Infectious DNA from coliphage T1

Summary Infectious DNA from phage T1 was inactivated by UV-light (2,537 Å). No effect of irradiation on the kinetics of the assay in a spheroplast system could be observed. UV-damaged molecules compete with unirradiated DNA for the infection. Infectious T1-DNA is subject to host-cell reactivation of UV-damage, the amount of which depends on the physiological conditions of the spheroplasts. Though UV-radiosensitivity of T1 particles is not influenced by the presence of the radical scavenging compound cysteamine, infectious DNA can be protected effectively by this chemical (0.01M) against UV-damage when HCR-negative spheroplasts are used for the assay. Incorporation of 5-bromouracil radiosensitizes infectious T1-DNA in the presence and absence of HCR. This effect can be eliminated when the DNA is irradiated in the presence of cysteamine. The mechanism of radioprotection is discussed.     

Multiplication of Bacteriophage P22 in Penicillin-Induced Spheroplasts of Salmonella typhimurium

The spheroplasts of Salmonella typhimurium (LT2) prepared by treatment with penicillin were capable of adsorbing phage P22 C(1). The normal multiplication of the phage took place, although the burst size was reduced to one-fourth of that in intact cells. Rate of incorporation of (14)C-thymidine into spheroplasts was increased severalfold on phage infection. Multiplication of C(+) also took place, but no lysogeny could be established in spheroplasts. Furthermore, spheroplasts prepared from cells lysogenized with wild-type phage, LT2 (C(+)), and a temperature-inducible C(2) mutant, LT2(tsC(2)), were not inducible. Unlike normal cells, both mitomycin C and actinomycin D interfered with the phage multiplication in spheroplasts. The spheroplast system offers great advantages in the study of the synthesis of nucleic acids and proteins in phage-infected LT2.     

DNA injection during bacteriophage T4 infection of Escherichia coli.

The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome. The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core. The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast. A membrane potential was not required for the process of DNA injection. On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison. Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed.     

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.     

Characteristics of chromosomal DNA isolated from Escherichia coli spheroplasts

The chromosomal DNA of Escherichia coli spheroplasts induced by penicillin G was studied biochemically and electron microscopically. Although the spheroplasts were unable to divide, they continued to synthesize chromosomal DNA for several hours even in the presence of penicillin G. Some differences were observed between the chromosomal DNA of the parent cells and that of the spheroplasts in sucrose gradient centrifugation and electron microscopy; two types of chromosomal DNA, a slower sedimenting form and a faster sedimenting form, were released from the gently lysed parent cells. The former was membrane-free folded chromosome and the latter was membrane-associated chromosome. In contrast, the chromosome from the spheroplast showed a single intermediate value of sedimentation coefficient between those of the chromosomal DNA from the parent cell. Cytochrome spreading for electron microscopy showed that the spheroplast chromosomal DNA formed an aggregated mass consisting of several chromosome-molecules of the parent cell.     

Natural penicillin resistance of Francisella tularensis

Altered viable forms of F. tularensis with spheroplast specific damages of the surface structures were isolated after the culture exposure to lithium chloride (0.5 and 1%). Study of natural penicillin resistance in the spheroplasts and bacterial forms of F. tularensis revealed their difference: the spheroplasts of the strains tested had a lower resistance to beta-lactam antibiotics than the bacterial forms while the activity of spheroplast beta-lactamase did not differ from that of the enzyme of the bacterial form and equalled 224 to 252 U/ml of the cell suspension. Therefore, on the model of the lithium-induced spheroplasts it appeared possible to show that the damages of the surface structures of the cell walls of F. tularensis changed the penicillin resistance level which was indicative of involvement of the F. tularensis cell walls in the phenomenon of the natural resistance to beta-lactams.     

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.     

Genetic recombination in fused spheroplasts of Providence alcalifaciens.

Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 X 10(-6) to 1.7 X 10(-7) per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane.     

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.     

Macromolecule synthesis in yeast spheroplasts

Conditions have been established for the preparation of spheroplasts of Saccharomyces cerevisiae which are able to increase their net content of protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), several-fold upon incubation in a medium stabilized with 1 m sorbitol. The rate of RNA and protein synthesis in the spheroplasts is nearly the same as that occurring in whole cells incubated under the same conditions; DNA synthesis occurs at about half the whole cell rate. The spheroplasts synthesize transfer RNA and ribosomal RNA. The newly synthesized ribosomal RNA is incorporated into ribosomes and polysomes. The polysomes are the site of protein synthesis in these spheroplasts. Greater than 90% of the total RNA can be solubilized by treatment of the spheroplasts with sodium dodecyl sulfate or sodium deoxycholate. These spheroplast preparations appear to be a useful subject for the study of RNA metabolism in yeast.     

Efficient transfection of the archaebacterium Halobacterium halobium.

We developed an efficient polyethylene glycol-mediated spheroplast transfection method for the extremely halophilic archaebacterium Halobacterium halobium. The 59-kilobase-pair linear phage phi H DNA molecule routinely produced between 5 X 10(6) and 2 X 10(7) transfectants per microgram of DNA. Between 0.5 and 1% of spheroplasts were transfected per microgram of luminal diameter H DNA. Under our conditions, survival and regeneration of H. halobium spheroplasts were also quite efficient, suggesting that this method will be useful for introducing other DNAs into these bacteria.     

Interaction of the isolated DNA of lambda phage with spheroplasts of E. coli treated with sturine]

The method of centrifugation in sucrose density gradient (30-55%) of the spheroplast membrane preparations treated and untreated with sturine and infected with phage lambda DNA demonstrated that sturine, treatment increased the phage lambda DNA absorption three-fold. About 50% of the lambda DNA molecules adsorbed by spheroplasts are bound with the cytoplasmic membrane of spheroplasts treated with sturine; 50% of the lambda DNA molecules are bound with the cell wall membrane on the sturine-untreated spheroplasts. The data obtained allow to conclude that the stimulating effect of sturine in E. coli spheroplasts transfection by lambda DNA is connected with redistribution of phage DNA absorbed on spheroplasts from the cell wall to the cytoplasmic membrane facilitating the penetration of DNA and its fastening on the membrane.     

Synthetic Capabilities of Plasmolyzed Cells and Spheroplasts of Escherichia coli

Effects of plasmolysis and spheroplast formation on deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, and phospholipid synthesis by Escherichia coli strain THU were studied. RNA and protein synthesis were severely diminished. DNA and phospholipid synthesis were inhibited, but less so; they could be partly restored. DNA synthesis could be restored by replacing thymine in the medium with thymidine, and phospholipid synthesis, by adding back small quantities of soluble cell extract. Plasmolysis effected marked reductions in rates of growth and macro-molecule synthesis, and temporarily reduced culture viability. Plasmolysis also caused an anomalous stimulation of phospholipid synthesis. Spheroplasts and plasmolyzed cells synthesized small amounts of ribosomal RNA that sedimented normally. However, this ribosomal RNA was very inefficiently packaged to ribosome subunits. Spheroplasts were unable to carry out induced synthesis of beta-galactosidase, and plasmolyzed cells were delayed in this function. Radioautographs examined in an electron microscope showed that DNA synthesis in plasmolyzed cells and spheroplasts was performed by a substantial fraction of the culture populations. That DNA and membrane were associated in the spheroplasts used in this study was suggested by formation of M-bands containing membrane and most of the cell's DNA. The results are discussed in terms of alterations of membrane structure and conformation attending plasmolysis and spheroplasting.     

Role of the Cytoplasmic Membrane in the Synthesis of Ribonucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Disrupted spheroplast preparations of Pseudomonas schuylkilliensis strain P contained fragments of cytoplasmic membrane and approximately 82% of the total cellular phospholipid. The protoplast-bursting factor (PB-factor), partially purified from pig pancreas, and a heat-treated pancreatic lipase fraction both inhibited ribonucleic acid (RNA) synthesis by disrupted spheroplasts but did not inhibit or only slightly inhibited RNA synthesis by intact cells or intact spheroplasts. The PB-factor preparation and the heat-treated pancreatic lipase fraction catalyzed partial (15 to 50%) deacylation of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine in disrupted spheroplasts but not in intact spheroplasts. Phospholipase A activity was demonstrated in the PB-factor preparation by use of isolated phospholipids as substrates. Treatment of disrupted spheroplasts with the PB-factor preparation caused a 70% inhibition in oxidative phosphorylation and RNA synthesis, but had little effect on electron transport. Addition of adenosine-5'-triphosphate or adenosine-5'-diphosphate and a mixture of ribonucleosides after treatment with the PB-factor preparation partially restored oxidative phosphorylation but did not relieve the inhibition in RNA synthesis. The most reasonable explanation for the latter observation appears to be that the concentrations of newly synthesized nucleotides retained by the preparations with partially deacylated membrane phospholipids were insufficient to permit the synthesis of RNA.     

Effect of penicillin on the morphology and reproduction ofAzotobacter chroococcum

The effect of penicillin on the morphology and reproduction of some strains ofA. chroococcum was studied on a number of solid media. When the growth was not entirely suppressed by the penicillin, filamentous cells and spheroplasts were formed. The formation of spheroplasts was stimulated by peptone. Gonidia were sometimes formed inside the spheroplasts and also inside giant cells. They were released from the cell after disruption or after lysis of the cell wall. In some cases they produced dwarf cells. Under certain conditions groups of gonidia present in a cell fused and formed one or more normal-looking cells inside the mother cell. Sometimes one or moreAzotobacter cells developed inside a spheroplast or at the site of a spheroplast with a lysed cell wall. Microcolonies consisting of small cocci representing gonidia and dwarf cells were also observed occasionally at the sites of spheroplasts with lysed cell walls. Occasionally tiny groups of small elements with a less marked structure were found at such sites, probably representing debris of lysed cells. The production of normal-looking cells inside filamentous cells was greatly stimulated on a medium containing 10 percent horse serum, with a drop of sterile water containing 200 or 250 I.U. penicillin added in the centre of the plate. The growth ofA. chroococcum was greatly retarded when the medium contained 10 U/ml penicillin and seemed to be checked entirely at concentrations of 20 U/ml penicillin or higher. Occasionally, however, even at concentrations of 100 and 300 U/ml penicillin, a few filamentous cells were found and also a few microcolonies, visible only through the microscope, consisting of gonidia or regenerative rods. By repeated exposure ofAzotobacter to penicillin populations could be obtained that were adapted to high concentrations of this antibiotic.     

Translocation of colicin from the receptor to the inner cell membrane: function of the peptidoglycan layer

Sensitivity of spheroplasts (prepared in two ways) of a colicin-sensitive strain, of colicinresistant and of colicin-tolerant mutants and of strains immune to colicins E1 and E2 was estimated and compared. Generally, the removal of the peptidoglycan layer brought about a slight nonspecific support for colicin translocation across the cell wall in sensitive,tolB tolerant and immune bacteria.tolB spheroplasts were colicin E1-sensitive, but E2-insensitive. Spheroplasts were always fragile and lysed spontaneously, especially those produced by lysozyme. Bacteria carryingtolA, tolQ andtolR mutations kept their colicin insensitivity as spheroplasts, just as the resistant ones. Bacteria rendered colicinogenic and hence colicin-immune turned to high colicin sensitivity in spheroplast form. The results indicate a change in plasma membrane associated with the spheroplast formation.     

Transfection of Escherichia coli Spheroplasts IV. Transfection of rec+ and rec? Spheroplasts by Native,Denatured, and Renatured T5 Bacteriophage DNA After Repair of Single-Strand Breaks by Polynucleotide Ligase

Transfection of Escherichia coli spheroplasts by native T5 phage DNA was not affected by treatment with polynucleotide ligase. Denatured T5 phage DNA infectivity, only 0.1% of the native DNA level, was increased slightly by polynucleotide ligase treatment. Renatured T5 phage DNA infectivity was also increased slightly by polynucleotide ligase treatment. To form an infective center with rec(+) spheroplasts, 1.6 to 2.1 native T5 phage DNA molecules were required; however, 1.4 T5 phage DNA molecules were required to form an infective center with recA(-)B(-) spheroplasts, and one molecule was sometimes sufficient for rec B(-) spheroplasts. Polynucleotide ligase treatment of T5 phage DNA had no effect on these parameters. Thus, the single-strand interruptions of T5 phage DNA are probably not essential to the survival of the parental T5 phage DNA, and T5 phage DNA, especially the denatured form, is highly sensitive to some nucleases in E. coli spheroplasts.     

Kinetic and Morphological Observations on Saccharomyces cerevisiae During Spheroplast Formation

A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed "prospheroplast." The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast.     

Responses of enzymically isolated aleurone cells of oat to gibberellin a(3)

Oat (Avena sativa L.) aleurone layer cells (spheroplasts) were isolated by maceration of the aleurone layer with a mixture of commercially available cellulase and pectinase. About 20% of the cells present in intact layers were released as spheroplasts and 79 +/- 9% of the spheroplast population was viable as judged by methylene blue staining. The spheroplasts became disorganized in solutions containing less than 0.4 md-mannitol. When the spheroplasts were incubated for 48 hours, total activities of acid p-nitrophenyl phosphatase and acid proteinase increased and protein levels decreased. These changes were not effected by gibberellin A(3). Isolated aleurone layers incubated under the same conditions as the spheroplasts showed reduced responses to gibberellin A(3). It is concluded that the necessary presence of an osmoticum limits the value of spheroplasts as a system for studying the mechanism of action of gibberellin A(3) in the aleurone cell.     

Reversion to bacillary forms of Serratia marcescens spheroplasts induced by carbenicillin. Scanning electron microscopic study

Bacterial cells of Serratia marcescens were easily induced to form spheroplasts in liquid medium by the addition of carbenicillin. The spheroplasts were unable to divide, but they were able to revert to the bacillary forms in liquid medium not containing carbenicillin. Four phases of the reversion sequence could be differentiated by scanning electron microscopy. (1) After 3 hr of incubation in carbenicillin-free medium, some projections arose out of the spheroplasts, and grew and elongated. (2) Their elongation resulted in a morphological change in the spheroplasts from spherical bodies to long irregular bacillary forms. (3) Further incubation caused several constricted areas in the bacillary form. (4) The long bacillary forms split along the constricted areas to become the parent bacillary forms of S. marcescens. When the long bacillary form that developed during the reversion was retreated with carbenicillin, it was immediately induced to become a spheroplast again.