The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization

N. Magarveya,1, J. Heb,1, K. A. Aidooc,1 and L. C. Vining1

Department of Biology, Dalhousie University, Halifax, Nova Scotia, CanadaB3H 4J11

Author for correspondence: L. C. Vining. Tel: +1 902 494 2040. Fax: +1 902 494 3736. e-mail: Leo.Vining{at}Dal.Ca


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The pdx-4 mutation in Streptomyces venezuelae ISP5230 confers a growth requirement for pyridoxal (pdx) and is a marker for the genetically mapped cluster of genes associated with chloramphenicol biosynthesis. A gene regulating salvage synthesis of vitamin B6 cofactors in S. venezuelae was cloned by transforming a pdx-4 mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complementation of the host’s pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction analysis indicated that the complementing plasmid had integrated into the chromosome; after excision by a second crossover, the plasmid failed to propagate. To avoid loss of the recombinant vector, a pdx-dependent Streptomyces lividans mutant, KAA1, with a phenotype matching that of S. venezuelae pdx-4, was isolated for use as the cloning host. Introduction of pIJ702 carrying an S. venezuelae genomic library into S. lividans KAA1, and selection of prototrophic transformants, led to the isolation of a stable recombinant vector containing a 2·5 kb S. venezuelae DNA fragment that complemented requirements for pdx in both S. venezuelae and S. lividans mutants. Sequence analysis of the cloned DNA located an intact ORF with a deduced amino acid sequence that, in its central and C-terminal regions resembled type-I aminotransferases. The N-terminal region of the cloned DNA fragment aligned closely with distinctive helix–turn–helix motifs found near the N termini of GntR family transcriptional regulators. The overall deduced amino acid sequence of the cloned DNA showed 73% end-to-end identity to a putative GntR-type regulator cloned in cosmid 6D7 from the Streptomyces coelicolor A3(2) genome. This location is close to that of pdxA, the first pdx marker in S. coelicolor A3(2) identified and mapped genetically in Sir David Hopwood’s laboratory. The S. venezuelae gene and S. coelicolor pdxA are postulated to be homologues regulating vitamin B6 coenzyme synthesis from pdx.

Keywords: chloramphenicol synthesis, pdx marker, regulator gene

Abbreviations: pdx, pyridoxal; pdn, pyridoxine; Am, apramycin; Ap, ampicillin; Cm, chloramphenicol; Ts, thiostrepton

The GenBank accession number for the sequence reported in this paper is AF286159.

a Present address: Department of Natural Products Research, Wyeth Ayerst Research, Pearl River, NY, USA.

b Present address: Pharmaceutics Department, Pharmacy University of Shenyang, 103 Wenhua Rd, Shenyang, P.R. China.

c Present address: Department of Education, Mount Saint Vincent University, Halifax, NS, Canada.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chloramphenicol (Cm) is a broad-spectrum antibiotic produced by Streptomyces venezuelae and related species (Vining & Westlake, 1984 ). Within its structure is a p-nitrophenylserinol moiety related to the phenylpropanoid amino acids in originating from the shikimate pathway, but biosynthetically distinct in being formed by reactions branching at chorismic acid to synthesize p-amino-substituted aromatic intermediates via 4-amino-4-deoxychorismic acid (Vining & Stuttard, 1994 ). The availability of genetically characterized mutants in the Cm biosynthesis pathway offered a potential avenue to gene cloning through complementation of defective hosts, but posed a need for positive selection procedures to efficiently identify complemented transformants. To avoid laborious screening procedures for antibiotic activity, advantage was taken of auxotrophic marker genes. Genetic loci of Cm biosynthesis (cml) genes as well as convenient markers have been mapped in S. venezuelae ISP5230 by conjugation and transduction (Vats et al., 1987b ). Conjugational mapping (Doull et al., 1986 ) indicated that a group of these genes (cml-2, 3, 5–9, 11, 12) is located in the same arc of the S. venezuelae chromosome as a cys gene (cys-28) and various pdx and arg genes. The location was supported by cotransductional analyses with actinophage SV1 (Vats et al., 1987b ). Since pdx markers cotransduced with cml genes at higher frequencies than with cys-28, the cml genes were grouped between the pdx and cys markers. The arg genes cotransduced at frequencies of 1·5–6% with cys-28, but were not cotransducible with pdx, indicating that they lay beyond cys-28, at the opposite end of the cml cluster from the pdx markers. Overall, the cotransduction data confirmed results from conjugational crosses, indicating that Cm biosynthesis genes are located in the region of the S. venezuelae ISP5230 chromosome between cys-28 and pdx-4. Additional fine-structure transductional mapping demonstrated that all of the genetically characterized cml loci lay between these two auxotrophic markers (Vats, 1987 ). The transduction data demonstrated that pdx-4 and cys-28 were cotransducible at a frequency of 0·5%, and since actinophage SVI can package only about 45 kb of DNA (Vats et al., 1987a ), this places a limit of about 45 kb on the length of the gene cluster for Cm biosynthesis.

Efforts to clone either cml or auxotrophic genes by complementing mutations in null-mutant hosts failed initially because plasmid vectors developed for other streptomycetes (Hopwood et al., 1985 ; Baltz & Seno, 1988 ) transformed S. venezuelae with very low efficiency. Thus pIJ941, developed from the Streptomyces coelicolor A3(2) plasmid SCP2 (Lydiate et al., 1985 ), transformed Streptomyces lividans successfully, but could not be used with S. venezuelae ISP5230 until a spontaneous deletion in the tra region generated pDQ101 (Aidoo et al., 1990 ). With pDQ101 carrying BglII-digested S. venezuelae genomic DNA inserts, transformation of pdx-4 and pdx-5 mutants yielded prototrophs, and cml mutants of S. venezuelae ISP5230 gave Cm-producing colonies. However, recombinant plasmids complementing the host mutations were rapidly lost from the transformants (Aidoo, 1989 ). Since Southern hybridizations indicated that loss of the recombinant vector was due to recombination between the host chromosome and homologous vector inserts, an S. lividans mutant, KAA1, with features similar to those of the S. venezuelae pdx-4 and pdx-5 mutants, was isolated for use as a cloning host. Transformation of S. lividans KAA1 with pIJ702 carrying segments of S. venezuelae genomic DNA yielded prototrophic colonies from which pIJ702 carrying a 2·5 kb DNA fragment that complemented the host’s requirement for pyridoxal (pdx) could be isolated.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Micro-organisms, plasmids and cultures.
Bacteria, phage and plasmids used are listed in Table 1. Streptomyces venezuelae strains were maintained on MYM agar (Stuttard, 1982 ) supplemented with apramycin (Am; 50 µg ml-1) and/or thiostrepton (Ts; 25 µg ml-1) when required. The minimal medium (MM agar) used to culture S. venezuelae in Petri plates contained the asparagine-salts solution of Hopwood (1967) with maltose instead of glucose as carbon source. Streptomyces lividans was maintained on K1 medium (g l-1): maltose, 10; yeast extract (Difco), 5; Casamino acids (Difco), 0·2; K2HPO4, 0·5; MgSO4 . 7H2O, 0·2; FeSO4 . 7H2O, 0·1; agar, 15. Cultures of S. venezuelae and S. lividans were grown as described by Aidoo et al. (1990) . Escherichia coli was grown in LB or 2x YT medium (Sambrook et al., 1989 ).


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Table 1. Bacterial strains and plasmids used

 
Isolation of pdx- mutants from S. lividans.
S. lividans TK24 was treated with nitrosoguanidine (NTG) as described by Delic et al. (1970) to allow 5–10% survival (90 min at 30 °C) and the spores were plated on MM agar supplemented with 1·0 µg pdx hydrochloride ml-1. Approximately 2500 colonies were screened by replica-plating on MM to detect pdx- colonies. The growth requirement in these putative pdx auxotrophs was confirmed by patching spores formed on MM+pdx medium to MM agar; the frequency was calculated to be 0·3%. From each mutagenic treatment, one strain showing good growth on MM+pdx but negligible growth on MM alone or on MM+pyridoxine (pdn) was chosen and spores were spread on MM containing either pdx (1·0 µg ml-1) or pdn (1·0 µg ml-1) to distinguish between requirements for these supplements. Strains able to grow only when MM was supplemented with pdx were classified as pdxH mutants (Dempsey, 1987 ). One such mutant strain (KAA1), for which the frequency of reversion to prototrophy was estimated as 10-8 per c.f.u., was chosen as a host for complementation cloning.

Detecting plasmid integration by cotransduction.
To verify plasmid integration when an auxotrophic S. venezuelae host was transformed with a vector carrying cloned homologous DNA, the site of the vector TsR marker gene in the chromosome of protoprophic transformant VS622 was investigated by co-transduction analysis. Because S. venezuelae VS622 was only weakly sensitive to actinophage SV1, the alternative infective agent actinophage SV9 (Stuttard, 1989 ) was used. Transductants recovered initially on MM agar were patched on MM containing sodium citrate to prevent phage superinfection. To ensure the initial growth of recipient strains necessary for phage infection, Ts was not used as the primary selective agent; instead, transductant colonies were allowed to sporulate on MYM agar, then screened for phenotypic expression of TsR by replica-plating on MYM agar supplemented with Ts. To assess how much DNA had integrated, transductants (VS625–VS628) were digested with SstI and the digests were probed with the [32P]dCTP-labelled TsR gene.

DNA manipulations.
The general procedures of Sambrook et al. (1989) were followed. The boiling method of Holmes & Quigley (1981) was used for screening plasmid DNA in multiple colonies. Otherwise, plasmid DNA was isolated from E. coli by the alkaline lysis method (Kieser, 1984 ). Streptomycete genomic DNA was obtained as described by Hopwood et al. (1985) , but the final aqueous DNA solution was extracted with chloroform containing 1% (v/v) cetyl trimethylammonium bromide.

Transformation.
Competent E. coli cells were prepared, transformed and plated on LB agar containing ampicillin (Ap; 0·1 mg ml-1), and 0·05 mg Am ml-1 where needed, as described by Sambrook et al. (1989) . To transform streptomycetes the general protoplasting procedure of Hopwood et al. (1985) was used; but was modified for S. venezuelae (Aidoo et al., 1990 ). Ligation mixtures containing the DNA digest and linearized vector were used to transform protoplasts of the S. venezuelae or S. lividans KAA1 host to TsR (Hopwood et al., 1985 ); transformant colonies were patched from selective regeneration medium.

Construction of genomic libraries.
For the library in pIJ702, genomic DNA from S. venezuelae ISP5230 was digested to completion with BamHI before ligation with vector DNA linearized with BglII and dephosphorylated with thermolabile bacterial alkaline phosphatase (BRL). The ligation mixture was used to transform S. lividans KAA1 to TsR. The library in pDQ101 was prepared similarly, but the genomic DNA was digested with BglII and ligated to the BamHI site in the vector. The ligation mixture was used to transform S. venezuelae to TsR.

Southern hybridization.
Plasmid and genomic DNA digested with restriction endonucleases was electrophoresed in a 0·8% agarose gel, transferred to a positively charged nylon membrane (Qiagen) and probed (Southern, 1975 ) with a [32P]dCTP-labelled DNA fragment. Hybridization was carried out at 65 °C in a solution containing 5x SSPE (1x SSPE is 0·18 M NaCl, 10 mM Na2HPO4 and 1 mM EDTA, pH 7·7), 5x Denhardt’s solution (Denhardt, 1966 ), 0·5% (w/v) SDS and 100 µg denatured salmon sperm DNA ml-1. Membranes were washed at 65 °C with SSPE solutions (twice with 2x, then once each with 1x and 0·1x) containing 0·1% SDS. Hybridization was detected with a CS phosphor-imaging screen, which was scanned for radioactivity with a molecular imager (Bio-Rad model GS525).

DNA sequencing.
The 2·5 kb BamHI fragment initially cloned in pDQ153 was detected in a {lambda} phage library of S. venezuelae genomic DNA by hybridization probing and reisolated. It was then cloned in both orientations in the BamHI site of the phagemid vector pBluescript II SK(+). Nested deletions were introduced (Henikoff, 1984 ) and phagemid DNA was sequenced by the dideoxy chain-termination procedure (Sanger et al., 1977 ). Some DNA fragments were also sequenced at the Dalhousie/NRC Joint Sequencing Facility with an ABI 373 automated sequencer.

Sequence analysis.
Nucleotide sequences were analysed with version 8.1 software developed by the Genetics Computer Group (GCG), University of Wisconsin (Devereux et al., 1984 ) ORFs were detected with the GCG CODONPREFERENCE program, as well as with FramePlot 3.1 (Ishikawa & Hotta, 1999 ; www.nih.go.jp/~jun/cgi-bin/frameplot.pl) and GeneRunner version 3.05 (Hastings Software). For multiple sequence alignments, the Gene Runner and internet-based CLUSTAL W (www.clustalw.genome.ad.jp/sit-bin/nph-clustalw) programs were used. The mean mol% G+C, the mol% G+C values at specific positions within reading frames, and the locations of start and stop codons were determined with FramePlot 3.1.

Construction and use of a replacement vector.
The vector used to disrupt ORF1 was prepared by digesting pJV151 with NcoI to remove a 1·6 kb fragment containing ORF1 and then ligating a 1·45 kb NcoI–NcoI AmR cassette (Paradkar & Jensen, 1995 ) into the NcoI site. Selection for a transformant with the AmR phenotype gave pJV152. The modified 2·5 kb insert in pJV152 was excised with BamHI and inserted at the BamHI site in the multiple cloning region of pHJL400. The construct was used to transform E. coli DH5{alpha}. Extraction of plasmid DNA from a transformant with an AmR ApR phenotype yielded pJV153, which conferred resistance to both Am (50 µg ml-1) and Ap (100 µg ml-1). When digested with BamHI, pJV153 released a 2·5 kb BamHI fragment. The plasmid was passaged through E. coli ET12567 (MacNeil et al., 1992 ) and used by the procedure of Aidoo et al. (1990) to transform S. venezuelae ISP5230 to TsR. Fifty transformants were patched on MYM agar containing both Am and Ts; spores were then replica-plated without selection on MYM agar to obtain AmR TsS colonies. The genotypes of strains were confirmed by Southern hybridization of BamHI-digested genomic DNA using the 1·45 kb AmR gene as a probe.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid integration during transformation of S. venezuelae
Complementing pdx mutations in S. venezuelae ISP5230 by transforming the mutants with pDQ101 carrying a library of wild-type genomic DNA fragments yielded several prototrophs (S. venezuelae VS622–VS624). Although the TsR phenotype associated with pDQ101 was lost rapidly during routine culture transfers, prototrophy persisted, implicating plasmid integration by recombination between chromosomal genes and homologous vector inserts, followed by vector excision and elimination. Consistent with this, a probe containing the TsR gene from Streptomyces azureus (Thompson et al., 1982 ) hybridized with the high-molecular-mass component of DNA extracted from TsR prototrophic transformants, but not from TsS segregants. After the extract had been digested with BclI, the probe hybridized with a 1·06 kb DNA fragment predicted to have originated from the TsR gene of pDQ101 integrated in the chromosome. None of the prototrophic VS622–VS624 transformants contained a stable recombinant plasmid that could be extracted and used to transform the host to TsR.

In earlier studies it had been shown that the pdx and cml double mutations in each of the S. venezuelae hosts VS248 (cml-11 pdx-2), VS253 (cml-11 pdx-3) and VS258 (cml-12 pdx-4) used for these transformations were co-transducible (Vats et al., 1987a ). However, selecting colonies for prototrophy after the hosts had been transformed to TsR did not yield strains restored in Cm biosynthesis. To test whether a recombinant plasmid had initially integrated into the chromosome at a pdx site, the location of the TsR gene in the chromosomes of S. venezuelae VS622–VS624 was investigated by co-transduction analysis. With VS622 as the donor phage, the TsR gene and wild-type alleles of pdx-2 and pdx-3 cotransduced at frequencies of 28·7 and 33·3%, respectively, indicating that vector DNA had integrated in the region containing the pdx sites. To assess how much DNA had integrated, transductant genomes were digested with SstI and the digests were probed with the [32P]dCTP-labelled TsR gene. Detection of a 4·0 kb signal (corresponding in size to the fragment containing the TsR gene) in each genomic digest indicated that the DNA introduced had included at least the pDQ101 vector.

Complementation cloning in S. lividans KAA1
Genomic DNA fragments obtained from S. venezuelae ISP5230 by complete digestion with BamHI were ligated to the streptomycete vector pIJ702 linearized with BglII. The ligation mixture was used to transform S. lividans KAA1 protoplasts to TsR, and approximately 7000 TsR colonies on regeneration medium were screened by replica-plating on MM and MM+pdx agar. Six prototrophic colonies were detected; on extraction each yielded a recombinant plasmid (pDQ153) containing a 2·5 kb DNA insert.

Recloning the pDQ153 insert for transformation of S. venezuelae and sequencing
Since BamHI could not be used to excise the S. venezuelae DNA insert from its hybrid BamHI/BglII cloning site in pDQ153, the 2·5 kb BamHI fragment was identified in, and reisolated from a genomic library of S. venezuelae DNA in {lambda} GEM-11 (Facey et al., 1996 ). The phage library was probed with labelled pDQ153 and the hybridizing fragment in a BamHI digest of DNA extracted from the single strongly labelled plaque was recloned in pHJL400 to give pJV154 and in pBluescript II SK(+) to give pJV151. A restriction map of the pJV151 insert is shown in Fig. 1. Transforming S. venezuelae pdx mutants with pJV154 restored prototrophy in S. venezuelae VS258 (pdx-4 cml-12) and VS499 (pdx-5 cml+), but not in VS248 (pdx-2 cml-11) nor in VS253 (pdx-3 cml-11). The fact that pDQ153 complemented S. lividans KAA1 implied that the mutation in this strain, and the pdx-4 and pdx-5 mutations in S. venezuelae strains VS258 and VS499, affected a similar host function. Other functions, not restored by pJV154, were presumably inactivated by the pdx-2 and pdx-3 mutations in S. venezuelae VS248 and VS253.



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Fig. 1. Plasmids used and partial restriction maps of their S. venezuelae DNA inserts. Abbreviations: Ba, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NcoI; Ps, PstI; Sa, SacI; Sl, SalI; Sm, SmaI; X, XhoI.

 
Subcloning pJV151
Digestion of pJV151 with SacI cleaved the 2·5 kb insert into segments of approximately 1·05 and 1·40 kb which, together with the parent insert, were subcloned in pHJL400 to give pJV155, pJV156 and pJV154, respectively (see Fig. 1). Failure of S. lividans KAA1, S. venezuelae VS258 (pdx-4) and S. venezuelae VS499 (pdx-5) to yield prototrophs after transformation with pJV156 indicated that the 1·40 kb fragment did not restore the function of defective regions in the S. lividans or S. venezuelae mutants. However, with pJV155 both S. venezuelae hosts gave prototrophic transformants, implying that functions lost with the pdx-4 and pdx-5 mutations were restored by the 1·05 kb SacI fragment. The heterologous S. lividans KAA1 host regained prototrophy only when the entire 2·5 kb fragment in pJV154 was introduced. The results suggest that function may have been restored in the S. venezuelae hosts by gene repair, whereas in S. lividans it required the introduction of an intact gene expressing the activity lost by mutation. Sensitivity of the S. venezuelae transformants to Ts implicated vector excision associated with a double crossover during homologous recombination.

Sequence and analysis of the cloned DNA
Both strands of the pJV151 insert were sequenced. The overall G+C content of the 2455 bp of S. venezuelae DNA cloned in the plasmid was 76·0 mol%, typical of streptomycete genes (Wright & Bibb, 1992 ). Frame and codon preference analysis of the nucleotide sequence detected one complete ORF (ORF1 in-frame -1), as well as two incomplete ORFs (ORF2 and ORF3 in-frame -3). The mean third-base G+C content in ORFs 1, 2 and 3 was 94·4, 97·7 and 95·6 mol%, respectively. ORF1 started at nt 2181–2179 with an ATG, preceded by a potential ribosome-binding site (GGA) at nt 2190–2188. The first in-frame stop codon downstream of the ATG was a TGA at nt 585–583, giving an ORF of 1596 bp, encoding a protein of 532 aa. ORF2 began with an ATG codon at nt 419–422. No RBS was detected and the sequence was truncated at the end of the cloned fragment. ORF3 began outside the cloned sequence and was assigned as an ORF from its very high third-codon G+C content, which declined steeply after an in-frame TGA codon at nt 2194–2192. Searching GenBank with BLASTX (Altschul et al., 1997 ) for protein sequences matching deduced amino acid sequences of the three ORFs showed that the ORF1 product contained two recognizable regions: from the C terminus to the centre, the sequence resembled class-I aminotransferases, while the N-terminal region contained a helix–turn–helix DNA-binding motif similar to that in GntR family transcriptional regulators. The products of the ORF2 and ORF3 partial sequences were similar to putative membrane proteins CAB 61673.1 and CAB 61675.1, respectively, encoded by ORFs in S. coelicolor A3(2) cosmid 6D7, which also contained an ORF closely matching ORF1 (see below).

Disruption of ORF1
To assess the role of ORF1 in S. venezuelae ISP5230, an allele replacement vector (pJV153; Fig. 2) was prepared by deleting a 1·6 kb NcoI segment from the ORF1 DNA cloned in pJV151 and introducing at that site a 1·45 kb NcoI cassette carrying the AmR gene (Paradkar & Jensen, 1995 ). From the resulting plasmid, pJV152, the region of DNA containing the AmR cassette flanked by outlying regions of ORF1 was excised as a 2·5 kb BamHI fragment and recloned in the BamHI site of the pHJL400 multiple cloning region, giving pJV153. The StreptomycesE. coli bifunctional vector pHJL400 is segregationally unstable in streptomycetes (Larson & Hershberger, 1986 ), facilitating gene disruption in S. venezuelae ISP5230 (Han et al., 1994 ). After passage through the DNA-non-methylating host E. coli ET12567 to avoid restriction (MacNeil et al., 1992 ), pJV153 was used to transform S. venezuelae protoplasts. The AmR TsR transformants (VS680) initially recovered were propagated without selection for TsR and then replica-plated on diagnostic media to identify AmR TsS colonies (VS681). Detection of 32P-labelled 8·1 and 2·5 kb BamHI fragments in genomic DNA from VS680 and VS681, respectively, by Southern hybridization with an AmR gene probe indicated that pJV153 had integrated into pdx-4 in the VS680 chromosome and, subsequently, excised its vector component during the second crossover giving VS681; allele exchange and segregation account for the disrupted copy of ORF1 in the latter strain. In its failure to grow on MM, but abundant growth on MM+pdx agar, VS681 resembled the S. venezuelae auxotrophs VS258 (pdx-4) and VS499 (pdx-5). Like these it yielded prototrophs (VS681P, VS258P and VS499P) when transformed with pJV154, and the prototrophs could be returned to auxotrophy by disruption with pJV153. Strains VS258P and VS499P segregated AmR TsS colonies that grew well on MM+pdx, but failed to grow on MM agar. The results indicated a relationship between expression of ORF1 and availability of pdx phosphate.



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Fig. 2. Construction of pJV153, the plasmid vector used to disrupt the putative pdxR in S. venezuelae ISP5230. Abbreviations are defined in the legend to Fig. 1.

 
Similarities between class-I aminotransferases, GntR regulators and ORF1
In a BLASTX comparison the deduced amino acid sequence of ORF1 showed 26% identity and 41% similarity to aspartate aminotransferase of the thermophilic archaeon Thermus aquaticus. The resemblance between the central to C-terminal region of the deduced amino acid sequence of ORF1 and a class-I aminotransferase consensus sequence was evident in a CLUSTAL W motif alignment (Fig. 3). Moreover, Prosite pattern detection with the GeneFinder program (Wu et al., 1996 ; 1998 ) recognized in the ORF1 protein a conserved region containing a potential pdx phosphate attachment site (see Fig. 3) that has been found in other class-I aminotransferases. In the N-terminal region of the deduced amino acid sequence of ORF1, GeneFinder detected a Prosite pattern motif for a helix–turn–helix. Although this motif is a common indicator of a DNA-binding site (Pabo & Sauer, 1984 ; Brennan & Matthews, 1989 ), the ORF1 sequence was distinctive in matching the helix–turn–helix signature of the GntR family of bacterial regulatory proteins (Haydon & Guest, 1991 ). The GntR family includes KorA, which regulates conjugal transfer of the broad-host-range Streptomyces plasmid pIJ101 (Kendall & Cohen, 1988 ), the MocR regulators of rhizopine catabolism in Rhizobium species (Rossbach et al., 1994 ), PtsJ, the putative phosphotransferase system regulator in Salmonella typhimurium (Titgemeyer et al., 1995 ) and ORFT2, postulated to regulate transcription in Rhodobacter sphaeroides (Neidle & Kaplan, 1992 ). BLAST searches comparing GenBank entries with the deduced amino acid sequence of S. venezuelae ORF1 indicated marked resemblances to MocR, PtsJ and ORFT2 (26–29% identity and 40–41% similarity). Sequencing of the genome of S. coelicolor A3(2) (http://www.Sanger.ac.uk/Projects/S_coelicolor/) has recently located ORF22, in cosmid 6D7, a sequence containing a GntR motif and showing 73% identity to S. venezuelae ORF1. A CLUSTAL W alignment of the N-terminal region of the ORF1 product with amino acid sequences from the above GntR-family regulators (Fig. 4) showed conserved regions with a consensus that, as predicted (Pabo & Sauer, 1984 ), favoured specific amino acids at certain positions (alanine at position 5, glycine at position 9, valine at position 15). The probable involvement of aminotransferase activity in reactions generating vitamin B6 coenzymes, considered with the evidence that ORF1 regulates gene transcription, suggests the gene designation pdxR.



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Fig. 3. CLUSTAL W comparison of the consensus sequence of type-1 aminotransferases with a pairwise Pfam alignment from the middle to the C-terminus of the deduced amino acid sequence of ORF1.

 


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Fig. 4. Alignment of the N-terminal region of the S. venezuelae pdxR product with putative GntR regulatory proteins: ORF1, deduced amino acid sequence of S. venezuelae pdxR cloned in pDQ153; Scoel, deduced amino acid sequence from ORF22 in cosmid 6D7 cloned from S. coelicolor A3(2); MocR, regulator of rhizopine catabolism in Rhizobium meliloti L5-30 (Rossbach et al., 1994 ); PtsJ, regulator of the phosphotransferase system in Salmonella typhimurium (Titgemeyer et al., 1995 ); YcdX, amino acid sequence deduced from YcdX in the surfactin biosynthesis operon of Bacillus subtilis (Cosmina et al., 1993 ); YrdX, protein of unknown function in Rhodobacter sphaeroides (Neidle & Kaplan, 1992 ). The boxed A, G and V are the residues most favoured as DNA binding sites in a helix–turn–helix structure.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Complementation cloning with a surrogate host
Compared with S. coelicolor A(3)2, where the genes for actinorhodin were successfully cloned in mutant hosts blocked in the pathway for actinorhodin biosynthesis, S. venezuelae ISP5230 appears to be exceptionally recombinogenic. Vectors carrying homologous chromosomal DNA fragments integrated into the mutant host chromosome so readily that the recombinant vector carrying cml genes could not be isolated. Similar difficulties were encountered by Aidoo et al. (1990) and Paradkar et al. (1993) during attempts to clone S. venezuelae genes by complementing auxotrophic mutants. Using an S. lividans TK24 auxotroph as a surrogate host avoided recombinant vector integration and provided the advantages of a well characterized host and established cloning methodology. A mutant S. lividans host with the same phenotype as S. venezuelae pdx-4 and a low frequency of reversion to prototrophy was readily obtained by mutagenizing spores with nitrosoguanidine. Because the growth requirements of the pdx mutants isolated from S. venezuelae ISP5230 and S. lividans TK24 were met by providing pdx, but not pdn, the lesion was presumed to be in the salvage pathway converting pdn to coenzymically active forms, rather than in the reactions for de novo biosynthesis of pdn (Dempsey, 1987 ). The inactivated gene was expected to be pdxH, encoding pyridoxol (phosphate) oxidase, which has been characterized in E. coli (Dempsey, 1966 ). The E. coli pdxH has been sequenced (GenBank accession no. M923510) and a homologue was recently detected in cosmid D10 from S. coelicolor A(3)2 (http://www.sanger.ac.uk/Projects/S_coelicolor ). However, S. venezuelae ORF1 clearly differed from the E. coli and S. coelicolor pdxH genes since neither was recognized in a BLASTX search of GenBank with ORF1 as the query sequence. This implied that DNA complementing pdx mutations in S. venezuelae did not contain pdxH and that this key gene of the vitamin B6 salvage pathway in the mutants was structurally intact.

Function of the ORF1 product
The absence of cross-feeding among pdx mutants supported analysis of the ORF1 sequence in implying that the mutations were not in structural genes for pdn biosynthesis or the formation of vitamin B6 coenzymes, and pointed to a role for the ORF1 gene in regulating the latter process. Similarities between the pdx-dependent phenotypes of S. venezuelae auxotrophs obtained by chemical mutagenesis and the phenotype of strain VS681, in which the native ORF1 allele had been replaced specifically with a disrupted copy, support a role for the gene in regulating the availability of pdx phosphate and related vitamin B6 coenzymes. This conclusion is strengthened by the presence in the C-terminal region of ORF1 of a pdx phosphate binding site within a sequence resembling aminotransferase genes. Regulation of aminotransferase activity through an N-terminal DNA-binding sequence with a characteristic operator motif is consistent with the organization of GntR transcriptional regulators (Haydon & Guest, 1991 ). The essential role of the aminotransferase component is emphasized by the results from subcloning ORF1, which demonstrated that only the C-terminal 1·05 kb DNA segment complemented pdx-4 and pdx-5 mutations in S. venezuelae

Location of an ORF1 homologue in S. coelicolor
Much of the S. coelicolor A3(2) genome has now been sequenced and the ORFs detected have been positioned in an ordered cosmid bank on restriction fragments, providing a physical map of the chromosome (Redenbach et al., 1996 ). Searching the currently available S. coelicolor A3(2) database with the WUBLAST program using TBLASTN (Altschul et al., 1997 ; accessible at http://www.sanger.ac.uk/cgi-bin/nph-BLAST_Server.html) gave a close match between the deduced amino acid sequences of ORF1 from S. venezuelae and ORF22 in cosmid 6D7 from S. coelicolor A3(2). Significantly, the S. coelicolor sequence included the signature motif for a GntR family transcriptional regulator. The location of cosmid 6D7 is close to the locus of pdxA, the first pdx gene isolated and initially mapped on the chromosome of S. coelicolor A3(2) by genetic procedures (Vivian & Charles, 1970 ; D. A. Hopwood, personal communication). This pdxA locus has been positioned as accurately as possible on the current chromosome map established by cosmid analysis and genome sequencing (Hopwood & Kieser, 1990 ; Redenbach et al., 1996 ). Its location near cosmid 6D7 in S. coelicolor corresponds well with the position of pdxR on the S. venezuelae chromosome in its proximity to the 2 o’clock map location of the cml cluster (Vats et al., 1987a ) on the circular genetic map of S. venezuelae ISP5230, constructed (Stuttard et al., 1987 ; Stuttard, 1988 ) to elicit evolutionary relationships between this species and Streptomyces coelicolor A3(2). An approximate alignment of selected alleles postulated to lie between 12 o’clock and 2 o’clock in the chromosomes of the two species (Fig. 5) suggests that the S. venezuelae gene cloned in ORF1 not only corresponds to both pdxA and cosmid gene 6D7.22 in S. coelicolor A3(2), but also that it is the pdx marker flanking the cml cluster.



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Fig. 5. Approximate alignment of the 12 o’clock to 2 o’clock regions of the genetic maps of S. venezuelae ISP5230 (Stuttard, 1988 ) and S. coelicolor A3(2) (Redenbach et al., 1996 ). S. coelicolor cosmids are underlined.

 

   ACKNOWLEDGEMENTS
 
We are grateful to Dr A. S. Paradkar, University of Alberta, for the NcoI cassette containing the AmR gene, and to Dr C. L. Hershberger, Eli Lilly & Company, for the vector pHJL400. We also thank Sir D. A. Hopwood for advice on the genetically mapped pdx genes of S. coelicolor A3(2). This work was supported by the Natural Sciences and Engineering Research Council of Canada.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 7 November 2000; revised 23 April 2001; accepted 26 April 2001.