Isolation of a Derivative ofEscherichia coli–Enterococcus faecalisShuttle Vector pAM401 Temperature Sensitive for Maintenance inE. faecalisand Its Use in Evaluating the Mechanism of pAD1par-Dependent Plasmid Stabilization

A derivative of theEscherichia coli–Enterococcus faecalisshuttle vector pAM401 was isolated by mutagenesis in anE. colimutator strain. This plasmid, designated pAM401ts, was more than an order of magnitude less stable at 38°C than at 30°C in theE. faecalishost strain JH2-2. TheE. faecalisplasmid pAD1-encodedparstability locus was cloned onto pAM401ts, and its effects on plasmid stability and host cell viability were assessed. It was found thatparstabilized pAM401ts at 38°C but also caused a substantial drop in cell viability three to four generations after a temperature shift from 30 to 38°C. After a maximum viability drop of 94%, culture growth recovered as plasmid-free cells began to accumulate. Provision of excess RNAII, the putativeparantidote,in transattenuated cell killing. These characteristics support a postsegregational killing mechanism forpar-mediated plasmid stabilization.     

Partitioning of plasmid R1 in Escherichia coli : II. Incompatibility properties of the partitioning system

A theoretical as well as an experimental study of the effect of the partitioning system on plasmid R1drd-19 incompatibility was performed. The theoretical numerical analysis (by computer) was based upon the following assumptions: (i) The partitioning (par) mechanism is independent of the replication (rep) and replication control (cop) mechanism. (ii) A par mutation causes random (binomial) distribution of plasmid copies between the daughter cells at cell division. (iii) In the par⁺ case, the plasmid copies are equipartitioned and selected randomly for partitioning. (iv) Selection of plasmid copies for replication is random. (v) Two different replication control systems were considered: Model 1 assumes that the plasmid copy number is set to exactly 2n before cell division, whereas in Model 2 exactly n copies are synthesized per cell per cell cycle. Numerical analysis was performed for the n values 2–8. The result was that in all cases (par⁺/par⁺, par⁺/par, par/par), steady states with respect to copy number distribution within the heteroplasmid population were rapidly (within five or six generations) established, giving constant loss rates. The rate of loss was slightly higher in the par/par case than in the other two. The two replication control models gave almost identical loss rates. In the par⁺/par case, the par⁺ plasmid had an advantage over the par plasmid. The experimental approach was to create heteroplasmid populations of Escherichia coli by introducing two genetically marked derivatives of plasmid R1drd-19 and then follow the reduction in the relative size of this population during exponential growth in LB medium. The loss rate was essentially the same in the par⁺/par⁺ and par⁺/par combination and slightly higher in the par/par case, suggesting that plasmid incompatibility mainly is caused by the replication and copy number control system. In the par⁺/par situation, the par⁺ plasmid had a pronounced advantage over the par plasmid. The par region of plasmid R1 (without the basic replicon) was cloned onto the vector pSF2124. A par (deletion) mutation was not complemented by par⁺. Plasmid pSF2124, which does not seem to carry a par system of its own, could use the R1 par system, adding to the conclusion that par is independent of rep and cop. Plasmids pSF2124 and R1drd-19 are completely compatible, whereas plasmid pSF2124 carrying the R1 par system and plasmid R1drd-19 showed a weak incompatibility although the copy numbers of the two plasmids were not affected in the heteroplasmid cells. Hence, the partitioning system causes incompatibility, but the effect is weak compared to that of the cop system. The result is consistent with some sort of assortment of plasmids before partitioning.     

Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli

Summary Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. II. Specific recombination functions involved in plasmid maintenance

Summary Clo DF13 plasmids that are present at high copy-number in bacterial cells, such as Clo DF13 cop1 Ts, cop2 and cop3 are not stably inherited in the progeny, when certain plasmid DNA regions have been deleted. We have localized two Clo DF13 DNA regions involved in stable maintenance through accurate partitioning (par) namely parA, located between 71% and 72% and parB, located between 45% and 50% on the Clo DF13 genome. The instability of these cop plasmids which is accompanied by the formation of high amounts of multimeric DNA molecules, could be abolished by the insertion of transposon Tn901 into the plasmid genome. In particular that part of Tn901, that encodes for the site-specific recombination/ resolution system, appeared to be essential for stabilizing plasmid molecules. Wild-type parA^- and/or parB^- Clo DF13 plasmids, in contrast to cop mutants lacking these regions, are stably maintained during subsequent cell division, indicating that other (host specified) functions contribute to plasmid stability. Analysis of the role of host recombination systems in plasmid partitioning revealed that the recA function has no influence and recBC contributes only weakly to plasmid stability. With respect to the recE pathway, however, we found that in a recE proficient host all plasmids, even those lacking parA and/or parB, are stably maintained, indicating that the function of parA and parB can be replaced not only by the site-specific resolution functions of transposon Tn901, but also by the recE system. The possible role of plasmid specified and host specified functions in plasmid partitioning will be discussed.     

Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene

Summary The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.     

The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility

The incompatibitity that pSC101-derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmid's replication origin. We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superheliclty, can function as a cis-acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy. A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101-specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower-copy-number plasmid. We propose a model by which the par locus can enchance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.     

Cloning of hok gene into anhydrotetracycline inducible pASK75 vector reveals potent antimicrobial effect of 19 amino acid long N-terminal fragment of hok peptide

An important toxin-antitoxin (TA) system hok/sok, encoded by R1 plasmid of Escherichia coli, is involved in the post segregation killing of cells that have lost the plasmid. The lethal properties of hok protein have been utilized for the environmental containment of microbes and the development of potential vaccine candidates. This study aimed to demonstrate the potent anti-microbial property of a 19 amino acid (AA) long N-terminal fragment of hok peptide. This was accomplished by designing a conditional suicide system based on hok gene expression cloned in an anhydrotetracycline (aTc) inducible vector – pASK75. Heat shock and electroporation were utilized for the transformation of Escherichia coli and Vibrio cholerae cells, respectively. The minimal induction concentration (MI_dC) of aTc, determined by analyzing the expression of green fluorescent protein cloned separately into pASK75 vector, was 30 ng/mL. As hok gene was synthesized de novo (using recombinant polymerase chain reaction) in our study, various random sized hok fragments were generated (as a result of the error-prone nature of Taq polymerase). The smallest hok fragment able to bring about effective antimicrobial killing was a 19 AA long N-terminal fragment of hok having the wild type sequence, except for the carboxy terminus AA residue. The MI_dC of aTc in our experiments was 6-fold lower than previously reported, making our bacterial clones suitable for use in mammalian systems as potential vaccine candidates. Based on our experiments, we hypothesize the 19 AA long N-terminal fragment of hok peptide to be the smallest possible hok fragment sufficient to bring about effective antimicrobial killing.     

Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli

Summary The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322. Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-d-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A. oxidans, is encoded on pAO1. Immunoprecipitation of ³⁵S-methionine-labelled E. coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E. coli maxicells revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A. oxidans. The 6-HDNO activity was constitutively expressed in E. coli cells, possibly from an A. oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192.     

A new efficient expression system for Bacillus and its application to production of recombinant phytase

A new expression system was developed for Bacillus subtilis.This system uses a shuttle vector (B. subtilis– Eschericia coli) carrying a phosphate starvation-inducible promoter (pst) and on a fed-batch cultivation strategy. The pst-promoter proved to be very strong and retain its tight regulation also when present on a multi-copy plasmid. The expression system developed showed promising results when applied to the production of recombinant Bacillusphytase – phytase activity at the end of cultivation reached 28.7 U ml^–1.     

High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 μM; for Staphylococcus aureus 2.31 μM, and for Enterococcus faecalis 5.54 μM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of β-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.     

A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production

A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neo ^r gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 10² to 5 × 10³ copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neo ^r gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.     

Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P_vgb of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P_vgb was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P_vgb provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P_vgb, while a construct containing P_vgb, the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P_vgb, vgb operon) or pKVpPV (P_vgb, vgb operon, par) slightly attenuated (30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P_vgb, egfp) or pKVpP-E (P_vgb, par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P_vgb, vgb operon, par, tod) or pKVpP-T (P_vgb, par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.     

Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.     

EXPRESSION OF THE DETOXIFYING GENE B1 IN ES‐CHERICHIA COLI AND SYNECHOCOCCUS1

Abstract We inserted the mosquito esterase B1 gene into the expression vector pRL‐439, which possesses the strong promoter PpsbA. The recombinant plasmid pRL‐Bl was used to transform E. coli HBlOl, and the positive clones were screened on LB medium plate containing 100 mg/mL ampicillin. The results of dot blotting and Southern hybridization demonstrated that these positive clones were transformed bacteria. Western blotting indicated that esterase B1 gene had been successfully expressed under the control of the PpsbA promoter in E. coli. A shuttle verruction‐B1 (pDGBl) was constructed by inserting B1‐cDNA from pRL‐Bl into polycloning site of plasmid pDc8. PDGBl was transferred into Synechoccus sp. PCC7942 through triparental conjugal transfer. Transformed single Synechococcus sp. PCC 7942 clone was obtained by neomycin screening, and large‐scale culture in liquid medium was carried out. Results of Southern blotting proved that pWB1 was transferred into Synechococcus sp. PCC 7942.     

Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4

The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-llke transposable elements suggesting an evolutionary retationship.     

Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus X vector

The human granulocyte macrophage colony-stimulating factor (GM-CSF) is a glycoprotein with important clinical applications for the treatment of neutropenia and aplastic anemia and reducing infections associated with bone marrow transplants. We evaluated the potential for using a potato virus X (PVX) viral vector system for efficient expression of the biologically functional GM-CSF protein in Nicotiana benthamiana leaves. The GM-CSF gene was cloned into PVX viral expression vector, driven with the CaMV 35S promoter. Gene transfer was accomplished by inoculating N. benthamiana leaves with the plasmid DNA of PVX vector containing the GM-CSF gene. The expression level of the recombinant GM-CSF protein was determined with ELISA and its size was confirmed by Western blot analysis. The results showed that: (1) leaf age significantly affects GM-CSF protein concentration with younger leaves accumulating 19.8 mg g⁻¹ soluble protein which is 2.6 times the concentration in older leaves, (2) recombinant protein accumulation within a given leaf declined slightly over time but was not significantly different between 7 and 11 days post-inoculation (dpi), and (3) the two leaves immediately above the inoculated leaves play an important role for GM-CSF accumulation in the younger leaves. Protein extracts of infected N. benthamiana leaves contained recombinant human GM-CSF protein in concentrations of up to 2% of total soluble protein, but only when the pair of leaves immediately above the inoculated leaves remained intact. The recombinant protein actively stimulated the growth of human TF-1 cells suggesting that the recombinant human GM-CSF expressed via PVX viral vector was biologically active.     

Hyperproduction of aspartase by a catabolite repression-resistant mutant of Escherichia coli B harboring multicopy aspA and par recombinant plasmids

Recombinant plasmids bearing the Escherichia coli K-12 aspartase gene (aspA) and the plasmid partition locus (par) were introduced into a catabolite repression-resistant strain of E. coli B, AT202, constructed by mutational and transductional methods. Plasmid pNK101(pBR322-aspA-par) was stably maintained in cells of AT202 even after 30 cell generations, while pYT471(pBR322-aspA), which bore no par locus, was lost at high frequencies from the host cells. Strain AT202 harboring pNK101 produced 3-fold and 80-fold more aspartase than the wild-type E. coli B harboring pNK101 and the wild-type E. coli B strain, respectively. The maximum amount of aspA product (aspartase) was 40–45% of the total cellular protein.     

Stable maintenance in chemostat-grownEscherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene

Plasmid stability was studied in antibiotic-free chemo-stat cultures . Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoRl/EcoRV region of the cloning vector pBR322 or in the HindIII]BamHl region of pACYCI84 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.Issued as NRCC publication No. 23992.     

Overexpression,purification and characterization of a recombinant secretary catalase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

A recombinant Bacillus subtilis strain (KN25) was generated for the large-scale preparation of catalase. The B. subtilis katA gene encoding for catalase was cloned into the shuttle vector PRB374, downstream of the constitutively active vegII promoter, followed by transformation of the B. subtilis strain WB600 with the plasmid. The transformant strain, KN25 secretes high levels (3,500 U/ml) of catalase, which facilitates its purification. Three simple purification steps yielded nearly homogeneous catalase, with ∼70% recovery. The purified recombinant catalase has a specific activity of 34,600 U/mg under optimal conditions, and is more resistant to acidic conditions than bovine liver catalase.     

腺病毒载体介导PDX-1在骨髓间充质干细胞中的表达

为研究PDX-1基因在骨髓间充质干细胞中的表达情况及生物学功能的发挥,构建了含PDX-1基因的重组腺病毒载体. 酶切PDX-1基因并连入穿梭质粒pAdTrack-CMV.用电穿孔法使穿梭质粒pAdTrack-CMV-PDX-1与病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组.利用脂质体介导重组腺病毒载体转染293细胞,包装出完整的腺病毒.分离、培养、扩增骨髓间充质干细胞.用重组腺病毒感染间充质干细胞.用荧光显微镜、RT-PCR、免疫荧光染色等方法检测PDX-1、胰岛素基因及蛋白质的表达,用放射免疫分析法检测转基因细胞分泌胰岛素情况.结果表明:通过测序、PCR、酶切等鉴定PDX-1基因已正确插入穿梭质粒中,并与病毒骨架质粒重组.重组腺病毒滴度为6.3×10⁷ PFU/ml.通过荧光显微镜观察证实重组腺病毒可高效感染骨髓间充质干细胞,经RT-PCR、免疫荧光染色证实转染pAd-PDX-1后培养7天的细胞中有PDX-1及胰岛素基因的表达.这些转基因的细胞向胞外分泌的胰岛素量为(15.21±3.50) mIU/L.