Dependence of transfection efficiency of calcium treated Escherichia coli cells on bacterial genotype and form of lambda DNA

Summary The transfecting activity of linear DNA is 100 times higher in calcium treated E. coli K 12 (ⁱ⁴³⁴) than in non-lysogenic strains: the levels of transfection are 1–2.10⁷ and 1–2.10⁵ infective centers per 1 g of DNA, respectively. The high efficiency of lysogenic cells transfection is not due to the spontaneously liberated helper phage. Evidently, it is called forth by transfecting DNA-prophage recombination or/and by inhibition of nuclease activity in lysogenic cells. Both ring forms DNA (supercoiled and open circles) show very low infectivity, if any, in calcinated cells.     

Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

Electron Microscopic Evidence for Linear Insertion of Bacteriophage MU-1 in Lysogenic Bacteria

Temperate bacteriophage Mu-1 was used to generate a lysogenic derivative of the F'lac episome of Escherichia coli. Intact, covalently circular molecules of F'lac and lysogenic F'lac Mu(+) deoxyribonucleic acid (DNA) were isolated and examined by electron microscopy. The mean contour lengths of F'lac and F'lac Mu(+) molecules were 37.6 +/- 0.4 mum and 53.2 +/- 0.4 mum, respectively. The mean difference, 15.6 mum, is similar to the mean contour length of 12.9 +/- 0.1 mum obtained for linear DNA molecules released by osmotic shock from mature phage Mu-1 virions. These results provide direct physical evidence that phage Mu-1 integrates by linear insertion of its genome into the DNA of lysogenic host bacteria. Chemical and physical analyses of phage Mu-1 DNA indicate that it is similar to E. coli DNA in respect of gross base composition, buoyant density, and melting temperature.     

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7.

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.     

Effect of experimental magnetic storm on the production of lambda phage.

1. Sharp fluctuation of the intensity of the vertical component of the MF amounting to +/- 0.1 Oe changing the sign over each 3 min causes variability of both lysogenic and indicator strains of E. coli. This testifies to an extremely low threshold of their magnetic susceptibility and to biological importance of fluctuations of natural parameters of the geomagnetic field as an ecological factor of the environment. 2. A change in the intensity of the vertical component of the MF, not any higher than +/-0.1 Oe, inhibits phage production in the lysogenic system of E. coli K = 12 lambda and is also reflected in the morphological peculiarities of negative phage colonies as well as in the phage-susceptibility of the E. coli indicator strain.     

Effect of spermidine on the RNA-A protein complex isolated from the RNA bacteriophage MS2.

The polyamine spermidine has recently been reported to be a substantial component of the RNA phage particle. Its effect on the isolated RNA-A protein complex of the phage MS2 is investigated here. This complex infects intact Escherichia coli cells via F-pili, as does the whole phage. It is shown that the infectivity of the complex on intact E. coli cells was enhanced by incubation with spermidine. Optimal stimulation (20-fold) of the complex infectivity was achieved by incubation with 3 x 10(-4) M spermidine for 20 to 30 min at 37 degrees C. This gave a more compact structure to the complex, as could be seen by its faster sedimentation in sucrose gradients. Although spermidine and Mg2+ are known to partially replace one another in several systems, no enhancement of the infectivity of the complex, but only its considerably faster sedimentation in sucrose gradients, occurred after incubation with 3 x 10(-4) M Mg2+. Only if the Mg2+ concentration was raised by more than one order of magnitude could increased infectivity of the complex be observed. At concentrations of spermidine and Mg2+ that maximally stimulated the infectivity of the complex on intact E. coli cells, no increase in infectivity of phenol-extracted RNA to E. coli spheroplasts was detected. From these in vitro results, the role of the polyamine spermidine in the RNA phage particle for the infecting, RNA-A protein complex molecules in phage infection is discussed.     

Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli.

Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed.     

Mapping of New Escherichia coli K and 15 Restriction Sites on Specific Fragments of Bacteriophage φX174 DNA

We have isolated several new phiX174 mutants which contain sites sensitive to restriction by Escherichia coli. One contains an E. coli 15 restriction site and three are double mutants containing an E. coli K site as well as the E. coli 15 site. The replicative form (RF) DNA of one of the mutants containing a K site has been shown to be restricted in spheroplasts of a K-12 strain. The infectivity of this RF, but not wild-type RF, has also been shown to be inactivated by an E. coli K extract and by purified K restriction enzyme in vitro. The product of the RF treated with purified K restriction enzyme in vitro is a full length linear molecule. The mutant sites have also been localized to specific regions of the phiX174 genome by a fragment mapping technique, making use of specific fragments of phiX174 RF DNA obtained by digestion with a specific endonuclease.     

Induction of Prophage SPO2 in Bacillus subtilis: Prophage Excision in the Absence of Bacterial or Bacteriophage DNA Synthesis

Bacillus subtilis lysogenic for SPO2 wild type was induced under conditions preventing synthesis of both bacterial and phage DNA. The infectivity of phage DNA in transfection is strongly decreased under these conditions, whereas the activity of single phage genes as measured by marker rescue with superinfecting phage is unaffected. DNA from induced cells was sedimented in neutral sucrose gradients. After induction, phage DNA was detected at a position in the gradients, which was different from the bulk of the bacterial DNA, corresponding to linear double-stranded DNA of about 25 x 10(6) daltons. Similar results were obtained with bacteria lysogenic for a SPO2 prophage carrying a DNA-negative mutation. No separation of phage and bacterial DNA activity was detected when chloramphenicol was present during the induction period. These experiments show that prophage SPO2 can excise from the bacterial chromosome without previous replication.     

Outer membrane protein A expression in Escherichia coli K1 is required to prevent the maturation of myeloid dendritic cells and the induction of IL-10 and TGF-beta

Dendritic cells (DCs) are professional APCs that direct both cellular and humoral immune responses. Escherichia coli K1 causes meningitis in neonates; however, the interactions between this pathogen and DCs have not been previously explored. In the present study, we observed that E. coli K1, expressing outer membrane protein A (OmpA), was able to enter, survive, and replicate inside DCs, whereas OmpA(-) E. coli was killed within a short period. Opsonization of OmpA(+) E. coli either with adult or cord serum did not affect its survival inside DCs. Exposure of DCs to live OmpA(+) E. coli K1 prevented DCs from progressing in their maturation process as indicated by failure to up-regulate costimulatory molecules, CD40, HLA-DR, and CD86. The distinct DC phenotype requires direct contact between live bacteria and DCs. The expression of costimulatory molecules was suppressed even after pretreatment of DCs with LPS or peptidoglycan. Furthermore, the suppressive effects of OmpA(+) E. coli on DCs were abrogated when the bacteria were incubated with anti-OmpA Ab. The inhibitory effect on DC maturation was associated with increased production of IL-10 as well as TGF-beta and decreased production of IL-6, TNF-alpha, IL-1beta, and IL-12p70 by DCs, a phenotype associated with tolerogenic DCs. These results suggest that the subversion of DC functions may be a novel strategy deployed by this pathogen to escape immune defense and persist in the infected host to reach a high degree of bacteremia, which is crucial for E. coli to cross the blood-brain barrier.     

Infectious DNA from coliphage T1

Summary DNA isolated from coliphage T1 is infective in spheroplasts of E. coli K12/1. The efficiency of the assay amounts to approximately 10^-4 plaque-forming units per DNA molecule of 32·10⁶ daltons. A linear relationship between DNA concentration and total phage yield or infective centers, respectively, holds for native DNA. For heat-treated DNA, however, the co-operation of 1.4 molecules is required for successful infection. Beyond a critical concentration of about 0.1g/ml a self-inhibiting effect of infectious T1-DNA is observed. Breakage by shearing and denaturation of the DNA-molecules destroy their infectious activity. Renaturation, however, restores infectivity to 60–90 per cent of the original activity. Heat treatment of T1-DNA in M/5 NCE buffer results in narrow-coiled, mismatched molecules with partially denatured regions. Though the efficiency of infection of such molecules is reduced by about 30 per cent, the critical concentration of T1-DNA shifts to higher values by a factor of ten, thus giving an increase in the total plaque yield of the system. The effect is explained by the transition of native into narrow-coiled molecular configuration.     

Induction of Phage Production in the Lysogenic Escherichia coli by Hydroxyurea

1) Hydroxyurea, a reversible DNA synthesis inhibitor, was used to study the mechanism of prophage λ induction in Escherichia coli K12. Induction of prophage was judged on two criteria: increase of phage-producing cells and loss of colony-forming ability of the cells. 2) Hydroxyurea induced an increase of phage-producing cells only in lysogenic strains known to be inducible with ultraviolet irradiation for prophage development and not in strains such as E. coli K12 (λind^–) or E. coli K12 recA (λ⁺). 3) When protein synthesis was inhibited, hydroxyurea did not increase phage-producing cells of lysogenic strains; it showed a bacteriocidal effect on lysogenic recA⁺ strains, but not on nonlysogenic strains. 4) The sensitivity of E. coli K12 recA to hydroxyurea was independent of whether or not the cells were lysogenic. 5) From the results it is suggested that certain steps leading to loss of colony-forming ability (i.e. prophage induction) do not require de novo protein synthesis but require the presence of the host recA⁺ gene.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

Effect of rpoS mutations on stress-resistance and invasion of brain microvascular endothelial cells in Escherichia coli K1

Escherichia coli K1 strains are predominant in causing neonatal meningitis. We have shown that invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for E. coli K1 crossing of the blood-brain barrier. BMEC invasion by E. coli K1 strain RS218, however, has been shown to be significantly greater with stationary-phase cultures than with exponential-phase cultures. Since RpoS participates in regulating stationary-phase gene expression, the present study examined a possible involvement of RpoS in E. coli K1 invasion of BMEC. We found that the cerebrospinal fluid isolates of E. coli K1 strains RS218 and IHE3034 have a nonsense mutation in their rpoS gene. Complementation with the E. coli K12 rpoS gene significantly increased the BMEC invasion of E. coli K1 strain IHE3034, but failed to significantly increase the invasion of another E. coli K1 strain RS218. Of interest, the recovery of E. coli K1 strains following environmental insults was 10-100-fold greater on Columbia blood agar than on LB agar, indicating that growing medium is important for viability of rpoS mutants after environmental insults. Taken together, our data suggest that the growth-phase-dependent E. coli K1 invasion of BMEC is affected by RpoS and other growth-phase-dependent regulatory mechanisms.     

Properties of Infectious T1 Deoxyribonucleic Acid

T1 deoxyribonucleic acid (DNA) infection of spheroplasts was characterized by the following. A small number of the DNA molecules initiated infectious centers, and a small number of the spheroplasts were infected by T1 DNA. Once a favorable encounter of T1 DNA with spheroplast occurred, a minimum of 20 to 30 min was required for T1 DNA to enter the spheroplast. The mature T1 particles produced in the infection of spheroplasts by T1 DNA were released in a burst, but the average burst size was quite small compared with a normal burst of the phage-infected bacteria. T1 DNA preparations, capable of causing viral growth in spheroplasts, did not require detectable amounts of protein for infectivity, were homogeneous in band and boundary sedimentation, and had a guanine plus cytosine content of 48% and a minimal molecular weight of 35 x 10(6). Denatured T1 DNA, like denatured lambdaDNA, did not infect spheroplasts. Renatured T1 DNA was not infectious; this was in marked contrast to renatured lambdaDNA.     

Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli.

The cellular content of all 20 aminoacyl-tRNA species was determined in small cultures of Escherichia coli by labeling cells with 3H-amino acids and extraction of 3H-amino acid-labeled nucleic acid by standard procedures. Of 3H-amino acid-labeled material, 25 to 90% was identified as 3H-aminoacyl-tRNA by the following criteria: sensitivity to base hydrolysis with expected kinetics; association of 3H counts released by base treatment of the 3H-amino acid-labeled nucleic acid with amino acid standards upon paper chromatography of the hydrolysate; and changes in the amount of 3H-amino acid-labeled nucleic acid recovered from cells as a function of time. Individual aminoacyl-tRNA content was determined with as few as 8 X 10(7) to 4 X 10(8) E. coli cells. Although the total number of aminoacyl-tRNA molecules per cell varied only by 10 to 20% among various strains of E. coli, some individual aminoacyl-tRNA families varied two- to threefold among strains. For a given amino acid, the number of aminoacyl-tRNA molecules per cell in E. coli strain K38 growing with a doubling time of 60 min varied from 730 (glutamyl-tRNA) to 7,910 (valyl-tRNA) with a mean value of 3,200. The total number of aminoacyl-tRNA molecules per cell (6.4 X 10(4)) in E. coli K38 was 5.5-fold higher than the number of ribosomes and was equal to 84% of the amount of elongation factor Tu molecules per cell. The ratio of aminoacyl-tRNA to synthetase for 10 amino acids varied from about 1 to 15 with a mean value of 4.7. The turnover of individual aminoacyl-tRNA families in E. coli cells was estimated to be in the range of 1.7 to 8.1 s-1 with a mean value of 3.7 s-1. An estimate of minimum in vivo molecular activity of aminoacyl-tRNA synthetases gives values of 2 to 48 s-1 for individual enzymes.     

Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.     

Expression of the genome of Mu-like phage D3112 specific for Pseudomonas aeruginosa in Escherichia coli and Pseudomonas putida cells

The behavior of Escherichia coli cells carrying RP4 plasmid which contains the genome of a Mu-like D3112 phage specific for Pseudomonas aeruginosa was studied. Two different types of D3112 genome expression were revealed in E. coli. The first is BP4-dependent expression. In this case, expression of certain D3112 genes designated as "kil" only takes place when RP4 is present. As a result, cell division stops at 30 degrees C and cells form filaments. Cell division is not blocked at 42 degrees C. The second type of D3112 genome expression is RP4-independent. A small number of phage is produced independently of RP4 plasmid but this does not take place at 42 degrees C. No detectable quantity of the functionally active repressor of the phage was determined in E. coli (D3112). It is possible that the only cause for cell stability of E. coli (D3112) or E. coli (RP4::D3112) at 42 degrees C in the absence of the repressor is the fact of an extremely poor expression of D3112. In another heterologous system, P. putida both ways of phage development (lytic and lysogenic) are observed. This special state of D3112 genome in E. coli cells is proposed to be named "conditionally expressible prophage" or, in short, "conex-phage", to distinguish it from a classical lysogenic state when stability is determined by repressor activity. Specific blockade of cell division, due to D3112 expression, was also found in P. putida cells. It is evident that the kil function of D3112 is not specific to recognize the difference between division machinery of bacteria belonging to distinct species or genera. Protein synthesis is needed to stop cell division and during a short time period this process could be reversible. Isolation of E. coli (D3112) which lost RP4 plasmid may be regarded as an evidence for D3112 transposition in E. coli. Some possibilities for using the system to look for E. coli mutants with modified expression of foreign genes are considered.     

Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores.

A study of mechanisms of electrotransfection using Escherichia coli (JM 105) and the plasmid DNA pBR322 as model system is reported. pBR322 DNA carries an ampicillin resistance gene: E. coli transformants are conveniently assayed by counting colonies in a selection medium containing 50 micrograms/ml ampicillin and 25 micrograms/ml streptomycin. Samples not exposed to the electric field showed no transfection. In the absence of added cations, the plasmid DNA remains in solution and the efficiency of the transfection was 2 x 10(6)/micrograms DNA for cells treated with a 8-kV/cm, 1-ms electric pulse (square wave). DNA binding to the cell membrane greatly enhanced the efficiency of the transfection and this binding was increased by milimolar concentrations of CaCl2, MgCl2, or NaCl (CaCl2 greater than MgCl2 greater than NaCl). For example, in the presence of 2.5 mM CaCl2, 55% of the DNA added bound to E. coli and the transfection efficiency was elevated by two orders of magnitude (2 x 10(8)/micrograms DNA). These ions did not cause cell aggregation. With a low ratio of DNA to cells (less than 1 copy/cell), transfection efficiency correlated with the amount of DNA bound to the cell surface irrespective of salts. When the DNA binding ratio approached zero, the transfection efficiency was reduced by two to three orders, indicating that DNA entry by diffusion through the bulk solution was less than 1%. Square pulses of up to 12 kV/cm and 1 ms were used in the electrotransfection experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method of estimating the effectiveness of ionizing radiation and magnetic fields for phage induction in lysogenic bacterial culture

Either the difference delta N of the content of free phage particles in the experiment and the control or K ratio of these values can be used to estimate the effectiveness of ionizing radiation or other agents inducing phage formation in a lysogenic bacterium culture. The estimation technique the results of which are nearly independent of the fluctuations in the number of phage particles in the control, the inductor dose being invariable, is the most adequate one. The induction of phage in E. coli K12 (lambda) culture by X-rays and magnetic field is an example illustrating that the K ratio, which can be called "the induction coefficient", is in a good agreement with the requirement mentioned above. A possible nature of the phenomenon observed is discussed.