Complete sequence and analysis of the stability functions of pPSX, a vector that allows stable cloning and expression of Streptomycete genes in Escherichia coli K12

Plasmid 62 (2009) 39–43

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Short Communication

Complete sequence and analysis of the stability functions of pPSX, a vector that allows stable cloning and expression of Streptomycete genes in Escherichia coli K12 Daniel S. Philip1, Derek S. Sarovich1, John M. Pemberton 1,* Department of Microbiology and Parasitology, University of Queensland, Brisbane 4072, Australia

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Article history: Received 19 November 2008 Revised 5 March 2009 Available online 20 March 2009 Communicated by Dr. Dhruba K. Chattoraj Keywords: Streptomyces E. coli Stable vector Complete sequence pPSX

a b s t r a c t The broad host range, cloning and expression vector pPSX has been completely sequenced and analysed. pPSX is 14.7 kb in length and contains the fusion of two continuous segments of the parental 34 kb, IncW plasmid pR388. pPSX appears to have retained at least three sets of gene/s which contribute in different ways to plasmid stability. The first of these parB, is a known participant in the partitioning of low-copy number plasmids. While the adjoining gene, orf35, has high homology with kfrA, a putative plasmid nucleoid organiser that is often associated with the ParAB family of proteins. The second set of genes; orfs 18, 19, 20, whose exact functions are not clear, have homology to the stability operons of both IncW and IncN plasmids. The third is the resolvase, resP, which may resolve plasmid multimers that can lead to plasmid instability. pPSX is a small, stable cloning vector good for cloning and expression of a wide range of genes, including those from streptomycetes. Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.

1. Introduction Broad host range plasmids were discovered nearly 40 years ago by Sykes and Richmond (1970). Subsequent research has demonstrated that these plasmids play a major role in the evolution and spread of a wide range of bacterial gene clusters involved in biologically significant traits which include multiple antibiotic resistance, pathogenicity, antibiotic synthesis and the degradation and recycling of man-made environmental pollutants (Gillings et al., 2008; Revilla et al., 2008). These plasmids have evolved such that their genes are expressed in a wide range of bacteria. A remarkable feature of the broad host range IncW plasmid pR388 (34 kb) is that it is absolutely stable in E. coli K12 in the absence of antibiotic selection pressure (Saro-

* Corresponding author. Fax: +61 7 33654620. E-mail address: [email protected] (J.M. Pemberton). 1 D.P. sequenced pPSX. D.S. kindly supplied pPSX. The remaining research and analysis was performed equally D.P. and J.P.

vich and Pemberton, 2007). pR388 is retained by E. coli K12 even when encoding the synthesis of the toxic antitumour antibiotic violacein (Sarovich and Pemberton, 2007). Violacein is an intensely purple-pigmented, antitumour antibiotic. The violacein gene cluster from Chromobacterium violaceum was the first antitumour antibiotic biosynthetic operon to be cloned and expressed in E. coli K12 (Pemberton et al., 1991). Violacein belongs to a group of potent antitumour antibiotics well known to medical science – the indolocarbazoles; which include staurosporine and rebeccamycin. Violacein synthesis is encoded by five genes: vioABCDE (August et al., 2000). VioB, is the indolocarbazole synthase (ICS), which catalyses the condensation of two tryptophan molecules to form the indolocarbazole core. All known indolocarbazole gene clusters, the majority of which occur in streptomycetes, have homologs of VioB (Kim et al., 2007). VioA, C and D are monooxygenases which modify this core by oxygenation or hydroxylation to produce the end-product violacein (August et al., 2000). The streptomycete encoded, rebeccamycin gene cluster was the second indolocarbazole gene cluster to be

0147-619X/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2009.03.002

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cloned and expressed in an E. coli heterologous host (Hyun et al., 2003). The expression of gene/s that encode toxic molecules, such as violacein, requires, first and foremost, a broad host range and stable cloning vector. Using three rounds of Sau3A partial digestion followed by ligation, the smallest stable derivative of pR388 was found to be 12 kb. A stable cosmid/bacterial artificial chromosome (BAC) cloning vector, pPSX (14 kb) (Sarovich and Pemberton, 2007), was constructed by the addition of the COS site from pHC79 (Hohn and Collins, 1980) and the multiple cloning site (MCS) of pUC18 (Yanisch-Perron et al., 1985) to the reduced pR388 core (Laravllaand and Fernandodela, 1988). Like pR388, pPSX is absolutely stable in E. coli K12. Surprisingly, a variety of streptomycete genes cloned into pPSX are expressed in E. coli K12 (Sarovich and Pemberton, 2007; Philip et al., 2008). This paper presents the complete sequence and analysis of pPSX which may provide an insight into those genes which contribute to its remarkable stability. 2. Results and discussion 2.1. Sequence and genetic map of pPSX pPSX is an extremely stable cloning vector based on a minimal replicon of pR388. The sequence data presented here (Fig. 1; Table 1; NCBI Accession No. FJ422118) shows

that pPSX results from the fusion of two continuous segments of pR388. Segment 1 covers the region from the middle of trwA to the middle of orf5, encoding the origin of vegetative replication, transposition and partitioning genes, as well as the genes for trimethoprim (dhfr) and sulfonamide (sul1) antibiotic resistance (see Table 1). Segment two is in the opposite orientation relative to segment one of pR388. This region encodes the segment from orf23 to oriT (see Table 1). The COS site from pHC79 and MCS from pUC18 have been inserted in orfA of segment one to produce the final cosmid/BAC vector, pPSX (Sarovich and Pemberton, 2007). During the construction of pPSX, more than half of trwA and orf5 were deleted due to the digestion process. Another gene, orfA, was cut in half by the insertion of the COS and MCS regions. To date, none of these genes have demonstrated functionality. 2.2. Stability functions pPSX appears to have retained at least three and perhaps four sets of gene/s which contribute in different ways to plasmid stability. The first of these, parB, is a known participant in the partitioning of low-copy number plasmids and plays a vital role in maintaining plasmid stability. In E. coli, this is achieved by ensuring that during cell division each of the daughter cells receives at least one copy of the plasmid (Gordon and Wright, 2000; Godfrin-Estevenon

Fig. 1. Map of the stable cosmid cloning vector pPSX. oriV, origin of vegetative replication; repA, replication protein; resP, resolvase; intl1, integrase; dhfr, dihydrofolate reductase confers trimethoprim resistance; MCS, multiple cloning site; COS, cohesive ends of lambda phage; qacED1, resistance to quaternary ammonium compounds; sul1, sulfonamide resistance; oriT, origin of transfer replication; parB, partition protein; Sequencing of pPSX was carried out at the Australian Genome Research Facility (AGRF).

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D.S. Philip et al. / Plasmid 62 (2009) 39–43 Table 1 Genes within segments one and two of pPSX. orf/Gene

Function

Reference

Segment one parB Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon orf35 Has high homology with kfrA from the salmonella enteric plasmid pIE321 (IncW) and some IncP1-beta plasmids (eg, pR751). It is associated with parA and parB oriV/repA Origin of vegetative replication/plasmid replication initiation protein resP A Resolvase gene. Most likely a homolog of the Tn21 resolvase gene orf38 Has high homology to an inner membrane protein of the plasmid R7 K from Providencia rettgeri DSM 1131 (Accession No. YP_001874893) IntI1 Site-specific recombinase dhfr Dihydrofolate reductase confers trimethoprim resistance ‘orfA No assigned phenotype/gene probably inactivated MCS Multiple cloning site from pUC18 COS Lambda COS site from pHC79 Resistance to quaternary ammonium compounds (QAC) qacED1 sul1 Sulfonamide resistance ‘orf5 Function unknown/probably inactive Segment two ‘trwA Relaxase enhancer, which has a DNA binding domain. It works in conjunction with TrwB and TrwC at the oriT site. Probably inactive oriT Origin of plasmid transfer replication orf18 Homolog of the stbA gene from pIE321 (IncW). In the conjugative, incN plasmids pKM101 and pCU1, this is part of the stability operon (stb operon) orf19 Homolog of the stbB gene from pIE321 (IncW). May be involved in auto regulation of the stb operon by repression of the stbAB promoter orf20 Homolog of the stbC gene from pIE321 (IncW). Insertion/deletion causes instability orf23 Shows homology to the methylase of Type I restriction/modification system of Burkholderia pseudomallei 1106a orf22 Function unknown orf21 Function unknown

et al., 2002). Plasmid partitioning systems are encoded in two closely linked genes parA, parB and a cis-acting centromere-like site parS. ParA is a Walker box or actin-like ATPase (Ebersbach and Gerdes, 2005; Funnell, 2005). ParB binds as a dimer to specific sequences within the cis site (Schumacher and Funnell, 2009). The partitioning complex determines the intracellular location of the plasmid. Just before partitioning, the plasmid copies move towards the poles and away from the site of cellular scission. After division, the plasmids occupy an equatorial position. Although pPSX contains a homolog of ParB there are no direct homologs of ParA or ParS. Next to parB is orf35, which exhibits high homology with kfrA, a putative plasmid nucleoid organiser that is often associated with the ParAB family of proteins (Adamczyk et al., 2006). Second, is the stability operon orf18, 19, 20, which has homology to the stability operons (staABC) of IncN and other incW plasmids (Paterson et al., 1999; Sota et al., 2006). While the exact functions encoded by orfs18, 19, 20 and their IncW and IncN plasmid homologs remain unknown, the protein encoded by orf18 has homology with the traD gene of the naphthalene catabolic plasmid pNAH7, which plays a role in plasmid mobilisation. In addition, the protein encoded by orf19 is a distant homolog of ParA ATPases. Since the origin of plasmid transfer (oriT), which allows pPSX to be mobilised from one cell to another by IncP plasmids such as RP4, is next to the orf18, 19, 20 operon it is possible that the orf18, 19, 20 genes play a role in both plasmid mobility and broad host range stability (Fernández-López et al., 2006). Third, the resolvase, ResP may play an important

Lukaszewicz et al., 2002 Adamczyk et al., 2006 Okumura and Kado, 1992 Fernández-López et al., 2006 Revilla et al., 2008 Collis et al., 1998 Swift et al., 1981 Revilla et al., 2008 Hohn and Collins, 1980 Paulsen et al., 1993 Sundstroem et al., 1988

Revilla et al., 2008; Moncalián and de la Cruz, 2004 Paterson et al., 1999 Paterson et al., 1999; Tabuchi et al., 1992 Paterson et al., 1999 Accession No. ABN88696

role in the resolution of multimeric forms of the plasmid. In other plasmids, the failure to resolve such multimers can lead to plasmid instability (LeBard et al., 2008). Finally, after the minimal stable replicon of pR388 had been isolated several attempts failed to reduce it further indicating orfs21, 22, 23 are required for stability of pPSX. Very little is known regarding the function of these genes. orf23 has homology with Type 1 modification DNA methylases from Burkholderia pseudomallei 1106a (Accession No. ABN88696), suggesting that this operon may encode a Type I restriction/modification system; however orfs21 and 22 do not show any homology to known restriction endonucleases or DNA specificity proteins. If this is a Type I restriction-modification system, then like some Type II restriction-modification systems, it could form a plasmid addiction system which ensures the host cell retains the plasmid (Kobayashi, 2001). 2.3. Down regulation of toxic genes Violacein is an antibiotic and E. coli K12 strains synthesising violacein grow more slowly than their non-synthesising counterparts. It would be anticipated that when E. coli K12 strains containing plasmids, which encode and express violacein synthesis, were subcultured there would be a strong selection for cells which have either lost the plasmid or have deleted the violacein genes. When E. coli K12 LE392 pPSX-vioABCDE is subcultured in the absence of selective pressure on PYE agar plates for 100 generations there is no detectable loss of the plasmid (<1 in 10,000)

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and all colonies show a uniform, but low production of violacein (Sarovich and Pemberton, 2007). This plasmid encoded, down regulation of violacein synthesis greatly reduces its toxic effects on the host cell and allows E. coli K12 LE392 to retain the plasmid. The mechanism of this down regulation and the genes responsible are not known. In contrast, E. coli K12 LE392 pUC18-vioABCDE, which overproduces violacein, is extremely unstable. When subcultured in PYE broth for 15 generations less than 10% of the population retained the plasmid. Subculturing E. coli K12 LE392 pUC18-vioABCDE in PYE broth containing 100 lg/ml of ampicillin for 100 generations resulted in cells that eventually produced little or no violacein. 2.4. Trimethoprim resistance and recovery of transformants The use of trimethoprim resistance as the selective marker in transformation/electroporation experiments involving pPSX can result in a marked increase in the recovery of transformants. When pPSX was transformed into E. coli K12 LE392, the recovery of trimethoprim resistant transformants was 1  105 per lg of pPSX DNA. By comparison, when pUC18 was transformed into E. coli K12 LE392, the recovery of ampicillin resistant transformants was about the same, at 2  105 per lg of pUC18 DNA. pPSX is 14.7 kb in size, has a copy number between 5 and 15 yet the frequency of transformation per lg of plasmid DNA is comparable to that of pUC18, a smaller (2.7 kb), much less stable cloning vector. Usually, as the size of the plasmid vector increases, the transformation frequency decreases. When pPSX (14.7 kb), pPSX-vioABCDE (22 kb) and pPSXTn5-vio+ (59 kb) were transformed into E. coli K12 LE392 and selection made for trimethoprim resistance, transformants were recovered at the rate of 1  105, 4  104 and 1  104 per lg of plasmid DNA, respectively. The reason for this high transformation frequency remains unknown. One possible explanation is that trimethoprim is entirely bacteriostatic and once the resistant dihydrofolate reductase encoded by pPSX is produced, folic acid is synthesised and growth resumes. This relatively high transformation frequency is an important feature of pPSX particularly if it is used to construct Bacterial Artificial Chromosome (BAC) libraries where insert DNAs are >100 kb and transformation frequencies low.

2.5. Broad host range expression of trimethoprim resistance pPSX retains the genes for trimethoprim resistance (dhfr), sulfonamide resistance (sul1) and resistance to quaternary ammonium compounds (qacED1). In conjugation experiments, the primary selective marker is trimethoprim resistance. When pPSX was mobilised from E. coli K12 LE392 pPSX using pED709, a conjugative, kanamycin sensitive, derivative of the IncP plasmid RP4, into a range of heterologous hosts, the frequency of transfer varied between 1 in 105 and 1 in 108 per donor cell (Table 2). In each case the heterologous host expressed trimethoprim resistance. Unlike E. coli K12, all these heterologous hosts only retain the plasmid under antibiotic selection, suggesting that the sta-

Table 2 Frequency of transfer of pPSX from E. coli K12 LE392 pED709 pPSX into various heterologous hosts. Recipent strains

Trimethoprim resistant exconjugants per donor cell

Aeromonas hydrophila JMP636 Agrobacterium radiobacter Paracoccus denitrificans JMP928 Pseudomonas stutzeri JMP783 Pseudomonas aeruginosa PAO1 Pseudomonas putida Ralstonia eutropha JMP228 Rhizobium leguminosarum Rhodobacter sphaeroides RS7009

1  108 4  107 1  106 2  105 1  105 1  107 4  106 3  106 1  105

Materials and methods pertaining to bacterial growth conditions and conjugation experiments have been published previously (Sarovich and Pemberton, 2007; Philip et al., 2008). For most recipient strains, exconjugants were selected on PYEA plates containing 100 lg/ml of trimethoprim. For P. aeruginosa and P. putida, exconjugants were selected on PYEA plates containing 1000 lg/ml trimethoprim. pED709 is a kanamycin sensitive derivative of the IncP plasmid pRP4.

bility functions of pPSX do not work efficiently in these hosts. 2.6. Regulation of antibiotic synthesis in E. coli K12 If a plasmid encodes both the synthesis of a toxic antibiotic and a strong addiction system, it might be anticipated that plasmids, which no longer produced the antibiotic, would have a selective advantage; however, this is not what is observed. On non-selective medium, E. coli K12 LE392 pPSX-vioABCDE not only shows absolute plasmid stability but a uniform but comparatively low production of the purple-pigmented violacein by all colonies compared with high levels of violacein synthesis by E. coli K12 LE392 pUC18-vioABCDE. This difference in the levels of violacein synthesis may be due to the difference in copy number between pPSX (5–15 copies per cell) and pUC18 (50–100 copies per cell). The level of violacein synthesis can also be affected by host genes. For E. coli K12 LE392 pPSX-vioABCDE, synthesis of the purple-pigmented violacein is visible on PYE agar plates in 24–36 h. In contrast, E. coli K12 DH5a pPSX-vioABCDE produces little visible purple-pigmented violacein; even after 7 days. This difference may be due to the stringent response gene relA; LE392 is RelA+ and DH5a is RelA. In Streptomyces coelicolor A3(2), deletion of the (p)ppGpp synthetase gene, relA, results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) (Sun et al., 2001). 2.7. Broad hot range instability and antibiotic synthesis In other bacteria, such as the high G+C Gram negative soil bacterium Sphingomonas sp., the presence of pPSX-vioABCDE results in hyper production of violacein. When pPSX-vioABCDE was mobilised from E. coli K12 LE392 pPSX-vioABCDE into Sphingomonas sp. JMP4092, by the conjugative plasmid pED709, the resultant Sphingomonas sp. JMP4092 pPSX-vioABCDE exconjugants grew slowly and overproduced violacein. The overproduction was so

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great that when JMP4092 pPSX-vioABCDE was subcultured onto a PYE Tp50 agar plate and incubated for 5 days at 35 °C it rapidly turned dark purple then black. After three serial subcultures of JMP4092 pPSX-vioABCDE on PYE agar, in the absence of antibiotic selection for the plasmid, <1% of colonies retained the plasmid. When the three serial subcultures of JMP4092 pPSX-vioABCDE were performed, on PYE agar in the presence of antibiotic (trimethoprim) selection for the plasmid, there was a gradual loss of the violacein producing colonies. The more toxic intense purple violacein producing colonies were replaced by blue and green colonies which produced the less toxic intermediates deoxyviolacein and proviolacein. These pathway variants carry mutations in vioD and vioC, respectively (Balibar and Walsh, 2006). Further subculturing resulted in the appearance of white colonies which failed to produce any colored violacein precursors. These colorless colonies may contain mutations in either vioA or vioB. Conversely, they may represent extensive deletions of the violacein cluster. In contrast, E. coli K12 LE392 pPSX-vioABCDE is absolutely stable, produces less violacein and even after five serial subcultures on PYE agar plates with or without antibiotic selection there is no variation in violacein synthesis and no plasmid loss. Finally, while the overall percentage of streptomycete genes cloned into pPSX and expressed in E. coli K12 is yet to be determined, the number of potential plasmid encoded genes responsible for this phenomenon has been narrowed down. With pPSX, screening for the expression of genes in E. coli K12 is not complicated by either the loss of the plasmid or loss of the insert under non-selective conditions. References Adamczyk, M., Dolowy, P., Jonczyk, M., Thomas, C.M., Jagura-Burdzy, G., 2006. The kfrA gene is the first in a tricistronic operon required for survival of IncP-1 plasmid R751. Microbiology 152 (6), 1621–1637. 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. Balibar, C.J., Walsh, C.T., 2006. In vitro biosynthesis of violacein from Ltryptophan by the enzymes VioA-E from Chromobacterium violaceum. Biochemistry 45 (51), 15444–15457. Collis, C.M., Kim, M.J., Stokes, H.W., Hall, R.M., 1998. Binding of the purified integron DNA integrase Intl1 to integron- and cassetteassociated recombination sites. Mol. Microbiol. 29 (2), 477–490. Ebersbach, G., Gerdes, K., 2005. Plasmid segregation. Mech. Annu. Rev. Genet. 39, 453–479. Fernández-López, R., Garcillán-Barcia, M.P., Revilla, C., Lázaro, M., Vielva, L., de la Cruz, F., 2006. Dynamics of the IncW genetic backbone imply general trends in conjugative plasmid evolution. FEMS Microbiol. Rev. 30 (6), 942–966. Funnell, B.E., 2005. Partition-mediated plasmid pairing. Plasmid 53, 119– 125. Gillings, M., Boucher, Y., Labbate, M., Holmes, A., Krishnan, S., Holley, M., Stokes, H.W., 2008. The evolution of class 1 integrons and the rise of antibiotic resistance. J. Bacteriol. 190 (14), 5095–5100. Godfrin-Estevenon, A.M., Pasta, F., Lane, D., 2002. The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli. Mol. Microbiol. 43 (1), 39–49. Gordon, G., Wright, A., 2000. DNA segregation in bacteria. Annu. Rev. Microbiol. 54, 681–708.

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