Radioactive DNA was prepared bybiosynthetic labeling: the bacteria were grown for ... was grown in 10 ml Spizizen medium supplemented with amino acids and 5 me (and 75 pgg) ... changes of 0.15 M sodium chloride containing 0.1 31 sodium citrate until no further radio- .... The photographs were taken at focal settings.
AN AUTORADIOGRAPHIC STUDY OF GENETIC TRANSFORMATION By GEORGE T. JAVOR* AND ALEXANDER ToMASZt THE ROCKEFELLER UNIVERSITY
Communicated by Rollin D. Hotchkiss, June 19, 1968
An estimation of the frequency of all competent cells (i.e., cells capable of absorbing DNA and undergoing transformation) can be made on the basis of some reasonable assumptions by comparing the frequencies of single- and doublemarker transformations for two unlinked markers.' Such estimations have been made, and it was suggested that in Hemophilus influenzae, under optimal conditions, a vast majority of the cells may be competent. In Bacillus subtilis, estimations of this kind consistently yielded a much lower figure (10-25% of the cells competent).2 On the other hand, in a third transformable organism, Pneumococcus, unreasonably high values (over 100%) were computed with this method.3 Recently, higher than 100 per cent values were reported for Bacillus subtilis also.4 Precise information with regard to the frequency of transformable bacteria in a cell population is of theoretical interest. In addition, such information is obviously mandatory for the interpretation of experiments in which biochemical and physiological properties of "highly competent" and "incompetent" cultures are compared. The main reason for undertaking an autoradiographic study of the transformation process is to obtain such precise information. The advantage of this method is that it registers exclusively the irreversible uptake of DNA. In addition, the autoradiographic method allows a direct morphological study of competent bacteria. Materials and Methods.-As recipient strains in the transformation experiments, the R6 (streptomycin- and micrococcini-sensitive) strain of Pneumococcus and the Mu8u5ul6 (adenine-, leucine-, rnethionine-) strain of Bacillus subtilis were used (the latter was kindly supplied by Dr. L. MAlindich, Public Health Research Institute, New York). The methods for the cultivation of bacteria, transformation, viability assays for Pneumococcus5 6 and B. subtilis7 were all published procedures. The competent state of pneumococci was induced by purified preparations of the "competence-activator" substance.8 Cell suspensions with different levels of competence were obtained by terminating (with subtilisin) the conversion of cells to competence at various times after the addition of activator. Competent B. suibtilis cells were prepared by the method of Anagnostopoulos and Spizizen (90 min of growth in the "phase-II" medium).7 "Low competence" cultures were prepared the same way, except that they were used immediately after transfer of the cells to the phase-II medium. Pneumococcal transforming DNA, carrying the streptomycin and micrococcin resistance markers, and B. subtilis transforming DNA labeled with the adenine+ leucine+ genes were prepared from the appropriate strains of the two organisms.9 Radioactive DNA was prepared by biosynthetic labeling: the bacteria were grown for at least 4-5 generations in the presence of tritiated precursors of DNA. A uracil-delendent, streptomycin-resistant strain of Pneumococcus was grown in 5 ml C-medium6 containing 2 me (and 26.2 /Ag) of uridine-6-H3. A thymine-requiring strain of B. subtilis'0 was grown in 10 ml Spizizen medium supplemented with amino acids and 5 me (and 75 pgg) of thymidine-(methyl-H3). The labeled bacteria were lysed (with deoxycholate in 1216
IIICROBIOLOG Y: JA VOR AND VOVL L. 60, 1968
TOJIASZ11217
Pneumococcus; with lvsozyme followed by sodium dodecylsulfate in B. subtilis), and the crude lysates were heated at 720C for 10 min. After centrifugation, the supernatant fluids were dialyzed (Visking tubing) for 4-6 days in the cold roonm against numerous changes of 0.15 M sodium chloride containing 0.1 31 sodium citrate until no further radioactivity was released through the dialysis membrane. The concentration of DNA in the dialyzed extracts was determined by alkaline hydrolysis followed by acid precipitation. The specific activities were: 3.14 X 107 dl)m//g DNA (Pneumococcus) and 2.85 X 107 dpm/ug DNA (B. subtilis). All DNA's were used within 2 weeks of their preparation. The saturating concentration of radioactive DNA and the optimal exposure time for the particular cell suspension were determined in preliminary experiments. In order to avoid complication arising from a possible inactivation of genetic markers by radioactive decay, the transformation frequencies to individual and double markers were determined in separate tubes with nonradioactive DNA. The bacteria that received radioactive DNA were treated with DNase, washed free of extracellular radioactivity, and concentrated by centrifugation. Small l)ortions were deposited on Millipore filters to determine cell-bound radioactivity. Droplets of the same suspension were mounted on glass slides and heat-fixed under an infrared lamp. After washing in distilled water, the slides were covered with a film of nuclear emulsion (ILFORD, K5) and processed for autoradiography."1 An average minimal decay time, i.e., the time required to obtain at least one radioactive decomposition per cell, was determined from the cell-bound radioactivity and microscopic cell counts. The approximate "efficiency of counting" of the radioautographic method was determined in a preliminary experiment with uridine-labeled cells. It was found to be 10-12%o. The figure of 10% efficiency was used in the transformation experiments: the slides were "exposed" in, the dark for times long enough to allow more than ten decomposition.s to occur per cell. After development and fixing, the autoradiograms were scanned with a phase contrast microscope (Zeiss, Ultraphot) at 2500X magnification. Photographs were made with a Polaroid camera. The frequency of competent bacteria was computed on the basis of the formula: (NA X NB)/(NAB X C) X 100 = frequency of competent cells (in per cent of viable or total cells).' NA, NB, and NAB represent the number of transfornmants to markers A, B, and AB. C stands for the number of cells. Results. Table 1 summarizes and Figures 1 and 2 illustrate the observations made in several experiments. Quantitative observations: Control experiments shown in Table 1 indicate that the silver grains associated with bacteria represent DNA molecules absorbed by the cells rather than the uptake of some radioactive "contaminant." The frequency of radioactive cells could be suppressed by the addition of nonradioactive heterologous (thymus) DNA or by pretreatment of the radioactive DNA with
D-Nase. The principal findings described in Table 1 can be summarized as follows: (1) The frequency of radioactive cells changed in parallel with the frequency of cells transformed for single genetic markers. In some experiments heterologous (B. subtilis) DNA labeled specifically with tritiated thymidine was used. The frequency of radioactive pneumococci here too changed in parallel with the frequency of single-marker transformants. (2) The table shows that in Pneumococcus the competent state can be shared by 100 per cent of the individual cells. (3) In our best experiment with B. subtilis (frequencies of transformation to adenine and leucine prototrophy were 1% and 0.5%0, respectively), 15 per cent of the cells have fixed radioactive DNA. (4) Computation of the extent of competence from transformation frequencies often yielded unreasonably high values in experiments with Pneumococcus. Experiment II demonstrates such a case in
1218
MICROBIOLOGY:
TABLE 1.
JA --OR AND TOMASZ
Estimation of the frequency of competent cells from autoradiographic data and from transformation frequencies. BIOASSAY METHODl
AUTORAnTnr.RAPRY METHnn
a
-
5
us U
0 o o 0 -4 lmca H
III
5. >
w
4
coccu cocu IPPneumo- co+
0No. Cells Transform d
Qu
a
II
mu m
cu .i0 U
vs w 00-
>
4.-uWt)0EO
-0 us
*zu
os
4
_
i
1.6x107 2.6x103 l.9x103
6U0
72
12
Pneumo-
co co-
300 300
5771207
100 36
1.35xlO8
coccus
Pneumo-
co
300 300
100
1.35xlO8
1.55x108 1.35x10
72
2.7xlO7
M~.IAa 0>Ia1
w 4
. 25x10
41
u 0 u R -. 6 ca
.
6
7.5xlO6 7.2xlO6 ----
0
5
ost
6 42x10 3 .2x10
567 230
U C
5
83
co
o
0
SK us
330 275
+
per ml Double Markers
Markers -
*.^Iu 4)0 r.0 4jW 06E04)u '5 u a 0 -o uE 8
0
co-
Pneumo-
Single s-4
Aa
55
coccus
coccus+
PROC. N. A. S.
3.2x102 3x102
3. 2x10
