Proc. Nat. Acad. Sci. USA Vol. 68, No. 8, pp. 1848-1852, August 1971 DNA Uptake during Genetic Transformation and the Growing Zone of the Cell Envelope (pneumococcus/electron microscopy/choline/ethanolamine/autoradiography) ALEXANDER TOMASZ, EVA ZANATI, AND RICHARD ZIEGLER The Rockefeller University, New York, N.Y. 10021 Communicated by Norton D. Zinder, May 26, 1971 ABSTRACT Continuous cellular incorporation of cho- (10), determination of CF-receptor activity (11), and the line molecules is essential for DNA binding to compe- tests for the competence-specific agglutinin (12) were all tent pneumococci. The choline molecules are incorporated into the cell surface at the equatorial region of the performed by published procedures. The concentration of CF cocci. As a working hypothesis, it is proposed that DNA during the "activation" assay was usually 100-500 Units molecules enter these bacteria at the growing zone(s) of (13) per ml. Lysis sensitivity of pneumococci was determined the cell envelope. as follows: 0.9 ml of the culture was pipetted into tubes pre- 0.1 Ml in the pared with ml of phosphate buffer (1 M, pH 8) and 50 The presence of choline residues teichoic acid portion of a solution of deoxycholate. The preparation of Bacillus of the pneumococcal cell envelope (1) is essential for several 5% is subtilis DNA labeled with [3H]thymidine and radioautography physiological functions of this bacterium. Not only choline of cell-bound DNA were described (14). Pneumococcal DNA required for growth (2), but biosynthetic replacement of carrying the streptomycin-resistance marker and labeled with choline in the cell surface by structural analogs (such as [3H]thymidine was prepared from a streptomycin-resistant, ethanolamine) causes several striking physiological changes thymidine-requiring strain of pneumococcus (15). is a in cell in the bacteria (2-5); one such change defect divi- Reversible and irreversible binding (16) of radioactive DNA sion that prevents the physical separation of daughter cells was determined as follows: to a 3-ml culture of bacteria The of new choline molecules from one another. incorporation (about 107 viable cells/ml), 3H-labeled transforming DNA into the cell surface takes place at a distinct growing zone, was added (0.1 Mg; 106 dpm/ml). After incubation at 30°C of We now localized at the equatorial region pneumococci (6). for 20 min, the tube was chilled on ice; a small aliquot was describe evidence which suggests that the cell-surface changes removed into growth medium containing DNase (1 Mg/ml) that make pneumococci capable of absorbing and integrating and later scored for the number of transformants. The extracellular DNA are in some way coupled to the functioning rest of the suspension was centrifuged in the cold (10,000 X of the growing zone of the cell envelope. g, 10 min) and, after two washes with 5-ml portions of cold MATERIALS AND METHODS growth medium, a portion of the suspension was pipetted into Cultures of the R36A strain of Diplococcus pneumoniae cold 10% TCA and the precipitate was collected on a glass- were used in all experiments at a concentration of 107-108 fiber filter to determine "reversibly bound" DNA. The rest cells/ml. The composition of a chemically defined (Cd) (7) of the cell suspension was treated with 5-10 ug/ml of pan- and a semisynthetic (C) (8) medium was described earlier. creatic DNase and 10 Mul/ml of snake venom (Russell snake An enriched defined medium (Cden) was prepared by sup- venom, Worthington) at 37°C for 10 min; after two washings plementing Cd medium with all the amino acids present in with growth medium, a portion of the suspension was pipetted acid-hydrolyzed casein, at the same concentrations as in the into cold 10% TCA and the precipitate was collected onto C medium. "Choline-phase" and "ethanolamine-phase" glass-fiber filters to determine DNA bound in a nuclease- bacteria were grown in Cden medium supplemented with resistant ("irreversible") form. A Hitachi, Perkin-Elmer 5 MLg/ml of choline or 40 Mg/ml of ethanolamine, respectively. type HU-1lC-1 electron microscope was used at 75 kV. All Bacterial growth was monitored with a Coleman nephe- chemicals used were analytical grade commercial products. lometer. Cells were transferred from one medium to another RESULTS by filtration (Millipore filters, pore size 0.45 Mm). Radioactive Two types of experimental designs were used. In one of these, tracers were used at 5 Mg/ml and 1 MCi/ml concentrations, cells growing in growth medium containing choline ("choline- unless otherwise noted. The incorporation of radioactive phase cells") were transferred to an amino-alcohol-free tracers into cold trichloroacetic acid (TCA)-insoluble mate- medium or to a medium containing ethanolamine in place of rial was measured by collecting the samples on glass-fiber choline. In the second type of experiment, pneumococci that disks (Schleicher and Schuell); after drying, the disks were were growing in ethanolamine-containing medium for several counted in a toluene-based scintillator in a scintillation generations ("ethanolamine-phase cells") were transferred spectrometer (Unilux, Nuclear-Chicago). The preparation and back to choline-containing medium. assay of competence factor (CF) (9) and transforming DNA For the interpretation of such experiments, it was essential first to determine the effect of choline deprivation on the Abbreviations: TCA, trichloroacetic acid; CF, competence factor. general metabolism of pneumococci. It was found that for 1848 Downloaded by guest on October 7, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) DNA Uptake and the Cell Envelope 1849 TABLE 1. Effect of choline deprivation on cellular response to CF [32P] DNA bound (cpm/ml Transformants Tube Medium of culture) per ml A Complete 140 3.2 X 106 B No choline 2 <102 6IC C Choline readded 30 1.4 X 101 IU, 30 Ea 6 CI Control bacteria (A), choline-starved cells in choline-free 0c10 6 0 (n medium (B), and in medium resupplemented with choline (C) C 25 were treated with CF for 20 min (8 ml of cells to 1 ml of CF); 0 \ I , 5-ml portions of the cultures then received 0.1 ml of 32P-labeled D 6 transforming DNA, 2.5 of after 20 min of incuba- -oU.0 1g choline, and, a}) 03 ' 0 20 tion, DNase (10 usg/ml); after 15 min of incubation at 370C, E 7. small aliquots were tested for transformability, the rest of the z I I 15 b suspensions were washed four times with 5 ml of medium, then x 1-ml portions were filtered onto Millipore membranes, dried, and E tested for cell-bound radioactivity by scintillation counting. o02- 010203 0 06 110 about one generation time after the removal of choline from the medium, the cells grew; the rates of mass increase and 5 of DNA, RNA, and protein syntheses were normal. Depletion of the intracellular pools of choline compounds was com- pleted during the first third of the period of growth in the absence of choline. In all experiments, the effect of choline Minutes deprivation on transformability was tested within this period of normal growth. FIG. 1. Decay of cellular response to CF during depletion of choline pools. Physiologically incompetent pneumococci were Choline deprivation experiments transferred from the low pH, Cden medium to fresh Cd.. medium A culture of physiologically incompetent bacteria growing in at pH 8, containing a limiting amount of choline (0.1 sg, 0.5 the enriched defined medium at low pH (6.8) was transferred A&Ci/ml). At frequent intervals after the transfer, 0.1-ml aliquots were removed to test incorporation of choline into cold TCA- to choline-free medium at the same pH. The culture was precipitable material (solid lines). 0.8-ml samples were pipetted halved; a control suspension was supplied with normal con- into tubes with 0.1 ml CF and, after 15 min of incubation, the bac- centration of choline (tube A), the second received no amino- teria were further diluted (0.1 ml into 1.0 ml) into C medium alcohol (tube B). After incubation at 370C for 30 min, the containing choline, transforming DNA, and subtilisin. Trans- pH of both cultures was adjusted to 8, and half of the suspen- formations were terminated after 10 min. Dashed lines indicate sion in tube B was transferred to a third test tube containing the number of transformants. A second "pulse" of choline (0.1 a normal concentration of choline (control for reversibility; ,pg; 0.5 ACi/ml) was added to the pH 8 culture after 40 min tube C). Each of the three cultures then received CF; after of incubation with the first pulse of choline. 20 min of treatment with CF, the level of competence was In another experiment of identical design, activatability and determined by incubation with transforming DNA. Table 1 binding of [3H] thymine-labeled transforming DNA was tested shows that (a) choline deprivation resulted in a virtual dis- during the incorporation of a single, nonradioactive choline pulse (0.1 Aig of choline/ml) at times indicated by A, B, C, and D. For appearance of "activatability" by CF, measured both as results, see Table 2. genetic transformation or as ability to bind radioactive DNA. (b) The inhibitory effect of choline deprivation was quickly reversible by the addition of choline to the same In another experiment, competent bacteria have been ex- medium in which the bacteria were starved. This result ex- posed to temporary choline starvation and/or ethanolamine cludes trivial artifacts such as "leakage" of proteolytic or treatment, subsequent to completion of DNA uptake (i.e., after nucleolytic enzymes into the medium by the choline-starved addition of DNase).