DOCSLIB.ORG

Methylation-Dependent Transcription Controls Plasmid Replication of the Clodf13 Cop-I(Ts) Mutant ARNOLD J

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Methylation-Dependent Transcription Controls Plasmid Replication of the Clodf13 Cop-I(Ts) Mutant ARNOLD J JOURNAL OF BACTERIOLOGY, Nov. 1986, p. 728-733 Vol. 168, No. 2 0021-9193/86/110728-06$02.00/0 Copyright C 1986, American Society for Microbiology Methylation-Dependent Transcription Controls Plasmid Replication of the CloDF13 cop-i(Ts) Mutant ARNOLD J. VAN PUTTEN,1* RONALD DE LANG,' EDUARD VELTKAMP,' H. JOHN J. NIJKAMP,' PIET VAN SOLINGEN,' AND JOHAN A. VAN DEN BERG2 Department of Genetics, Biological Laboratory, Vrije Universiteit, 1081 HV Amsterdam,1 and Gist-brocades, 2600 MA Delft,2 The Netherlands Received 1 April 1986/Accepted 10 July 1986 The CloDF13 cop-l(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42°C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-l(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42°C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA H synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed. The bacteriocinogenic plasmid CloDF13 (9,957 base pairs) creased in comparison with that of the wild-type plasmid. originates from Enterobacter cloacae but is also stably However, at 42°C the plasmid copy number increases dras- maintained in Escherichia coli. In this latter host the plasmid tically. At this latter temperature the mutant plasmid shows is present to the extent of about 10 copies per cell (27). A a "runaway" behavior. Furthermore, cells harboring this small DNA region located between 1.8 and 11.5% of the mutant plasmid are killed at 42°C. CloDF13 genome is essential for autonomous replication (25, We have studied the implications of the cop-i(Ts) muta- 31). This region contains the origin of replication (oriV) and tion at the molecular level. Our data show that the regulation the information for two transcripts involved in plasmid of DNA replication is altered owing to increased RNA II replication, namely, a preprimer RNA of about 580 nucleo- synthesis. This increase in transcription results in a decrease tides (RNA II) and a small 108-nucleotide repressor RNA in the amount of active RNA I repressor molecules synthe- (RNA I) (30). RNA I is complementary to RNA II and is sized from the opposite strand. involved in the control of the processing of RNA II (11). The Recently, we noticed that the runaway replication of control of replication of CloDF13 strongly resembles that of CloDF13 cop-J(Ts) derivatives was quite variable in dif- other small multicopy plasmids like ColEl (28) and pMB1 ferent hosts. We demonstrate that in the absence of Dam (8), particularly with respect to the RNA I-RNA II interac- methylation no increase in plasmid copy number and no tion. Moreover, convergent transcription has been described killing effect is observed at elevated temperatures, and that also for other plasmid replication systems (32). the regulation of DNA replication is restored in this back- Several nonconditional copy number mutants of CloDF13 ground. Analysis of the region surrounding the cop-i(Ts) and related plasmids have been described. The majority of mutation reveals the presence of three putative Dam these mutations map within the region essential for autono- methylation sites (GATC) (10), one of which is disrupted by mous replication, particularly in the part encoding RNA I the cop-J(Ts) mutation (24) (Fig. 1). As shown in this paper (12, 17, 18, 24, 33). However, in the case of ColEl mutations the absence of the Dam methylase leads at 42°C to a outside this region were found that influenced the plasmid decreased primer synthesis, attended with a restoration of copy number (29). It turned out that the presence of a the regulation by RNA I. The significance of Dam methyla- replication regulatory protein, designated Rop (9), on the tion for CloDF13 plasmid replication is discussed. pMB1 and ColEl genome was responsible for this effect. In the case of CloDF13 no evidence could be obtained for the presence of a Rop-like regulatory protein (A. J. van Putten MATERIALS AND METHODS and G. Cesareni, unpublished results). Bacterial strains. The E. coli K-12 strains used in this Beside nonconditional mutants, also some conditional study were N3012 (thr leu thi lacY rpsL minA minB supE copy control mutants of nonconjugative plasmids were de- [2]), C600 (14), 71/18 [A(lac pro) F lacJi ZAM15 pro' (16)]; scribed previously. Those mutants are thermosensitive for GM48 (dam-3 dcm-6 leu thi thr lacY galK galT ara fnuA their control of plasmid replication (3, 17, 33). A conditional tsx-78 supE44 [13]). E. coli HB101 was used for plasmid CloDF13 copy mutation, designated cop-J(Ts), is located constructions. within the region involved in the initiation of preprimer Media, buffers, and chemicals. Cells were grown in synthesis, a region that overlaps with the transcription Lennox broth (LB) medium. LA plates contain LB medium terminator of the cloacin operon (3, 24) (Fig. 1). At 30°C the with 1 to 2% agar. Antibiotics were added at the following plasmid copy number of this mutant is only slightly in- final concentrations: ampicillin (50 to 100 jig/ml) and tetra- cycline (15 ,ug/ml). * Corresponding author. Plasmids and phages. The phages used in this study were 728 VOL. 168, 1986 METHYLATION CONTROLS CloDF13 PLASMID REPLICATION 729 , T -35 homology fT CX G T A T A --'-G-C cop mutation-I(TSA)* A - T T-A * C-G T - A T - A 3' 669 terminator RNA 694 T-A5A744 * *o - H S* CTAGTTTGGTGGAGGGGTCCACCAAAAAAGCAAATGTCCCGTTTTCTAATGCGCGTCTTTT CTAGAAAAGATGACTTGG initiation RNA II -10 homology terminator cloacin operon FIG. 1. Nucleotide sequence of the region surrounding the RNA II promoter. The terminator structures of the cloacin operon and the RNA I transcript are also shown. Symbols: (*) Dam methylation site, (0) Dcm methylation site. X132 (9) and XBG37 and 4BG38 (this paper). The plasmids (kb) BglII-BamHI-PvuII DNA fragment cloned into used are listed in Table 1. pBR322. Plasmid pPA153-209 containing the cop-J(Ts) ori- In vitro construction and characterization of recombinant gin of plasmid CloDF13 was constructed by P. M. Andreoli phages and plasmids. For the construction of the recombi- by ligating the 1.74-kb PstI-AvaI fragment of plasmid nant phages 4BG37 and 4)BG38 as well as the plasmids pVU209 (3) to the 2.2-kb PstI-AvaI fragment of plasmid pVU401, pVU402, and pVU403 we made use of standard pAT153 (29). Plasmid pGB210 was constructed by inserting recombinant DNA techniques. DNA fragments containing a 0.4-kb chromosomal E. coli DNA fragment into the PstI the promoter-terminator to be studied were cloned either in site of plasmid pPA153-209. Ampicillin-susceptible trans- HindIll-linearized X132 DNA or SmaI-linearized pKG1800 formants were selected by the method of Boyko et al. (6). DNA. Plaques obtained from the transfection of X132 DNA Plasmid pDAMA was constructed by deleting the 0.5-kb were screened by the method of Benton and Davis (4). BamHI-BamHI fragment from plasmid pDAM118. This frag- Occasionally, clones that were known to contain the pro- ment is a central part of the dam gene. Relevant restriction moter fragments in the right orientation were identified by maps and characteristics of the plasmids used in this study directly plating on McConkey plates and screening for red are given in Fig. 2. plaques. Recombinant plasmids were analyzed after the Determination of I8-galactosidase and galactokinase activi- isolation of plasmid DNA and subsequent digestion with ties. The P-galactosidase activity was determined by the restriction enzymes. The orientation of the cloned fragments procedure of Miller (16). The galactokinase activity in cells was verified by DNA sequence analysis by using the dideoxy harboring pKG1800 derived plasmids was assayed by the chain-termination method (22). We observed no influence of method of Adhya and Miller (1). the cloning of promoter-terminator fragments on plasmid Quantitation of plasmid amplification. The amount of plas- copy number or growth rate of plasmid-containing cells. mid amplification was analyzed by comparing the standard Plasmid pDAM118 containing the DNA adenine methyl- plasmid preparation with and without induction at the ase gene (dam) was described by Brooks et al. (7). Plasmid nonpermissive temperature. The following procedure was pDCM containing the entire EcoRII modification system was used: transformants were grown overnight at 28°C in 10 ml of kindly provided by T. Gingeras. It consists of a 5.4-kilobase LB medium and suspended in 100 ml of fresh LB medium at an optical density at 600 nm of 0.2. The cells were grown at TABLE 1. Plasmids used in this study Eco RI Bam HI/Bgill Source or EcoRI Plasmid Description reference PstI PM pVU206 Clo DF13 cop-l(Ts) 24 derivative pGB 210 Pst (4.3 kb) p DCM pPA153-209 Hybrid plasmid containing P. M. Andreoli kb) Cop_tS1 ~~~(8.1 the CloDF13 cop-l(Ts) Amps, S.Tet tR,cop-i Is Aval AMpR, Tets, dcmr origin pGB210 This study pDAM118 pBR322 derivative 7 copit containing the DNA adenine Eco RI Eco RI methylase gene Bam Hi pDAMA& This study Barn Hi pDCM pBR322 derivative T.
Load more

Recommended publications
  • Effect of Plasmid Copy Number and Lac Operator Sequence on Antibiotic-Free Plasmid Selection by Operator-Repressor Titration in Escherichia Coli
    Research Article J Mol Microbiol Biotechnol 2004;7:197–203 DOI: 10.1159/000079828 Effect of Plasmid Copy Number and lac Operator Sequence on Antibiotic-Free Plasmid Selection by Operator-Repressor Titration in Escherichia coli Rocky M. Cranenburgh Kathryn S. Lewis Julian A.J. Hanak Cobra Biomanufacturing Plc, The Science Park, Keele , Staffs., UK Key Words Introduction Recombinant protein · Metabolic burden · DNA vaccine · lac repressor · DAP · DH1 lacdapD The use of antibiotics and antibiotic resistance genes in the production of recombinant biotherapeutics is unde- sirable for a number of reasons. These include the risk of Abstract antibiotic gene transfer to pathogenic organisms [David- The Escherichia coli strain DH1 lacdapD enables plasmid son, 1999], plasmid loss due to plasmid size and metabol- selection and maintenance that is free from antibiotics ic stress [Smith and Bidochka, 1998] or antibiotic break- and selectable marker genes. This is achieved by using down during culture and the risk of contamination of the only the lac operator sequence as a selectable element. product with antibiotics [Baneyx, 1999]. Strategies have This strain is currently used to generate high copy num- been developed to avoid the use of antibiotics, including ber plasmids with no antibiotic resistance genes for use plasmids enabling complementation of a host auxotrophy, as DNA vaccines and for expression of recombinant pro- or possessing post-segregational killing (PSK) mechanisms teins. Until now these have been limited to pUC-based [Hanak and Cranenburgh, 2001]. However, these share plasmids containing a high copy number pMB1-derived another major disadvantage with the antibiotic-selected origin of replication, and the principle lacO1 and auxil- plasmids: the metabolic burden associated with constitu- iary lacO3 operators.
    [Show full text]
  • The Effects of Sub-Inhibitory Levels of Chloramphenicol on Pbr322
    Journal of Experimental Microbiology and Immunology (JEMI) Vol. 7:82-88 Copyright © April 2005, M&I UBC The Effects of Sub-Inhibitory Levels of Chloramphenicol on pBR322 Plasmid Copy Number in Escherichia coli DH5α Cells SARAH BEGBIE, GIULIO DOMINELLI, KIA DUTHIE, JOANNA HOLLAND, MARISSA JITRATKOSOL Department of Microbiology and Immunology, UBC While humans use antibiotics at inhibitory levels to prevent microbial growth, antibiotics are normally found at sub-inhibitory concentrations in nature. The role and effects of sub-inhibitory levels of antibiotics is one of the key areas of research in microbial science today. In this experiment, the effects of sub-inhibitory levels of chloramphenicol on pBR322 plasmid copy number in Escherichia coli DH5α cells were examined. E. coli DH5α cells with pBR322 plasmid were grown in different concentrations of chloramphenicol ranging from 1 to 5 µg/ml. The cells were enzymatically lysed and the DNA extracted through a phenol:chloroform:isoamyl alcohol extraction. The total bacterial DNA was run on a 0.8% agarose gel; using the ratio of genomic DNA to pBR322 DNA, the plasmid copy numbers per genome were calculated. It was found that 1 µg/ml chloramphenicol quantities had no significant affect when compared to the control; however, 3-5 µg/ml of chloramphenicol caused pBR322 concentrations to double. Due to assay insensitivity, there was no significant difference between the 3 µg/ml and 5 µg/ml samples. ___________________________________________________________ Chloramphenicol is a commonly used bacterial cultures prior to overnight incubation. bacteriostatic antibiotic that reversibly binds to the 50S Following growth in the presence of the antibiotic, subunit (L10 protein) and inhibits peptidyl transferase yield of plasmid DNA was analyzed and compared.
    [Show full text]
  • Characterization of Copy Number Mutants of Plasmid Psc101
    J. Gen. App!. Microbiol., 43, 309-316 (1997) Characterization of copy number mutants of plasmid pSC1O1 Abdul Wadood, Mitsuko Dohmoto, Shigeki Sugiura, and Kazuo Yamaguchi* Institute for Gene Research, Kanazawa University, Kanazawa 920, Japan (Received September 19,1997; Accepted November 28, 1997) We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 83rd and 115th codons in the rep gene and an increase in the copy number by 4- to 8-fold. Although the in vivo and in vitro repressor activities of these mutated Rep proteins were quite different from each other, the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that ex- cess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mu- tated rep genes were retained in the same plasmids, the copy number of these plasmids was de- creased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy num- ber regulation. Key Words copy number; DNA replication; dominance test; plasmid; pSC101 Plasmid pSC101 (Cohen and Chang, 1977), 9.2 1995). Furthermore, overproduction of the Rep protein kilobase pairs (kb) in size (Yamaguchi and Yama- can inhibit the replication of plasmid pSC101 (Ingmer guchi, 1984b), is present at approximately 5 copies and Cohen, 1993).
    [Show full text]
  • Effect of RNA I Gene Dosage on Plasmid Copy Number and Incompatability DAVID R
    JOURNAL OF BACTERIOLOGY, May 1983, p. 809-818 Vol. 154, No. 2 0021-9193/83/050809-10$02.00/0 Copyright C 1983, American Society for Microbiology Characterization and Complementation of pMB1 Copy Number Mutant: Effect of RNA I Gene Dosage on Plasmid Copy Number and Incompatability DAVID R. MOSER AND JUDITH L. CAMPBELL* Department of Chemistry, California Institute of Technology, Pasadena, California 91125 Received 2 July 1982/Accepted 20 January 1983 A 16-base-pair insertion has been identified as the mutation responsible for the high-copy-number phenotype of the plasmid copy number mutant pFH118. The mutation is located near the plasmid origin of replication in a region ofthe genome that encodes two overlapping RNA transcripts. One of these transcripts, RNA I, acts as a negative regulator of plasmid replication. The second transcript is the precursor to the primer for the initiation of DNA synthesis. We demonstrate through complementation that the pFH118 DNA overproduction phenotype is a consequence of the reduced effectiveness of the mutant RNA I at inhibiting plasmid replication and not a consequence of an altered target site on the primer precursor. In addition, a series of plasmids containing multiple RNA I-coding genes was constructed for investigating the effects of RNA I gene dosage on plasmid copy number and incompatibility. The results of this study strongly support the inhibitor dilution model of plasmid copy control with RNA I as the plasmid-specified inhibitor responsible for both copy number control and incom- patibility. The stable inheritance of multicopy bacterial number, were found to have deleted the inser- plasmids at defined copy numbers implies a tion sequence and produced a normal RNA I.
    [Show full text]
  • RNAII Transcribed by IPTG-Induced T7 RNA Polymerase Is Non-Functional As a Replication Primer for Colel-Type Plasmids in Escherichia Coli Michael Y
    1995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 10 1691-1695 RNAII transcribed by IPTG-induced T7 RNA polymerase is non-functional as a replication primer for ColEl-type plasmids in Escherichia coli Michael Y. Chao, Ming-Chung Kan and Sue Lin-Chao* Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China Received February 17, 1995; Revised and Accepted March 30, 1995 ABSTRACT advantageous for the expression of certain gene products would be a cloning vector that has an inducible high copy number. RNAII, an RNA species encoded by ColEl-type plas- Several RI-derived plasmids that show temperature-dependent mids, serves as a primer for plasmid DNA replication. regulation of copy number have been described (7); these Previous work has shown that overproduction of plasmids replicate at a low copy number at 30°C and exhibit RNAII transcribed by Escherichia coliRNA polymerase 'runaway replication' at 370C, resulting in an exponential results in elevated plasmid copy number. To produce increase in the concentration ofplasmid DNA. However, the host a plasmid in which the elevation of its copy number is cells carrying these plasmids lyse under 'runaway' conditions. inducible, we placed transcription of RNAII under the Recent work by Bacharov et al. (8) has shown that IPTG- control of a bacteriophage T7 late promoter regulated induced overproduction of RNAII transcribed from a strong by IPTG-inducible T7 RNA polymerase. During induc- synthetic promoter by E.coli RNA polymerase results in a 4-fold tion of T7 RNA polymerase by IPTG, we found that increase in plasmid copy number (8).
    [Show full text]
  • A Plasmid System with Tunable Copy Number
    bioRxiv preprint doi: https://doi.org/10.1101/2021.07.13.451660; this version posted July 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 A Plasmid System with Tunable Copy Number 2 Miles V. Rouches and Yasu Xu 3 Field of Biophysics, Cornell University, Ithaca, NY 14853, USA ∗ 4 Louis Cortes and Guillaume Lambert 5 School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA 6 (Dated: July 13, 2021) Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis.
    [Show full text]
  • A Plasmid System with Tunable Copy Number
    1 A Plasmid System with Tunable Copy Number 2 Miles V. Rouches and Yasu Xu 3 Field of Biophysics, Cornell University, Ithaca, NY 14853, USA 4 Louis Cortes and Guillaume Lambert⇤ 5 School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA 6 (Dated: July 16, 2021) Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic e↵ects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the e↵ects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number enables the precise control of gene expression and highlight the importance of tuning plasmid copy number as a tool for the optimization of synthetic biological systems. 7 INTRODUCTION 38 cellular growth and metabolism. Specifically, we first in- 39 troduce a strategy for finely tuning plasmid copy number 40 8 Plasmids are an invaluable tool in biotechnology and in a way that is dependent on the concentration of an ex- 41 9 genetic engineering due to their small size and the ease ogenous inducer molecule.
    [Show full text]
  • Absolute and Relative QPCR Quantification of Plasmid Copy
    Journal of Biotechnology 123 (2006) 273–280 Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli Changsoo Lee, Jaai Kim, Seung Gu Shin, Seokhwan Hwang ∗ SchoolofEnvironmentalScienceandEngineering,PohangUniversityofScienceandTechnology,San31 Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Republic of Korea Received 31 May 2005; received in revised form 28 August 2005; accepted 10 November 2005 Abstract Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5␣ harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid ␤-lactamase gene (bla) and for the chromosomal d-1- deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8–3.9% and 4.7–5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15–20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E.
    [Show full text]
  • Plasmid Copy-Number Control and Better-Than-Random Segregation Genes of Psm19035 Share a Common Regulator
    Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator Ana B. de la Hoz*, Silvia Ayora*, Izabela Sitkiewicz†, Silvia Ferna´ ndez*, Renata Pankiewicz†, Juan C. Alonso*‡, and Piotr Ceglowski† *Department of Microbial Biotechnology, Centro Nacional de Biotecnologı´a,Consejo Superior de Investigaciones Cientı´ficas,28049 Madrid, Spain; and †Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland Communicated by Donald R. Helinski, University of California, San Diego, CA, November 10, 1999 (received for review July 10, 1999) Transcription initiation of the copy-number control and better-than- (RNA III) that induces transcriptional attenuation within the leader random segregation genes of the broad-host-range and low-copy- region of the rep mRNA (11, 15–17). In the absence of Cop, the number plasmid pSM19035 are subjected to repression by the auto- increased (derepressed) transcription of rep mRNA interferes, in regulated pSM19035-encoded ␻ product in Bacillus subtilis cells. The cis, with initiation of transcription of RNA III (convergent tran- promoters of the copS (Pcop1 and Pcop2), ␦ (P␦), and ␻ (P␻) genes scription). Thus, the Cop protein limits the amounts of rep mRNA have been mapped. These promoters are embedded in a set of either either directly (as a repressor) or indirectly, by affecting the intensity seven copies of a 7-bp direct repeat or in a block consisting of two of transcription necessary to produce RNA III. The factor(s) that 7-bp direct repeats and one 7-bp inverted repeat; the blocks are would control the synthesis of Cop has not been yet described.
    [Show full text]
  • Genetic Control of Cole1 Plasmid Stability That Is Independent of Plasmid Copy Number Regulation
    UC Office of the President Recent Work Title Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation. Permalink https://escholarship.org/uc/item/8rp9q6cj Journal Current genetics, 65(1) ISSN 0172-8083 Authors Standley, Melissa S Million-Weaver, Samuel Alexander, David L et al. Publication Date 2019-02-01 DOI 10.1007/s00294-018-0858-0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Current Genetics https://doi.org/10.1007/s00294-018-0858-0 ORIGINAL ARTICLE Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation Melissa S. Standley1 · Samuel Million‑Weaver1,2 · David L. Alexander1,3 · Shuai Hu1 · Manel Camps1 Received: 26 March 2018 / Revised: 8 June 2018 / Accepted: 12 June 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA relA strain as a sensitized model to study factors affecting plasmid stability in the context of recombinant gene expression. We find that in this model, plasmid stability can be restored by two types of genetic modifications to the plasmid origin of replication (ori) sequence: point mutations and a novel 269 nt duplication at the 5′ end of the plasmid ori, which we named DAS (duplicated anti-sense) ori.
    [Show full text]
  • Copy Number of Cole1-Type Plasmids Decreases When Rop Is Supplied in Trans on a Separate Plasmid
    Journal of Experimental Microbiology and Immunology (JEMI) Vol. 21: 1 – 8 Copyright © July 2017, M&I UBC Copy Number of ColE1-type Plasmids Decreases When rop is Supplied in trans on a Separate Plasmid Jose Emmanuel Gozon Gana, Zuroon Hou-Hang Ho, Shraavan Raveendran, Nai Chun Shao Department of Microbiology & Immunology, University of British Columbia Previous studies have shown that in Escherichia coli co-transformed with two different plasmids, the expression of the Rop protein from one plasmid can act in trans to decrease the copy number of the other plasmid, an effect referred to as plasmid exclusion. These studies relied on previous findings on ColE1-type plasmids showing that copy number is primarily regulated by the RNA II transcript, which serves as a primer for plasmid replication, the RNA I transcript, which binds and sequesters RNA II, and the Rop protein, which stabilizes the RNA I : RNA II hybrid. However, these studies, which used the pUC19, pBR322, and pCAWK vectors, did not account for the influence of an RNA II point mutation that was present in pUC19, but absent in both pBR322 and pCAWK. Hence, we decided to use the pAPA3 plasmid in lieu of pUC19, to control for the effect of the RNA II point mutation. We hypothesized that by controlling for the RNA II point mutation with the use of pAPA3 plasmid, the presence of Rop would result in greater levels of exclusion of the lower copy number plasmids in co-transformed cells. To test this hypothesis we generated co-transformants in Escherichia coli DH5α containing either pBR322 or pCAWK and pAPA3 plasmids.
    [Show full text]
  • Isolation and Characterization of Novel Mutations in the Psc101 Origin That Increase Copy Number Received: 29 September 2017 Mitchell G
    www.nature.com/scientificreports OPEN Isolation and characterization of novel mutations in the pSC101 origin that increase copy number Received: 29 September 2017 Mitchell G. Thompson1,2,3, Nima Sedaghatian1,2, Jesus F. Barajas2,4, Maren Wehrs1,2, Accepted: 11 January 2018 Constance B. Bailey1,2, Nurgul Kaplan1,2,4, Nathan J. Hillson1,2,4, Aindrila Mukhopadhyay1,2 & Published: xx xx xxxx Jay D. Keasling1,2,5,6,7 pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identifed multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identifed in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cell in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fuorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication.
    [Show full text]