Department of Biological Sciences, University of Alberta, Edmonton, Canada T6G 2E9
Correspondence
Susan E. Jensen
susan.jensen{at}ualberta.ca
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ABSTRACT |
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Present address: CanBioCin, Inc., Suite 1015, 8308-114 St., Edmonton, Canada AB T6G 2E1.
Present address: Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, USA.
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INTRODUCTION |
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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.
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METHODS |
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Recombinant DNA techniques.
Plasmid isolation, transformation, ligation, end-labelling of DNA using [-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 NdeIBamHI 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 BamHINruI fragment. The reassembled ccaR gene was recloned into pBluescript II pSK(+) as a HindIIIXhoI fragment to pick up appropriate restriction sites, and finally cloned into pT7-7 as a NdeIBamHI fragment to yield pTK-1.
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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 NdeIXbaI fragments, and cloned into pDA1100 under the control of the gylP1/P2 promoters, to give pDA1102 and pDA1103 (Fig. 1
).
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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 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 0·60·8 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 ccaR : : tsr, a ccaR mutant in which the 1·4 kb BamHINruI 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 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 probeprobe 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).
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RESULTS |
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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 15). In contrast, cultures carrying pTK-2 did not show detectable CcaR production (lanes 610). 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
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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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 7476 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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DISCUSSION |
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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 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 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 TGGAAT17 bpAAACAT while that of TSP-2 is TCCCGA18 bpGTTCTT. 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
-butyrolactone autoregulator receptor protein from S. clavuligerus adds support to the possibility that expression of ccaR may be regulated by a
-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
-lactam metabolites in S. clavuligerus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 April 2004;
revised 28 July 2004;
accepted 10 September 2004.
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