the two operons was under the control of VB. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Virginiae butanolide; Peptide synthethase; l-lysine 2-aminotransferase; AMP ligase; Lysine cyclodeaminase; Cytochrome P450 hydroxylase
Abbreviations: aa, amino acid; ACP, acyl carrier protein; barA, VB receptor gene in Streptomyces virginiae; BlmS, an enzyme for bluensomycin biosynthesis from Streptomyces bluensis; DegT, pleiotropic regulatory protein of Mathanobacterium thermoautotrophicum; DhbE, 2,3-dihydroxybenzoate:AMP ligase in siderophore biosynthesis of Bacillus subtilis; EntE, dihydroxybenzoic acid-activating enzyme in enterbactin biosynthesis of Escherichia coli; EryK, an erythromycin C-12 hydroxylase in erythromycin biosynthesis of Saccharopolyspora erythraea; FkbL, lysine cyclodeaminase in ascomycin biosynthesis of Streptomyces hygroscopicus var. ascomyceticus; HPA, hydroxypicolinic acid; NikC, a l-lysine 2-aminotransferase in nikkomycin D biosynthesis of Streptomyces tendae; PPS, peptide synthetase; RapL, lysine cyclodeaminase in rapamycin biosynthesis of S. hygroscopicus; SnbA, 3-hydroxypicolinic acid:AMP ligase in pristinamycin I biosynthesis; SpsC, protein for spore coat polysaccharide biosynthesis from B. subtilis; StrS, an enzyme for N-methyl-l-glucosamine biosynthesis from Streptomyces glaucescens; varR, a regulatory gene for VS resistance gene; VB, an autoregulator virginiae butanolide from S. virginiae; visA, homolog of l-lysine 2-aminotransferase gene in nikkomycin D biosynthesis; visB, homolog of 3-hydroxypicolinic acid:AMP ligase gene in pristinamycin I biosynthesis; visC, homolog of lysine cyclodeaminase gene in rapamycin biosynthesis; visD, homolog of cytochrome P450 hydroxylase gene in erythromycin biosynthesis; VS, a cyclohexadepsipeptide antibiotic virginiamycin S produced by S. virginiae; VM1, a macrolide antibiotic virginiamycin M1; YbtE, salicyl:AMP ligase in yersiniabactin biosynthesis of Yersinia pestis * Corresponding author. Tel.: 181-6-6879-7433; fax: 181-6-6879-7432. E-mail address: [email protected] (T. Nihira).
1. Introduction Virginiamycin S (VS), a cyclohexadepsipeptide antibiotic produced by Streptomyces virginiae, belongs to a virginiamycin family group A. VS is synthesized in vivo by stepwise condensation of seven amino-acid precursors: d-a-aminobutyric acid, N-methyl-l-phenylalanine, l-4-oxopipecolic acid, 3-hydroxypicolinic acid, l-phenylglycine derived from phenylalanine, l-threonine and l-proline (Paris et al., 1990; Yamada et al., 1997). In S. virginiae, VS is coproduced with a macrolide antibiotic virginiamycin M1 (VM1), a member of virginiamycin family group B, with which VS shows strong synergistic bactericidal activity against a wide range of gram-positive bacteria (Cocito, 1979). VS, like many other secondary metabolites of peptidyl nature, has been postulated to be synthesized by large multifunctional enzymes called non-ribosomal peptide synthetases (NRPSs) (Kleinkauf and von Dohren, 1996). In the NRPS system, each amino acid is activated as an aminoacyl adenylate and is linked to the enzyme as a thioester with a phosphopantetheinyl group. An elongation reaction then occurs by transferring the activated carboxyl to the amino
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00424-9
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group in the next amino acid, thus executing N-to-C stepwise condensation. NRPSs contain several modules on a single polypeptide. Each of these modules can catalyze activation, condensation and a modification reaction specific to one kind of amino or hydroxy acid (Turgay et al., 1992). Each module is usually composed of an activation domain (500 aa), an acyl carrier protein domain (80 aa), and an elongation domain (350 aa), in addition to an occasional N-methylation domain (350 aa), epimerization domain (500 aa), and/or C-terminal thioesterase domain. The virginiamycins biosynthesis in S. virginiae is triggered by nM concentrations of virginiae butanolide (VB), a representative g-butyrolactone autoregulator of streptomycetes (Yamada et al., 1997). A signal cascade that regulates the virginiamycin biosynthesis starts from the binding of VB to the specific VB receptor, BarA (Kinoshita et al., 1997; Nakano et al., 1998). A 10-kb region containing the barA gene is judged to be a regulator-island consisting of 6 regulatory genes governing the virginiamycin biosynthesis and virginiamycin resistance. To learn more about the genes involved in virginiamycin production, a 6.5-kb region downstream of the regulator-island was cloned, and four plausible VS biosynthetic genes were identified as being under the control of VB. 2. Materials and methods 2.1. Strains, growth conditions, and plasmids S. virginiae (strain MAFF 10-06014; National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba, Japan) was grown at 288C as described previously. VB-C6 was added after 7 h of cultivation to a final concentration of 300 nM (Kinoshita et al., 1997). Escherichia coli DH5a strain (Hanahan, 1983) and pUC19 were used for the genetic manipulation in E. coli, and for preparing templates for DNA sequencing. DNA manipulation in E. coli was performed as described by Sambrook and Fritsch (1989). 2.2. Chemicals All chemicals were of reagent or high-performance liquid chromatography grade and were purchased from either Nacalai Tesque (Osaka, Japan), Takara Shuzo (Shiga, Japan) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The RNA ladder was obtained from GIBCO BRL (Gaithersburg, MD). a-[ 32P]dCTP was purchased from ICN Biomedicals Inc. (Costa Mesa, CA). Virginiamycin M1 and S were purified by a previously described method (Lee et al., 1998). 2.3. DNA sequencing and sequence analysis The nucleotide sequence was determined for both strands using an ALF red DNA sequencer (Amersham Pharmacia Biotech., Tokyo, Japan). Sequencing reactions were carried
out with a Thermo Sequencing Kit (Amersham Pharmacia Biotech.) according to the manufacturer’s instructions. Homology searches were carried out using the programs BLAST (Altschul et al., 1990). The nucleotide sequence data reported in this paper has been deposited in to the GenBank/DDBJ data bank with the accession number AB072568. 2.4. RNA preparation and Northern blot analysis Total RNA was isolated by the method of Hopwood et al. (1985), and was quantified by absorbance at 260 nm. RNA (10 mg per lane) was electrophoresed on a 1.2% agarose gel and transferred to Hybond-N 1 (Amersham Pharmacia Biotech.) according to the manufacturer’s recommendations. Hybridization was carried out at 658C for 1 h in Rapid-hyb buffer (Amersham Pharmacia Biotech.), followed by washing of the blot three times at 508C for 10 min with 2 £ SSC (1 £ SSC contains 0.015 M sodium citrate and 0.15 M NaCl pH 7.7) containing 0.1% sodium dodecyl sulfate. The SalI-SalI, AgeI-BamHI, AgeI-NotI and SalI-SalI fragments were used as specific probes for visA, visB, visC and visD, respectively (Fig. 1). The DNA fragments were labeled with a-[ 32P]dCTP by using the Random Primer DNA Labeling Kit (version 2,Takara Shuzo Co.) according to protocols supplied by the manufacturer. 2.5. Transcriptional start sites determination The visB-visA transcriptional start site was elucidated by S1 nuclease mapping with a 338-bp probe generated by PCR with pSV401 as a template using 32P-5 0 end labeled primer 5 0 -CGGTAGCGCTCGGCGGCATCAGGGG-3 0 and the unlabeled primer 5 0 -CGCTCTCCACAGC CCGTGTCACGTCGC-3 0 (complementary to nucleotides 150 to 125 or to nucleotides 2288 to 2257 relative to the putative visB start codon). pVS401 was constructed by inserting a 1708bp BamHI-SalI fragment containing the intergenic region between visB and visC (Fig. 1) in pUC19. For the S1 nuclease reaction (total volume of 30 ml), 30 mg of S. virginiae RNA from the 12-h cell was hybridized to the 32P-end labeled probe in sodium trichloroacetic acid buffer (3 M NaTCA in 40 mM PIPES and 1 mM EDTA pH 7) at 658C in a water bath for 15 min, after which the temperature setting was allowed to fall slowly to 458C, where it was maintained for 15 h. The hybridized probe was treated for 1 h at 378C with 300 ml of S1 nuclease digestion solution containing 75 units of S1 nuclease (Takara), 280 mM NaCl, 30 mM sodium acetate pH 4.4, 4.5 mM zinc acetate and 20 mg/ml partially cleaved denatured non-homologous DNA. The reaction was terminated by adding 75 ml termination solution (2.5 M ammonium acetate and 50 mM EDTA). Primer extension analysis was employed to determine the visC-visD transcriptional start site. The primer 5 0 CGCTCTCCACAGCCCGTGTCACGTCGC-3 0 (complementary to nucleotides 152 to 126 relative to the putative visC start codon) was 5 0 -end labeled with 32P as described
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Fig. 1. (A) Gene organization in the 6.3-kb SphI-SalI region downstream of the varS-varR operon in S. virginiae. Probes used for Northern blot analysis are indicated by banded boxes. #1: visA probe (SalI-SalI fragment). #2: visB probe (AgeI-BamHI fragment). #3: visC probe (AgeI-NotI fragment). #4: visD probe (SalI-SalI fragment). (B) Frame analysis of the 6.3-kb SphI-SalI region.
by Sambrook and Fritsch (1989). The probe was annealed to S. virginiae RNA from the 12-h cells, and was reverse-transcribed by Rous-associated virus 2 (RAV-2) reverse transcriptase (Takara). Each 32P-labeled primer was used for making a sequence ladder with a BcaBEST Dideoxy Sequencing Kit (Takara) using the pVS401 as a template. The ladder, the S1 nuclease mapping product, and the primer-extended product were separated on a 6% polyacrylamide-8 M urea gel.
3. Results 3.1. Sequence of the varR downstream region and identification of the visA, visB, visC and visD gene To search for genes involved in virginiamycin S biosynthesis, the nucleotide sequence was determined for a 6.3-kb region downstream of a repressor gene (varR) of a VS transporter gene (varS) (Namwat et al., 2001). Four plausible open reading frames (ORFs) with typical Streptomyces codon usage (Bibb et al., 1984; Wright and Bibb, 1996) were identified and designated as visA, visB, visC and visD (Fig. 1). Homology analysis of the deduced products showed significant similarities with enzymes
involved in the biosynthesis of several cyclopeptolide antibiotics, suggesting that visA, visB, visC and visD were all involved in the biosynthesis of virginiamycin S. 3.1.1. visA encoded l-lysine 2-aminotransferase The first ORF (visA, 1,260 bp) is located 287 bp downstream of varR in the opposite direction of the transcription (Fig. 1). The absence of typical promoter sequences and the presence of a palindromic sequence (DG ¼ 243.1 Kcal/ mol) followed by 6T residues after the TGA stop codon suggested that visA was transcribed with its upstream gene(s). The deduced VisA protein (419 amino acids, Mr of 45,546, pI of 6.02) was highly similar to pyridoxal-phosphate dependent dehydrases and aminotransferases. Its highest homology was with NikC (53% identity, 65% similarity), a l-lysine 2-aminotransferase involved in nikkomycin D biosynthesis of Streptomyces tendae (Bruntner and Bormann, 1998). Moderate homologies of 29–36% identity were observed with StrS involved in the N-methyl-l-glucosamine biosynthesis of Streptomyces glaucescens (Accession number CAA07383), BlmS involved in the bluensomycin biosynthesis of Streptomyces bluensis (Accession number AAD28515), and SpsC involved in the biosynthesis of spore coat polysaccharide of Bacillus subtilis (Accession number P39623). Lys residue has been postu-
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lated as a pyridoxal-phosphate binding site in this group of aminotransferases, and is conserved as Lys-190 in VisA. 3.1.2. visB encoded 3-hydroxypicolinic acid:AMP ligase The second ORF (visB, 1,656 bp) is located upstream of visA with the same orientation of transcription, and the 3 0 end 4 bp of visB overlaps with the 5 0 -end of visA (Fig. 1). A typical Shine-Dalgarno sequence (AAGGA) was found six nucleotides upstream of the visB start codon. The deduced VisB protein (551 amino acids, Mr of 59,758, pI of 5.48) showed its highest homology with SnbA (66% identity, 72% similarity), a 3-hydroxypicolinic acid:AMP ligase involved in pristinamycin I biosynthesis in Streptomyces pristinaespiralis (de Crecy-Lagard et al., 1997). Homologies of 41– 50% identity were observed with the following enzymes: DhbE, a 2,3-dihydroxybenzoate:AMP ligase involved in siderophore biosynthesis in B. subtilis (Accession number P40871); EntE, a dihydroxybenzoic acid-activating enzyme involved in enterobactin biosynthesis in E. coli (Accession number 226890); and YbtE, a salicyl:AMP ligase involved in yersiniabactin biosynthesis in Yersinia pestis (Accession number AAC69591). Further sequence analysis indicated that VisB belongs to a superfamily of adenylate-forming enzymes, in particular to the subfamily composed of coumarate-CoA ligases, acetyl-CoA synthetases, and firefly luciferase (de Crecy-Lagard et al., 1997). The main characteristic of this subfamily is that the members contain the conserved sequences for ATP binding and amino acid adenylation, but lack the acyl carrier protein (ACP) domain. VisB showed a good match to the proposed consensus sequences for cores A, C, and E–I, but deviated substantially from those for cores B and D (de Crecy-Lagard et al., 1997). The amino-acid sequence 192-FLLSGGTTALPK-203 located in core sequence C showed a high degree of similarity to the consensus ATP binding site [FLIVMY]-xx[STG]-[STAG]-[G-ST]-[STEI]-[SG]-x-[PASLIVM]-[KR] (Prosite: PDOC00427). 3.1.3. visC encoded lysine cyclodeaminase The third ORF (visC, 888 bp) is located 236 bp upstream of visB with the opposite direction of transcription (Fig. 1). The deduced VisC protein (295 amino acids, Mr of 31,366, a pI of 5.47) showed a high degree of homology to FkbL (48% identity, 59% similarity), a lysine cyclodeaminase involved in the biosynthesis of polyketide ascomycin from Streptomyces hygroscopicus var. ascomyceticus (Wu et al., 2000) and to RapL (47% identity, 58% similarity), also a lysine cyclodeaminase involved in the biosynthesis of polyketide rapamycin from S. hygroscopicus (Khaw et al., 1998). 3.1.4. visD encoded cytochrome P450 hydroxylase The fourth ORF (visD, 1,209 bp) is located 347 bp downstream of visC with the same direction of transcription (Fig. 1). A typical Shine-Dalgarno sequence (AAGGA) was found seven nucleotides upstream of the visD start codon. The presence of an inverted repeat in the 3 0 region of visD
(DG ¼ 219.2 kcal/mol) as a plausible transcriptional terminator was observed. The deduced VisD protein (402 amino acids, Mr of 44,631, a pI of 4.98) showed a high degree of homology to EryK (43% identity, 56% similarity), an erythromycin C-12 hydroxylase involved in the biosynthesis of erythromycin from Saccharopolyspora erythraea (Stassi et al., 1993) and to cytochrome P450 tylI (34% identity, 47% similarity), also a hydroxylase involved in the biosynthesis of tylosin from Streptomyces fradiae (Accession number S49051). Multiple alignment of the deduced VisD protein with the members of the cytochrome P450 monooxygenase revealed that significant identity exists at a carboxyl-terminal motif [ 342AFGHGIHYCLGGPLARL 359E] and at a motif from residues 236–244 [ 236LLLMAGHV 244S]: the Cys residue in the former motif has been identified to be the fifth axial ligand for the heme prosthetic group, and the latter motif has been considered to be the O2 binding pocket (Stassi et al., 1993). 3.2. Transcriptional analysis of the visA, visB, visC and visD gene To clarify the expression pattern of these genes during the cultivation of S. virginiae, Northern blot analysis was performed using visA, visB, visC and visD as probes toward RNA samples from 7- to 14-h cultivation (see Fig. 1 for the position of probes). No transcription was observed until 10 h of cultivation, but at 12 h of cultivation, i.e. 1 h after VB production (at 11 h) or 2 h before virginiamycin production (14 h) (Lee et al., 1998), clear transcriptions for all the visA, visB, visC and visD genes were detected simultaneously (Fig. 2). The transcription should be regarded as highly temporal, because all the transcripts became barely detectable at 14 h of cultivation. The visA transcription appeared as 3.0- and 1.3kb transcripts, while the visC transcription appeared as 2.7and 1.3-kb transcripts. Because the 3.0-kb visA-transcript was far larger than visA alone (1260 bp) and because a visB probe, rather than the varR probe covering the visA-downstream region, hybridized to the same band (Fig. 2B), it was concluded that visA was transcribed mainly as the bicistronic visB-visA transcript. This conclusion agreed well with the lack of typical promoter sequences in the 5 0 -untranslated region of visA and the presence of a plausible transcriptional terminator in the 3 0 -region of visA (3.1.1 and 3.1.2). Likewise, visC was concluded to be co-transcribed with the downstream visD as a 2.7-kb transcript, because the same 2.7-kb band was detected with a visD probe (Fig. 2D), although the transcript detected by the visD probe was rather degraded. The 1.3-kb band seen below the bicistronic transcript with the visA and the visC probe, respectively, seem to be artifacts arising from the vast excess of 16S rRNA which saturates the membrane just above the signal and out-compete the binding of degraded bicistronic transcripts. The effect of VB on the transcription of visA, visB, visC and visD was also investigated by adding external VB at 7 h of cultivation (Figs. 2A,C). Each of the two transcripts was
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Fig. 2. Northern blot analysis on visA (A); visB (B); visC (C); and visD (D) during cultivation of S. virginiae. Total RNA was extracted from cells cultivated at 288C for the indicated period. Left half of A and C: cultivation without VB addition. Under the experimental conditions employed the production of VB and virginiamycin started at 11 and 14 h of cultivation, respectively, as shown by arrows above the lanes. Right half of A and C: cultivation with VB added at 7 h. To determine the effect of VB on the transcription of these genes, VB was added to the culture at 7 h to a final concentration of 64 ng/ml. B and D: cultivation without VB addition. A: visA probe. B: visB probe. C: visC probe. D: visD probe.
detected at 8 h of cultivation (only 1 h after VB addition), suggesting that the transcription of all four genes is under direct control of the VB-BarA system.
priate positions, respectively (Bourn and Babb, 1995), whereas -35 elements were not found. These promoters seemed to be recognized by alternative sigma factors.
3.3. Transcriptional start sites of the visB-visA and visCvisD transcript
4. Discussion
S1 nuclease mapping or primer extension analysis was carried out with RNA from 12-h culture to localize the transcriptional start site (tss) of visB-visA and visC-visD transcripts, respectively (Fig. 3). The visB-visA transcript had a single tss at A situated 64-bp upstream from the ATG initiation codon of visB. The tss for visC-visD was determined to be at C, which itself was situated 51-bp upstream from the ATG initiation codon of visC. Both the promoters have the plausible 210 sequence at the appro-
In this study, four genes (visA, visB, visC and visD) plausibly involved in VS biosynthesis were identified about 3.5 kb downstream of barA encoding a VB-specific receptor. Both the visA and visC products were highly homologous to enzymes that cyclize l-lysine: VisA homologous to llysine 2-aminotransferase to form 1-piperideine 2carboxylic acid (Bruntner and Bormann, 1998) and VisC to l-lysine cyclodeaminase important for the production of l-pipecolic acid (Khaw et al., 1998). Because feeding
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Fig. 3. Transcriptional start site analysis on (A) the visB-visA transcript; and (B) the visC-visD transcript. Lanes A, C, G, and T, DNA sequencing ladder generated with the corresponding primer; lane S, S1 nuclease-treated sample; lane P, sample from primer extension reaction. The S1 nuclease reaction and the primer extension reaction were carried out with total RNA prepared from 12-h culture of S. virginiae. The transcriptional start sites are indicated by arrows and by white letters in black boxes. (C) The intergenic region between the start codon of visB and visC. Transcriptional start sites, white letters in black boxes; start codons, double underlining; Shine-Dalgano (SD) sequence, wavy underlining; 210 elements, single underlining. The indicated nucleotide positions are relative to the corresponding transcriptional start sites.
experiments have revealed that two molecules of l-lysine are incorporated into VS (one as 3-hydroxypicolinic acid and another as 4-oxopipecolic acid moiety) (Reed et al., 1989), it is reasonable to assume that both the visA and visC genes are involved in converting l-lysine into the necessary precursors for VS biosynthesis, although several more enzymatic reactions should apparently take place to convert 1-piperideine 2-carboxylic acid into the final precursors. Although the detailed pathway of the precursor biosynthesis is not known at present, visA can be postulated to participate in providing 3-hydroxypicolinic acid, and visC in providing pipecolic acid (Fig. 4). The visB product was
highly homologous to 3-hydroxypicolinic acid:AMP ligase, which activates 3-hydroxypicolinic acid to an adenylated intermediate (Fig. 4). The apparent operon structure of visB with visA provides further support that both the visA and visB genes are involved in providing 3-hydroxypicolinic acid moiety in VS. On the other hand, the visD product was highly homologous to cytochrome P450 monooxygenases which have been reported to participate in sitespecific oxidation of aglycon moieties of macrolide antibiotics. Because pipecolic acid moiety has been proposed to be oxidized after the formation of macrocyclic ring (Stassi et al., 1993), the visD product can be postulated to participate
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Fig. 4. The proposed function of VisA, VisB, VisC and VisD in the biosynthesis of virginiamycin S. Me: methyl. 3-HPA: 3-hydroxypicolinic acid. l-Thr: lthreonine, d-AmBu: d-a-aminobutyric acid. l-Pro: l-proline. l-MePhe: N-methyl-l-phenylalanine. l-PheGly: l-phenylglycine.
in the oxidation of pipecolic acid moiety to form 4-oxopipecolic acid moiety. Northern blot analysis of these genes also supported their involvement in VS biosynthesis, since the transcription of all four genes occurred simultaneously only 2 h prior to the production of VS. The rapid emergence of the visB-visA and the visC-visD transcripts by the internal or external VB suggests direct transcriptional regulation by the VB-BarA system (Kinoshita et al., 1997), although no obvious BarA binding site (BARE) was detected in the intergenic region between visB and visC. Detailed analyzes of the initiation and rapid termination of the transcription are underway in our laboratory. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search toll. J. Mol. Biol. 215, 403–410.
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Characterization of virginiamycin S biosynthetic genes from Streptomyces virginiae Wises Namwat a, Yuji Kamioka a, Hiroshi Kinoshita a, Yasuhiro Yamada b, Takuya Nihira a,* a
b
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Applied Biological Science, Faculty of Engineering, Fukuyama University, 1 Gakuenmachi, Fukuyama, Hiroshima 729-0292, Japan Received 17 September 2001; received in revised form 27 December 2001; accepted 21 January 2002 Received by A.M. Campbell
Abstract Streptomyces virginiae produces g-butyrolactone autoregulators (virginiae butanolide, VB), which control the biosynthesis of virginiamycin M1 and S. A 6.3-kb region downstream of the virginiamycin S (VS)-resistance operon in S. virginiae was sequenced, and four plausible open reading frames (ORFs) (visA, 1,260 bp; visB, 1,656 bp; visC, 888 bp; visD, 1209 bp) were identified. Homology analysis revealed significant similarities with enzymes involved in the biosynthesis of cyclopeptolide antibiotics: VisA (53% identity, 65% similarity) to l-lysine 2-aminotransferase (NikC) of nikkomycin D biosynthesis, VisB (66% identity, 72% similarity) to 3-hydroxypicolinic acid:AMP ligase of pristinamycin I biosynthesis, VisC (48% identity, 59% similarity) to lysine cyclodeaminase of ascomycin biosynthesis, and VisD (43% identity, 56% similarity) to erythromycin C-22 hydroxylase of erythromycin biosynthesis. Northern blotting as well as high-resolution S1 analysis of the ORFs revealed that they were transcribed as two bicistronic transcripts, namely 3.0-kb visB-visA and another 2.7-kb visCvisD transcript, with promoters locating upstream of visB and visC, respectively. Transcription of the two operons was observed only 1 h after the VB production, which was 2 h before the virginiamycin production. Furthermore, prompt induction of the transcription was observed as a result of external VB addition, suggesting that the expression of the two operons was under the control of VB. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Virginiae butanolide; Peptide synthethase; l-lysine 2-aminotransferase; AMP ligase; Lysine cyclodeaminase; Cytochrome P450 hydroxylase
Abbreviations: aa, amino acid; ACP, acyl carrier protein; barA, VB receptor gene in Streptomyces virginiae; BlmS, an enzyme for bluensomycin biosynthesis from Streptomyces bluensis; DegT, pleiotropic regulatory protein of Mathanobacterium thermoautotrophicum; DhbE, 2,3-dihydroxybenzoate:AMP ligase in siderophore biosynthesis of Bacillus subtilis; EntE, dihydroxybenzoic acid-activating enzyme in enterbactin biosynthesis of Escherichia coli; EryK, an erythromycin C-12 hydroxylase in erythromycin biosynthesis of Saccharopolyspora erythraea; FkbL, lysine cyclodeaminase in ascomycin biosynthesis of Streptomyces hygroscopicus var. ascomyceticus; HPA, hydroxypicolinic acid; NikC, a l-lysine 2-aminotransferase in nikkomycin D biosynthesis of Streptomyces tendae; PPS, peptide synthetase; RapL, lysine cyclodeaminase in rapamycin biosynthesis of S. hygroscopicus; SnbA, 3-hydroxypicolinic acid:AMP ligase in pristinamycin I biosynthesis; SpsC, protein for spore coat polysaccharide biosynthesis from B. subtilis; StrS, an enzyme for N-methyl-l-glucosamine biosynthesis from Streptomyces glaucescens; varR, a regulatory gene for VS resistance gene; VB, an autoregulator virginiae butanolide from S. virginiae; visA, homolog of l-lysine 2-aminotransferase gene in nikkomycin D biosynthesis; visB, homolog of 3-hydroxypicolinic acid:AMP ligase gene in pristinamycin I biosynthesis; visC, homolog of lysine cyclodeaminase gene in rapamycin biosynthesis; visD, homolog of cytochrome P450 hydroxylase gene in erythromycin biosynthesis; VS, a cyclohexadepsipeptide antibiotic virginiamycin S produced by S. virginiae; VM1, a macrolide antibiotic virginiamycin M1; YbtE, salicyl:AMP ligase in yersiniabactin biosynthesis of Yersinia pestis * Corresponding author. Tel.: 181-6-6879-7433; fax: 181-6-6879-7432. E-mail address: [email protected] (T. Nihira).
1. Introduction Virginiamycin S (VS), a cyclohexadepsipeptide antibiotic produced by Streptomyces virginiae, belongs to a virginiamycin family group A. VS is synthesized in vivo by stepwise condensation of seven amino-acid precursors: d-a-aminobutyric acid, N-methyl-l-phenylalanine, l-4-oxopipecolic acid, 3-hydroxypicolinic acid, l-phenylglycine derived from phenylalanine, l-threonine and l-proline (Paris et al., 1990; Yamada et al., 1997). In S. virginiae, VS is coproduced with a macrolide antibiotic virginiamycin M1 (VM1), a member of virginiamycin family group B, with which VS shows strong synergistic bactericidal activity against a wide range of gram-positive bacteria (Cocito, 1979). VS, like many other secondary metabolites of peptidyl nature, has been postulated to be synthesized by large multifunctional enzymes called non-ribosomal peptide synthetases (NRPSs) (Kleinkauf and von Dohren, 1996). In the NRPS system, each amino acid is activated as an aminoacyl adenylate and is linked to the enzyme as a thioester with a phosphopantetheinyl group. An elongation reaction then occurs by transferring the activated carboxyl to the amino
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00424-9
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group in the next amino acid, thus executing N-to-C stepwise condensation. NRPSs contain several modules on a single polypeptide. Each of these modules can catalyze activation, condensation and a modification reaction specific to one kind of amino or hydroxy acid (Turgay et al., 1992). Each module is usually composed of an activation domain (500 aa), an acyl carrier protein domain (80 aa), and an elongation domain (350 aa), in addition to an occasional N-methylation domain (350 aa), epimerization domain (500 aa), and/or C-terminal thioesterase domain. The virginiamycins biosynthesis in S. virginiae is triggered by nM concentrations of virginiae butanolide (VB), a representative g-butyrolactone autoregulator of streptomycetes (Yamada et al., 1997). A signal cascade that regulates the virginiamycin biosynthesis starts from the binding of VB to the specific VB receptor, BarA (Kinoshita et al., 1997; Nakano et al., 1998). A 10-kb region containing the barA gene is judged to be a regulator-island consisting of 6 regulatory genes governing the virginiamycin biosynthesis and virginiamycin resistance. To learn more about the genes involved in virginiamycin production, a 6.5-kb region downstream of the regulator-island was cloned, and four plausible VS biosynthetic genes were identified as being under the control of VB. 2. Materials and methods 2.1. Strains, growth conditions, and plasmids S. virginiae (strain MAFF 10-06014; National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba, Japan) was grown at 288C as described previously. VB-C6 was added after 7 h of cultivation to a final concentration of 300 nM (Kinoshita et al., 1997). Escherichia coli DH5a strain (Hanahan, 1983) and pUC19 were used for the genetic manipulation in E. coli, and for preparing templates for DNA sequencing. DNA manipulation in E. coli was performed as described by Sambrook and Fritsch (1989). 2.2. Chemicals All chemicals were of reagent or high-performance liquid chromatography grade and were purchased from either Nacalai Tesque (Osaka, Japan), Takara Shuzo (Shiga, Japan) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The RNA ladder was obtained from GIBCO BRL (Gaithersburg, MD). a-[ 32P]dCTP was purchased from ICN Biomedicals Inc. (Costa Mesa, CA). Virginiamycin M1 and S were purified by a previously described method (Lee et al., 1998). 2.3. DNA sequencing and sequence analysis The nucleotide sequence was determined for both strands using an ALF red DNA sequencer (Amersham Pharmacia Biotech., Tokyo, Japan). Sequencing reactions were carried
out with a Thermo Sequencing Kit (Amersham Pharmacia Biotech.) according to the manufacturer’s instructions. Homology searches were carried out using the programs BLAST (Altschul et al., 1990). The nucleotide sequence data reported in this paper has been deposited in to the GenBank/DDBJ data bank with the accession number AB072568. 2.4. RNA preparation and Northern blot analysis Total RNA was isolated by the method of Hopwood et al. (1985), and was quantified by absorbance at 260 nm. RNA (10 mg per lane) was electrophoresed on a 1.2% agarose gel and transferred to Hybond-N 1 (Amersham Pharmacia Biotech.) according to the manufacturer’s recommendations. Hybridization was carried out at 658C for 1 h in Rapid-hyb buffer (Amersham Pharmacia Biotech.), followed by washing of the blot three times at 508C for 10 min with 2 £ SSC (1 £ SSC contains 0.015 M sodium citrate and 0.15 M NaCl pH 7.7) containing 0.1% sodium dodecyl sulfate. The SalI-SalI, AgeI-BamHI, AgeI-NotI and SalI-SalI fragments were used as specific probes for visA, visB, visC and visD, respectively (Fig. 1). The DNA fragments were labeled with a-[ 32P]dCTP by using the Random Primer DNA Labeling Kit (version 2,Takara Shuzo Co.) according to protocols supplied by the manufacturer. 2.5. Transcriptional start sites determination The visB-visA transcriptional start site was elucidated by S1 nuclease mapping with a 338-bp probe generated by PCR with pSV401 as a template using 32P-5 0 end labeled primer 5 0 -CGGTAGCGCTCGGCGGCATCAGGGG-3 0 and the unlabeled primer 5 0 -CGCTCTCCACAGC CCGTGTCACGTCGC-3 0 (complementary to nucleotides 150 to 125 or to nucleotides 2288 to 2257 relative to the putative visB start codon). pVS401 was constructed by inserting a 1708bp BamHI-SalI fragment containing the intergenic region between visB and visC (Fig. 1) in pUC19. For the S1 nuclease reaction (total volume of 30 ml), 30 mg of S. virginiae RNA from the 12-h cell was hybridized to the 32P-end labeled probe in sodium trichloroacetic acid buffer (3 M NaTCA in 40 mM PIPES and 1 mM EDTA pH 7) at 658C in a water bath for 15 min, after which the temperature setting was allowed to fall slowly to 458C, where it was maintained for 15 h. The hybridized probe was treated for 1 h at 378C with 300 ml of S1 nuclease digestion solution containing 75 units of S1 nuclease (Takara), 280 mM NaCl, 30 mM sodium acetate pH 4.4, 4.5 mM zinc acetate and 20 mg/ml partially cleaved denatured non-homologous DNA. The reaction was terminated by adding 75 ml termination solution (2.5 M ammonium acetate and 50 mM EDTA). Primer extension analysis was employed to determine the visC-visD transcriptional start site. The primer 5 0 CGCTCTCCACAGCCCGTGTCACGTCGC-3 0 (complementary to nucleotides 152 to 126 relative to the putative visC start codon) was 5 0 -end labeled with 32P as described
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Fig. 1. (A) Gene organization in the 6.3-kb SphI-SalI region downstream of the varS-varR operon in S. virginiae. Probes used for Northern blot analysis are indicated by banded boxes. #1: visA probe (SalI-SalI fragment). #2: visB probe (AgeI-BamHI fragment). #3: visC probe (AgeI-NotI fragment). #4: visD probe (SalI-SalI fragment). (B) Frame analysis of the 6.3-kb SphI-SalI region.
by Sambrook and Fritsch (1989). The probe was annealed to S. virginiae RNA from the 12-h cells, and was reverse-transcribed by Rous-associated virus 2 (RAV-2) reverse transcriptase (Takara). Each 32P-labeled primer was used for making a sequence ladder with a BcaBEST Dideoxy Sequencing Kit (Takara) using the pVS401 as a template. The ladder, the S1 nuclease mapping product, and the primer-extended product were separated on a 6% polyacrylamide-8 M urea gel.
3. Results 3.1. Sequence of the varR downstream region and identification of the visA, visB, visC and visD gene To search for genes involved in virginiamycin S biosynthesis, the nucleotide sequence was determined for a 6.3-kb region downstream of a repressor gene (varR) of a VS transporter gene (varS) (Namwat et al., 2001). Four plausible open reading frames (ORFs) with typical Streptomyces codon usage (Bibb et al., 1984; Wright and Bibb, 1996) were identified and designated as visA, visB, visC and visD (Fig. 1). Homology analysis of the deduced products showed significant similarities with enzymes
involved in the biosynthesis of several cyclopeptolide antibiotics, suggesting that visA, visB, visC and visD were all involved in the biosynthesis of virginiamycin S. 3.1.1. visA encoded l-lysine 2-aminotransferase The first ORF (visA, 1,260 bp) is located 287 bp downstream of varR in the opposite direction of the transcription (Fig. 1). The absence of typical promoter sequences and the presence of a palindromic sequence (DG ¼ 243.1 Kcal/ mol) followed by 6T residues after the TGA stop codon suggested that visA was transcribed with its upstream gene(s). The deduced VisA protein (419 amino acids, Mr of 45,546, pI of 6.02) was highly similar to pyridoxal-phosphate dependent dehydrases and aminotransferases. Its highest homology was with NikC (53% identity, 65% similarity), a l-lysine 2-aminotransferase involved in nikkomycin D biosynthesis of Streptomyces tendae (Bruntner and Bormann, 1998). Moderate homologies of 29–36% identity were observed with StrS involved in the N-methyl-l-glucosamine biosynthesis of Streptomyces glaucescens (Accession number CAA07383), BlmS involved in the bluensomycin biosynthesis of Streptomyces bluensis (Accession number AAD28515), and SpsC involved in the biosynthesis of spore coat polysaccharide of Bacillus subtilis (Accession number P39623). Lys residue has been postu-
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lated as a pyridoxal-phosphate binding site in this group of aminotransferases, and is conserved as Lys-190 in VisA. 3.1.2. visB encoded 3-hydroxypicolinic acid:AMP ligase The second ORF (visB, 1,656 bp) is located upstream of visA with the same orientation of transcription, and the 3 0 end 4 bp of visB overlaps with the 5 0 -end of visA (Fig. 1). A typical Shine-Dalgarno sequence (AAGGA) was found six nucleotides upstream of the visB start codon. The deduced VisB protein (551 amino acids, Mr of 59,758, pI of 5.48) showed its highest homology with SnbA (66% identity, 72% similarity), a 3-hydroxypicolinic acid:AMP ligase involved in pristinamycin I biosynthesis in Streptomyces pristinaespiralis (de Crecy-Lagard et al., 1997). Homologies of 41– 50% identity were observed with the following enzymes: DhbE, a 2,3-dihydroxybenzoate:AMP ligase involved in siderophore biosynthesis in B. subtilis (Accession number P40871); EntE, a dihydroxybenzoic acid-activating enzyme involved in enterobactin biosynthesis in E. coli (Accession number 226890); and YbtE, a salicyl:AMP ligase involved in yersiniabactin biosynthesis in Yersinia pestis (Accession number AAC69591). Further sequence analysis indicated that VisB belongs to a superfamily of adenylate-forming enzymes, in particular to the subfamily composed of coumarate-CoA ligases, acetyl-CoA synthetases, and firefly luciferase (de Crecy-Lagard et al., 1997). The main characteristic of this subfamily is that the members contain the conserved sequences for ATP binding and amino acid adenylation, but lack the acyl carrier protein (ACP) domain. VisB showed a good match to the proposed consensus sequences for cores A, C, and E–I, but deviated substantially from those for cores B and D (de Crecy-Lagard et al., 1997). The amino-acid sequence 192-FLLSGGTTALPK-203 located in core sequence C showed a high degree of similarity to the consensus ATP binding site [FLIVMY]-xx[STG]-[STAG]-[G-ST]-[STEI]-[SG]-x-[PASLIVM]-[KR] (Prosite: PDOC00427). 3.1.3. visC encoded lysine cyclodeaminase The third ORF (visC, 888 bp) is located 236 bp upstream of visB with the opposite direction of transcription (Fig. 1). The deduced VisC protein (295 amino acids, Mr of 31,366, a pI of 5.47) showed a high degree of homology to FkbL (48% identity, 59% similarity), a lysine cyclodeaminase involved in the biosynthesis of polyketide ascomycin from Streptomyces hygroscopicus var. ascomyceticus (Wu et al., 2000) and to RapL (47% identity, 58% similarity), also a lysine cyclodeaminase involved in the biosynthesis of polyketide rapamycin from S. hygroscopicus (Khaw et al., 1998). 3.1.4. visD encoded cytochrome P450 hydroxylase The fourth ORF (visD, 1,209 bp) is located 347 bp downstream of visC with the same direction of transcription (Fig. 1). A typical Shine-Dalgarno sequence (AAGGA) was found seven nucleotides upstream of the visD start codon. The presence of an inverted repeat in the 3 0 region of visD
(DG ¼ 219.2 kcal/mol) as a plausible transcriptional terminator was observed. The deduced VisD protein (402 amino acids, Mr of 44,631, a pI of 4.98) showed a high degree of homology to EryK (43% identity, 56% similarity), an erythromycin C-12 hydroxylase involved in the biosynthesis of erythromycin from Saccharopolyspora erythraea (Stassi et al., 1993) and to cytochrome P450 tylI (34% identity, 47% similarity), also a hydroxylase involved in the biosynthesis of tylosin from Streptomyces fradiae (Accession number S49051). Multiple alignment of the deduced VisD protein with the members of the cytochrome P450 monooxygenase revealed that significant identity exists at a carboxyl-terminal motif [ 342AFGHGIHYCLGGPLARL 359E] and at a motif from residues 236–244 [ 236LLLMAGHV 244S]: the Cys residue in the former motif has been identified to be the fifth axial ligand for the heme prosthetic group, and the latter motif has been considered to be the O2 binding pocket (Stassi et al., 1993). 3.2. Transcriptional analysis of the visA, visB, visC and visD gene To clarify the expression pattern of these genes during the cultivation of S. virginiae, Northern blot analysis was performed using visA, visB, visC and visD as probes toward RNA samples from 7- to 14-h cultivation (see Fig. 1 for the position of probes). No transcription was observed until 10 h of cultivation, but at 12 h of cultivation, i.e. 1 h after VB production (at 11 h) or 2 h before virginiamycin production (14 h) (Lee et al., 1998), clear transcriptions for all the visA, visB, visC and visD genes were detected simultaneously (Fig. 2). The transcription should be regarded as highly temporal, because all the transcripts became barely detectable at 14 h of cultivation. The visA transcription appeared as 3.0- and 1.3kb transcripts, while the visC transcription appeared as 2.7and 1.3-kb transcripts. Because the 3.0-kb visA-transcript was far larger than visA alone (1260 bp) and because a visB probe, rather than the varR probe covering the visA-downstream region, hybridized to the same band (Fig. 2B), it was concluded that visA was transcribed mainly as the bicistronic visB-visA transcript. This conclusion agreed well with the lack of typical promoter sequences in the 5 0 -untranslated region of visA and the presence of a plausible transcriptional terminator in the 3 0 -region of visA (3.1.1 and 3.1.2). Likewise, visC was concluded to be co-transcribed with the downstream visD as a 2.7-kb transcript, because the same 2.7-kb band was detected with a visD probe (Fig. 2D), although the transcript detected by the visD probe was rather degraded. The 1.3-kb band seen below the bicistronic transcript with the visA and the visC probe, respectively, seem to be artifacts arising from the vast excess of 16S rRNA which saturates the membrane just above the signal and out-compete the binding of degraded bicistronic transcripts. The effect of VB on the transcription of visA, visB, visC and visD was also investigated by adding external VB at 7 h of cultivation (Figs. 2A,C). Each of the two transcripts was
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Fig. 2. Northern blot analysis on visA (A); visB (B); visC (C); and visD (D) during cultivation of S. virginiae. Total RNA was extracted from cells cultivated at 288C for the indicated period. Left half of A and C: cultivation without VB addition. Under the experimental conditions employed the production of VB and virginiamycin started at 11 and 14 h of cultivation, respectively, as shown by arrows above the lanes. Right half of A and C: cultivation with VB added at 7 h. To determine the effect of VB on the transcription of these genes, VB was added to the culture at 7 h to a final concentration of 64 ng/ml. B and D: cultivation without VB addition. A: visA probe. B: visB probe. C: visC probe. D: visD probe.
detected at 8 h of cultivation (only 1 h after VB addition), suggesting that the transcription of all four genes is under direct control of the VB-BarA system.
priate positions, respectively (Bourn and Babb, 1995), whereas -35 elements were not found. These promoters seemed to be recognized by alternative sigma factors.
3.3. Transcriptional start sites of the visB-visA and visCvisD transcript
4. Discussion
S1 nuclease mapping or primer extension analysis was carried out with RNA from 12-h culture to localize the transcriptional start site (tss) of visB-visA and visC-visD transcripts, respectively (Fig. 3). The visB-visA transcript had a single tss at A situated 64-bp upstream from the ATG initiation codon of visB. The tss for visC-visD was determined to be at C, which itself was situated 51-bp upstream from the ATG initiation codon of visC. Both the promoters have the plausible 210 sequence at the appro-
In this study, four genes (visA, visB, visC and visD) plausibly involved in VS biosynthesis were identified about 3.5 kb downstream of barA encoding a VB-specific receptor. Both the visA and visC products were highly homologous to enzymes that cyclize l-lysine: VisA homologous to llysine 2-aminotransferase to form 1-piperideine 2carboxylic acid (Bruntner and Bormann, 1998) and VisC to l-lysine cyclodeaminase important for the production of l-pipecolic acid (Khaw et al., 1998). Because feeding
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Fig. 3. Transcriptional start site analysis on (A) the visB-visA transcript; and (B) the visC-visD transcript. Lanes A, C, G, and T, DNA sequencing ladder generated with the corresponding primer; lane S, S1 nuclease-treated sample; lane P, sample from primer extension reaction. The S1 nuclease reaction and the primer extension reaction were carried out with total RNA prepared from 12-h culture of S. virginiae. The transcriptional start sites are indicated by arrows and by white letters in black boxes. (C) The intergenic region between the start codon of visB and visC. Transcriptional start sites, white letters in black boxes; start codons, double underlining; Shine-Dalgano (SD) sequence, wavy underlining; 210 elements, single underlining. The indicated nucleotide positions are relative to the corresponding transcriptional start sites.
experiments have revealed that two molecules of l-lysine are incorporated into VS (one as 3-hydroxypicolinic acid and another as 4-oxopipecolic acid moiety) (Reed et al., 1989), it is reasonable to assume that both the visA and visC genes are involved in converting l-lysine into the necessary precursors for VS biosynthesis, although several more enzymatic reactions should apparently take place to convert 1-piperideine 2-carboxylic acid into the final precursors. Although the detailed pathway of the precursor biosynthesis is not known at present, visA can be postulated to participate in providing 3-hydroxypicolinic acid, and visC in providing pipecolic acid (Fig. 4). The visB product was
highly homologous to 3-hydroxypicolinic acid:AMP ligase, which activates 3-hydroxypicolinic acid to an adenylated intermediate (Fig. 4). The apparent operon structure of visB with visA provides further support that both the visA and visB genes are involved in providing 3-hydroxypicolinic acid moiety in VS. On the other hand, the visD product was highly homologous to cytochrome P450 monooxygenases which have been reported to participate in sitespecific oxidation of aglycon moieties of macrolide antibiotics. Because pipecolic acid moiety has been proposed to be oxidized after the formation of macrocyclic ring (Stassi et al., 1993), the visD product can be postulated to participate
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Fig. 4. The proposed function of VisA, VisB, VisC and VisD in the biosynthesis of virginiamycin S. Me: methyl. 3-HPA: 3-hydroxypicolinic acid. l-Thr: lthreonine, d-AmBu: d-a-aminobutyric acid. l-Pro: l-proline. l-MePhe: N-methyl-l-phenylalanine. l-PheGly: l-phenylglycine.
in the oxidation of pipecolic acid moiety to form 4-oxopipecolic acid moiety. Northern blot analysis of these genes also supported their involvement in VS biosynthesis, since the transcription of all four genes occurred simultaneously only 2 h prior to the production of VS. The rapid emergence of the visB-visA and the visC-visD transcripts by the internal or external VB suggests direct transcriptional regulation by the VB-BarA system (Kinoshita et al., 1997), although no obvious BarA binding site (BARE) was detected in the intergenic region between visB and visC. Detailed analyzes of the initiation and rapid termination of the transcription are underway in our laboratory. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search toll. J. Mol. Biol. 215, 403–410.
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