Department of Biological Sciences, CW405 Biological Sciences Building, University of Alberta, Edmonton, Alberta, CanadaT6G 2E91
Author for correspondence: Brenda K. Leskiw. Tel: +1 780 492 1868. Fax: +1 780 492 9234. e-mail: brenda.leskiw{at}ualberta.ca
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ABSTRACT |
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Keywords: Streptomyces, differentiation, antibiotic production, tRNA, mistranslation
Abbreviations: GFP, green fluorescent protein
The GenBank accession number for the sequence reported in this paper is AF436078.
a Present address: Cubist Pharmaceuticals, 3002386 East Mall Road UBC, Vancouver, BC, Canada V6T 1Z3.
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INTRODUCTION |
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In S. coelicolor, bldA encodes the principal leucyl tRNA for translation of rare UUA codons. In its absence the UUA codons in the messages for the pathway-specific activators of pigmented antibiotic production, ActIIORF4 and RedZ for actinorhodin and undecylprodigiosin biosynthesis, respectively, are not efficiently translated. In both cases the genes are transcribed in bldA mutants (White & Bibb, 1997 ), however the ActIIORF4:ErmE fusion protein is not detected (Gramajo et al., 1993
); transcription of redZ-dependent genes is severely reduced (White & Bibb, 1997
); and neither antibiotic is produced. The replacement of the single TTA codon in actII-orf4, or the actII-orf4:ermE fusion, with the synonymous leucine codon TTG, restores actinorhodin production (Fernández-Moreno et al., 1991
) and results in detectable ActII-orf4:ErmE fusion protein (Gramajo et al., 1993
) in bldA mutants, respectively, providing further support for the suggestion that bldA targets are not efficiently translated in bldA mutant strains. Moreover, several TTA-containing reporter genes, including ampC, lacZ and carB (Leskiw et al., 1991b
), are either not expressed or are expressed at severely reduced levels in bldA mutants.
There is some evidence to indicate that the bldA gene may be involved in the temporal regulation of gene expression in Streptomyces. Although the S. coelicolor bldA tRNA is transcribed from a vegetative promoter and expressed throughout growth, it appears to be regulated at the post-transcriptional level. In contrast to a tRNA for the abundant leucine codon CUC, the mature, 5'-processed bldA tRNA shows growth-phase dependent accumulation, being expressed at low levels until the onset of secondary metabolism and morphological differentiation and reaching maximal levels of accumulation late in growth (Leskiw et al., 1993 ; Trepanier et al., 1997
). Consistent with the low level of the
in vegetative cultures, a TTA-containing reporter gene, ampC, showed delayed production of ß-lactamase in S. lividans (Leskiw et al., 1991b
), and a subtilisin-inhibitor-encoding ssi gene, that had been engineered to contain two TTA codons, showed reduced expression, particularly during early growth, in S. lividans (Ueda et al., 1993
). This inability of young Streptomyces cultures to efficiently translate UUA-containing mRNAs suggests that the bldA tRNA may function to modulate the expression of key genes involved in sporulation and antibiotic production, as well as genes involved in other late developmental and metabolic processes. However, in contrast, studies by Gramajo et al. (1993)
found that functional bldA tRNA was present throughout growth in liquid cultures and did not show the temporal pattern of accumulation observed in both solid and in liquid grown cultures by Leskiw et al. (1993)
. Therefore, there is still no firm evidence that bldA regulates translation of UUA-containing mRNAs.
In spite of the evidence that the bldA tRNA is required for translation of TTA codons in genes required for sporulation, antibiotic production and other late functions, there is a growing body of evidence to indicate that under certain conditions TTA codons can be translated in the absence of bldA. The TTA-containing reporter genes aad and hyg were found to be only partially dependent on bldA for expression; S. coelicolor and S. lividans bldA mutant strains containing either gene on a low copy phage vector exhibited high levels of resistance to spectinomycin and hygromycin, respectively, when grown on the nutritionally complex R2YE medium, but not on minimal mannitol medium (Leskiw et al., 1991b ). In high copy number, actII-orf4 or redZ restore production of actinorhodin or undecylprodigiosin, respectively, to bldA strains when grown on the complex R2YE medium but not on the simple defined SMMS medium (White & Bibb, 1997
). In addition, redZ promoter mutations that serve to increase transcription have a similar effect on undecylprodigiosin production in bldA mutants (Guthrie et al., 1998
). As well, tetracenomycin C is still produced in an S. lividans bldA mutant at one sixth the level seen in a bldA+ strain, indicating that the single UUA codon in the O-methyltransferase (encoded by tcmP), is mistranslated (Decker & Hutchinson, 1993
). Taken together, these results indicate that mistranslation of UUA codons is occurring at some low level. Moreover, the medium-dependent expression of TTA-containing genes suggests that the level of mistranslation is dependent on environmental conditions.
In an effort to further investigate the general role of bldA in the regulation of gene expression in Streptomyces, bldA was identified and analysed in Streptomyces clavuligerus. In addition to the ß-lactam antibiotic cephamycin C, S. clavuligerus also produces the potent ß-lactamase inhibitor clavulanic acid (Reading & Cole, 1977 ), and the biosynthetic pathways for these two compounds are co-ordinately regulated by the product of the ccaR regulatory gene (Alexander & Jensen, 1998
; Perez-Llarena et al., 1997
). Since this pathway-specific regulatory gene contains a single TTA codon, it was expected that disruption of the bldA gene would result in loss of production of both cephamycin C and clavulanic acid. We show here that although the bldA mutant strain was sporulation defective, disruption of the bldA gene had no effect on translation of CcaR or on production of cephamycin C and clavulanic acid.
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METHODS |
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Liquid cultures for the isolation of plasmid DNA, the measurement of cephamycin and clavulanic acid production and/or production of S. clavuligerus cell extracts were prepared by inoculating spores into TSB supplemented with 1% (w/v) soluble starch (TSBS) for S. clavuligerus, and 1% (v/v) glycerol (TSBG) for S. lividans. The cultures were then incubated on a rotary shaker for 4872 h. Production cultures were prepared by subculturing the seed cultures into fresh TSBS or TSBG and incubating for a further 2448 h. Surface grown cultures for the measurement of cephamycin and clavulanic acid production were prepared by inoculating S. clavuligerus spores onto the surface of cellophane discs on starch-asparagine (SA) medium (Aharonowitz & Demain, 1978 ) and incubating for 24168 h at 28 °C. In the case of the constructed bldA mutant, mycelial fragments (same number of c.f.u.) were used rather than spores.
Liquid cultures for Western analysis of CcaR expression in S. coelicolor were prepared by inoculating mycelia scraped from the surface of cellophane discs on R2YE plates into 25 ml TSB supplemented with 0·5% (w/v) glucose or 0·75% (w/v) glycerol. The cultures were then incubated on a rotary shaker at 30 °C and 250 r.p.m. for 48 h.
Surface-grown cultures for the isolation of RNA were prepared as described above for the measurement of antibiotic production. Liquid-grown cultures for the isolation of RNA were prepared by inoculating mycelial fragments (final OD600 of 0·03) of both the S. clavuligerus wild-type and bldA mutant strains into 100 ml TSBS and incubating at 28 °C and 250 r.p.m. for 2472 h.
Escherichia coli cultures were maintained on LB or 2YT agar and grown in 2YT broth at 37 °C (Sambrook et al., 1989 ). Plasmid-containing cultures were supplemented with ampicillin (100 µg ml-1; Sigma) or Apralan (50 µg ml-1; Provel) as appropriate.
Transformation of Streptomyces strains.
Protoplasts were prepared and transformed as described by Hopwood et al. (1985) for S. lividans and S. coelicolor, with modifications by Bailey & Winstanley (1986)
for S. clavuligerus. Selection of transformants was carried out as described by Hopwood et al. (1985)
. Because of the highly active restrictionmodification system in S. clavuligerus, covalently closed circular DNA for transformation into this strain was either isolated from a dam dcm E. coli strain (ET12567) or first passaged through S. lividans.
Recombinant DNA techniques.
Standard DNA manipulations, such as restriction endonuclease digestion, ligation, generation of blunt ends, random primer labelling, E. coli transformation and preparation of single-stranded phage or phagemid DNA were all done as described by Sambrook et al. (1989) . Isolation of chromosomal and plasmid DNA from Streptomyces strains was done as described by Hopwood et al. (1985)
. Plasmid cloning vectors, oligonucleotide primers and plasmid constructs used for sequence analysis, gene disruption, complementation or expression studies are listed in Tables 1
and 2
.
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Sequence analysis of the S. clavuligerus bldA gene.
The bldA-hybridizing 1·5 kb SalI fragment was subcloned from pAU102 by digestion with NcoI, XmaI and SalI restriction enzymes alone or in combination, followed by purification of the fragments from polyacrylamide gels by crushing and soaking, and finally ligation into pUC119 or pUC120. All of the recombinant plasmids were transformed into E. coli MV1193 for generation of single-stranded template and all of the templates were sequenced using universal primer. Synthetic oligonucleotide primers were also used to sequence the gaps. Both strands were sequenced over the entire 1·5 kb region.
Generation of a bldA null mutant strain.
To create a bldA null mutation in S. clavuligerus, the sequence 5' of the bldA gene was first purified as a 540 bp SalINcoI fragment from pAU102 by crushing and soaking. The fragment containing sequence 3' of the bldA gene was generated by PCR using pAU102 as template DNA, universal sequencing primer and a specific oligonucleotide NTR5 (see Table 2), which is complementary to the sequence immediately 3' of the bldA tDNA and containing a 5' non-homologous extension with a KpnI restriction site. Following amplification, the PCR product was digested with BamHI and KpnI to generate a 739 bp fragment. The replacement fragment, a 1·45 kb KpnINcoI fragment containing the gene for apramycin resistance (apr), was purified using Gene Clean (Bio101) from pUC120Ap(Nco) (Table 1
). The 5' and 3' bldA flanking fragments, as well as the apr fragment, were ligated simultaneously into SalI/BamHI-digested pUC119, and transformed into E. coli MV1193 competent cells. Plasmid DNA was isolated from Apralan-resistant transformants, and the presence of bldA flanking sequences was verified by restriction analysis and Southern hybridization (not shown). All of the transformants had identical restriction patterns so one representative was chosen and designated pAU103. The entire 2·8 kb gene replacement insert was then removed from pAU103 as a HindIIIEcoRI fragment, ligated into similarly digested pIJ486 and transformed into S. lividans TK24. Plasmid DNA was isolated from thiostrepton resistant (ThioR), Apralan resistant (AprR) transformants and the integrity of the recombinant plasmid was verified by restriction analysis. Again, one representative, designated pAU104, was chosen for transformation into S. clavuligerus NRRL 3585, and ThioR transformants were selected. The ThioR transformants, which could result from freely replicating plasmid or by a single cross-over into the chromosome, were then propagated for several rounds of liquid culture without selection to allow plasmid loss. A double cross-over event, with subsequent loss of the vector sequences and resulting in AprR, thiostrepton sensitive (ThioS) cultures, was monitored by separately plating aliquots of the broth cultures on MYM agar plates containing Apralan or thiostrepton. When the sample plates indicated that the cultures were ThioS and AprR, aliquots were plated onto MYM agar without antibiotic. Isolated colonies were patched onto a master plate without selection, and these master plates were used to replica plate to MYM agar containing either thiostrepton or Apralan for identification of AprR, ThioS colonies. Since the colonies were not derived from a single cell or spore, they were transferred to ISP#3 sporulation medium (Difco) and the small quantities of spores produced at the periphery of the bald colonies were collected to generate single spore isolates. To confirm that the bldA gene had been deleted, chromosomal DNA was obtained from several isolates, digested with SalI, and analysed by Southern blot using the [32P]dCTP random primer-labelled apramycin resistance gene fragment and, after stripping (using a boiling solution of 0·1% SDS), with similarly labelled bldA DNA as probe. The bldA probe was generated by PCR using pAU102 as template and NTR9 (corresponding to the region spanning the NcoI site 127147 nt upstream of the 5' end of the bldA tDNA, and containing an EcoRI site engineered onto the 5' end) and NTR10 (complementary to the 3' end of the bldA tDNA) as primers (see Table 2
).
Creation of complementation constructs.
A high-copy-number bldA complementation vector, designated pAU105, was generated by ligating a 305 bp bldA fragment into EcoRI/XbaI-digested pIJ486. The 305 bp bldA fragment was generated by PCR using the primers NTR8 (complementary to the region 4564 nt downstream of the 3' end of the tDNA, and containing an XbaI site engineered onto the 5' end) and NTR9 (see above and Table 2). To create a single-copy complementation construct, the bldA gene was also cloned into the Streptomyces
C31 att-site integrating E. coli/Streptomyces shuttle vector, pSET152. In this case, a larger, 655 bp bldA-containing fragment, extending an additional 360 bp upstream of the 305 bp fragment described above, was used for the complementation. The 655 bp fragment was generated by PCR using the primers NTR8 and NTR20 (located in the region 489506 nt upstream of the tDNA and including an EcoRI site engineered onto its 5' end; Table 2
). The 655 bp bldA gene fragment was ligated along with a 1·1 kb EcoRIXbaI thiostrepton resistance gene fragment from pAU5 (Giebelhaus et al., 1996
) into the EcoRI site of pSET152 and transformed into E. coli DH5
. The isolated recombinant plasmid was designated pSET152(bldA+tsr). As a control, the thiostrepton resistance gene alone was isolated as a BclI fragment from pAU5 and ligated into the BamHI site of pSET152 to generate pSET152(tsr). In both pSET152(bldA+tsr) and pSET152(tsr), the thiostrepton resistance gene was in the same orientation as the lacZ gene, and the bldA gene in pSET152(bldA+tsr) was in the opposite orientation to ensure that any bldA expression was from its own promoter and not the result of read-through expression from either the tsr or lacZ promoter. Both plasmids were transformed into E. coli ET12567, a dam dcm strain for generation of unmethylated plasmid DNA, and then into the S. clavuligerus bldA deletion mutant,
bldA4-1.
Creation of a ccaR expression construct.
An expression construct, pDA1102, carrying the ccaR gene from S. clavuligerus under the control of the glycerol regulatable promoters (gylP1P2) from S. coelicolor was prepared by digesting the gylP1P2-bearing integrative plasmid, pMT3226 (generously provided by C. Smith, University of Manchester), with XbaI and BamHI to remove the xylE reporter gene. The xylE gene was replaced by a pair of oligonucleotide linkers, DylSB1 and DylSB2 (Table 2) that self anneal to give a short stretch of DNA sequence bounded by XbaI and BamHI sites. The inserted sequence corresponds to the region between the gylP1P2 promoters and the gylA ORF in S. coelicolor, except for minor changes to introduce restriction sites including an NdeI site at the position normally occupied by the gylA start codon, and a single base insertion in the region of the RBS. This construct (pDA1100) allows genes engineered to have NdeI sites at their start codons to be positioned appropriately behind the gylP1P2 promoters and RBS.
The ccaR expression construct was then prepared by PCR amplification of ccaR from S. clavuligerus using Taq DNA polymerase and the primer Dyl30 to introduce an NdeI site at the start codon, and a pre-existing primer, Dyl12, to introduce an EcoRI site immediately downstream of the gene (Table 2). The amplified ccaR gene was passaged through pSL1180 and pBluescript II SK to replace the EcoRI site with the required XbaI site, and then cloned into the NdeI and XbaI sites of pDA1100 to give pDA1102. The fidelity of the amplified ccaR gene was confirmed by sequence analysis. For the purposes of this study, the start codon of ccaR is defined as the ATG codon at nt 2566 of the deposited sequence (AF073897), rather than the GTG codon at nt 2584 as originally suggested by Perez-Llarena et al. (1997)
.
RNA isolation and analysis.
RNA was extracted from surface-grown or liquid cultures as described elsewhere (Leskiw et al., 1993 ). Northern blot analysis of bldA and leuU transcripts was performed as previously described (Trepanier et al., 1997
) [the probes for bldA, leuU and the 5S rRNA control were the oligonucleotides BKL5, BKL42 and BKL53 (Table 2
), respectively, except that the hybridization temperature for the bldA probe was 37 °C]. Northern blot analysis of ccaR was performed according to Williams & Mason (1985)
as previously described (Leskiw et al., 1993
). For detection of ccaR transcripts, the probe was a [32P]dCTP random primer-labelled 574 bp NcoISacI ccaR gene fragment. Hybridization was carried out at 50 °C in 50% formamide hybridization solution. Washes were carried out at 50 °C with a final wash at 60 °C. As a control for RNA loading, the membrane was stripped (at 65 °C in a solution containing 5 mM Tris/HCl, pH 8·0, 2 mM EDTA and 0·1xDenhardts solution) and then probed with the 32P end-labelled 17-mer oligonucleotide BKL54 (Table 2
) to detect 16S rRNA transcripts. Hybridization and washing were carried out at 55 °C according to Procedure B described by Hopwood et al. (1985)
. The signals were detected using a phosphorimager (Molecular Dynamics model 445 SI).
Antibiotic quantification and cell extract preparation.
Cultures of S. clavuligerus growing on SA or in TSBS medium were sampled at various times between 24 and 168 h. The same cultures used for RNA isolation were also used for determination of antibiotic production. When liquid cultures were used, culture filtrates were stored at -20 °C and bioassayed (100 µl aliquots on 1 cm filter paper discs) as previously described (Jensen et al., 1982 ) within 1 week for cephamycin and clavulanic acid production. Antibiotic production from S. clavuligerus grown on the surface of cellophane discs was assayed by removing a plug of agar from under the cellophane disc with a No. 4 cork borer (7 mm diameter), and placing the plug on the surface of the MYM bioassay plate for 1 h to allow the antibiotic to diffuse. The plug was then removed and the plate was overlaid with 5 ml soft MYM (0·6% agar) supplemented with an aliquot of E. coli ESS for assay of cephamycin C, and S. aureus N2, as well as 3 µg penicillin G ml-1 (omitted from the controls), for assay of clavulanic acid.
When S. clavuligerus cell extracts were to be analysed, the mycelia were harvested from TSBS cultures by filtration, washed with TDE buffer (50 mM Tris/HCl, pH 7·2, 0·1 mM DTT, 0·01 mM EDTA) and then resuspended in 1/5 original volume of TDE. Washed mycelia were disrupted by sonication (three 15 s pulses; microprobe, power setting 3; Branson Sonifier 450, Branson Ultrasonic) and then centrifuged for 15 min at 4 °C. Cell extracts were assayed for protein content (Bradford, 1976 ) using the microassay procedure described by the supplier of the reagent (Bio-Rad) and stored at -70 °C.
When cell extracts were to be analysed from S. coelicolor strains containing pDA1102, the mycelia were harvested from TSB (+0·5% glucose or 0·75% glycerol as described above) cultures by centrifugation, washed with 500 µl TDE buffer and then resuspended in 500 µl TDE. Washed mycelia were disrupted by sonication (two 20 s pulses; microprobe, power setting 3; Branson Sonifier 450, Branson Ultrasonic) and then centrifuged for 10 min at 4 °C. Cell extracts were then assayed for protein content and stored as described above.
Western blot analysis.
Ten to thirty micrograms of cell extract protein were separated on 10 or 15% SDS-polyacrylamide gels. The proteins were transferred to PVDF membranes (Immobilon-P; Millipore) using a Bio-Rad Transblot apparatus. The membranes were blocked in 5% (w/v) BSA or skimmed milk (Difco) in Tris-buffered saline containing 0·1% Tween 20 for 1 h at 25 °C. Proteins were detected using either the ECL (enhanced chemiluminescence) Western system reagents and protocols (Amersham) or the Renaissance Western Blot Chemiluminescence Reagent (NEN Life Science Products). The CcaR primary antibody (Alexander, 1998 ) was used at 1:5000 dilution. Donkey anti-rabbit IgGhorseradish peroxidase conjugate (Amersham) was used as secondary antibody (1:5000 or 1:10000 dilution).
Green fluorescent protein (GFP) reporter gene analysis.
The gfpmut3 gene (Cormack et al., 1996 ) containing three TTA codons was cloned into the high copy number Streptomyces vector pIJ486, under the control of the upregulated and constitutively expressed ermE* promoter (Bibb et al., 1985
) and designated pAU101 (H. E. Markus & B. K. Leskiw, unpublished). pAU101 was introduced into S. lividans and S. clavuligerus wild-type and corresponding bldA mutant strains, and the colonies were assessed for GFP expression under a microscope using a mercury lamp and a FITC filter.
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RESULTS |
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The nucleotide sequence corresponding to the mature, processed form of the S. clavuligerus bldA gene product (tDNA) was found between nt 681 and 764 on the 1·5 kb SalI fragment. No other tRNA genes are located in this region of the chromosome. Comparison of the putative S. clavuligerus bldA tRNA with the S. coelicolor bldA tRNA showed 91% identity between the two tRNAs with a single base gap (Fig. 1
). With two exceptions, the sequence differences are confined to the variable loop and a single base difference is seen in the T-loop at position 57. Although the tRNAs differ at this position, they both contain a purine which is typically conserved in tRNAs. The S. clavuligerus bldA tDNA is 84 nt in length compared to the S. coelicolor bldA tDNA which is 87 nt. The differences in length are due to an extra base in the variable loop and the gene encoded 3' CCA terminus in the S. coelicolor sequence. The sequence of the 1·5 kb SalI fragment has been deposited in GenBank under accession no. AF436078.
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The bldA gene was also cloned into the Streptomyces C31 att-site integrating, E. coli/Streptomyces shuttle vector pSET152, to ensure that a single copy of the bldA gene was sufficient to complement the mutant phenotype. In this case, a larger, 655 bp bldA-containing fragment, extending an additional 360 bp upstream of the 305 bp fragment described above, was used for the complementation. The larger size was chosen because although the 305 bp bldA fragment used for the high-copy complementation restored the wild-type phenotype, it was later determined that the S. clavuligerus bldA gene may contain additional promoters not included on this fragment (Trepanier, 1999
). The recombinant plasmid, designated pSET152(bldA+tsr), and the control plasmid, pSET152(tsr), were introduced into S. clavuligerus
bldA4-1. After selection of thiostrepton-resistant transformants, integration of both vectors into the S. clavuligerus chromosome was verified by Southern analysis (not shown) of chromosomal DNA digests using separately the bldA and tsr gene fragments as probes. As expected, the presence of the bldA gene in single copy in the S. clavuligerus chromosome was sufficient to complement the bldA sporulation deficient phenotype, although S. clavuligerus
bldA4-1 containing pSET152(bldA+tsr) did not sporulate as vigorously as the wild-type strain or the strain complemented with the bldA gene at high copy number. Whether this was due to a positional effect of integration of the single-copy bldA gene at the attB site, rather than at its native location, or due to the presence of a bldA promoter more than 487 bp upstream of the bldA tDNA, was not further investigated.
Antibiotics and the bldA tRNA both show growth-phase dependent accumulation in S. clavuligerus
Since the processed, mature form of the bldA tRNA showed growth-phase dependent accumulation in S. coelicolor (Leskiw et al., 1993 ; Trepanier et al., 1997
), it was of interest to determine if the bldA tRNA would show the same pattern of accumulation in S. clavuligerus. RNA was isolated in duplicate at various time points between 24 and 72 h post-inoculation from liquid cultures, and between 30 and 168 h post-inoculation from surface-grown cultures of S. clavuligerus NRRL 3585 and S. clavuligerus
bldA4-1. The RNA samples were isolated from two independent time courses and subjected to Northern blot analysis. Representative results are shown in Fig. 3
. Similar to analysis of the bldA tRNA from S. coelicolor (Leskiw et al., 1993
; Trepanier, 1999
; Trepanier et al., 1997
), the bldA tRNA showed a temporal pattern of accumulation in wild-type S. clavuligerus, with higher levels of the tRNA being seen in older, differentiating cultures. This was in contrast to the pattern of accumulation of the processed form of an abundant leucyl-tRNA (encoded by leuU), which was present at relatively constant levels throughout growth. As expected, the bldA tRNA was undetectable in the
bldA4-1 mutant strain.
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Not all UUA codons are efficiently mistranslated in the S. clavuligerus bldA mutant
The production of cephamycin C and clavulanic acid in the S. clavuligerus bldA mutant in the absence of elevated levels of the ccaR mRNA, either from additional cloned copies of ccaR or overexpression of the transcript from the single chromosomal copy, suggested one of two possibilities. Firstly, that the UUA codon in the ccaR message was being efficiently mistranslated, or secondly, that only a very few copies of the regulator were needed to activate transcription of the antibiotic biosynthetic pathway genes, and that low-level mistranslation of the ccaR mRNA was sufficient to achieve a threshold level. To distinguish between these two possibilities, cell extracts prepared from 24 and 48 h liquid cultures of the wild-type and bldA mutant strains, as well as a negative control strain S. clavuligerus ccaR::apr with a disrupted copy of ccaR in the chromosome, were subjected to Western analysis to determine the CcaR expression levels. Equivalent amounts (10 µg) of cell extract protein were probed with antiserum specific for CcaR and the results are shown in Fig. 4. Barely detectable levels of CcaR were present in the 24 h cultures of both the wild-type and bldA strains (not shown), and both strains expressed the same high level of CcaR by 48 h post-inoculation.
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DISCUSSION |
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The UUA codon in the mRNA of the pathway-specific regulator of cephamycin C and clavulanic acid biosynthesis is mistranslated in an S. clavuligerus bldA deletion mutant
As has been the case in all constructed bldA mutants from other Streptomyces species, deletion of the bldA-encoded tRNA from the S. clavuligerus chromosome blocked aerial mycelium formation. However, deletion of the tRNA did not appear to have any effect on translation of the TTA-containing ccaR gene, encoding the pathway-specific regulator of cephamycin C and clavulanic acid. In contrast, the expression of a heterologous reporter gene, gfp-mut3, containing 3 TTA codons, was severely dependent on bldA for expression. The difference in bldA dependence of ccaR and gfp-mut3 in S. clavuligerus, together with the growing body of evidence that suggests that some bldA targets are mistranslated under some conditions, leads us to propose that the context of the TTA codon is an important determinant for the frequency of mistranslation. Since ccaR is the first example thus far of a target that is apparently independent of bldA when expressed at its wild-type copy number, it seems reasonable to assume that the TTA codon in this context is mistranslated efficiently. Likewise, the target genes that have been demonstrated to be dependent on bldA for expression [gfp-mut3, carB (Epp et al., 1987 ; Leskiw et al., 1991b
), hyg (Leskiw et al., 1991b
; Zalacain et al., 1986
), actII-orf2 (Fernández-Moreno et al., 1991
), actII-orf4 (Fernández-Moreno et al., 1991
), strR (Distler et al., 1987
), redZ (White & Bibb, 1997
), celA2 (Garda et al., 1997
), lipR (Servin-Gonzalez et al., 1997
), lacZ, ampC and aad (Burland et al., 1995
; Hollingshead & Vapnek, 1985
; Kalnins et al., 1983
; Leskiw et al., 1991b
)] must not be mistranslated efficiently, at least under the conditions tested. In an effort to elucidate what this context effect might be, the sequence within one ribosomal binding unit [12 codons (Chen & Inouye, 1990
)] of the TTA codon in the bldA independent ccaR gene was compared to the TTA-surrounding sequences in the 12 target genes (see above for list) known to be bldA dependent (available as supplementary data at http://mic.sgmjournals.org). While the relative position of some rare codons has been shown to have a severe effect on translation when they are located in the first 2550 codons, and a more moderate effect when located further away from the 5' end (Chen & Inouye, 1990
), this does not appear to be a major factor in the translation of TTA codons in Streptomyces. ccaR, with a TTA codon at position 38 (see GenBank accession no. AF073897), is bldA independent while lipR, with a TTA codon at position 831, is bldA dependent.
Another factor known to result in different levels of mistranslation is the relative rate of translation at TTA codons. Since minor and rare codons are typically translated at slower rates than major codons (Sorensen et al., 1989 , 1990
), it is possible that the presence or absence of minor codons in the vicinity of TTA codons could alter the rate of translation and subsequently increase or decrease the level of TTA codon mistranslation. However, the number of minor codons preceding the TTA codon (within one ribosomal binding unit) has no apparent effect on the level of mistranslation as lipR contains no minor codons in this region, and gfp-mut3 contains six to eight minor codons within 12 codons upstream of each of its three TTA codons. Likewise, the presence or absence of a minor codon immediately 5' or 3' of the TTA codon appears to have no effect.
Since individual nucleotides flanking rare codons have been linked to frameshifting in a number of systems (Belcourt & Farabaugh, 1990 ; Brierley et al., 1992
; Farabaugh et al., 1993
; Jacks et al., 1988
; Lindsley & Gallant, 1993
; Shpaer, 1986
), the nature of the nucleotides immediately preceding and following the TTA codons was also examined. While no apparent pattern was obvious for the nucleotide 5' of the TTA codon, the nucleotide 3' of the TTA codon appears to be non-random with respect to C versus G and T versus A distribution. With one exception, TTA codons in the nine Streptomyces genes assayed for bldA dependence are followed by C or T. Interestingly, the only case where this is not true is in the single known bldA independent target, ccaR, where a G is immediately 3' of the TTA codon. The TTA-containing non-streptomycete genes that have been assayed for expression in S. coelicolor bldA mutants show a more random distribution of nucleotides following TTA codons; however, all of these bldA dependent targets contain at least one TTA codon in the context TTAC or TTAT. This observation raises the intriguing possibility that bldA dependence is determined by whether in-frame mistranslation or frameshifting occurs when a ribosome stalls at a hungry TTA codon. In the case of the bldA target, ccaR, the sequence TTA G appears to be mistranslated in-frame, as evidenced by bldA independent expression of apparently wild-type levels of full length CcaR. This implies that the G 3' of the TTA codon must somehow prevent or suppress a +1 frameshift. The +1 reading frame in this sequence is the TAG (UAG) amber stop codon which is not expected to allow the required pairing with a tRNA to introduce a +1 frameshift. Similarly, the sequence TTA A, which encodes an ochre stop codon in the +1 frame also should not allow a +1 frameshift. Therefore, the sequence TTA R (G or A) is predicted to be mistranslated in-frame by imperfect pairing with another tRNA, most likely
which has been shown to translate UUA codons efficiently in E. coli in the absence of competition from the cognate tRNA (Takai et al., 1994
). On the other hand, the sequence TTA Y (C or T), which encodes tyrosine in the +1 frame, is predicted to allow a +1 frameshift to occur. In this case, the frameshifting presumably occurs preferentially over in-frame mistranslation since all bldA targets containing the sequences TTA Y are bldA dependent. Two possible explanations for why frameshifting might be favoured over in-frame mistranslation are: the codonanticodon interaction might be stronger with the tyrosinyl tRNA in the +1 frame than it is with the non-cognate
in the zero frame, and/or tyrosinyl-tRNA might be more abundant than the non-cognate leucyl-tRNAs required for in-frame mistranslation. Although it may seem surprising that these predicted frameshift products have not been detected thus far, most studies to date have tested for product by function and have not looked for non-functional product, since it was generally assumed that the majority of peptides were prematurely terminated at hungry TTA codons. Although most frameshift products are expected to terminate prematurely at +1 frame stop codons, many of these products should be distinct in size from truncated proteins that would be produced by ribosomal fall-off at TTA codons.
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ACKNOWLEDGEMENTS |
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Received 25 June 2001;
revised 23 October 2001;
accepted 30 October 2001.