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pPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli
Plasmid 60 (2008) 53–58
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pPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli q Daniel S. Philip, Derek S. Sarovich, John M. Pemberton * Department of Microbiology and Parasitology, University of Queensland, Research Road, St. Lucia, Brisbane, Qld 4072, Australia
a r t i c l e
i n f o
Article history: Received 22 December 2007 Revised 24 February 2008 Available online 11 April 2008 Communicated by Julian Rood Keywords: Streptomyces E. coli Stable vector PCR Gene expression pPSY
a b s t r a c t pPSY is a 12 kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10 kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and subcloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent L-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividans amyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli. Crown copyright Ó 2008 Published by Elsevier Inc. All rights reserved.
1. Introduction The actinomycetes are a diverse collection of Gram-positive filamentous bacteria that include the prolific antibiotic producers, the streptomycetes. Whole genome sequencing and analysis reveal that each streptomycete genome contains more than 20 gene clusters devoted to the synthesis of chemotherapeutic agents such as antibiotics, as well as multiple copies of genes encoding extracellular enzymes such as amylases (Omura et al., 2001; Bentley et al., 2002; Ikeda et al., 2003; Oliynyk et al., 2007). Functional analysis of these genes has conventionally relied on their cloning and expression in suitable heterologous hosts, usually by using another streptomycete, such as Streptomyces lividans or Streptomyces albus (Gullon et al., 2006; Connell, 2001). q
All authors have contributed equally to this work. * Corresponding author. Fax: +61 7 33654620. E-mail address: [email protected] (J.M. Pemberton).
Since streptomycetes are difficult to culture and genetically manipulate (Bentley et al., 2002) an alternative option is to clone and express their genes in Escherichia coli. Attempts to clone and directly express streptomycete genes from their native promoters in E. coli K12 are generally unsuccessful. Nevertheless, previous studies have revealed that a significant group of streptomycete antitumour antibiotics, the indolocarbazoles, can be synthesised in E. coli (Pemberton et al., 1991; August et al., 2000; Hyun et al., 2003). The pathway encoding the purple pigmented indolocarbazole violacein was the first antitumour antibiotic pathway to be cloned and expressed in E. coli (Pemberton et al., 1991). The violacein gene cluster was cloned from the high G+C (65%) Gram-negative bacterium Chromobacterium violaceum. Of the more than 50 indolocarbazole producing strains of bacteria, which encode indolocarbazole synthases (ICS), almost all are Gram-positive streptomycetes (Sanchez et al., 2006). Indeed, the streptomycete indolocarbazole gene cluster encoding the antitumour
0147-619X/$ - see front matter Crown copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2008.02.003
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antibiotic rebeccamycin, from the streptomycete Lechevalieria aerocolonigenes, became the second such cluster to be expressed in E. coli (Hyun et al., 2003). In each of these examples, the genes were transcribed by E. coli from the native promoters. Recent research from this laboratory demonstrated that a key consideration of any strategy for the cloning and heterologous expression of indolocarbazole pathways in E. coli was the stability of the cloning vector (Sarovich and Pemberton, 2007). In this study we constructed the cosmid/ BAC cloning vector, pPSX, which remained highly stable in E. coli in the absence of selection pressure, even when the vector encoded the synthesis of the toxic antitumour antibiotic violacein. The rapid accumulation of large amounts of genomic sequence data provides the opportunity to gain direct access to genes of known function via PCR. While pPSX has proven very successful for the construction of highly stable cosmid clone banks containing large inserts of streptomycete DNA, a different vector was required for the less complicated cloning of PCR products. Using a similar approach to that previously described for the development of pPSX, we created an amplicon cloning vector (pPSY), which remained highly stable in the absence of antibiotic selection, overcoming the inherent instability of Colicin E1 (ColE1)based cloning vectors. To test the properties of pPSY, three single gene phenotypes of known function from high G+C bacteria were amplified by PCR, cloned into pGEM-T Easy and subsequently sub-cloned into pPSY. The stability and
expression of the pGEM-T Easy and pPSY plasmid constructs were tested in E. coli. Of the three single gene phenotypes, two were from the Gram-positive S. lividans and the other from the Gram-negative C. violaceum. 2. Materials and methods 2.1. Bacterial strains and growth conditions Table 1 outlines the bacterial strains and plasmids used in this study. Media and growth conditions have been published previously (Sarovich and Pemberton, 2007; Kidd and Pemberton, 2002). 2.2. Bacterial conjugation Bacterial conjugation was performed in the manner described previously (Sarovich and Pemberton, 2007). 2.3. DNA extraction Genomic DNA was extracted with a WizardÒ SV 96 Genomic DNA Purification System (Promega, Annandale, Australia) as per manufacturer’s instructions. Plasmid DNA was extracted with the Quantum Prep Plasmid MiniPrep Kit (BioRad) according to the manufacturer’s specifications. 2.4. Quantitative ultraviolet (UV) exposure survivability test Whole plate irradiation was employed to quantitatively measure strain UV resistance. Strains were grown overnight in PYE broth to achieve a thick cell suspension and 5 mL of the suspension was poured into an empty Petri dish. About 100 lL of sample was aseptically removed
Table 1 Bacterial strains and plasmids used in this study Bacterial strain
Genotype or description
Source
Escherichia coli DH5a Q358 LE392 S17-1
Host Host Host Host
Hanahan (1983) Karn et al. (1980) Borck et al. (1976) Simon et al. (1983)
Pseudomonas stutzeri ATCC 17588 JMP783
Wild-type isolate Restrictionless mutant derived from ATCC 17588; Rifr, Smr
Palleroni et al. (1970) Sarovich and Pemberton (2007)
Streptomyces lividans ATCC19844
Wild-type isolate
American Type Culture collection
General use cloning vector; Apr Amplicon cloning vector; Apr Amplicon cloning vector, vioA gene inserted; Apr Amplicon cloning vector, recA gene inserted; Apr Amplicon cloning vector, amyA gene inserted; Apr pUC18 with violacein cluster cloned into the BamHI site of the MCS; Apr Cosmid cloning vector, BamHI site removed, Tn5-Vio+ inserted; Kmr , Apr Parental plasmid 33 kb, incW; Tpr Amplicon cloning vector, smaller derivative of pR388, 12 kb; Tpr pR388 smaller derivative. Violacein synthesis genes inserted via transpositional escape from pHC79DBamHI::Tn5-Vio+; Kmr, Tpr pR388 smaller derivative. Contains vioA gene; Tpr 4.5 kb cloning vector; Cmr pPR510 containing vioBCDE cloned into the EcoRI site of the MCS; Cmr pR388 smaller derivative. Contains recA from S. lividans; Tpr pR388 smaller derivative. Contains recA from S. lividans and pGEM-T Easy; Tpr Contains amyA from S. lividans; Tpr
Yanisch-Perron et al. (1985) Promega This study This study This study Sarovich and Pemberton (2007) Sarovich and Pemberton (2007) Laravllaand and Fernandodela (1988) This study This study
Plasmids pUC18 pGEM-T Easy pGEM-T-vioA pGEM-T-recA pGEM-T-amyA pJP1000 pHC79DBamHI::Tn5-Vio+ pR388 pPSY pPSY::Tn5-Vio+ pPSY-vioA pR510 pR510-vioBCDE pPSY-recA pPSY-pGEM-recA pPSY-amyA
strain strain strain strain
for for for for
cloning experiments cloning experiments cloning experiments mobilisation experiments
This study Quigley and Reeves (1987) This study This study This study This study
Abbreviations: Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance; Tpr, trimethoprim resistance; Rifr, rifampicin resistance; Smr, streptomycin resistance; MCS, Multiple cloning site; Vio+, violacein synthesis.
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D.S. Philip et al. / Plasmid 60 (2008) 53–58 from the Petri dish at time zero, serially diluted in sterile 0.9% saline and plated on PYE agar for the purpose of a viable count. The Petri dish was subsequently exposed to UV for 10 s. Following this exposure 100 lL of sample was again aseptically removed, serially diluted and plated on PYE agar for a viable count. These steps were repeated until the cells had been exposed to UV for a total of 1 min. Viable counts were wrapped in aluminium foil to prevent photoreactivation. Cells were incubated overnight prior to assessing viable counts (measured as colony forming units). 2.5. Plasmid constructions
2.6. Stability testing Plasmid stability testing was performed according to the method of Sarovich and Pemberton (2007). 2.7. Sequencing Sequencing was carried out by the Australian Genome Research Facility.
3. Results
Unless otherwise stated, all DNA manipulations were performed according to standard protocols (Sambrook et al., 1989). PCR was performed using 0.02 U/lL PhusionTM High-Fidelity DNA Polymerase (Finnzymes, Finland) according to the manufacturer’s instructions, with the addition of 3% dimethyl sulphoxide. Strategies for constructing plasmids were as follows: (i) pPSY: pPSY was constructed by removing genes from pR388 that were non-essential for the maintenance and stability of the plasmid. The final plasmid was 12 kb and could be extracted from E. coli in relatively large amounts comparable to those obtained with pPSX (Sarovich and Pemberton, 2007). (ii) pPSY::Tn5-Vio+: pPSY::Tn5-Vio+ was constructed by transpositional escape of Tn5-Vio+ from pHC79::Tn5-Vio+ onto pPSY. The plasmid pHC79::Tn5-Vio+ is unstable in E. coli and in the absence of selection pressure is rapidly lost. When E. coli LE392 pPSY, pHC79::Tn5-Vio+ was subcultured for 100 generations in PYE broth supplemented with Km, all the survivors were Km resistant, produced violacein, were Tp resistant but Ap sensitive. Analysis of plasmid DNA from these clones showed that the pHC79::Tn5Vio+ was lost and that Tn5-Vio+ had transposed onto pPSY. (iii) pGEM and pPSY plasmids carrying PCR-amplified genes: The primers used to amplify the individual genes are listed in Table 2. The PCR thermocycling protocol followed a two-step procedure and was as follows: 98 °C for 5 min, followed by 35 cycles of 98 °C for 30 s and 72 °C for 1 min with a final extension of 72 °C for 6 min. The resulting fragment was visualised using agarose gel electrophoresis to verify correct band size. The amplicon was A-tailed and cloned into pGEM-T Easy as per manufacturer’s instructions. The presence of the gene in pGEM-T Easy was determined by restriction analysis and sequencing. It was sub-cloned into the EcoRI site of pPSY, transformed into E. coli DH5a and transformants were selected by plating on PYE agar supplemented with Tp. (iv) pGEM-T-vioA and pPSY-vioA: Successful transfer of vioA into both pGEM and pPSY was verified by restriction analysis, gene expression and sequencing. (v) pGEM-T-recA, pPSY-recA and pPSY-pGEM-recA: Two different plasmid constructs were generated by the EcoRI digestion and ligation of pPSY and pGEM-T-recA; pPSY-recA and pPSY-pGEMrecA. pGEM-T-recA was subjected to DNA sequencing, which confirmed the presence of the recA gene from S. lividans and verified the correct orientation with regard to the lac promoter of pGEM-T Easy. (vi) pGEM-T-amyA and pPSY-amyA: Successful transfer of amyA into both pGEM and pPSY was verified by sequencing, restriction analysis and the ability to degrade starch (Kidd and Pemberton, 2002). (vii) pR510-vioBCDE: The violacein genes (vioBCDE) were excised from pUC18-vioBCDE by SphI and inserted into the SphI site of pR510 (Quigley and Reeves, 1987). Sequencing determined the orientation of these genes with respect to the lac promoter.
3.1. Construction and characterisation of the PCR amplicon cloning vector, pPSY Following construction of pPSY, it was necessary to establish its properties including stability, transmissibility, restriction sites and its utility as a stable cloning and expression vector. 3.2. Stability of pPSY Initial tests demonstrated that pPSY was highly stable in E. coli in the absence of antibiotic selection. After 100 generations of growth in non-selective PYE broth there was no detectable loss (<0.1%) of the plasmid as determined by trimethoprim resistance (Tpr) and the ability to extract plasmid DNA. Violacein is toxic to the host cell and when encoded by ColE1 vectors such as pUC18 and pHC79 greatly reduces their stability (August et al., 2000). In contrast, when pPSY encoded violacein synthesis it was highly stabile. 3.3. Transmissibility of pPSY When E. coli pPSY was mated with an E. coli recipient strain there was no detectable (<1 in 109 exconjugants/donor cell) transfer, indicating that genes essential for conjugation had been removed. However pPSY and pPSY::Tn5-Vio+ were mobilised from the mobilising E. coli strain S17-1 to another E. coli strain and to Pseudomonas stutzeri at frequencies of 1 in 104 and 1 in 105 per donor cell, respectively. Subcultured in the absence of antibiotic selection pressure pPSY-Tn5::Vio+ is highly stable in E. coli, but highly unstable (70% loss after 15 generations) in P. stutzeri. 3.4. Restriction map of pPSY A restriction map of pPSY was created using a combination of digests with EcoRI, HindIII, BamHI, PstI and SphI (Fig. 1). The restriction pattern showed that the vector was approximately 12 kb in size and possessed a single
Table 2 Primers used in this study Gene vioA recA amyA
Primer 1 0
Primer 2 0
0
vioA5 (5 -GCGGGCGTGAGTTGACTA-3 ) recA50 (50 -GAAGCGATCGAATCAAGCAAACC-30 ) amyA50 (50 -AGGGGTTGACCGTGGTTGAAG-30 )
The target DNAs used were: vioA, pJP1000; recA and amyA, S. lividans.
vioA30 (50 -GAAATGGATGCGTGGAAAAT-30 ) recA30 (50 -GTTCGTCGGGTCACGGGTCA-30 ) amyA30 (50 -GTCGTCCTTGCGGAGGTACTTG-30 )
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gle gene phenotypes recA (recombinase A) and amyA (alpha amylase A), of S. lividans. 3.6. Cloning and expression of recA from S. lividans ATCC19844 using pPSY
Fig. 1. pPSY, a smaller derivative of pR388 for use as an amplicon cloning vector. The 34 kb, IncW plasmid R388 was subjected to four rounds of partial digestion, self-ligation, transformation and stability selection to remove non-essential genes. Abbreviations: oriV, origin of vegetative replication; repA, replication; tnpR, putative resolvase; L-IR, left inverted repeat; intl1, site specific recombinase encoded by Class I integron; dfrB2, dihydrofolate reductase (which confers trimethoprim resistance); orfA, conserved hypothetical protein.
EcoRI site that could have potential utility as a unique cloning site. 3.5. Use of pPSY for cloning and expression of vioA amplified by PCR It was of interest to determine whether single gene phenotypes isolated from PCR amplification would be stably expressed when cloned into pPSY. The first gene chosen for this purpose was vioA, an L-tryptophan amino acid oxidase from the violacein indolocarbazole biosynthetic pathway. A key feature of the violacein pathway is that individual genes can be isolated from the core pathway and subsequently used in complementation experiments to detect expression. Cultures of E. coli pPSY-vioA and E. coli pPR510-vioBCDE are white. Neither strain has a complete violacein pathway. pPSY-vioA was transformed into E. coli pPR510-vioBCDE, and transformants selected on Cm Tp plates. All the E. coli pPSY-vioA, pPR510-vioBCDE transformants produced violacein and turned purple indicating that vioA was expressed and it complemented vioBCDE to produce a functional indolocarbazole biosynthetic pathway. This confirmed that single genes from Gram-negative bacteria could be amplified by PCR, cloned first into pGEM-T Easy then sub-cloned into pPSY and stably expressed in E. coli. While pPSY-vioA was highly stable, pGEM-T-vioA was highly unstable with less than 1% of the cells retaining the plasmid after 100 generations of growth in non-selective medium. These observations were extended to the sin-
Database analysis revealed that the nucleotide sequence for the open reading frame (ORF) of recA from various streptomycetes was strongly conserved. However, this sequence conservation did not extend to the upstream promoter region. As a result, the functional primer pair only covered the translational start region and the recA open reading frame. In E. coli DH5a the RecA mutation not only prevents homologous recombination but also makes this strain highly sensitive to UV irradiation (Howard-Flanders et al., 1969). When pGEM-recA was transformed into DH5a it showed a marked increase in UV resistance compared with the control, indicating that the S. lividans recA gene was a functional homologue of the E. coli recA gene (data not shown). However, since the S. lividans recA gene does not have its native promoter, it must be transcribed from the lac promoter of pGEM-T Easy. When the recA gene was sub-cloned from pGEM-T Easy into pPSY two different constructs were obtained. First, the recA gene without the lac promoter(pPSY-recA) and with the lac promoter(pPSY-pGEM-recA) remaining situated upstream of the recA gene. Quantitative testing of DH5a pPSY-recA showed no increase in UV resistance compared with DH5a (data not shown). In contrast the pPSY-pGEM-recA plasmid showed increased UV resistance, demonstrating that the recA is expressed in E. coli when it is situated downstream of a suitable promoter sequence (Fig. 2). Although pPSY-pGEM-recA contains both a stable and an unstable vector within the same plasmid, it retains the high stability of pPSY. This reinforces our observations that pPSY remains highly stable regardless of the nature of the DNA insert. In contrast pGEM-T-recA was highly unstable, where <1% of cells retained the plasmid after 100 generations. 3.7. Amplification, cloning and expression of the amylase gene, amyA, from S. lividans in E. coli Genomic sequencing of streptomycetes has revealed that they carry multiple copies of amylase genes (Bentley et al., 2002); one of the most common is amlB from Streptomyces coelicolor and Streptomyces avermitilis. Using the known sequences from S. coelicolor and S. avermitilis a set of primers were designed which successfully amplified the amylase ORF (amyA) and its upstream region from S. lividans. Sequencing of pGEM-T-amyA revealed that cloned PCR product was 1787 bp (GenBank Accession No. EU352611). BLAST analysis showed that there was an ORF of 1524 bp which encoded a hypothetical protein containing 508 amino acids, with very high homology (>90%) to known a-amylases from a variety of streptomycetes including S. coelicolor and S. avermitilis. In addition, the amyA amplicon contained 151 bp of DNA upstream from the starting point of the ORF. Since
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Fig. 2. UV resistance survival graph. The two strains, Escherichia coli DH5a pPSY-pGEM-recA and the negative control E. coli DH5a, were tested for their ability to survive UV exposure. Strains were exposed to UV for 10-s intervals with subsequent viable counts performed. The quantitative UV exposure test was performed in triplicate with percent survival representing an average from the three experiments performed.
the amylase activity encoded by amyA was strongly expressed regardless of its orientation in relation to the lac promoter of pGEM-T Easy, this suggests that the 151 bp region upstream contains a promoter(s), which is active in E. coli. When amyA was transferred into the EcoRI site of pPSY, it was strongly expressed regardless of its orientation. This strengthens the view the PCR product not only encodes the amyA ORF but also a native promoter active in E. coli. Again testing shows that pPSY-amyA is highly stable while pGEM-T-amyA is highly unstable. 4. Discussion Escherichia coli K12 is the heterologous host of choice for most biologists. For over 60 years the physiology and genetics of this Gram-negative bacterium have been studied in minute detail. While a wide range of bacterial genes have been cloned and expressed in E. coli, genes from the prolific antibiotic producing streptomycetes are not readily expressed in this host. The list of possible reasons for this lack of expression is very long; however, one major problem is cloning vector instability. In particular, ColEI-based vectors, such as pBR322 and pUC18, are inherently unstable and even more unstable when they encode the synthesis of molecules toxic to the E. coli host. Nevertheless two gene clusters encoding the indolocarbazole antitumour antibiotics, violacein and rebeccamycin, are expressed in E. coli from their own promoters (Pemberton et al., 1991; August et al., 2000; Hyun et al., 2003). The vector instability which occurs when the violacein gene cluster is inserted into ColE1, based cloning vectors (pUC18/pHC79/pGEM-T Easy) has been overcome by the construction of the cosmid/BAC vector pPSX. This vector is highly stable in the absence of antibiotic selection pressure, even when it encodes the synthesis of toxic products (Sarovich and Pemberton, 2007). In the current study a second vector, pPSY, was constructed to provide a rapid, simple and effective method for the stable cloning and expression in E. coli of DNA sequences obtained by PCR. In view of the anticipated difficulties in the cloning and expression of streptomycete genes in E. coli, three single
gene phenotypes were amplified, cloned into pPSY, and tested for expression. All three genes were isolated from high G+C bacteria; vioA from Gram-negative C. violaceum; recA and amyA from Gram-positive S. lividans. Surprisingly, all three genes were strongly and stably expressed in E. coli using pPSY. The first gene used in this study, vioA, is an L-amino oxidase essential for the synthesis of the indolocarbazole antitumour antibiotic violacein. The demonstration that individual indolocarbazole genes such as vioA can be accessed by PCR and stably expressed in E. coli from their own promoters using pPSY, will allow the cloning and expression of many other indolocarbazole genes. It is now a relatively simple matter to use these genes and pPSY to assemble synthetic indolocarbazole pathways in E. coli to stably produce a range of novel indolocarbazole antitumour antibiotics. In the relatively few instances where single gene phenotypes from the high G+C Gram-positive streptomycetes have been cloned and expressed in E. coli, expression is usually achieved by cloning the gene next to a strong, inducible promoter such as the lac promoter of E. coli. When the recombinase A (recA) gene of S. lividans, lacking its native promoters, was cloned into pGEM-T Easy it was only expressed when placed in the correct orientation next to the Lac promoter. When sub-cloned into pPSY without its native promoter or the Lac promoter it was not expressed regardless of the orientation. This lack of expression reinforces our observation that there are no strong promoters on either side of the EcoRI site of pPSY, which would drive transcription through this site. Genes cloned into this site would require their native promoters to be functional in E. coli. For most streptomycete genes, it has been assumed that detectable expression does not occur from native promoters. In an initial attempt to identify and characterise native streptomycete promoters, Bourn and Babb (1995) identified eight Classes (A–H). Among the many promoters examined in this study there were at least three amylase gene promoters. All three fell into Class C, which is represented by a strongly conserved (TTGAC)-35 region and a much less conserved -10 region.
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When amyA and its native promoter(s) were amplified and cloned into pGEM-T Easy it was strongly expressed regardless of its orientation in respect to the lac promoter, suggesting that transcription was occurring from the native promoter(s). This conclusion was strengthened when amyA was expressed in E. coli when sub-cloned into pPSY. Expression was independent of the orientation of amyA in the EcoRI site of pPSY. An examination of the 151 bp directly upstream from the amyA ORF shows that it contains a Class C type streptomycete promoter, indicating that this promoter is active in E. coli. Due to ongoing genomic sequencing projects, it is now possible to access sequence data for a wide range of streptomycete single and multiple gene phenotypes. Future studies will reveal just how many of these genes can be cloned into pPSY and expressed in E. coli from their native promoters. References August, P.R., Grossman, T.H., Minor, C., Draper, M.P., Macnell, I.A., Pemberton, J.M., Call, K.M., Holt, D., Osbourne, M.S., 2000. Sequence analysis and functional characterisation of the violacein biosynthetic pathway from Chromobacterium violaceum. J. Microb. Biotechnol. 2, 513–519. Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H., Kieser, T., Larke, L., Murphy, L., Oliver, K., O’Neil, S., Rabbinowitsch, E., Rajandream, M.A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J., Hopwood, D.A., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147. Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S., Murray, N.E., 1976. The construction in vitro of transducing derivatives of phage lambda. Mol. Gen. Genet. 146, 199–207. Bourn, W.R., Babb, B., 1995. Computer assisted identification and classification of streptomycete promoters. Nucleic Acids Res. 23, 3696–3703. Connell, N.D., 2001. Expression systems for use in actinomycetes and related organisms. Curr. Opin. Biotechnol. 12 (5), 446–449. Gullon, S., Olano, C., Abdelfattah, M.S., Brana, A.F., Rohr, J., Mendez, C., Salas, J.A., 2006. Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin. Appl. Environ. Microbiol. 72 (6), 4172–4183.
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pPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli q Daniel S. Philip, Derek S. Sarovich, John M. Pemberton * Department of Microbiology and Parasitology, University of Queensland, Research Road, St. Lucia, Brisbane, Qld 4072, Australia
a r t i c l e
i n f o
Article history: Received 22 December 2007 Revised 24 February 2008 Available online 11 April 2008 Communicated by Julian Rood Keywords: Streptomyces E. coli Stable vector PCR Gene expression pPSY
a b s t r a c t pPSY is a 12 kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10 kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and subcloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent L-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividans amyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli. Crown copyright Ó 2008 Published by Elsevier Inc. All rights reserved.
1. Introduction The actinomycetes are a diverse collection of Gram-positive filamentous bacteria that include the prolific antibiotic producers, the streptomycetes. Whole genome sequencing and analysis reveal that each streptomycete genome contains more than 20 gene clusters devoted to the synthesis of chemotherapeutic agents such as antibiotics, as well as multiple copies of genes encoding extracellular enzymes such as amylases (Omura et al., 2001; Bentley et al., 2002; Ikeda et al., 2003; Oliynyk et al., 2007). Functional analysis of these genes has conventionally relied on their cloning and expression in suitable heterologous hosts, usually by using another streptomycete, such as Streptomyces lividans or Streptomyces albus (Gullon et al., 2006; Connell, 2001). q
All authors have contributed equally to this work. * Corresponding author. Fax: +61 7 33654620. E-mail address: [email protected] (J.M. Pemberton).
Since streptomycetes are difficult to culture and genetically manipulate (Bentley et al., 2002) an alternative option is to clone and express their genes in Escherichia coli. Attempts to clone and directly express streptomycete genes from their native promoters in E. coli K12 are generally unsuccessful. Nevertheless, previous studies have revealed that a significant group of streptomycete antitumour antibiotics, the indolocarbazoles, can be synthesised in E. coli (Pemberton et al., 1991; August et al., 2000; Hyun et al., 2003). The pathway encoding the purple pigmented indolocarbazole violacein was the first antitumour antibiotic pathway to be cloned and expressed in E. coli (Pemberton et al., 1991). The violacein gene cluster was cloned from the high G+C (65%) Gram-negative bacterium Chromobacterium violaceum. Of the more than 50 indolocarbazole producing strains of bacteria, which encode indolocarbazole synthases (ICS), almost all are Gram-positive streptomycetes (Sanchez et al., 2006). Indeed, the streptomycete indolocarbazole gene cluster encoding the antitumour
0147-619X/$ - see front matter Crown copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2008.02.003
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antibiotic rebeccamycin, from the streptomycete Lechevalieria aerocolonigenes, became the second such cluster to be expressed in E. coli (Hyun et al., 2003). In each of these examples, the genes were transcribed by E. coli from the native promoters. Recent research from this laboratory demonstrated that a key consideration of any strategy for the cloning and heterologous expression of indolocarbazole pathways in E. coli was the stability of the cloning vector (Sarovich and Pemberton, 2007). In this study we constructed the cosmid/ BAC cloning vector, pPSX, which remained highly stable in E. coli in the absence of selection pressure, even when the vector encoded the synthesis of the toxic antitumour antibiotic violacein. The rapid accumulation of large amounts of genomic sequence data provides the opportunity to gain direct access to genes of known function via PCR. While pPSX has proven very successful for the construction of highly stable cosmid clone banks containing large inserts of streptomycete DNA, a different vector was required for the less complicated cloning of PCR products. Using a similar approach to that previously described for the development of pPSX, we created an amplicon cloning vector (pPSY), which remained highly stable in the absence of antibiotic selection, overcoming the inherent instability of Colicin E1 (ColE1)based cloning vectors. To test the properties of pPSY, three single gene phenotypes of known function from high G+C bacteria were amplified by PCR, cloned into pGEM-T Easy and subsequently sub-cloned into pPSY. The stability and
expression of the pGEM-T Easy and pPSY plasmid constructs were tested in E. coli. Of the three single gene phenotypes, two were from the Gram-positive S. lividans and the other from the Gram-negative C. violaceum. 2. Materials and methods 2.1. Bacterial strains and growth conditions Table 1 outlines the bacterial strains and plasmids used in this study. Media and growth conditions have been published previously (Sarovich and Pemberton, 2007; Kidd and Pemberton, 2002). 2.2. Bacterial conjugation Bacterial conjugation was performed in the manner described previously (Sarovich and Pemberton, 2007). 2.3. DNA extraction Genomic DNA was extracted with a WizardÒ SV 96 Genomic DNA Purification System (Promega, Annandale, Australia) as per manufacturer’s instructions. Plasmid DNA was extracted with the Quantum Prep Plasmid MiniPrep Kit (BioRad) according to the manufacturer’s specifications. 2.4. Quantitative ultraviolet (UV) exposure survivability test Whole plate irradiation was employed to quantitatively measure strain UV resistance. Strains were grown overnight in PYE broth to achieve a thick cell suspension and 5 mL of the suspension was poured into an empty Petri dish. About 100 lL of sample was aseptically removed
Table 1 Bacterial strains and plasmids used in this study Bacterial strain
Genotype or description
Source
Escherichia coli DH5a Q358 LE392 S17-1
Host Host Host Host
Hanahan (1983) Karn et al. (1980) Borck et al. (1976) Simon et al. (1983)
Pseudomonas stutzeri ATCC 17588 JMP783
Wild-type isolate Restrictionless mutant derived from ATCC 17588; Rifr, Smr
Palleroni et al. (1970) Sarovich and Pemberton (2007)
Streptomyces lividans ATCC19844
Wild-type isolate
American Type Culture collection
General use cloning vector; Apr Amplicon cloning vector; Apr Amplicon cloning vector, vioA gene inserted; Apr Amplicon cloning vector, recA gene inserted; Apr Amplicon cloning vector, amyA gene inserted; Apr pUC18 with violacein cluster cloned into the BamHI site of the MCS; Apr Cosmid cloning vector, BamHI site removed, Tn5-Vio+ inserted; Kmr , Apr Parental plasmid 33 kb, incW; Tpr Amplicon cloning vector, smaller derivative of pR388, 12 kb; Tpr pR388 smaller derivative. Violacein synthesis genes inserted via transpositional escape from pHC79DBamHI::Tn5-Vio+; Kmr, Tpr pR388 smaller derivative. Contains vioA gene; Tpr 4.5 kb cloning vector; Cmr pPR510 containing vioBCDE cloned into the EcoRI site of the MCS; Cmr pR388 smaller derivative. Contains recA from S. lividans; Tpr pR388 smaller derivative. Contains recA from S. lividans and pGEM-T Easy; Tpr Contains amyA from S. lividans; Tpr
Yanisch-Perron et al. (1985) Promega This study This study This study Sarovich and Pemberton (2007) Sarovich and Pemberton (2007) Laravllaand and Fernandodela (1988) This study This study
Plasmids pUC18 pGEM-T Easy pGEM-T-vioA pGEM-T-recA pGEM-T-amyA pJP1000 pHC79DBamHI::Tn5-Vio+ pR388 pPSY pPSY::Tn5-Vio+ pPSY-vioA pR510 pR510-vioBCDE pPSY-recA pPSY-pGEM-recA pPSY-amyA
strain strain strain strain
for for for for
cloning experiments cloning experiments cloning experiments mobilisation experiments
This study Quigley and Reeves (1987) This study This study This study This study
Abbreviations: Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance; Tpr, trimethoprim resistance; Rifr, rifampicin resistance; Smr, streptomycin resistance; MCS, Multiple cloning site; Vio+, violacein synthesis.
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D.S. Philip et al. / Plasmid 60 (2008) 53–58 from the Petri dish at time zero, serially diluted in sterile 0.9% saline and plated on PYE agar for the purpose of a viable count. The Petri dish was subsequently exposed to UV for 10 s. Following this exposure 100 lL of sample was again aseptically removed, serially diluted and plated on PYE agar for a viable count. These steps were repeated until the cells had been exposed to UV for a total of 1 min. Viable counts were wrapped in aluminium foil to prevent photoreactivation. Cells were incubated overnight prior to assessing viable counts (measured as colony forming units). 2.5. Plasmid constructions
2.6. Stability testing Plasmid stability testing was performed according to the method of Sarovich and Pemberton (2007). 2.7. Sequencing Sequencing was carried out by the Australian Genome Research Facility.
3. Results
Unless otherwise stated, all DNA manipulations were performed according to standard protocols (Sambrook et al., 1989). PCR was performed using 0.02 U/lL PhusionTM High-Fidelity DNA Polymerase (Finnzymes, Finland) according to the manufacturer’s instructions, with the addition of 3% dimethyl sulphoxide. Strategies for constructing plasmids were as follows: (i) pPSY: pPSY was constructed by removing genes from pR388 that were non-essential for the maintenance and stability of the plasmid. The final plasmid was 12 kb and could be extracted from E. coli in relatively large amounts comparable to those obtained with pPSX (Sarovich and Pemberton, 2007). (ii) pPSY::Tn5-Vio+: pPSY::Tn5-Vio+ was constructed by transpositional escape of Tn5-Vio+ from pHC79::Tn5-Vio+ onto pPSY. The plasmid pHC79::Tn5-Vio+ is unstable in E. coli and in the absence of selection pressure is rapidly lost. When E. coli LE392 pPSY, pHC79::Tn5-Vio+ was subcultured for 100 generations in PYE broth supplemented with Km, all the survivors were Km resistant, produced violacein, were Tp resistant but Ap sensitive. Analysis of plasmid DNA from these clones showed that the pHC79::Tn5Vio+ was lost and that Tn5-Vio+ had transposed onto pPSY. (iii) pGEM and pPSY plasmids carrying PCR-amplified genes: The primers used to amplify the individual genes are listed in Table 2. The PCR thermocycling protocol followed a two-step procedure and was as follows: 98 °C for 5 min, followed by 35 cycles of 98 °C for 30 s and 72 °C for 1 min with a final extension of 72 °C for 6 min. The resulting fragment was visualised using agarose gel electrophoresis to verify correct band size. The amplicon was A-tailed and cloned into pGEM-T Easy as per manufacturer’s instructions. The presence of the gene in pGEM-T Easy was determined by restriction analysis and sequencing. It was sub-cloned into the EcoRI site of pPSY, transformed into E. coli DH5a and transformants were selected by plating on PYE agar supplemented with Tp. (iv) pGEM-T-vioA and pPSY-vioA: Successful transfer of vioA into both pGEM and pPSY was verified by restriction analysis, gene expression and sequencing. (v) pGEM-T-recA, pPSY-recA and pPSY-pGEM-recA: Two different plasmid constructs were generated by the EcoRI digestion and ligation of pPSY and pGEM-T-recA; pPSY-recA and pPSY-pGEMrecA. pGEM-T-recA was subjected to DNA sequencing, which confirmed the presence of the recA gene from S. lividans and verified the correct orientation with regard to the lac promoter of pGEM-T Easy. (vi) pGEM-T-amyA and pPSY-amyA: Successful transfer of amyA into both pGEM and pPSY was verified by sequencing, restriction analysis and the ability to degrade starch (Kidd and Pemberton, 2002). (vii) pR510-vioBCDE: The violacein genes (vioBCDE) were excised from pUC18-vioBCDE by SphI and inserted into the SphI site of pR510 (Quigley and Reeves, 1987). Sequencing determined the orientation of these genes with respect to the lac promoter.
3.1. Construction and characterisation of the PCR amplicon cloning vector, pPSY Following construction of pPSY, it was necessary to establish its properties including stability, transmissibility, restriction sites and its utility as a stable cloning and expression vector. 3.2. Stability of pPSY Initial tests demonstrated that pPSY was highly stable in E. coli in the absence of antibiotic selection. After 100 generations of growth in non-selective PYE broth there was no detectable loss (<0.1%) of the plasmid as determined by trimethoprim resistance (Tpr) and the ability to extract plasmid DNA. Violacein is toxic to the host cell and when encoded by ColE1 vectors such as pUC18 and pHC79 greatly reduces their stability (August et al., 2000). In contrast, when pPSY encoded violacein synthesis it was highly stabile. 3.3. Transmissibility of pPSY When E. coli pPSY was mated with an E. coli recipient strain there was no detectable (<1 in 109 exconjugants/donor cell) transfer, indicating that genes essential for conjugation had been removed. However pPSY and pPSY::Tn5-Vio+ were mobilised from the mobilising E. coli strain S17-1 to another E. coli strain and to Pseudomonas stutzeri at frequencies of 1 in 104 and 1 in 105 per donor cell, respectively. Subcultured in the absence of antibiotic selection pressure pPSY-Tn5::Vio+ is highly stable in E. coli, but highly unstable (70% loss after 15 generations) in P. stutzeri. 3.4. Restriction map of pPSY A restriction map of pPSY was created using a combination of digests with EcoRI, HindIII, BamHI, PstI and SphI (Fig. 1). The restriction pattern showed that the vector was approximately 12 kb in size and possessed a single
Table 2 Primers used in this study Gene vioA recA amyA
Primer 1 0
Primer 2 0
0
vioA5 (5 -GCGGGCGTGAGTTGACTA-3 ) recA50 (50 -GAAGCGATCGAATCAAGCAAACC-30 ) amyA50 (50 -AGGGGTTGACCGTGGTTGAAG-30 )
The target DNAs used were: vioA, pJP1000; recA and amyA, S. lividans.
vioA30 (50 -GAAATGGATGCGTGGAAAAT-30 ) recA30 (50 -GTTCGTCGGGTCACGGGTCA-30 ) amyA30 (50 -GTCGTCCTTGCGGAGGTACTTG-30 )
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gle gene phenotypes recA (recombinase A) and amyA (alpha amylase A), of S. lividans. 3.6. Cloning and expression of recA from S. lividans ATCC19844 using pPSY
Fig. 1. pPSY, a smaller derivative of pR388 for use as an amplicon cloning vector. The 34 kb, IncW plasmid R388 was subjected to four rounds of partial digestion, self-ligation, transformation and stability selection to remove non-essential genes. Abbreviations: oriV, origin of vegetative replication; repA, replication; tnpR, putative resolvase; L-IR, left inverted repeat; intl1, site specific recombinase encoded by Class I integron; dfrB2, dihydrofolate reductase (which confers trimethoprim resistance); orfA, conserved hypothetical protein.
EcoRI site that could have potential utility as a unique cloning site. 3.5. Use of pPSY for cloning and expression of vioA amplified by PCR It was of interest to determine whether single gene phenotypes isolated from PCR amplification would be stably expressed when cloned into pPSY. The first gene chosen for this purpose was vioA, an L-tryptophan amino acid oxidase from the violacein indolocarbazole biosynthetic pathway. A key feature of the violacein pathway is that individual genes can be isolated from the core pathway and subsequently used in complementation experiments to detect expression. Cultures of E. coli pPSY-vioA and E. coli pPR510-vioBCDE are white. Neither strain has a complete violacein pathway. pPSY-vioA was transformed into E. coli pPR510-vioBCDE, and transformants selected on Cm Tp plates. All the E. coli pPSY-vioA, pPR510-vioBCDE transformants produced violacein and turned purple indicating that vioA was expressed and it complemented vioBCDE to produce a functional indolocarbazole biosynthetic pathway. This confirmed that single genes from Gram-negative bacteria could be amplified by PCR, cloned first into pGEM-T Easy then sub-cloned into pPSY and stably expressed in E. coli. While pPSY-vioA was highly stable, pGEM-T-vioA was highly unstable with less than 1% of the cells retaining the plasmid after 100 generations of growth in non-selective medium. These observations were extended to the sin-
Database analysis revealed that the nucleotide sequence for the open reading frame (ORF) of recA from various streptomycetes was strongly conserved. However, this sequence conservation did not extend to the upstream promoter region. As a result, the functional primer pair only covered the translational start region and the recA open reading frame. In E. coli DH5a the RecA mutation not only prevents homologous recombination but also makes this strain highly sensitive to UV irradiation (Howard-Flanders et al., 1969). When pGEM-recA was transformed into DH5a it showed a marked increase in UV resistance compared with the control, indicating that the S. lividans recA gene was a functional homologue of the E. coli recA gene (data not shown). However, since the S. lividans recA gene does not have its native promoter, it must be transcribed from the lac promoter of pGEM-T Easy. When the recA gene was sub-cloned from pGEM-T Easy into pPSY two different constructs were obtained. First, the recA gene without the lac promoter(pPSY-recA) and with the lac promoter(pPSY-pGEM-recA) remaining situated upstream of the recA gene. Quantitative testing of DH5a pPSY-recA showed no increase in UV resistance compared with DH5a (data not shown). In contrast the pPSY-pGEM-recA plasmid showed increased UV resistance, demonstrating that the recA is expressed in E. coli when it is situated downstream of a suitable promoter sequence (Fig. 2). Although pPSY-pGEM-recA contains both a stable and an unstable vector within the same plasmid, it retains the high stability of pPSY. This reinforces our observations that pPSY remains highly stable regardless of the nature of the DNA insert. In contrast pGEM-T-recA was highly unstable, where <1% of cells retained the plasmid after 100 generations. 3.7. Amplification, cloning and expression of the amylase gene, amyA, from S. lividans in E. coli Genomic sequencing of streptomycetes has revealed that they carry multiple copies of amylase genes (Bentley et al., 2002); one of the most common is amlB from Streptomyces coelicolor and Streptomyces avermitilis. Using the known sequences from S. coelicolor and S. avermitilis a set of primers were designed which successfully amplified the amylase ORF (amyA) and its upstream region from S. lividans. Sequencing of pGEM-T-amyA revealed that cloned PCR product was 1787 bp (GenBank Accession No. EU352611). BLAST analysis showed that there was an ORF of 1524 bp which encoded a hypothetical protein containing 508 amino acids, with very high homology (>90%) to known a-amylases from a variety of streptomycetes including S. coelicolor and S. avermitilis. In addition, the amyA amplicon contained 151 bp of DNA upstream from the starting point of the ORF. Since
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Fig. 2. UV resistance survival graph. The two strains, Escherichia coli DH5a pPSY-pGEM-recA and the negative control E. coli DH5a, were tested for their ability to survive UV exposure. Strains were exposed to UV for 10-s intervals with subsequent viable counts performed. The quantitative UV exposure test was performed in triplicate with percent survival representing an average from the three experiments performed.
the amylase activity encoded by amyA was strongly expressed regardless of its orientation in relation to the lac promoter of pGEM-T Easy, this suggests that the 151 bp region upstream contains a promoter(s), which is active in E. coli. When amyA was transferred into the EcoRI site of pPSY, it was strongly expressed regardless of its orientation. This strengthens the view the PCR product not only encodes the amyA ORF but also a native promoter active in E. coli. Again testing shows that pPSY-amyA is highly stable while pGEM-T-amyA is highly unstable. 4. Discussion Escherichia coli K12 is the heterologous host of choice for most biologists. For over 60 years the physiology and genetics of this Gram-negative bacterium have been studied in minute detail. While a wide range of bacterial genes have been cloned and expressed in E. coli, genes from the prolific antibiotic producing streptomycetes are not readily expressed in this host. The list of possible reasons for this lack of expression is very long; however, one major problem is cloning vector instability. In particular, ColEI-based vectors, such as pBR322 and pUC18, are inherently unstable and even more unstable when they encode the synthesis of molecules toxic to the E. coli host. Nevertheless two gene clusters encoding the indolocarbazole antitumour antibiotics, violacein and rebeccamycin, are expressed in E. coli from their own promoters (Pemberton et al., 1991; August et al., 2000; Hyun et al., 2003). The vector instability which occurs when the violacein gene cluster is inserted into ColE1, based cloning vectors (pUC18/pHC79/pGEM-T Easy) has been overcome by the construction of the cosmid/BAC vector pPSX. This vector is highly stable in the absence of antibiotic selection pressure, even when it encodes the synthesis of toxic products (Sarovich and Pemberton, 2007). In the current study a second vector, pPSY, was constructed to provide a rapid, simple and effective method for the stable cloning and expression in E. coli of DNA sequences obtained by PCR. In view of the anticipated difficulties in the cloning and expression of streptomycete genes in E. coli, three single
gene phenotypes were amplified, cloned into pPSY, and tested for expression. All three genes were isolated from high G+C bacteria; vioA from Gram-negative C. violaceum; recA and amyA from Gram-positive S. lividans. Surprisingly, all three genes were strongly and stably expressed in E. coli using pPSY. The first gene used in this study, vioA, is an L-amino oxidase essential for the synthesis of the indolocarbazole antitumour antibiotic violacein. The demonstration that individual indolocarbazole genes such as vioA can be accessed by PCR and stably expressed in E. coli from their own promoters using pPSY, will allow the cloning and expression of many other indolocarbazole genes. It is now a relatively simple matter to use these genes and pPSY to assemble synthetic indolocarbazole pathways in E. coli to stably produce a range of novel indolocarbazole antitumour antibiotics. In the relatively few instances where single gene phenotypes from the high G+C Gram-positive streptomycetes have been cloned and expressed in E. coli, expression is usually achieved by cloning the gene next to a strong, inducible promoter such as the lac promoter of E. coli. When the recombinase A (recA) gene of S. lividans, lacking its native promoters, was cloned into pGEM-T Easy it was only expressed when placed in the correct orientation next to the Lac promoter. When sub-cloned into pPSY without its native promoter or the Lac promoter it was not expressed regardless of the orientation. This lack of expression reinforces our observation that there are no strong promoters on either side of the EcoRI site of pPSY, which would drive transcription through this site. Genes cloned into this site would require their native promoters to be functional in E. coli. For most streptomycete genes, it has been assumed that detectable expression does not occur from native promoters. In an initial attempt to identify and characterise native streptomycete promoters, Bourn and Babb (1995) identified eight Classes (A–H). Among the many promoters examined in this study there were at least three amylase gene promoters. All three fell into Class C, which is represented by a strongly conserved (TTGAC)-35 region and a much less conserved -10 region.
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When amyA and its native promoter(s) were amplified and cloned into pGEM-T Easy it was strongly expressed regardless of its orientation in respect to the lac promoter, suggesting that transcription was occurring from the native promoter(s). This conclusion was strengthened when amyA was expressed in E. coli when sub-cloned into pPSY. Expression was independent of the orientation of amyA in the EcoRI site of pPSY. An examination of the 151 bp directly upstream from the amyA ORF shows that it contains a Class C type streptomycete promoter, indicating that this promoter is active in E. coli. Due to ongoing genomic sequencing projects, it is now possible to access sequence data for a wide range of streptomycete single and multiple gene phenotypes. Future studies will reveal just how many of these genes can be cloned into pPSY and expressed in E. coli from their native promoters. References August, P.R., Grossman, T.H., Minor, C., Draper, M.P., Macnell, I.A., Pemberton, J.M., Call, K.M., Holt, D., Osbourne, M.S., 2000. Sequence analysis and functional characterisation of the violacein biosynthetic pathway from Chromobacterium violaceum. J. Microb. Biotechnol. 2, 513–519. Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H., Kieser, T., Larke, L., Murphy, L., Oliver, K., O’Neil, S., Rabbinowitsch, E., Rajandream, M.A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J., Hopwood, D.A., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147. Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S., Murray, N.E., 1976. The construction in vitro of transducing derivatives of phage lambda. Mol. Gen. Genet. 146, 199–207. Bourn, W.R., Babb, B., 1995. Computer assisted identification and classification of streptomycete promoters. Nucleic Acids Res. 23, 3696–3703. Connell, N.D., 2001. Expression systems for use in actinomycetes and related organisms. Curr. Opin. Biotechnol. 12 (5), 446–449. Gullon, S., Olano, C., Abdelfattah, M.S., Brana, A.F., Rohr, J., Mendez, C., Salas, J.A., 2006. Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin. Appl. Environ. Microbiol. 72 (6), 4172–4183.
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