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: p-aminobenzoate synthase genes, primary and secondary metabolism
Abbreviations: ADC, 4-amino-4-deoxychorismic acid; PABA, p-aminobenzoic acid; PAPA, p-aminophenylalanine; Am, apramycin; Cm, chloramphenicol; Ts, thiostrepton; Vio, viomycin
The GenBank accession number for the sequence reported in this paper is AF189258.
a Present address: Microbiology Department, University of Minnesota, 1030 Mayo Building, 420 Delaware Street SE, Minneapolis, MN 55455, USA.
b Present address: Pharmaceutics Department, Pharmacy University of Shenyang, 103 Wenhua Rd, Shenyang, P.R. China.
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INTRODUCTION |
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METHODS |
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Transformation.
Competent E. coli cells were prepared and transformed as described by Sambrook et al. (1989) . Protoplasts of S. lividans were prepared, transformed and regenerated by standard methods for streptomycetes (Hopwood et al., 1985
); for S. venezuelae these methods were modified (Aidoo et al., 1990)
. Procedures for conjugal transfer of plasmids from E. coli to streptomycetes were essentially those of Flett et al. (1997)
.
DNA manipulations.
phage DNA, E. coli plasmid DNA and single-stranded DNA templates for sequencing were prepared as described by Sambrook et al. (1989)
. Genomic and plasmid DNA were isolated from streptomycetes by the methods of Hopwood et al. (1985)
. For rapid screening of plasmids in E. coli the Sekar (1987)
procedure was adopted. DNA fragments excised in agarose gel slices were extracted with the QIAEX II kit (Qiagen).
Amplification of S. venezuelae chromosomal DNA by PCR.
The primers used were 5'-ATGATCGTGGACCTSGACCGSAACGAC-3' (sun-3) and 5'-CTGCGGCTCSAGCTCGTCGATGATCTCCAT-3' (sun-4). The reaction mixture (total volume 100 µl) consisted of: dNTPs, 10 µl (44x1·25 mM); primer sun-3, 1 µl (268 pmol); primer sun-4, 1 µl (182 pmol); chromosomal DNA, 100 ng; 10xPromega PCR buffer, 10 µl; and water to 100 µl. It was aliquotted (20 µl) in thin-walled 0·5 ml microcentrifuge tubes and layered with mineral oil (ca 20 µl). Each aliquot was mixed with 1 U Taq DNA polymerase and heated to 80 °C, then thermal cycling was started (model PTC-100; MJ Research). The programme for the first 7 cycles was: 96 °C for 1 min (denaturing), 67 °C for 1 min (annealing) and 72 °C for 1 min (extension). For the next 30 cycles denaturing and annealing times were each reduced to 45 s. Amplified S. venezuelae DNA was inserted into the SmaI site of pUC18 (SureClone Kit; Pharmacia Biotech) and sequenced by the dideoxy chain-termination method (Sanger et al., 1977 ).
Construction of an S. venezuelae genomic library in GEM-11.
Genomic DNA (50100 µg) from S. venezuelae was partially digested at 37 °C in a 100 µl reaction mixture containing 3 U Sau3AI. The incubation time was adjusted to optimize the yield of 923 kb fragments; digestion was terminated with EDTA (20 mM). The DNA recovered was fractionated by sucrose-gradient centrifugation and 3'-CTAG-5' overhangs of the 923 kb fragments were partially filled-in by incubation with the Klenow fragment of DNA polymerase I in the presence of dGTP and dATP. GEM-11 arms digested with XhoI (giving 3'-AGCT-5' overhangs) were partially filled in with dTTP and dCTP; the modified fragments and arms, after incubation with T4 DNA ligase and a
phage packaging system (Promega), were used to infect E. coli LE392. The phage library was amplified as described by Sambrook et al. (1989)
and stored at -70 °C in SM buffer containing 7% (v/v) DMSO.
Construction of disruption vectors.
The vector used to disrupt pabAB has been described by Brown et al. (1996) . To disrupt the putative pabB in pJV305, a 1·5 kb DNA fragment containing an apramycin resistance (AmR) gene (Paradkar & Jensen, 1995
) was inserted into the Eco72I site of ORF1 to give pJV307. The disrupted ORF1 in pJV307 was then recloned as pJV323 in pHJL400. To construct a disruption vector containing the viomycin resistance (VioR) gene, the 2·0 kb Streptomyces vinaceus DNA fragment containing the gene (Thompson et al., 1982
) was excised from pJV230 (Chang, 1999
) and introduced into pJV305 at the Eco72I site in ORF1, giving pJV308. The 5·8 kb BamHI fragment containing ORF1 disrupted with the VioR gene was recloned from pJV308 into pHJL400, giving pJV324. Since protoplast procedures failed to give VioR transformants of VS629 with pJV324, the 5·8 kb BamHI insert of pJV324 was subcloned in the conjugal vector pJV326 to give pJV325. Conjugal transfer of pJV325 from E. coli into S. venezuelae VS629 using methodology developed by Mazodier et al. (1989)
and Flett et al. (1997)
yielded single- and double-crossover mutants VS1003 and VS1004.
DNA sequencing and analysis.
The 3·8 kb NcoI fragment of S. venezuelae ISP5230 DNA from recombinant phage YSB1 was subcloned (both orientations) into pBluescript II SK(+), giving pJV305 and pJV306. Nested overlapping deletions were introduced into the inserts by the Henikoff (1984) procedure and the DNA was used to transform E. coli DH5
. Inserts in plasmid DNA extracted from the transformants were sequenced (ABI Prism model 373 DNA Sequencer). To detect ORFs by codon third position mol% G+C bias and codon usage, the CODONPREFERENCE programme (GCG Wisconsin Package, version 9.0; Devereux et al., 1984
) was used. Restriction enzyme sites were located with GeneRunner (Hastings Software) and similarities between derived amino acid sequences and proteins in GenBank were assessed from BLAST searches (Altschul et al., 1997
). Sequences were aligned using CLUSTAL W (Higgins et al., 1996
); the MacVector software of the Oxford Molecular Group was used for phylogenetic analysis.
Hybridization.
For phage library screening (Hopwood et al., 1985) and Southern analyses (Southern, 1975
), DNA samples bound to nylon membranes were incubated with 32P-labelled probes at 65 °C overnight in hybridization solution (Sambrook et al., 1989
). The membranes were washed at room temperature with 2xSSC, and then twice at 65 °C with 0·1xSSC (each solution containing 0·1% SDS).
Cm production.
Strains of S. venezuelae were grown and analysed for Cm production by methods described by Brown et al. (1996) . The Cm content of filtered broths was determined by HPLC.
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RESULTS |
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Expression of genes for PABA biosynthesis
The 3·82 kb NcoI fragment of S. venezuelae ISP5230 DNA excised from YHB1 was blunt-ended and subcloned in the EcoRV site of pBluescript II SK(+) as pJV305 and pJV306 (alternative orientations; see Fig. 3
). To determine whether their inserts expressed enzymes for PABA biosynthesis, each of these plasmids was used to transform the E. coli PABA auxotrophs AB3292 and AB3295, which are defective in pabA and pabB functions, respectively. Plasmid pJV305 contained ORFs 1 and 2 oppositely oriented from the vector lacZ, whereas in pJV306, ORFs 1 and 2 were in the same orientation as lacZ (see Fig. 4
). To assess whether PABA synthase genes were expressed, the growth response of the E. coli pab mutants and transformants on medium supplemented with sulfanilamide was determined. Expression was inferred from enhanced sulfanilamide resistance. The higher sulfanilamide resistance in mutants transformed with pJV306 than in those transformed with pJV305 (Table 2
) implied that the cloned DNA was transcribed more efficiently when ORF1/ORF2 and lacZ were oriented in the same direction. This would be consistent with expression of the cloned E. coli insert from the vector lacZ promoter and with little or no expression from a native promoter. Comparable results have been reported for S. griseus pabAB, which was shown by Gil & Hopwood (1983)
to be expressed in E. coli only from the vector promoter.
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DISCUSSION |
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The probable identity of ORF1 and ORF2 with pabB and pabA, respectively, is indicated by comparisons of their deduced amino acid sequences with proteins in the GenBank database and by the ability of the ORFs to complement pab mutants in E. coli and S. lividans. However, the absence of a decisive loss of sulfanilamide resistance in S. venezuelae ISP5230 when pabB is disrupted implies that this gene set is not, as anticipated, primarily associated with PABA biosynthesis. The conclusion is supported by the negligible change in sulfanilamide resistance following disruption of pabB in the pabAB mutant VS629 to yield a strain inactivated in both of its known ADC synthase genes. It is possible that the weak PABA synthase activity in VS629 is due, at least in part, to incomplete inactivation of pabAB, so the effect of disrupting pabB is masked by the hosts residual PabAB activity. Contributing to this might be the high normal level of pabAB expression implied by the exceptionally high resistance of S. venezuelae to sulfanilamide. Aidoo et al. (1990) postulated that this high sulfanilamide resistance reflects a large ADC pool associated with the level of pabAB expression needed to support Cm biosynthesis. Spillover from the pool in the wild-type, or residual activity in the pabAB mutant might outweigh a relatively weak contribution to PABA synthesis from pabB and obscure the response to pabB disruption. However, the presence in S. venezuelae ISP5230 of a third gene set involved in supplying most of the PABA needed to support growth must also be considered.
Anthranilate and ADC synthase component I genes of S. venezuelae ISP5230 and S. coelicolor A3(2)
Information currently available from the S. coelicolor A3(2) genome sequencing project (http://www.sanger.ac.uk/Projects/S_coelicolor) has located four genes encoding putative anthranilate synthase component I proteins on cosmids cloned from the chromosome: cosmid 6E10 contains trpE1, cosmid E8 contains trpE2, cosmid 4G6 contains trpE3 and cosmid L11 contains an undesignated putative trpE. No PABA synthase component I genes have yet been detected. In a BLASTP search of GenBank for sequence similarity to the translated TrpE1, the highest score was obtained with the anthranilate synthase of S. venezuelae ISP5230 associated with the primary metabolic pathway for tryptophan biosynthesis (Lin et al., 1998 ). This result is consistent with similarities between genes of the primary metabolic tryptophan biosynthetic pathways in S. venezuelae and S. coelicolor, and with the presence of another primary metabolic trp gene (trpD) on the overlapping cosmid 6G1 (Redenbach et al., 1996
). Similar BLASTP searches of GenBank with the translated TrpE2 and TrpE3 sequences ranked only anthranilate synthases in the 30 highest scoring protein sequences. Evidence that trpE2 and trpE3 are tryptophan biosynthesis genes associated with secondary metabolism supports these search results: trpE2 is involved in biosynthesis of tryptophan residues for the lipopeptide calcium-dependent antibiotic of S. coelicolor A3(2) (Chong et al., 1998
), while the presence of three other trp genes (trpA, trpB and trpC) distinct from the primary metabolic trp genes on the same cosmid (4G6) as trpE3 implies an involvement with secondary metabolic tryptophan biosynthesis activity. Therefore, three of the four genes predicted to encode anthranilate synthases appear to be unambiguously assigned, but assignment of the cosmid L11 gene as trpE is less certain. A BLASTP search of GenBank with the translated sequence of this gene ranked the PabB sequences of Mycobacterium tuberculosis, Xanthomonas albilineans and Pseudomonas aeruginosa ahead of TrpE sequences in order of similarity and also gave high scores to the PabBs of enteric bacteria and Vibrio cholerae. The gene on S. coelicolor cosmid L11 may, therefore, encode an ADC synthase rather than an anthranilate synthase.
In support of this conclusion, BLASTP searches of the S. coelicolor A3(2) genome with deduced amino acid sequences of the YH1, YH2 and YH3 PCR products amplified from the S. venezuelae chromosome showed that the cosmid L11 gene product resembled the YH1 and YH3 sequences more closely (63% similarity, 53% identity) than it resembled the YH2 sequence (53% similarity, 35% identity). Since the deduced YH1 and YH3 amino acid sequences matched PabAB and PabB, respectively, whereas the deduced YH2 sequence matched TrpE, the results indicate that the S. coelicolor gene in cosmid L11 is more likely to be pabB than trpE. In agreement with the assignments of trpE genes in the other S. coelicolor cosmids, the product of trpE3 in cosmid 4G6 closely resembled (75% similarity, 70% identity) the deduced amino acid sequence of the YH2 PCR product, indicating that YH2 is amplified from a trpE3 counterpart in S. venezuelae. The product of trpE2 in cosmid E8 was less similar (65% similar, 55% identical amino acids) to the YH2 PCR product, confirming that YH2 is unlikely to have been amplified from a trpE2 counterpart. Although these sequence comparisons suggested that the pabB cloned from S. venezuelae corresponds to the S. coelicolor A3(2) gene in cosmid L11, a preferential relationship could not be demonstrated through BLASTP matching of S. coelicolor A3(2) genomic proteins with the translated sequence of S. venezuelae pabB. The highest scoring sequence was anthranilate synthase component I encoded by trpE2 in cosmid E8 (59% similar, 46% identical amino acids), followed by the anthranilate synthase of trpE3 in cosmid 4G6 (54% similar, 39% identical amino acids), and then the product of the putative trpE in cosmid L11 (48% similar, 37% identical amino acids). However, similar rankings were obtained when the products of B. subtilis pabB or S. venezuelae pabAB were used to query the S. coelicolor A3(2) genomic proteins, suggesting that such sequence comparisons are not reliable indicators of functional similarity. The indication that the putative pabB/pabA gene set cloned from S. venezuelae encodes proteins with weak PABA synthase activity, but appreciable anthranilate activity is, nevertheless, noteworthy in that it could account for the observation (Lin et al., 1998 ) that disruption of the primary metabolic trpE(G) in S. venezuelae ISP5230 did not create an auxotrophic requirement for tryptophan. The analysis of S. coelicolor and S. venezuelae sequences presented above bears on the evidence from gene disruptions that implicates a third and so far undetected set of genes encoding PABA biosynthesis in S. venezuelae ISP5230 and suggests that this set may be related to the putative trpE cloned from S. coelicolor A3(2) in cosmid L11.
Phylogenetic analysis of genes in the pabB/trpE superfamily
The shared evolutionary origin of genes for PABA and anthranilate synthases, postulated from sequence data by Goncharoff & Nichols (1984) , was demonstrated further in the results of a CLUSTAL W alignment of 35 PabB and TrpE sequences. The dendrogram showing sequence relationships (Fig. 7
) indicated a superfamily derived from an ancestral sequence that diverged to form three initial groups. One (Group A) contains six TrpEs, the second (Group B) contains two TrpEs and the third (Group C) has both TrpEs and PabBs. The product of pabB cloned from S. venezuelae ISP5230 is present in Group C; its sequence shows early divergence from subgroups containing either TrpE or other PabB sequences and it has no closely related family member of recent origin.
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ACKNOWLEDGEMENTS |
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Received 7 November 2000;
revised 29 March 2001;
accepted 26 April 2001.