A biochemical analysis of the interaction of DNA gyrase with the bacteriophage Mu, pSC101 and pBR322 strong gyrase sites: the role of DNA sequence in modulating gyrase supercoiling and biological activity

Replication of bacteriophage Mu DNA, a process requiring efficient synapsis of the prophage ends, takes place within the confines of the Escherichia coli nucleoid. Critical to ensuring rapid synapsis is the function of the SGS, a strong gyrase site, located at the centre of the Mu genome. Replacement of the SGS by the strong gyrase sites from pSC101 or pBR322 fails to support efficient prophage replication. To probe the unique SGS properties we undertook a biochemical analysis of the interaction of DNA gyrase with the Mu SGS, pSC101 and pBR322 sites. In binding and cleavage assays the order of efficacy was pSC101 > Mu SGS > pBR322. However, in supercoiling assays the Mu SGS (cloned into pUC19) exhibited a strong enhancement of gyrase-catalysed supercoiling over pUC19 alone; the pSC101 site showed none and the pBR322 site gave a moderate improvement. Most striking was the Mu SGS-dependent increase in processivity of the gyrase reaction. This highly processive supercoiling coupled with efficient binding may account for the unique biological properties of the SGS. The results emphasize the importance of the DNA substrate as an active component in modulating the gyrase supercoiling reaction, and in determining the biological roles of specialized gyrase sites.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium

The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5α and E. ColiJM109, respectively. Batch fermentation of E. coliDH5α-PUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize CoIE1-origin plasmid replication.     

Stable inheritance of shuttle vectors based on plasmid pIM13 in a mutant strain of Clostridium acetobutylicum.

New shuttle vectors for Clostridium acetobutylicum were constructed, using as replicons the Gram-positive plasmid pIM13, and derivatives of the Gram-negative plasmid pBR322, including pUC19. These vectors transformed C. acetobutylicum at a high frequency (up to 10(6) transformants per microgram DNA) by PEG-mediated protoplast transformation. A mutant host strain, NI-4082, was isolated on the basis of its ability to maintain plasmid pIM13 stably in the absence of selection pressure. The shuttle vectors showed no segregational or structural instability in this mutant strain. Moreover, the results suggested a relationship between segregational instability and the multimerization of pIM13 in C. acetobutylicum. The host/vector system described possessed all the properties required for efficient gene cloning in this species.     

The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing

A series of nic- cloning vectors have been constructed analogous to the pUC plasmids but which are smaller in size and carry more extensive polylinker regions within the lacZ' gene. The vectors pMTL20 and pMTL21 carry six additional sites (AatII, MluI, NcoI, BglII, XhoI and StuI) to those present in pUC18 and pUC19, while pMTL22 and -23 possess eleven new cloning sites (ActII, MluI, NcoI, BglII, XhoI, StuI, NaeI, EcoRV, ClaI, NdeI and NruI). More importantly, the relative order of the restriction sites within the polylinker of these latter vectors has been totally rearranged, relative to pUC18 and pUC19, to facilitate the conversion of DNA fragments with incompatible ends to fragments with compatible termini. The availability of such DNA fragments is a crucial requirement when M13 templates are generated for dideoxy sequencing by the sonication procedure. Derivatives of these vectors have also been constructed which demonstrate improved segregational stability by incorporation of the pSC101 par locus. During the construction of these new vectors data were obtained which demonstrated that the pUC and pMTL plasmids contain a previously unreported single base pair difference within the RNA I/RNA II region (compared to pBR322) responsible for a three-fold increase in plasmid copy number. The pUC and pMTL plasmids were also shown to be functionally nic-, thus affording the lowest categorisation in genetic manipulation experiments.     

Dissection of the bacteriophage Mu strong gyrase site (SGS): significance of the SGS right arm in Mu biology and DNA gyrase mechanism

The bacteriophage Mu strong gyrase site (SGS), required for efficient phage DNA replication, differs from other gyrase sites in the efficiency of gyrase binding coupled with a highly processive supercoiling activity. Genetic studies have implicated the right arm of the SGS as a key structural feature for promoting rapid Mu replication. Here, we show that deletion of the distal portion of the right arm abolishes efficient binding, cleavage, and supercoiling by DNA gyrase in vitro. DNase I footprinting analysis of the intact SGS revealed an adenylyl imidodiphosphate-dependent change in protection in the right arm, indicating that this arm likely forms the T segment that is passed through the cleaved G segment during the supercoiling reaction. Furthermore, in an SGS derivative with an altered right-arm sequence, the left arm showed these changes, suggesting that the selection of a T segment by gyrase is determined primarily by the sequences of the arms. Analysis of the sequences of the SGS and other gyrase sites suggests that the choice of T segment correlates with which arm possesses the more extensive set of phased anisotropic bending signals, with the Mu right arm possessing an unusually extended set of such signals. The implications of these observations for the structure of the gyrase-DNA complex and for the biological function of the Mu SGS are discussed.     

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

In this study, we have demonstrated that the type and feeding regimen of amino acids have a significant impact on the quality as well as the quantity of DNA vectors produced. Nutrient pool and factorial design experiments were carried out in order to identify the amino acids involved in increased biomass and induction of plasmid amplification. Leucine, glycine, and histidine were responsible for increased biomass and leucine starvation in the presence of histidine was implicated in plasmid amplification. Supercoiling of the plasmid was optimized using a dual feeding strategy. As a result of this, a fed-batch fermentation strategy for the production of a 6.9 kb plasmid, pSVß, in Escherichia coli DH5α was developed. In batch fermentation, a maximum plasmid yield of 39.4 mg/L equivalent to 11.3 mg/g dry cell weight (DCW) was achieved with casein hydrolysate limitation. About 90% of plasmid was in the supercoiled (SC) form after 31 hr of fermentation but only remained so for a short period, leading to a very brief window for harvesting cells at scale. Subsequently, a fed-batch fermentation using a dual feeding strategy was employed. A mean maximum plasmid yield of 44 mg/L equivalent to 9.1 mg plasmid/g DCW was achieved. After 25 hr, 90% of plasmid was in the SC form and remained at this level for the remaining 10 hr of the fermentation, allowing adequate time for the harvesting of cells without the loss of supercoiling of product. This study emphasized that optimizing fermentation strategy and identifying the essential nutrients are beneficial for bioprocessing of plasmid DNA for therapeutic applications.     

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.     

Effect of high density cultivation on plasmid copy number in recombinantEscherichia coli cells

Summary The copy number of plasmid harboringE. coli K 12 strains was examined in fed batch cultivations in semisynthetic and synthetic media. Under conditions of high cell density (45–50 g dry weight/I) the plasmid copy number reached a maximum level between 200 and 400 copies per cell. A decrease of phosphate concentration in the medium was obtained similar to the increase of copy number. A high segregational and structural stability of vectors used in this work was observed.     

A protein factor which reduces the negative supercoiling requirement in the Mu DNA strand transfer reaction is Escherichia coli integration host factor

We have examined the supercoiling requirement for the in vitro Mu DNA strand transfer reaction and found that optimal efficiency requires a high level (sigma = -0.06) of donor plasmid superhelicity. At in vivo levels of supercoiling (sigma = -0.025) the reaction does not occur. Using an unreactive donor plasmid with a near physiological level of supercoiling, we identified an Escherichia coli protein factor which has the novel property of reducing the donor plasmid supercoiling requirement for the in vitro Mu DNA strand transfer reaction by 40%. This protein, which we named supercoiling relief factor was purified to near homogeneity and found to be identical to integration host factor (IHF), a protein known to induce site specific bends in DNA. The dramatic reduction in the supercoiling requirement was promoted by about 1.5 IHF dimers/donor substrate molecule. At these low levels of IHF, the HU requirement for the reaction was also reduced; a synergistic effect of the two proteins resulted in a greater than 10-fold stimulation of the reaction under appropriate conditions. Furthermore, at high concentrations of IHF, HU could be completely eliminated from the reaction.     

Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.     

Plasmid maintenance in Bacillus stearothermophilus is strain-dependent

We studied the segregational stability of plasmids based on pTB913, a 4.5-kb rolling-circle plasmid derived from the thermophilic Bacillus plasmid pTB19. In Bacillus stearothermophilus the stability of pTB913 derivatives appeared to be strain-dependent. In strain CU21 large amounts of single-stranded pTB913 DNA were found and the plasmid was highly unstable at 57 degrees C. In strain NUB3621, however, very low amounts of single-stranded plasmid DNA were formed and pTB913-based replicons were only slightly unstable at 57 degrees C. The NUB3621/pTB913 host-vector system seems appropriate for molecular cloning. A RepA-based replicon, also derived from pTB19 but replicating by a theta mechanism, was highly unstable in B. stearothermophilus NUB3621.     

Transfecting pDNA to E. coli DH5α using bovine serum albumin nanoparticles as a delivery vehicle

We describe the formulation of bovine serum albumin nanoparticles (BSA‐NPs) by the coacervation method using surfactants. Plasmids (pUC18, pUC18egfp and pBBR1MCS‐2) isolated from E. coli were incorporated into the BSA matrix by incubating in albumin solution prior to formulation of NPs. Plasmid incorporation was calculated by % yield, entrapment efficiency, DNA loading capacity and release of entrapped DNA by comparing with blank NPs. BSA‐DNA binding studies were carried out by using fluorescence spectroscopy and Fourier Transform Infra Red Spectroscopy (FT‐IR). The surface charge distribution of the NPs loaded with plasmid was calculated using zeta potential. The photoluminescence of BSA‐NPs was quenched when loaded with pDNA, confirming the interaction of DNA with BSA. Altogether, these results provide evidences for the excellent DNA carrying efficiency of BSA‐NPs without loss of plasmid's integrity. The NPs were used to transfect E. coli DH5α strain lacking ampicillin resistance. They, however, showed ampicillin resistance subsequent to transfection with plasmid encoding ampicillin resistance gene. Effect of transfection was confirmed by confocal microscopy and by the isolation of the plasmid by agarose gel electrophoresis from the transfected bacterial culture. This study clearly demonstrates the efficacy of BSA‐NPs as delivery vehicle for pDNA transfection. Copyright © 2014 John Wiley & Sons, Ltd.     

New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites

We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.     

Behaviour of bacteriophage Mu-based IncP suicide vector plasmids in Rhizobium spp.

Abstract The ability of P-type plasmids, containing bacteriophage Mu, to survive in Rhizobia was examined under conditions in which the plasmids were not directly selected. The plasmid pRP4::Mu::Tn7 survived in all tested strains of Rhizobia and Agrobacteria . The surviving plasmids were analysed for expression of plasmid coded properties and for presence of both bacteriophage Mu and Tn7 DNA. A large variation in plasmid size and coding capacity was observed. In the parent plasmid Tn7 is inserted into the Mu DNA. The majority of surviving plasmids had lost Mu DNA but the Tn7 DNA was retained. Analysis of surviving plasmids which contained Mu DNA indicated that a small deletion of Mu is sufficient to allow plasmid survival. Deleted derivatives of pJB4J1 were also isolated in Rhizobium under similar conditions. The potential of these suicide vectors as potential vectors for transposon mutagenesis in Rhizobium is discussed.     

The insertion of large pieces of foreign genetic material reduces the stability of bacterial plasmids

The stability of genetically engineered bacterial plasmids under continuous culture fermentation is of crucial importance in the application of microbiology to many processes of potential industrial importance. In order to determine the effect of inserting large pieces of foreign DNA on the stability of bacterial plasmids we have studied the behavior of pAT153 with DNA inserts of various sizes derived from cytomegalovirus. Foreign DNA up to 2 kb in length had no effect on stability, whereas the insertion of an 8-kb fragment resulted in a transient instability. This instability was overcome by the spontaneous appearance of leu+ cells in the culture. Insertion of a 21-kb DNA fragment resulted in a rapid loss of plasmid, which was not prevented by the appearance of leu+ cells. In all cases copy number analyses indicated that plasmid loss was due to segregational instability, probably because the plasmid placed an unacceptable metabolic load on the cell.     

Effect of dilution rate on the stability of TOL plasmid pTK0 in Pseudomonas putida PPK1

Continuous culture fermentation of Pseudomonas putida PPK1, containing the naturally occurring TOL plasmid pTK0, was carried out in either benzoate- or succinate-limited chemostats. The apparent stability of the plasmid decreased with increasing the dilution rate. An established model was used to predict the contribution of segregational instability and growth rate difference to the apparent plasmid stability.     

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

Background  

Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed.     

Plasmid DNA fermentation strategies: influence on plasmid stability and cell physiology

In order to provide sufficient pharmaceutical-grade plasmid DNA material, it is essential to gain a comprehensive knowledge of the bioprocesses involved; so, the development of protocols and techniques that allow a fast monitoring of process performance is a valuable tool for bioprocess design. Regarding plasmid DNA production, the metabolic stress of the host strain as well as plasmid stability have been identified as two of the key parameters that greatly influence plasmid DNA yields. The present work describes the impact of batch and fed-batch fermentations using different C/N ratios and different feeding profiles on cell physiology and plasmid stability, investigating the potential of these two monitoring techniques as valuable tools for bioprocess development and design. The results obtained in batch fermentations showed that plasmid copy number values suffered a pronounced increase at the end of almost all fermentation conditions tested. Regarding fed-batch fermentations, the strategies with exponential feeding profiles, in contrast with those with constant feeding, showed higher biomass and plasmid yields, the maximum values obtained for these two parameters being 95.64 OD₆₀₀ and 344.3 mg plasmid DNA (pDNA)/L, respectively, when using an exponential feed rate of 0.2 h⁻¹. Despite the results obtained, cell physiology and plasmid stability monitoring revealed that, although higher pDNA overall yields were obtained, this fermentation exhibited lower plasmid stability and percentage of viable cells. In conclusion, this study allowed clarifying the bioprocess performance based on cell physiology and plasmid stability assessment, allowing improvement of the overall process and not only plasmid DNA yield and cell growth.