Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus

Liru Wang{dagger}, Kapil Tahlan, Tracy L. Kaziuk, Dylan C. Alexander{ddagger} and Susan E. Jensen

Department of Biological Sciences, University of Alberta, Edmonton, Canada T6G 2E9

Correspondence
Susan E. Jensen
susan.jensen{at}ualberta.ca


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus. Previous sequence analyses of the ccaR gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli. A protein product was only obtained from the ATG construct. Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S. clavuligerus ccaR mutant. Bioassays showed that only the ATG construct could complement the ccaR mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC. The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon. Inspection of the region upstream of ccaR by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.


Abbreviations: SARP, Streptomyces antibiotic regulatory protein; TSP, transcription start point

{dagger}Present address: CanBioCin, Inc., Suite 1015, 8308-114 St., Edmonton, Canada AB T6G 2E1.

{ddagger}Present address: Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The production of cephamycin C, clavulanic acid and four other clavam metabolites in Streptomyces clavuligerus is accomplished by biosynthetic enzymes encoded by three separate clusters of genes (Tahlan et al., 2004a, b). The ccaR gene, located within the one of these clusters (Alexander & Jensen, 1998; Perez-Llarena et al., 1997), encodes a positive-acting transcriptional regulator that controls the production of both cephamycin C and clavulanic acid. CcaR is a member of the growing class of regulatory proteins referred to as Streptomyces Antibiotic Regulatory Proteins or SARPs (Wietzorrek & Bibb, 1997). The importance of CcaR in the production of cephamycin C and clavulanic acid has been established by showing loss of production in ccaR mutant strains (Alexander & Jensen, 1998; Perez-Llarena et al., 1997), and increased production when ccaR is overexpressed in wild-type S. clavuligerus (Perez-Llarena et al., 1997). However, the detailed mechanism by which ccaR regulates clavulanic acid and cephamycin C production has been more difficult to determine at the molecular level, and studies to date have generated conflicting results (Kyung et al., 2001; Santamarta et al., 2002).

While the ccaR gene has been located and sequenced, uncertainty remains regarding the translational and transcriptional regulatory signals that control its expression. Initial studies identified an ATG codon as the start codon for ccaR (Walters et al., 1994). However, a GTG codon present in-frame with the ATG but 18 bp downstream has also been proposed (Perez-Llarena et al., 1997). Because of the central role that CcaR plays in regulating cephamycin C and clavulanic acid production, there is interest in understanding how CcaR regulates the production of these valuable metabolites. CcaR is present at low levels in wild-type S. clavuligerus, and so engineered production of CcaR in Escherichia coli or Streptomyces species offers the best means to generate large amounts of protein for studies of its regulatory properties. However, this requires knowledge of the transcriptional and translational regulatory signals that control the expression of the ccaR gene. In this study we identify the start codon for ccaR and use S1 nuclease protection and primer extension studies to determine the location of the transcription start point(s) for the gene.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains were grown and maintained on LB medium (Sambrook et al., 1989) at 37 °C. S. clavuligerus strains were maintained on ISP medium 4 (Difco) at 28 °C. AS-1 medium supplemented with 10 mM MgCl2 (Bierman et al., 1992) was used for the isolation of S. clavuligerus exconjugants following intergeneric conjugation. Minimal liquid (NMMP) (Kieser et al., 2000) and complex [trypticase soy broth, TSB (Difco)] media, with and without added glycerol, were used to grow S. clavuligerus {Delta}ccaR : : tsr mutants carrying pDA1100, pDA1102 and pDA1103 constructs to assess complementation of the ccaR mutation. Spores stored at –70 °C in 20 % glycerol (w/v) were inoculated into TSB+1 % soluble starch and incubated for 40 h at 28 °C at 250 r.p.m. on a rotary shaker before they were subcultured at 2 % (v/v) into TSB or NMMP media for production of cephamycin C. S. clavuligerus {Delta}ccaR : : tsr mutants complemented with pDA1006 and pDA1006*, and S. clavuligerus wild-type strains for production of RNA used in SI nuclease protection and primer extension studies were grown in a complex soy flour-based medium (Mosher et al., 1999) using incubation conditions described above.


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Table 1. Bacterial strains and plasmids used in this study

 
For E. coli, media were supplemented with ampicillin (100 µg ml–1) or apramycin (Apralan, Provel, 50 µg ml–1), as appropriate. For S. clavuligerus, media were supplemented with apramycin (25 µg ml–1) or thiostrepton (5 µg ml–1), as appropriate.

Recombinant DNA techniques.
Plasmid isolation, transformation, ligation, end-labelling of DNA using [{gamma}-32P]dATP and other molecular biology procedures commonly used with E. coli were as described by Sambrook et al. (1989). Manual sequencing of DNA was performed using the Thermo Sequenase Radiolabelled Terminator Cycle Sequencing Kit (USB) and the reaction mixtures were fractionated by electrophoresis on 6 % denaturing polyacrylamide gels. Commonly used molecular biology procedures for Streptomyces species were as described by Kieser et al. (2000). Plasmids were introduced into S. clavuligerus either by protoplast transformation, as described previously (Paradkar & Jensen, 1995), or by conjugal plasmid transfer. Conjugation was carried out as described by Kieser et al. (2000), except that exconjugants were selected on AS-1 agar medium supplemented with 10 mM MgCl2 (Bierman et al., 1992) and antibiotics as appropriate.

Generation of ccaR expression vectors.
The oligodeoxyribonucleotide primers used in this study are listed in Table 2. Primers DYL30 (forward) and SEJ25 (reverse) were used in a PCR-mediated process to amplify a mutant form of ccaR with an NdeI site at the potential ATG start codon at the 5' end of the gene, and an EcoRI site just beyond the 3' end of the gene. PCR product was obtained using Vent DNA polymerase (New England Biolabs; 92 °C, 45 s; 57 °C, 60 s; 72 °C, 90 s for 30 cycles) in a reaction mixture containing 2 % DMSO, with pDA150 as template. The resulting PCR product was cloned as a NdeI–BamHI fragment into pSL1180. To reduce the possibility of unintended PCR-introduced mutations, only the 5' end of the PCR-amplified ccaR gene was used for subsequent manipulations. Digestion at a vector-derived SpeI site and an Eco47III site internal to ccaR (position +114 bp relative to the putative ATG start codon) excised the 5' end of the newly amplified gene. It was used to replace the corresponding fragment from a wild-type copy of ccaR that had been cloned previously into pBluescript II pSK(+) as a BamHI–NruI fragment. The reassembled ccaR gene was recloned into pBluescript II pSK(+) as a HindIII–XhoI fragment to pick up appropriate restriction sites, and finally cloned into pT7-7 as a NdeI–BamHI fragment to yield pTK-1.


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Table 2. Oligodeoxyribonucleotides used in this study

NdeI sites engineered into primers at the two potential start codons are underlined. Mutations introduced to change the potential GTG start codon to GTC are shown in bold type.

 
pTK-2 was constructed exactly as pTK-1, except that DYL34 (forward) and SEJ25 (reverse) were used to amplify a mutant form of ccaR with a NdeI site at the potential GTG (now changed to ATG) start codon near the 5' end of the gene.

pDA1100 is an integrative vector based on pMT3226 that was prepared by digesting with BamHI/XbaI to remove the xylE reporter gene, and replacing it with a synthetic DNA fragment that comprises 39 bp of double-stranded sequence flanked on each end by 5' overhanging GATC sequences. The synthetic fragment closely approximates the region upstream of glpF (previously called gylC) from the start codon to the gylP1P2 promoter, but modified to introduce a BamHI site at the gylP1P2 end, an NdeI site at the glpF start codon and an XbaI site immediately downstream (Fig. 1). In S. coelicolor, glpF is located just downstream from gylR, and its expression is regulated by GylR and glycerol (Hindle & Smith, 1984). pDA1100 maintains this organization and sequence as closely as possible. Mutant versions of ccaR with NdeI sites at either the potential ATG or GTG (changed to ATG) start codons were excised from the pBluescript II pSK(+) constructs, which gave rise to pTK-1 and pTK-2, respectively (see above), as NdeI–XbaI fragments, and cloned into pDA1100 under the control of the gylP1/P2 promoters, to give pDA1102 and pDA1103 (Fig. 1).



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Fig. 1. Plasmid constructs used for expression of ccaR under control of the gylP1/P2 promoter. Terminator fragments, tmmr and tfd, are shown as grey boxes; the gylP1/P2 fragment is shown as an open box; gylR, xylE and ccaR genes are shown as open arrows. B, BamHI; Xh, XhoI; Xb, XbaI; N, NdeI.

 
Primers LWA1, LWA2, LWA3 and LWA4 were used in a PCR-mediated process to mutate the GTG codon located near the 5' end of ccaR to GTC. With LWA1 (reverse) and LWA2 (forward) as primers and pDA150 as template, a 381 bp PCR product was amplified using the Expand High Fidelity PCR system (Roche; 95 °C, 30 s; 62 °C, 30 s; 72 °C, 1 min for 30 cycles) (Fig. 2a). The product was combined with a 922 bp PCR product amplified using primers LWA3 (forward) and LWA4 (reverse) and the same pDA150 template, and then the mixture of the two products was used with primers LWA2 (forward) and LWA4 (reverse) in a PCR reaction with no added template to give a 1·23 kb PCR fragment (Fig. 2b, c). The 1·23 kb fragment was made blunt by treatment with T4 polymerase I, digested with BamHI (LWA2 contains a BamHI site), and then ligated to BamHI/EcoRV-digested pSET152, giving pLW3.



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Fig. 2. Conservative mutation of the potential GTG start codon in ccaR to GTC. (a) Primers LWA1, LWA2, LWA3 and LWA4 were used to amplify two overlapping DNA fragments (381 bp and 922 bp) from the ccaR gene and upstream region by PCR. The primers LWA1 and LWA3 are mutagenic, resulting in conversion of the putative GTG start codon to a GTC codon. (b) The two amplified fragments were used as template with primers LWA2 and LWA4 in a second PCR reaction. (c) Amplified product (1·23 kb) containing the mutant form of ccaR.

 
pDA1006 carries ccaR cloned as a 1·4 kb BamHI–NruI fragment of S. clavuligerus DNA into the EcoRI–EcoRV sites of pSET152 after passage through pSL1180 to pick up compatible restriction sites for cloning. To prepare pDA1006*, a ccaR expression construct equivalent to pDA1006, except with the GTG potential start codon changed to GTC, the 300 bp KpnI–NruI fragment of pDA1006 which encompasses the 3' end of ccaR and downstream regions was used to replace the corresponding 144 bp KpnI–EcoRV fragment of pLW3.

Expression of ccaR in E. coli.
CcaR was expressed from either pTK-1 or pTK-2 in E. coli. In both plasmids ccaR was expressed under the control of the {pi}10 T7 promoter in the expression vector pT7-7. The plasmids were introduced by electrotransformation into E. coli BL21(DE3), which carries the gene for T7 RNA polymerase under the control of the IPTG-inducible lac promoter. Cultures were grown to an OD600 of 0·6–0·8 before induction with IPTG to a final concentration of 0·4 mM. Samples of cultures were harvested at intervals just prior to, and after induction with IPTG. Cells were resuspended to 1/10 original culture volume in SDS-PAGE sample buffer. After heating for 5 min at 100 °C, 5 and 15 µl amounts were separated on a 12 % SDS polyacrylamide gel and proteins were visualized by staining with Coomassie Brilliant Blue.

Expression of ccaR in S. clavuligerus.
pDA1102, pDA1103 and pDA1100 were transformed into S. clavuligerus {Delta}ccaR : : tsr, a ccaR mutant in which the 1·4 kb BamHI–NruI fragment that encompasses ccaR is deleted and replaced by a thiostrepton resistance gene (Alexander & Jensen, 1998). Transformants were cultivated in both TSB and NMMP growth media supplemented with various concentrations of glycerol, and assayed for the production of cephamycin C. Cells harvested at each sample time were also washed with 0·85 % NaCl, resuspended in 50 mM Tris/HCl, pH 7·2, 0·01 mM EDTA and 0·1 mM DTT, and then disrupted by sonication. After centrifugation for 5 min at 10 000 g, the resulting cell extracts were used for Western analysis.

The pDA1006 and pDA1006* complementation constructs were introduced into the {Delta}ccaR : : tsr strain by conjugation and with apramycin selection for exconjugants.

Western blot analysis.
Forty-microgram amounts of cell extract protein were separated by duplicate SDS-PAGE (12·5 %) then electroblotted onto Immobilon-P PVDF membranes (Millipore) and analysed as described previously (Alexander & Jensen, 1998). Primary anti-CcaR antibodies (Alexander & Jensen, 1998) were used at a dilution of 1 : 4000. Horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Amersham Life Sciences) was used as secondary antibody at a dilution of 1 : 5000.

S1 nuclease protection and primer extension studies.
The transcription start points (TSPs) for ccaR were determined by S1 nuclease protection analyses using previously described methods (Kieser et al., 2000), and by primer extension analysis. The S1 probe used to locate TSP-1 was amplified by PCR using primers LWA5 (forward) and BKL96 (reverse) with pDA1006 as template, and the Expand High Fidelity PCR system (Roche; 94 °C, 45 s; 65 °C, 45 s; 72 °C, 45 s for 10 cycles followed by 94 °C, 45 s; 70 °C, 45 s; 72 °C, 45 s for 15 cycles). The resulting probe fragment extended from –193 bp to +22 bp relative to the ccaR translation start point, and included 10 bp of non-S. clavuligerus-derived sequence to enable full-length protection of the probe to be distinguished from probe–probe reannealing. A second S1 protection assay was also performed to investigate the possibility of an additional upstream TSP for ccaR. The primers ccaR-UP-Forward and ccaR-UP-Reverse were used to amplify a probe using pDA1006 as template as described above. This second S1 probe extended from –412 bp to –133 bp relative to the translation start point.

Primer extension analysis was performed using the C. therm. Polymerase for reverse transcription in a two-step RT-PCR procedure according to the manufacturer's instructions (Roche), with the following changes. Twenty microlitre reactions were set up using 5 pmol of the end-labelled reverse primer BKL96 and 40 units of RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) with extension at 55 °C for 60 min and termination at 80 °C for 10 min. For both S1 nuclease protection and primer extension analyses, the reverse primers were used along with the template plasmid pDA1006 to prepare sequencing ladders, which were electrophoresed alongside the reaction products for size estimation.

Cephamycin C bioassay.
The production of cephamycin C by S. clavuligerus was estimated by an agar diffusion bioassay with E. coli ESS as the indicator strain (Jensen et al., 1982).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of ccaR in E. coli
The ccaR gene from S. clavuligerus has been cloned and sequenced, but the location of the translational start point is unclear. Two different plasmid constructs, pTK-1 and pTK-2, were prepared to examine the expression of ccaR in E. coli from the putative ATG and GTG start codons. pTK-1 contains a mutant ccaR gene with an NdeI site introduced by PCR at the ATG codon. pTK-2 contains a mutant ccaR gene with an NdeI site introduced at the GTG codon, thereby changing it to an ATG codon. In both cases, the NdeI site was used to position the gene precisely behind an optimized E. coli ribosome-binding site and {pi}10 T7 promoter in the plasmid vector pT7-7. The 5' ends of the ccaR genes in pTK-1 and pTK-2 were sequenced to ensure that no errors were introduced by the PCR procedure, and none were found.

Cultures of E. coli BL21 (DE3) carrying either pTK-1 or pTK-2 were grown in LB medium at 37 °C until mid-exponential phase, then induced by the addition of IPTG to a concentration of 0·4 mM, followed by continued incubation at either 21 or 37 °C. Samples of cultures taken before IPTG induction and at 3 h post-induction were harvested by centrifugation, resuspended to 1/10 of the original culture volume in SDS-PAGE sample buffer, heated for 5 min at 100 °C, and analysed by electrophoresis on a 12 % SDS polyacrylamide gel followed by staining with Coomassie Brilliant Blue. Fig. 3 shows that CcaR accumulated in cultures carrying pTK-1 (lanes 1–5). In contrast, cultures carrying pTK-2 did not show detectable CcaR production (lanes 6–10). To confirm that the absence of CcaR in pTK-2-bearing cells was not due to loss of the expression vector, plasmids were isolated from cell samples taken at each time point. All cultures showed the presence of plasmid, indicating that both pTK-1 and pTK-2 were maintained in the cells (data not shown). Furthermore, to ensure that the failure of pTK-2 to support the production of CcaR was not due to a mutation in the upstream untranslated region of the gene, pTK-2 isolated from a cell sample taken at the end of the expression period was subjected to DNA sequence analysis. No mutations were seen in the upstream region extending well beyond the {pi}10 T7 promoter region. Expression of ccaR from pTK-1 yielded CcaR protein, regardless of whether the cultures were incubated at 37 or 21 °C after induction. However, further analysis showed that the recombinant CcaR protein was located almost exclusively in the insoluble fraction of the cells, regardless of the expression temperature (data not shown).



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Fig. 3. Production of CcaR in E. coli cultures carrying either pTK-1 or pTK-2. SDS-PAGE analysis was used to assess CcaR production in cultures induced with 0·4 mM IPTG and grown for 3 h. Lanes 1–5, pTK-1; lanes 6–10, pTK-2. Lanes 1 and 6 are uninduced cultures. Lanes 2, 4, 7 and 9 represent 50 µl amounts of induced cells; lanes 3, 5, 8 and 10 represent 150 µl amounts of induced cells. Lanes 2, 3, 7 and 8 represent cells grown at 21 °C after induction; lanes 4, 5, 9 and 10 represent cells grown at 37 °C after induction. Lane 11 is empty; lane 12 contains molecular mass markers.

 
Expression of ccaR in S. clavuligerus
The expression of ccaR from both the potential ATG and GTG start codons was also investigated in a ccaR mutant strain ({Delta}ccaR : : tsr) of S. clavuligerus. Strains of S. clavuligerus {Delta}ccaR : : tsr carrying pDA1100, pDA1102 and pDA1103 were examined for the ability of the plasmid constructs to complement the mutated chromosomal copy of ccaR and thereby restore production of cephamycin C. The entire ccaR gene has been deleted from S. clavuligerus {Delta}ccaR : : tsr and replaced with a thiostrepton resistance gene, which rules out the possibility that complementation could occur by recombination and repair of the original mutated ccaR gene in the chromosome. In each case, the ccaR gene was expressed under the control of a glycerol-regulatable promoter, and complementation was tested following growth in both minimal (NMMP) and complex (TSB) media, in the presence and absence of glycerol (Table 3). Cephamycin C production was only seen in wild-type S. clavuligerus and in the {Delta}ccaR : : tsr mutant strain complemented with pDA1102, the ccaR expression construct originating at the ATG codon, and then only when grown in the presence of glycerol. While glycerol was essential for production of CcaR from pDA1102, increasing concentrations of glycerol in the growth medium did not necessarily increase the production of cephamycin C in either wild-type S. clavuligerus or the {Delta}ccaR : : tsr mutant strain. At 2·0 % glycerol (w/v) in NMMP and at 1·25 % glycerol in TSB, cephamycin C production was actually reduced compared to that at lower concentrations of glycerol. No cephamycin C production was detected from the S. clavuligerus {Delta}ccaR : : tsr mutant alone or carrying pDA1103, from the ccaR expression construct originating at the GTG codon, or from pDA1100, a control construct lacking a ccaR insert, in any of the growth media tested, or at any concentration of glycerol.


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Table 3. Cephamycin C production by wild-type S. clavuligerus and S. clavuligerus {Delta}ccaR : : tsr (pDA1102)

ND, Not done. Data are from single cultures assayed singly, but the entire experiment was repeated three separate times. Glycerol concentrations are % w/v.

 
CcaR production in complemented cells was also examined by Western analysis using anti-CcaR antibodies. Wild-type S. clavuligerus, S. clavuligerus {Delta}ccaR : : tsr (pDA1102) and {Delta}ccaR : : tsr (pDA1103) were grown in TSB±0·75 % glycerol (w/v), and cell extracts from 48 h cultures were analysed. CcaR protein was only detected in wild-type and S. clavuligerus {Delta}ccaR : : tsr (pDA1102), and in the latter case, only when the cells were grown in the presence of glycerol (Fig. 4). S. clavuligerus {Delta}ccaR : : tsr (pDA1102) produced an amount of CcaR protein approximately equal to that seen in the wild-type, whereas the {Delta}ccaR : : tsr (pDA1103) strain showed no detectable CcaR protein, consistent with the cephamycin C bioassay results.



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Fig. 4. Western analysis of S. clavuligerus wild-type, and {Delta}ccaR : : tsr mutants carrying the integrative vectors pDA1100, pDA1102 and pDA1103. Forty microgram amounts of cell-extract protein from each strain, harvested after 48 h growth in either TSB or TSB+0·75 % glycerol (w/v), were subjected to Western analysis using anti-CcaR polyclonal antisera. The ability of each strain to produce cephamycin C was estimated qualitatively by bioassay. Lanes 1–3, {Delta}ccaR : : tsr mutants carrying pDA1102, pDA1103 and pDA1100, respectively, and grown in the absence of glycerol; lanes 4–6, the same strains, but grown in the presence of glycerol; lane 7, wild-type culture grown in the absence of glycerol; lane 8, 1 µg purified CcaR protein. NA, Not applicable.

 
Complementation of S. clavuligerus {Delta}ccaR : : tsr mutants by a GTG to GTC mutant form of ccaR
Cephamycin C production was restored in {Delta}ccaR : : tsr mutants by complementation with pDA1102. However, expression from this construct is driven by the non-native gylP1P2 promoter, which might influence the choice of start codon. Furthermore, the ATG expression construct still contains the downstream GTG codon, and so it is possible that translation could be initiating at the GTG codon within this construct rather than at the ATG codon, as intended. To eliminate these complicating factors, a pSET152-based ccaR expression construct (pDA1006*) was prepared, in which ccaR is expressed from its native promoter. In this construct, a conservative mutation has been introduced to change the GTG codon near the 5' end of ccaR into a GTC codon, thereby precluding its use as a start codon. The corresponding vector, pDA1006, carries the wild-type form of the ccaR gene. These two complementation constructs were conjugated into the {Delta}ccaR : : tsr mutant strain. Exconjugants were cultured in TSBS medium for 48 h and then bioassayed for the production of cephamycin C. S. clavuligerus {Delta}ccaR : : tsr (pDA1006) and {Delta}ccaR : : tsr (pDA1006*) produced 125 % and 96 % of the wild-type level of cephamycin C, respectively. In contrast, a control plasmid, pDA1000, consisting of pSET152 carrying only the tsr gene, had no ability to restore cephamycin C production in the mutant. These results indicate that in vivo expression of ccaR in S. clavuligerus also initiates from the ATG rather than the GTG codon.

Identification of the TSP for ccaR
No promoter was evident upstream of the ATG start codon of ccaR. To locate the promoter region for ccaR, RNA isolated from wild-type S. clavuligerus grown for 24, 48, 72 and 96 h at 28 °C in soy medium was subjected to S1 nuclease protection analysis using a ccaR-specific probe. Soy medium supports high-level production of cephamycin C, clavulanic acid and the other clavam metabolites, and so should give correspondingly high levels of ccaR expression. A triplet of prominent bands indicating a TSP (TSP-1) at 74–76 bp upstream from the ATG start codon was observed when a DNA fragment extending from –193 to +22 bp relative to the ATG start codon of ccaR was used as the probe (Fig. 5a). In addition, a very faint band corresponding to a larger protected probe fragment was also observed, indicating a possible second TSP further upstream (Fig. 5a). To obtain better resolution, a second probe was designed to protect a region further upstream of ccaR, from –412 bp to –133 bp, and overlapping the first probe. When this second probe was employed, very weak protection of multiple bands was observed, and so the second ccaR TSP could not be located conclusively (data not shown). Primer extension analysis was also used to confirm results obtained by S1 protection assays. RNA isolated from wild-type S. clavuligerus grown in soy medium for 72 h was subjected to analysis using the reverse primer BKL96, the same primer used to prepare the S1 probe to map TSP-1. By primer extension, TSP-1 was mapped to a single nucleotide, 74 bp upstream of the ccaR ATG start codon (Fig. 5b). A faint band corresponding to the second TSP (TSP-2) was also observed and was located 173 bp upstream of the ccaR ATG start codon (Fig. 5b).



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Fig. 5. Determination of the ccaR TSPs. (a) S1 protection analysis of RNA isolated from wild-type S. clavuligerus grown in soy medium, using a ccaR-specific probe. Lanes 1–4, RNA isolated after 24, 48, 72 and 96 h growth; lane C, probe digested with S1 nuclease; lane P, undigested probe. (b) Primer extension analysis of RNA isolated from wild-type S. clavuligerus grown in soy medium. Lane 1, RNA isolated after 72 h growth and subjected to analysis. In both (a) and (b), the TSPs detected by S1 nuclease protection and primer extension analysis are indicated by an asterisk. Lanes G, A, T and C form the sequencing ladder generated using the reverse primer (BKL96). (c) Determination of the ccaR TSPs. Diagrammatic representation of the ccaR promoter region. The filled arrow represents the ccaR coding sequence, including the start codon. TSP-1 and TSP-2 giving rise to the ccaR transcripts are shown in bold type and the solid arrows represent the mapped ccaR transcripts. The –10 and –35 promoter regions identified are also indicated.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Heterologous expression of ccaR in E. coli from a strong T7-based promoter gave protein product only when constructs starting at the ATG codon were prepared. The ATG-based expression construct gave large amounts of CcaR protein, visible as a prominent band on SDS-PAGE 3 h post-IPTG induction. This suggests that ATG is the normal start codon for ccaR expression. The inability to detect any protein product from the GTG-based expression construct also suggests that, if truncated CcaR protein was produced, it did not persist in the cells long enough to be detected by PAGE analysis. Although CcaR was produced in large amounts from the ATG-based expression construct, essentially all of the protein was in an insoluble form, regardless of the incubation temperature.

Expression of ccaR in S. clavuligerus gave results consistent with those seen in E. coli. CcaR protein was only produced from expression constructs initiating translation at the ATG codon. These results indicate that not only is ATG the true start codon for ccaR expression, but also that the extreme amino terminus of the protein must be important for the structural integrity of the protein. Truncation by as few as six amino acid residues results in disappearance of the protein from cell extracts to the limits of detection by Western analysis. CcaR is an example of a class of pathway-specific antibiotic regulatory proteins referred to as SARPs. The amino terminal region has been suggested as the location of the DNA-binding fold that characterizes this group of regulatory proteins. Truncation may interfere with this important structural element and destabilize the protein.

Expression of ccaR in S. clavuligerus restored cephamycin C production in the {Delta}ccaR : : tsr mutant to near wild-type levels. Although the level of expression of ccaR in S. clavuligerus was modest, the gylP1P2 promoter was nonetheless seen to be both active, and glycerol-regulated in S. clavuligerus. The presence of a TTA codon in ccaR may limit expression under the gylP1P2 promoter, although previous studies have shown that expression of ccaR is relatively insensitive to the availability of bldA tRNA in S. clavuligerus (Trepanier et al., 2002).

While the ATG-based expression construct supported CcaR production in S. clavuligerus, whereas the GTG-based construct did not, it was still possible that expression might be regulated differently when driven by the natural ccaR promoter. However, a mutant form of ccaR under the control of its own promoter, but carrying a conservative mutation that changed the GTG codon to GTC, was still fully able to complement the {Delta}ccaR : : tsr mutant and restore cephamycin C production. Therefore, these results provide strong evidence to indicate that the ATG codon is the natural start codon for the ccaR gene.

The ccaR promoter was investigated using SI nuclease protection analysis to locate the transcription start point to a group of three residues –74 to –76 bp upstream from the ATG start codon. A second TSP located further upstream was seen as a faint band in some S1 nuclease protection analyses. Primer extension analysis further localized TSP-1 to a G residue at –74 bp. TSP-2 was also located by primer extension analyses to an A residue at –173 bp. The predicted promoter region associated with TSP-1 is TGGAAT–17 bp–AAACAT while that of TSP-2 is TCCCGA–18 bp–GTTCTT. Both of these promoters show only marginal similarity to other promoters associated with cephamycin and clavulanic acid production (Kovacevic et al., 1990; Paradkar et al., 1998; Petrich et al., 1992, 1994).

A feature commonly associated with SARPs is the presence of heptameric sequences repeated at 11 bp (or multiples of 11 bp) intervals in the promoter regions of target genes (Wietzorrek & Bibb, 1997). A consensus sequence for these heptameric repeats has been identified, but no such repeats were evident in the ccaR promoter region, despite reports that CcaR regulates its own production (Santamarta et al., 2002). However, if more than one regulatory element is interacting with the promoter, this may result in deviations from this general architecture. In this regard, Folcher et al. (2001) have defined an autoregulatory response element (ARE) sequence motif located upstream of a number of SARP-type genes, and associated with expression of the genes under the control of an A factor-like autoregulatory system. As part of that study, a potential ARE motif was identified in the region upstream of ccaR, and a recent report of the cloning of a gene encoding a {gamma}-butyrolactone autoregulator receptor protein from S. clavuligerus adds support to the possibility that expression of ccaR may be regulated by a {gamma}-butyrolactone-based quorum-sensing system (Kim et al., 2002). However, the ARE-type motif identified by Folcher et al. (2001) is located more than 800 bp upstream of ccaR TSP-1 and on the opposite strand relative to the promoter, which calls its significance into question. Additional study will be required to substantiate the involvement of an A factor-like autoregulatory system in the production of {beta}-lactam metabolites in S. clavuligerus.


   ACKNOWLEDGEMENTS
 
This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada. K. T., T. L. K. and D. C. A. were supported by studentships from the Alberta Heritage Foundation for Medical Research.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 16 April 2004; revised 28 July 2004; accepted 10 September 2004.



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