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
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ABSTRACT |
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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.
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INTRODUCTION |
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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 hosts requirement for pyridoxal (pdx) could be isolated.
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METHODS |
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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 (VS625VS628) 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 Denhardts 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 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 NcoINcoI 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
. 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.
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RESULTS |
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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 VS622VS624 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 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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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 21812179 with an ATG, preceded by a potential ribosome-binding site (GGA) at nt 21902188. The first in-frame stop codon downstream of the ATG was a TGA at nt 585583, giving an ORF of 1596 bp, encoding a protein of 532 aa. ORF2 began with an ATG codon at nt 419422. 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 21942192. 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 helixturnhelix 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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DISCUSSION |
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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 oclock 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 oclock and 2 oclock 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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ACKNOWLEDGEMENTS |
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Received 7 November 2000;
revised 23 April 2001;
accepted 26 April 2001.