The role of outer membrane proteins of E. coli K12 in the cell wall permeability for plasmid DNA

The Escherichia coli K12 mutant having the increased efficiency for plasmid DNA transformation has been shown to possess the different protein composition of the outer membrane of the cellular wall, as compared with that of the wild type strain. Correlation between the level of calcium-dependent plasmid transformation and the portion of infections DNA bound with cytoplasmic membranes is demonstrated for the Escherichia coli cells mutant for outer membrane structure and ability to be transformed by plasmid DNA.     

Inducible cell lysis system for the study of natural transformation and environmental fate of DNA released by cell death.

Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKL02 consists of lysis genes S, R, and Rz from bacteriophage lambda under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKL02 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. calcoaceticus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. calcoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage phi X174 and causes lysis of E. coli and P. stutzeri bacteria by activating cellular autolysins. Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKL01-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made.     

Plasmid DNA binds to the core oligosaccharide domain of LPS molecules of E. coli cell surface in the CaCl2-mediated transformation process

In the standard procedure for artificial transformation of E. coli by plasmid DNA, cellular competence for DNA uptake is developed by suspending the cells in ice-cold CaCl2 (50-100 mM). It is believed that CaCl2 helps DNA adsorption to the lipopolysaccharide (LPS) molecules on E. coli cell surface; however, the binding mechanism is mostly obscure. In this report, we present our findings of an in-depth study on in vitro interaction between plasmid DNA and E. coli LPS, using different techniques like absorption and circular dichroism spectroscopy, isothermal titration calorimetry, electron and atomic force microscopy, and so on. The results suggest that the Ca(II) ions, forming coordination complexes with the phosphates of DNA and LPS, facilitate the binding between them. The binding interaction appears to be cooperative, reversible, exothermic, and enthalpy-driven in nature. Binding of LPS causes a partial transition of DNA from B- to A-form. Finer study with the hydrolyzed products of LPS shows that only the core oligosaccharide domain of LPS is responsible for the interaction with DNA. Moreover, the biological significance of this interaction becomes evident from the observation that E. coli cells, from which the LPS have been leached out considerably, show higher efficiency of transformation, when transformed with plasmid-LPS complex rather than plasmid DNA alone.     

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.     

Optimization of electrotransformation conditions for Propionibacterium acnes

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.     

Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells.

The effect of various metabolic inhibitors (carbonylcyanid-m-chlorophenylhydrazone, nigericin, valinomycin, dicyclocarbodiimide, arsenate, NaF, etc.) and lipid-soluble synthetic ions (tetraphenylphosphonium bromide and tetraphenylboron sodium) on deoxyribonucleic acid (DNA) entry during transformation of Ca2+-treated Escherichia coli cells with plasmid DNA and on cell viability was investigated. In contrast to intact cells, Ca2+-treated E. coli cells were permeable to nigericin, valinomycin, and the other drugs tested. The inhibitors differentially affected [14C]proline active transport, and whereas some drugs inhibited transformation, the effects did not correlate with the effects on transport. The most potent inhibitors of transformation were nigericin, dicyclocarbodiimide, and tetraphenylboron sodium. Carbonylcyanid-m-chlorophenylhydrazone, tetraphenylphosphonium bromide, and valinomycin were relatively inactive. Tetraphenylboron sodium- and nigericin-treated cells bound were plasmid [14C]DNA in the deoxyribonuclease-resistant form than the control and other sample cells. Nevertheless, te penetration of exogenous plasmid DNA into the cell was greatly reduced, at least in case of nigericin. Unlike the other drugs, nigericin and dicyclocarbodiimide drastically affected the cell viability, the former within very short times of interaction. It is concluded that proton motive force does not play any significant role in DNA entry into Ca2+-treated E. coli cells. The results also suggest that adenosine 5'-triphosphate is not required for DNA entry either. The inhibitory effect of certain drugs is discussed in terms of structural perturbations induced by the drugs in cell envelope membranes.     

Probing of DNA polymorphic structure in the cell with osmium tetroxide

It is shown that osmium tetroxide, 2,2'-bipyridine can be applied as a probe of DNA structure in a bacterial cell. Using this probe we demonstrate (a) presence of structural distortions at the junctions between the right-handed B and left-handed Z DNA in a recombinant plasmid pRW751 and (b) unusual structure of the d(A-T)16 insert in pAT32 plasmid in E. coli cells and in in vitro.     

Transformation of Escherichia coli in foodstuffs.

The plasmid transfer by transformation of Escherichia coli in 12 foods was investigated under conditions commonly found in processing and storage of food. Transformation occurred in all foods with frequencies of at least 10(-8) when a simplified standard transformation protocol with non-growing cells was applied. Higher rates (ca. 10(-7)) were found in milk, soy drink, tomato and orange juice. Furthermore, E. coli became transformed at temperatures below 5 degrees C, i.e. under conditions highly relevant in storage of perishable foods. In soy drink this condition resulted in frequencies which were even higher than those determined after application of a temperature shift to 37 degrees C. The transformation of cells growing in milk and carrot juice at a constantly kept temperature of 37 degrees C provides evidence for the potential of E. coli to become transformed naturally. With purified DNA frequencies were determined in these substrates of ca. 2.5 x 10(-7) and 2.5 x 10(-8), respectively. Similar frequencies were also obtained in milk containing the crude nucleic acids of homogenised cell suspensions of E. coli (pUC18). Moreover, the release of plasmid DNA from E. coli during food processing and the subsequent uptake of this DNA by growing E. coli cells was shown to take place after homogenisation in milk indicating a horizontal plasmid transfer by transformation of E. coli.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism

AIMS: The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries. METHODS AND RESULTS: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population. CONCLUSIONS: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation. SIGNIFICANCE AND IMPACT OF THE STUDY: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.     

Effect of the molecular weight of DNA on the effectiveness of plasmid transformation of E. coli K12

The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA. Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.     

The effect of the oligomerization of pBR322 on the level of transformation in Escherichia coli

The ability of the known Escherichia coli strain JC3881 recB recC recF sbc15 to produce oligomeric and multimeric forms of pBR322 underlies the study presented. The individual oligomeric forms of pBR322 were isolated from the agarose gel. The plasmid forms were used for electron microscopic control and also introduced into the system of E. coli competent cells. The E. coli transformation level of different forms of plasmid DNA rose from monomers to pentamers. CCC forms of the plasmid possessed high efficiency of the E. coli cell transformation. The systems of the host recombination are to be significant in the process of plasmid oligomerization.     

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.     

少量制备大肠杆菌感受态细胞条件探索

目的:为了获得重复性好、转化率高的少量制备感受态细胞的方法,利用不同生长时期的大肠杆菌感受态细胞,进行转化比较。方法:根据普通实验室的实验条件,常规方法提取质粒,氯化钙法转化不同生长时期的大肠杆菌感受态细胞,比较转化率。结果:大肠杆菌感受态细胞的转化率与OD值显著相关,在OD600nm为0.39和0.55时转化率最高,在OD600nm为0.28~0.55之间均可得到理想的转化效果。结论:少量制备感受态细胞方法操作中无需添加任何保护试剂和细胞复苏培养,操作简便、重复性好,实验成本低廉。     

Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity

Genetic transformation of intact cells of Saccharomyces cerevisiae, achieved by incubating the cells with plasmid DNA in the presence of PEG, could be enhanced, not only by pretreatment of the cells with Li+ and 2-mercaptoethanol, as has been reported previously, but also by pretreatment with proteolytic enzymes. The efficiency of transformation with 2-mercaptoethanol rose dramatically when the pretreated cells had been handled in osmotically stabilized media. Following all the pretreatments the cells became leaky for nucleic acids as detected by the presence of endogenous RNAs in the medium. The pretreatments evidently facilitated the passage of transforming DNA across the cell wall.     

Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA

The F plasmid of Escherichia coli was used to study the genetic background of the control circuit in the bacteria that co-ordinates DNA replication and cell division of the host cells. When DNA replication of the F plasmid was blocked by growing cells carrying an amber-suppressible replication-defective F plasmid mutant under restrictive conditions, the cells continued to divide for about one generation until F plasmid was supposedly diluted to one copy per cell, and then they stopped dividing and formed non-septated filamentous cells. These observations suggest that completion of a round of replication is a necessary and sufficient condition of F DNA synthesis in the cell division of F+ bacteria; i.e. cell division of the F+ bacteria is coupled with DNA replication of the F plasmid. The observation that Giemsa-stainable materials in the filamentous cells were clustered in the center indicates that partitioning of chromosomal DNA (and presumably of F plasmid DNA) is also coupled with plasmid DNA replication. The function necessary for this coupling is carried by the 42.84-43.6 F (BamHI-PstI) segment, which is located outside the region essential for replication of the F plasmid. The nucleotide sequence demonstrates the existence of two open reading frames in this region, which encode polypeptides of 72 and 101 amino acids, respectively. These two reading frames are most likely to be transcribed as a single polycistronic message in the direction from the BamHI site at 42.84 F to the PstI site at 43.6 F. The expression of this "operon" is likely to be controlled by plasmid DNA replication.     

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results that have been reported for human cells, UV irradiation of transfecting DNA did not stimulate the genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with the UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. However, transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. We conclude that the responses of recipient cells to UV-damaged transfecting plasmids depend both on the type of recipient cell and the characteristics of the genetic sequence used for transfection.     

Natural plasmid transformation in<Emphasis Type="Italic">Escherichia coli</Emphasis>

Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli.     

Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions.

Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations. During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells. Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant. Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid. The results suggest that transport of DNA across the inner membrane is a limiting step in transformation. After transformation the bulk of labeled plasmid DNA remained associated with outer membranes. However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present. Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+. This parallels their relative efficiencies in promoting transformation. Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation.