A determination of the efficiency of uptake of SP50 DNA by competent cells of B. subtilis

Summary Phage SP50 excludes phage SPP1 both in infection and in transfection of B. subtilis. The dependence of the efficiency of exclusion on the concentration of SP50 DNA shows that one SP50 DNA molecule within a competent cell is sufficient to exclude SPP1 phage development. The concentration dependence allows a determination of the efficiency of uptake of SP50 DNA by competent cells. Only 1 out of 200 SP50 DNA molecules in the transfection mixture will become biologically active in excluding SPP1 phage development in the competent cell.     

Effect of Cycloheximide on Development of Mitochondria in Germinating Pea Cotyledons

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 µg protamine sulfate/ml, 0.15 m sucrose, and 10 m m MgSO₄ in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 × 10⁵ PFU/µg DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.     

Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae by HPlcl phage, or transfection by DNA from the phage, were examined. Physical separation of a large fraction of the intracellular phage DNA from the bulk of the host DNA was achieved by lysis of infected or transfected cells with digitonin, followed by low-speed centrifugation. The small amount of bacterial DNA remaining with the phage DNA in the supernatants could be distinguished from phage DNA by its ability to yield transformants. After infection by whole phage, three forms of intracellular phage DNA were observable by sedimentation velocity analysis: form III, the slowest-sedimenting one; form II, which sedimented 1.1 times faster than III, and form I, which sedimented 1.6 times faster than III. It was shown by electron microscopy, velocity sedimentation in alkali, and equilibrium sedimentation with ethidium bromide, that forms I, II and III are twisted circles, open circles, and linear duplexes, respectively.After the entry of phage DNA into wild-type cells in transfection, the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. Some of the fast-sedimenting molecules are presumably concatemers and are generated by recombination. In strain rec₁^? the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in wild-type cells. In strain rec₂^? there is little degradation of phage DNA, and the proportion of fast-sedimenting molecules is much smaller than in wild-type cells. Since rec₁^? and rec₂^? are transfected with much lower efficiency than wild type, our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for more efficient transfection.     

High-frequency Protoplast-transfection of Streptomyces parvulus 2297 with Actinophage R4 DNA

High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16~24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3 × 10^?5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.     

Calcium Requirement for Protoplast Transfection Mediated by Polyethylene Glycol of Lactobacillus casei by PL-1 Phage DNA

The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca²⁺ , Sr²⁺ increased the transfection rate as well, but Ba²⁺, Mn²⁺, and Mg²⁺ did not. Co²⁺ and Zn²⁺ inhibited transfection. The simultaneous use of Ca²⁺ and Mg²⁺ increased the transfection efficiency. Impairment of transfection caused by lack of Ca²⁺ could not be reversed by the addition of Ca²⁺ later. A decrease in the Ca²⁺ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.     

Photosynthesis and phage: early studies on phosphorus metabolism in photosynthetic microorganisms with 32P,and how they led to the serendipic discovery of 32P-decay suicide of bacteriophage

During my PhD thesis research (1946–1949), I explored the effects of light on the uptake of ³²P-labeled inorganic phosphate (P_i) by cells of photosynthetic bacteria and microalgae, and the dynamics of P turnover between low and high molecular weight cell constituents. The results were interpreted as evidence for the conversion of light energy to the chemical energy of phosphorylated compounds. The experimental results also suggested to me that the precursors of the P in DNA bacteriophages of Escherichia coli must be low molecular weight phosphorylated compounds present within the host cells and led to the design of an experiment to determine the conservation of ³²P of an infecting phage particle in its numerous progeny. The experiment envisaged was never conducted because phage labeled with ³²P of high specific activity showed unexpected loss of viability. Thus, by serendipity, ‘suicide’ of phage due to ³²P-β decay was discovered. ³²P-decay ‘suicide’ provided a technique that was useful for analysis of phage genetic structure and replication. This memoir describes the unusual circumstances leading to the decisive role of serendipity in revealing an extraordinary phenomenon. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transfection of Escherichia coli by Mu DNA

Summary Infectivity of Mu DNA was demonstrated in Ca⁺⁺-treated Escherichia coli cells that lacked the nucleases Exo V and Endo I. The efficiency of transfection is about 10^-7 per phage equivalent. Infectivity is destroyed by denaturation of Mu DNA, and cannot be restored by renaturation.     

Studies on transducing phage M-1 for Rhizobium japonicum D211

Phage M-1 produced clear plaques with a halo in the lawn of Rhizobium japonicum D211. A one step growth curve of phage M-1 showed a latent period of 3 h, burst size of 55 and rise period of 2 h. The inactivation of phage M-1 was found to be dependent upon the concentraion of d-glucosomanine. The neutralization kinetics of phage M-1 by antiphage serum gave a K value (velocity constant) of 83.1 min^–1. Transduction of str and kan was studied in the presence of antiphage serum and d-glucosamine. Cotransduction of different antibiotic resistance markers suggested that the system can be further explored for high resolution mapping in R. japonicum.Abbreviations YM yeast mannitol medium - PFU plaque forming unit - moi multiplicity of infection - EOP efficiency of plating     

Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity.

Summary The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10³-to 10⁴-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage . Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of Ral^–, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.     

Inactivation of transfecting DNA by physical and chemical agents: influence of genotypes of phage lambda and host cells

Inactivation of λ₁₁c and its purified DNA by UV irradiation, γ-rays of ¹³⁷Cs (in conditions of indirect action), nitrous acid, hydroxylamine and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied. The biological activity of isolated phage DNA was measured by the calcium transfection procedure. 14 different recipient strains of Escherichia coli K12 were used, including mutants deficient in excision and recombination repair (uvrA6, uvrB5, uvrC34, polA1, recA13, recC38, recD34, recA13B21C22, recA56uvrA6, exrA and recB21C22sbcB15).Whole phage was more resistant to the action of γ-rays than was isolated DNA. On the other hand, the chemical agents HNO₂ and MNNG inactivated phage much faster than isolated DNA. Of all mutations of the host cell only polA1 considerably increased the sensitivity of phage DNA to UV irradiation, γ-rays and MNNG. The mutations uvr^? affected the inactivation kinetics under UV action. In all other cases the genotype of the host cell was indifferent for the inactivation kinetics of phage DNA, even if it belonged to recombination deficient mutant λ red3 int6 (in which only UV and γ inactivation was studied). Possible reasons for the low efficiency of the host-cell repair toward the damage caused to λ DNA by different agents are discussed.     

Cell cycle propagation is driven by light–dark stimulation in a cultured symbiotic dinoflagellate isolated from corals

Endosymbiosis is an intriguing plant–animal interaction in the dinoflagellate–Cnidaria association. Throughout the life span of the majority of corals, the dinoflagellate Symbiodinium sp. is a common symbiont residing inside host gastrodermal cells. The mechanism of regulating the cell proliferation of host cells and their intracellular symbionts is critical for a stable endosymbiotic association. In the present study, the cell cycle of a cultured Symbiodinium sp. (clade B) isolated from the hermatypic coral Euphyllia glabrescens was investigated using flow cytometry. The results showed that the external light–dark (L:D) stimulation played a pivotal role in regulating the cell cycle process. The sequential light (40–100 μmol m⁻² s⁻¹ ~ 12 h) followed by dark (0 μmol m⁻² s⁻¹ ~ 12 h) treatment entrained a single cell cycle from the G₁ to the S phase, and then to the G₂/M phase, within 24 h. Blue light (~450 nm) alone mimicked regular white light, while lights of wavelengths in the red and infrared area of the spectrum had little or no effect in entraining the cell cycle. This diel pattern of the cell cycle was consistent with changes in cell motility, morphology, and photosynthetic efficiency (F _v /F _m ). Light treatment drove cells to enter the growing/DNA synthesis stage (i.e., G₁ to S to G₂/M), accompanied by increasing motility and photosynthetic efficiency. Inhibition of photosynthesis by 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea (DCMU) treatment blocked the cell proliferation process. Dark treatment was required for the mitotic division stage, where cells return from G₂/M to G₁. Two different pools of adenylyl cyclase (AC) activities were shown to be involved in the growing/DNA synthesis and mitotic division states, respectively. Communicated by Biology Editor Dr Michael Lesser     

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Summary The binding and uptake of nick-translated ³²P-labeled pBR322 by Anacystis nidulans 6301 have been characterized. Both processes were considerably enhanced in permeaplasts compared to cells. The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells. Uptake of DNA by permeaplasts was unaffected by: Mg²⁺ or Ca²⁺, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH₄Cl. ATP at 2.5–10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A. nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range. In contrast to transformation of A. nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark. The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.     

The formation,vacuolar localization,and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures

The metabolism of salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7–¹⁴C]SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O--D-glucose (SAG), methylsalicylate 2-O--D-glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [¹⁴C]SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A₁ (a specific inhibitor of the vacuolar H⁺-ATPase) and dissipation of the transtonoplast H⁺-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several -glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis–Menten type saturation kinetics with a K_m and V_max value of 11 M and 205 pmol min^–1 mg^–1, respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H⁺-antiport-type mechanism.     

Influence of culture density,pH, organic acids and divalent cations on the removal of nutrients and metals by immobilized Anabaena doliolum and Chlorella vulgaris

The potential of alginate-immobilized Anabaena doliolum and Chlorella vulgaris was assessed for removal of nutrients (NO _inf3 ^sup- and NH _inf4 ^sup+ ) and metals (Cr₂O _inf7 ^sup2- and Ni²⁺) at different biomass concentrations (0.05, 0.1, 0.25, 0.49 and 1.22 g dry wt l^-1) and pH values (4 to 10). Though uptake of all these substances was higher in concentrated algal beads (0.25, 0.49 and 1.22 g dry wt l^-1), their rate of uptake was significantly (P<0.001) lower than that of low (0.05 g dry wt l^-1) cell density beads. For A. doliolum, there was no significant difference in uptake rates for beads having densities of 0.05 and 0.1 g dry wt l^-1. Chlorella vulgaris, however, showed maximum efficiency at 0.1 g dry wt l^-1. Uptake of both the nutrients and the metals was maximal at pH 7 followed by pH 8, 6, 9, 10, 5 and 4. Of the different substances (organic acids and divalent cations) used, humic acid was most efficient in decreasing metal uptake. Mg²⁺ was, however, more efficient than Ca²⁺ in decreasing Ni²⁺ uptake. Immobilized algae with a cell density of 0.1 g dry wt l^-1 were the most efficient for nutrient and metal removal at pH 6 to 8.     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

Transfection of protoplasts from Streptomyces lividans 66 with actinophage SH10 DNA

Summary The slightly modified procedure for the transformation of protoplasts of S. coelicolor A3 (2) with SCP2 plasmid DNA and polyethylenglycol (PEG) (Bibb et al., 1978) was extented to infection of protoplasts of S. lividans 66 with actinophage SH10 DNA (Klaus et al., 1979).Maximal yield of transfected protoplasts was obtained at 20% PEG, 3 mM sodium-citrate and 150 mM NaCl final concentrations. The efficiency of transfection was determined to be about 2×10^-8 to 2×10^-7. The average value of competent protoplasts was about 1–2×10^-4 of regenerating protoplasts.In comparison with outgrowing spores infected with phage particles the average burst size of transfected protoplasts was reduced from 100 to 10 pfu/infected cell, the latent period prolonged from 45 min to 120 min and the rise period was not affected.     

The Kinetics of Polyethylenimine-Mediated Transfection in Suspension Cultures of Chinese Hamster Ovary Cells

The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO) cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture medium at various times after the initiation of transfection inhibited further cellular uptake of PEI–DNA particles. Using this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio of 2:1 (w/w) and a cell density of 2 × 10⁶ cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the rate and the level of PEI–DNA uptake in serum-free minimal medium were found to be dependent on the PEI–DNA ratio, the cell density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian cells for the purpose of large-scale transient recombinant protein production.     

Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.     

An Haemophilus influenzae mutant which inhibits the growth of HP1c1 phage

Summary A strain of Haemophilus influenzae, called hpm ^- inhibits the growth of phage HP1c1 but not S2. This inhibition is overcome by HP1c1ph mutants. Phage HP1c1 adsorbs normally to hpm ^- cells but only a small fraction of infected cells produce phage with a normal burst size or become lysogenic. When hpm ^- strains lysogenic for HP1c1 are induced, 100% of the cells yield phage. There is no degradation of phage DNA after infection of hpm ^- cells and HP1c1 can normally grow when its DNA is introduced into hpm ^- by transfection. The most probable explanation is that in hpm ^- cells the penetration of phage DNA is blocked. The hpm ^- property behaves as as unstable mutation.     

Structure of transfection-active histone H1/DNA complexes

Relationships between the structure of transfecting complexes of histone H1 and DNA and their transfection efficiency were studied. Transfection activity proved to be connected to complex aggregates. Low speed centrifugation of the complexes resulted in loss of the transfection activity. The complexes/aggregates were active with high efficiency in a broad range of weight input ratios r _i (0.1<r _i<30). Using atomic force microscopy (AFM), the complexes were imaged at negative, nearly electroneutral and positive charge conditions. Electroneutral complexes at r _i=1 showed a multitude of different complex forms. Fibrillar, network-like and branched structures were frequently present in one complex. Strongly positive charged complexes had a toroidal appearance. All these different forms contributed to the high transfection efficiency. Cellular uptake is supposed to be by phagocytosis.