w u z <!
(I)
0::
0
(f) (I)<(
6.520
T03 DNA (see Appendix):
M Na
=
7.39 X 104Thus the molecular weight of the sodium salt of T03 DNA observed in this experiment was 17.8 X 106. The average value of the molecular weight obtained from two determinations was 16.7 X 106.
Length and molecular weight of T03 DNA.
When particles of bacteriophage T03 in a high osmotic strength environment (4 M ammonium acetate) are removed from that environment and placed in a low osmotic strength environment (distilled water) some of the particles osmotically shock and burst, releasing their DNA into the surroundingmedium. This DNA can be trapped in a cytochrome c film, shadowed and examined in the electron microscope (44). An electron micrograph of T03 DNA prepared in this manner is shown in Figure 16.
The average of 12 measurements of the length of different T03 DNA molecules was 11.7
t
0.3 µ. The measured length of individual T03 DNA molecules ranged from 11.25 µ to 12.2 µ. Assuming 1.92 X 106 molecular weight units per µ, the molecular weight of the DNA of T03 is 22.5 X106. The single DNA molecule of phage T03 has a mass of 3.73 X lo- 11 µg and a volume of 3.7 X lo-5 µ3.
The process of osmotically shocking the phage and trapping the DNA in a cytochrome c film yields three distinct configurations for the DNA molecule. The free random coil form, the compact form that crosses back over itself in a typical flower shape, and the form in which the DNA has not been completely released from the phage head. The first two forms are illustrated in Figure 16, and the 3rd form is illustrated
Figure 16
Electron micrograph of T03 and T03 DNA (treated with cytochrome c and shadowed with platinum) , X 40,200.
in Figure 17. In some preparations it appeared that the DNA was being extruded through the tail while in others the tail was absent or it was attached to the end of the DNA molecule.
Base composition of T03 DNA.
A photograph of a paper chromatograph of formic acid hydrolyzed T03 phage is shown in Figure 18. The spots that absorbed ultraviolet
light are outlined. The spots representing the bases present in the
phage DNA are at the left. Control spots of adenine, guanine, thymine and cytosine are at the right. The two spots marked F are fluorescent spots that were present when whole phage was hydrolyzed but were not present when phage DNA was analyzed. The positions of the four bases present in the phage DNA match the positions of the control spots. The ultraviolet absorption spectra of the four bases present in T03 DNA matched the spectra of the corresponding control bases.
The results of the base analysis of T03 DNA are contained in Table 2. Based upon 3 determinations the DNA of T03 contains 29.0
+
0.6% adenine, 19.4
±
0.2% guanine, 30.8±
0.8% thymine and 20.8±
0.3%cytosine. The guanine plus cytosine content is 40.2%. The ratio of adenine to thymine is 0.94 and that of guanine to cytosine is 0.93.
The melting temperature of DNA has been found to be a function of its base composition (13). To utilize a marker DNA in the Tm determination a modified form of the equation of Marmur and Doty (13) was used. The difference in the Tm and the difference in the guanine plus cytosine content between two samples of DNA are related according to the equation:
Figure 17
Electron micrograph of the DNA of bacteriophage T03 being extruded from the tail of the phage (treated with cytochrome c and shadowed with platinum), X 93,500.
1-1J3 T~.3
0
f:/ / ' •/' 'i tc: \ ~ 8 y !
CONTROLS
T C.
Figure 18
Photograph of a paper chromatogram of hydrolyzed bacteriophage T03.
The spots that absorb ultraviolet light have been outlined. Spots
representing the bases; A, adenine; G, guanine; T, thymine; C, cyto- sine are shown to the right and spots from T03 are on the left. The spots marked F are fluo.rescent.
..
TABLE 2
Base composition of T03 DNA as determined by chromatographic separation of the bases (averages obtained from 3 determinations).
Base A G T
c
Total
Mole percent
A/T
=
0.94 G/C = 0.9329.0
-t
0.619.4
t
0.2 30.8 + 0.8-
20.8 + 0.3
-
100.0
Molar ratio 1.00 0.69 1.06
o.
72Purines (A+G)/Pyrimidines (T+c)
=
0.94 (GC) = 40.2%where 6Tm is the difference of the melting temperature of the marker DNA and of the DNA of unknown composition,
and 6CX:. is the percent difference of the
guanine plus cytosine content of the marker DNA and of the DNA of unknown composition.
The Tm of Escherichia coli DNA was found to be 91.5°C and for T03 DNA it was 88.5°c. The 6CX:. for these two samples of DNA is therefore:
6CX:.
=
2.44(3.0)=
7.3%Since E. coli DNA contains 50% G
+
C it follows that T03 DNA contains 42.7% G+
C.The buoyant density of DNA also depends upon its base composition in a known manner (52). If the density of T03 DNA is computed according to the methods of Schildkraut, Marmur and Doty, using their buoyant den- sities of 1.710 for Escherichia coli DNA and 1.731 for Micrococcus lyso- deikticus DNA, a density of 1.702 is obtained for T03 DNA. The density of T03 DNA cor~esponds to a guanine plus cytosine content of 43%.
The base composition of T03 DNA obtained by three independent meth- ods is presented in Table 3. Analysis by chromatographic separation of the bases gave 40.3% guanine plus cytosine. The melting temperature indicated a composition of 42.7% guanine plus cytosine. Finally, the buoyant density of T03 DNA corresponded to a guanine plus cytosine content of 43%.
Separation of the two complementary strands of T03 DNA.
According to the generally accepted Watson-Crick model for the
TABLE 3
Base composition of T03 DNA, sunnnary.
Method
Heat Denaturation, Tm Buoyant Density
Chromatography
Mole percent G
+
C42.7 43 40.2
structure of deoxyribonucleic acid (67, 68), individual molecules of DNA are composed of two complementary polynucleotide chains. For every adenine on one chain there is a thymine on the other chain. For every cytosine there is a guanine on the other chain. This model dictates that the mole fraction of adenine must equal the mole fraction of thymine. The same relationship also must hold between guanine and cytosine. However, no such relationships between the bases are nec- essary for bases in a single polynucleotide chain. The usual base equivalence does not hold for the single stranded DNA of phage 0Xl74
(54). The two strands of the DNA of bacteriophage
a
have been shown to have different but complementary base compositions (55). The heat denatured DNA from phagea
shows two bands in a cesium chloride density gradient (56), indicating that the two strands have different buoyant densities. The two strands of the DNA of the thermophilic bacteriophage TP-84 have different buoyant densities in cesium chloride. These twostrands also have different but complementary base compositions (23).
As was illustrated in Figures 13 and 14, native DNA from phage T03 bands as a single peak at a density of 1.695 in a cesium chloride density gradient. Heat denatured T03 DNA forms two bands at densities of 1.707 and 1.715 as is indicated in the photographs in Figure 19, experiments 1 A and 2 A, and the densitometer tracings in Figure 20, experiments 1 A and 2 A. When the two separated strands are mixed together and annealed, the renatured DNA forms a single band at a density of 1.699. Photographs of the renatured band are shown in Figure 19, experiments 1 B and 2 B, and densitometer tracings are
illustrated in Figure 20, experiments 1 B and 2 B. In experiment 1,
1 A 1 B 2 A 2 B p .. Figure 19 Photographs of bands formed by DNA in a cesium chloride density gradient at 44,770 rpm. 1 A, T03 DNA and T03 heat denatured DNA; 1 B, the material in 1 A, annealed and rebanded. 2 A, T03 heat denatured DNA and Micrococcus lysodeikticus DNA; 2 B, the material in 2 A, annealed and rebanded.
IA
18
2A
28
1.695 1.699 1.707 1.715 1.726 DENSITY
Figure 20
Densitometer tracings of photographs of bands formed by DNA in a cesium chloride density gradient at 44,770 rpm. 1 A, T03 DNA and T03 heat denatured DNA; 1 B, the material in 1 A, annealed and rebanded. 2 A, T03 heat denatured DNA and Micrococcus lysodeikticus DNA; 2 B, the material in 2 A, annealed and rebanded.
as illustrated in Figures 19 and 20, native T03 DNA was used to provide a density reference. The renatured DNA shows as a shoulder on the heavy side of the native peak in Figure 19 (1 B) and Figure 20 (1 B).
In experiment 2, as illustrated in Figures 19 and 20, Micrococcus lyso- deikticus DNA was used to provide a density reference. It is apparent that native T03 DNA is dissociated by heat denaturation into two sub- units whose buoyant densities differ. These two subunits can be annealed together under conditions that are optimal for the renaturation of the separated complementary strands of the DNA double helix.
In the experiment from which Figures 19 (2 A) and 20 (2 A) were taken, a series of photographs taken after equilibrium was reached indicated that a shoulder was forming on the light side of the band of the light strand of T03 DNA. This shoulder appeared at the density of the renatured T03 DNA shown in Figures 19 (2 B) and 20 (2 B). In the region between the light and heavy peaks the concentrations of the two strands were high enough to promote some renaturation of the LNA. As the renaturation process was completed for an individual DNA molecule, the renatured DNA molecule would move in the gradient to a position corresponding to its new buoyant density. The renatured molecules thus migrated through the light strand peak region and increased the ultra- violet absorbance accordingly. The band representing the light strand
is slightly darker than the band representing the heavy strand.
Several attempts were made to increase the height of the peaks, as seen in experiment 1 of Figures 19 and 20, by increasing the concen- tration of DNA. All attempts failed due to extensive renaturation of the DNA during the experiment. The use of the 30 rrnn centrifuge cell,
to increase the length of the light path through the cell, solved this problem by allowing the same concentration of heat denatured T03 DNA to be used as was used in experiment 1. In experiment 2 a 30 rran cell was used. · The light path and hence the absorbance of ultraviolet light, by the DNA in the bands, increased by a factor of 2.5.
The separation of the strands was observed only when a fresh pre- paration of heat denatured DNA was used. One preparation that had been heat denatured and stored at 4°C for 1 week showed extensive renaturation.
Additional observations.
During the course of this study several phenomena were observed that were not really pertinent to the problems being investigated at that time. They were not studied in detail and are thus listed here as observations that might be of interest.
The purification procedure for T03 involves centrifuging the lysate to remove the bacterial cell debris. When this cell debris was examined with a hand spectroscope, two dark absorption bands were observed, one at a wavelength of 570 mµ and the other at a wavelength of 533 mµ. These bands are a characteristic of a reduced cytochrome.
Much lighter bands were present when uninfected bacteria from a control culture were examined. Apparently the cell debris from a phage lysate contains a higher concentration of cytochrome than the bacteria from an uninfected culture. It is not known if the presence of large amounts of cytochrome is the result of induction of a bacterial cytochrome by the phage infection, the synthesis of phage specific cytochrome or just the uncontrolled synthesis of cytochrome that was set off by the phage infection.
While performing some experiments to determine the effect that various ions and sugars have on bacterial growth and phage infection,
it was observed that a medium in which sodium ion was replaced by potassium ion and glucose by sucrose, gave much better adsorption of T03 to its host than the usual TYNGC medium. However, no burst occurred during the 35 minutes of the experiment. In another experiment in which just the sodium ion was replaced by potassium a normal latent period was observed. Potassium or sucrose may enhance adsorption, but apparently sucrose either prevents or delays the burst.
It was found that a bacterial culture that had been grown over- night, well into the stationary phase of growth, gave uniform plaques when used as a lawn for titering phage preparations. The bacterial concentration in the lawn in these experiments was the same as in the usual phage assay. The apparent titer of the phage stock being assayed increased by 60% when stationary phase bacteria were used in the lawn.
This increase in efficiency of plating was discovered too late to be of any use in the experiments reported here.
The optimum growth temperature of the host organism in TYNGC medium was found to be 60°c. The division times at various temperatures were:
so
0c, 90 min; 60°c, 20 min; 65°c, 21 min; 70°c, 70 min.DISCUSSION
Bacteriophage T03 is a virus that infects the thermophilic bac- terium, Bacillus stearothermophilus, at a relatively high temperature of 60°c. The physical dimensions of phage T03 are similar to those of the E. coli phage
A
(57). The size of T03 places it in a range between the larger phages of E. coli such as the T-even phages (57, 58) and the small phages of E. coli such as 0Xl74 (59). T03 is strikingly similar to phage a (60), a bacteriophage that infects Bacillus megaterium. The morphology of other bacteriophages for Bacillus stearothermophilus is not known in detail. Phage 0µ-4 is known to be very small with a re- ported diameter of 10 mµ (22), however, electron micrographs of this phage have not been published. Phage TP-1 has a head diameter of approximately 65 mµ and a tail 240 mµ long and 12 mµ wide (14). T03 is thus in a size range in between those two thermophilic phages.The regular hexagonal appearance of the head of T03 probably indi- cates that the head has the form of an icosahedron, however, more de-
/
tailed work is necessary to verify this. Although the head appears as a regular hexagon in all three staining procedures used (PTA negative, uranyl acetate positive and uranyl acetate negative) the actual size of the head varies considerably with the staining procedure. The length of the head when stained negatively with PTA was 65 mµ. The length when stained positively with uranyl acetate was 47 mµ. With a uranyl acetate negative stain it was 57 mµ long. The heads appeared swollen and there were many ghosts in the PTA preparations and the head outline was quite indefinite in the uranyl acetate positive stained preparations. Since the head outline was well defined and no ghosts
were present in the uranyl acetate negative stained preparations, measurements for the head are those taken from preparations stained
in this manner.
The details of the structure of the tail of T03 were revealed by the uranyl acetate negative staining procedure. Further study is necessary to determine if the tail has a helical form as is probably the case of the E. coli phages, T2 (61) and TS (62).
As would be expected T03 is more thermostable than are phage of mesophiles. In broth, for example, the half life of the E. coli phage Tl is about 12 minutes at 65°C (69), as compared to 120 minutes for T03. Another E. coli phage, T7, is less stable, having a half life of less than one minute at 60°C. The half life of T03 at 60°C is 400 minutes. At 65°C T03 is about 4 times as stable as the thermophilic· phage TP-1 (14) . The thermophilic phage isolated by Onodera was
reported to be stable at l00°C (21). Phage T03 of course is not stable at this high temperature.
The rather normal melting temperature of the T03 DNA, taken togeth- er with the high 6Ha for heat inactivation of the phage, indicate that the proteins of the T03 are primarily responsible for the thermostabil- ity characteristics (25) of T03. Many proteins of mesophilic organisms are denatured rapidly at temperatures above 40 C. 0 The relative thermo-
stability of the proteins of T03 is an indication that they have rather unique structural characteristics. These characteristics might con- sist of a unique amino acid composition, as is the case of the ~-amylase
of Bacillus stearothermophilus, or of a folding arrangement of the polypeptides that is particularly resistant to thermal denaturation.
Thus a further study of the proteins of T03 could yield some insight into the relationship between the primary amino acid sequence and the tertiary structure of proteins in general.
The study of the enzyme that is responsible for lysis of a T03 infected bacterium would also yield valuable information with regard to the relationship between structure and function of enzymes in general.
Further study of the genetics and structure of T03 "lysozyme" would be even more meaningful in view of current research on the genetic control of the structure of T4 phage lysozyme (63, 64) and on the structural characteristics of egg white lysozyme (70, 71). However, T03 "lysozyme"
may not be a lysozyme at all. In strict terms lysozyme is N-acetylmura- mide glycanohydrolase and is known as muramidase. It has not been shown that T03 "lysozyme" is a muramidase. Until the enzymatic charac- teristics of T03 "lysozyme" are known, its relationship to T4 lysozyme should be taken as rather preliminary. This however in no way reduces the significance of this phage enzyme for it is of great interest in and of itself.
The poor adsorption of the T03 to the bacterial cell wall under all of the conditions studied may indicate that the present host is not the true host for this phage. On the other hand it may also mean that the proper envirorunent for good adsorption has not been found.
That the latter might be the case is strengthened by the fact that adsorption could be improved by utilizing various cation and sugar combinations in the media. This is also indicated by the finding that stationary phase bacteria serve as a better lawn than log phase bac- teria. The effect that temperature has on adsorption could probably
be investigated to advantage once a system is found that allows good adsorption. Since no burst was found when potassium ion and sucrose were used in the media, their precise effect on the infected cell could
be fruitfully investigated. This could presumably yield a good method for synchronizing the maturing process.
The deoxyribonucleic acid of T03 has proven to be extremely inter- esting. The determination of the molecular weight from the shape of the band in a cesium chloride density gradient indicates that this method can be used to determine DNA molecular weights to within 20%, provided the DNA is homogeneous with regards to density. This con- clusion was also reached by Studier in his study on the molecular weight of the DNA of bacteriophage T7 (37). The 20% inaccuracy of
this method also indicates that independent measurements of the molecu- lar weight should be made in conjunction with any made by band width.
Any density heterogeniety that was present in the sample of T03 DNA, that was used for the band molecular weight determination, would have broadened the band. The molecular weight that was determined from this broadened band would have been less than the actual molecular weight of native T03 DNA. Any degradation of the DNA in the sample would yield a similar result, a lower molecular weight than the molecular weight of native T03 DNA. The true molecular weight of T03 DNA must there- fore be greater than or equal to the molecular weight that was deter- mined from the band width.
The measurement of the length of the DNA molecule probably gives the best estimate of the actual molecular weight of a particular DNA molecule that can be made at the present time (45). The largest error
that is present in this type of determination is the calibration of
~
the magnification; this amounts to about 10%. The length measurements using the map measure could be reproduced to within 3%.
The base composition of T03 DNA, as determined from the buoyant density and the Tm of T03 DNA, is probably more accurate than the
composition that was determined in the spectrophotometric base analysis.
The reason for this is the relative simplicity of the buoyant density and thermal denaturation experiments and the relative control that is possible when compared to the chromatographic isolation of the bases and the spectrophotometric determination of the base composition. This does not mean however, that these two methods should supplant the
latter, for when a new system is being investigated, the isolation and identification of the bases is invaluable for the detection of bases other than the four that are usually present in DNA. The small incon- sistancy that exists in the base composition, as determined by the Tm and buoyant density measurements on the one hand and the spectrophoto- metric base analysis on the other hand, could be an indication of the presence of small quantities of other bases in addition to the four that were detected. Any base, other than adenine, guanine, thymine and cytosine, that is present in T03 DNA would have to be present in small amounts because of the agreement between the base compositions that were determined from the Tm and the buoyant density. It should be mentioned that at the concentrations 'utilized in the chromatography, a base, other than adenine, guanine, thymine or cytosine, that was present in small amounts, would not have been recognized.
The increase in the melting temperature of T03 DNA with an increase