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Jo 8. Donnellan, Jr.
Biology Division, auk Ridge National Laboratory, Oak Ridge, Tennessee
B. & Mags and A. S. Levinson
Ploneering Aesearch Division, U. S. Arny Matick Laboratories, Matick, Massachusetts

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*Research sponsored in part by the U. 8. Atomic Energy Commdssion under contract
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RNA 16 thought to play several roles in protein synthesis. Ribosomes
+
and ribosomal RNA are looked upon as the sites of protein synthesis while
the role of soluble or transfer RNA 18 to carry activated amino acids prior
to their incorporation into protein. (see, for example, Cold Spring Harbor
Symposia for quantitative Biology, 26, 1961). Jacob and Monud (1961) proposed
the existence of a third type of RNA involved in coding the amino acid
sequence of induced enzymes. This latter type of RNA, called messenger
or mRNA, should have a base sequence 11ke the DNA of the induced cell and,
.
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.
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at least in the case of induced enzyme synthesis, should turn over rapidly.
The presence of a type of RNA having these properties has been shown in many
systems (see, Volkin, 1963). Since bacterial spore formation and germination
are thought to involve considerable induced enzyme syathesis, it would seem
usefu to investigate the types of nucleic acids syathesized during these
4
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periods
NA
Mtz-James (1955) studied the phosphorus containing fractions of germinating
bacterial spores and observed an increase, during germination, in the total
RNA phosphorus. Woese (1961) observed the appearance of RNA particles having
unusual sedimentation properties during germination and postgerminative
development while woese and Forro (1960) proposed the turnover of a large
part of the spore RNA a8 & result of their studies on the kinetics of RNA
.
Agar
synthesis during this period.
The absence of DNA-like RNA in bacterial spores
has been suggested by Doi and Igarashi (1964) from their studies on the
fractionation of spore nucleic acids on methylated albumin-keselguhr colums .
These authors showed that spore RNAs differ only quantitatively from the
RNAS in vegetative cells. The present paper represents a study of the synthes 16
2242
of nucleic acido found in bacterial spores during germination and postgermd native
development.
Materials and Methods
Spores of the Marburg strain of Bacillus subtilis were prepared from
a chemically defined medium as described by Donnellan, Nags, and Levinson
(1964). Spores were separated from vegetative cells and debris by a
polyethylene glycol-potassium phosphate system similar to that described
by Sachs and Alderton (1961). After several water washes, the spores were
lyophilized and stored in vacuo. Microscopic examination indicated the preparations
contained greater than 95% free spores .
Por studies on germination and postgerminative development the synthetic
medium described by Donnellan, Nags and Levinson (1964) was used. Suspensions
containing a known weight of spores were incubated on a reciprocal shaker
at 35°C. At appropriate time intervals, aliquots were removed from the
suspensions , diluted and the turbidity measured at 440 mu in a Beckoman DU
spectrophotometer. At simlar intervals smears of the cultures were prepared
on microscope slides and later scored for spores, germinated spores, emerged
cells and dividing cells as previously described (Donnellan, Nage, and Levinson,
1964). Figures 1 and 2 show the results of measurements of turbidity and
microscopic examination of several samples of spores and germinating spores
during germination and postgerminative development. The points represent
averages of cultures measured at simdlar times during this period. :
At appropriate times during germination and postgerminative development
a suspension of cells was poured over a frozen salts solution to stop further
growth. The celle were collected by centrifugation and washed at 4°c.
Subsequent extractions and precipitations were al80 carried out at 4°C.
Samples were ground in a mortar with acid washed sand and the nucleic
acids extracted with phenol by a method similar to that of Gierer and
Schramm (1956). Dae tenth mm. MgCl, and 0.2% Dupanol in 20 mm tris
buffer (på 7.4) were present during the grinding and extraction. The
nucleic acids were precipated two or three times in 70% ethanol. Following
the last ethanol precipitation the nucleic acids were dissolved in 50 mm
sodium phosphate buffer (PR 667) containing 0.1 M Naci. 1.9 to 5.7 ml
of this solution containing approximately 2 mgm of nucleic acid (50
optical density units) were applied to methylated albumin-kieselguhr
colume. Columns 0.8 cm in diameter by 14 cm high were prepared using
the simplified technique of Monier et al. (1962). Elution was performed
using a linear NaCl gradient from 0.1 M to 1.2 M in 50 mm sodium phosphate
buffer (pH 6.7) with the colums maintained at 20°c. 3.5 ml fractions
were collected and analyzed in a Beckman DU spectrophotometer for W absorption
at 255 mu. In radioactivity studies, 1 to 2 mc of $20, were added for the
five minutes immediately prior to the collection of cells. One ml samples
of fractions eluting from the colums were counted in a liquid scintillation
counter wing a Dioxane, Naphthalene, Cellosolve, PPO, POPOP scintillation
fluid (Bruno and Christian, 1961).
Results and Discussion
Figure 3 18 a typical elution pattern of nucleic acids from a methylated
albumin-kieselguhr colum. In this case the spores had been grown for
165 min before they were fractionated and analyzed. RNA determinations
by the orcinol method (schneider 1957) and DNA measurements by the Keck
modification of the Ceriotti reaction (Keck 1956) indicate that peaks I,
III, and IV contain RNA while peak II contains DNA. Sueoka and Cheng (1962)
have shown that peak I contains transfer RNA wille peaks III and IV contain
168 and 238 ribosomal RNA, respectively. A series of elution patterns was
obtained for samples fractionated after various periods of germination and
postgerminative development. The relative amounts of transfer RNA,
ribosomal RNA and DNA were determined by suming the optical densities of
the fractions in each peak and subtracting a baseline contribution for a
similar number of fractions. From the relative amount of each nucleic acid
in the elution patterns and the total yield of nucleic acids per mg of
spore inoculum, the amounts of each type of nucleic acid per mg of spore
inoculum were determined after various periods of growth. These data
are shown in Mgure 4. It is clear that the synthesis of DNA 18 delayed
for about 120 minutes at the start of germination and postgerminative
development, while the synthesis of ribosomal RNA commences immediately.
These results are similar to those reported by Fitz-James (1955), Woese
(1961) and Woese and Forro (1960). In addition, these data indicate that
the synthesis of transfer RNA commences at the same time as synthesis of
ribosomal RNA.
The relative amounts of ribosomal RNA ( rRNA.) and transfer RNA ( 8 RNA.)
in the elution patterns were bed to determine the ratio of rRNA to BRNA
at various times dwing germination and postgermd nation development, as
shown in Figure 5. From these data there appears to be a differential
synthesis of these two types of RNA.
Figure . 6 18 a typical elution pattern of extracts from spores exposed
to peo, for 5 minutes after growing for 60 minutes. For growth periods
less than 60 minutes the spores are synthesizing a large amount of RNA that
elutes after peak IV as shown by the broadening of the radioactivity peak
in the 238 region. In addition, a large amount of material eluting in
the region of peak III (168 ribosomal RNA) 18 synthesized during this period.
50% growth periods of 90 minutes or longer the material formed during the
five minute p32 pulse appears similar to the total nucleic acids of the
cell (Figure 7). Midgley and McCarthy (1962) have shown in vegetative
bacteria that a 5 minute p32 pulse yields elution patterns of radioactivity
closely approximating the pattern of the total nucleic acids of the cell
as shown in Figure 7. At all times during germination and postgerininative
development pulses shorter than 5 minutes yield elution patterns with
radioactivity in regions beyond the 235 ribosomal RNA peak as observed in
several vegetative bacterial cells (Midgley and McCarthy, 1962) and in
cells of B. subtilis Just prior to sporulation (Doi and Igarashi, 1964).
The nature of the radioactive material eluting in fractions 10 to 25
(0.15 M NaCl) and the material eluting between the transfer RNA and DNA
peaks was not determined in these studies.
From these studies we canclucle that a differential synthesis of nucleic
acids occurs during germination and postgerminative development of bacterial
spores.
Initially and almost immediately in germination the synthesis of
ribosomal and transfer RNAs occur, fallowed at 120 minutes by the synthesis
of DNA. The data of Figure 5 do not permit a more accurate determination
of the kinetics of the change in the ratio of ribosomal RNA to transfer RNA.
From these data 1t 18 not clear whether this ratio increases gradually
throughout the early stages of germination or whether the change occurs as
late as 120 minutes when DNA synthesis commences. It is probably a coincidence
that, in this system, DNA synthesis starts at the same time as the cells
emerge, although other types of metabolic activity, such as an increase
in the rate of oxygen uptake, have been shown to coincide with emergence
(Mandels et al. 1956).
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In addition, early in germination, types of RNA are produced which
differ from those formed later in germination and during vegetative cell
growth. This difference is either in the amount of material produced
during the 5 minute labeling interval as seen in peak III of Figure 6
or the difference is in the elution characteristic of this material as
seen by the broadening of peak IV in Figure 6. Since bacterial spores
apparently do not contain DNA-like RNA (Doi and Igarashi, 1964), it would
not be surprising that germinating spores must produce larger amounts of
this material than vegetative cells. Different interpretations for the
appearance of the type of RMA observed in these studies are, of course,
possible.
References
Bruno, G. A. and Christian, J. E. 1961. Determination of carbon-14 in
aqueous bicarbonate solutions by liquid scintilation-counting
techniques. Application to biological fluids. Anal. Chem. 33 1216-1218.
Doi, R. H. and Igarashi, R. T. 1964. Ribonucleic acids of Bacillus subtilis
spores and sporulating cells. J. Bacteriol. 87 323-328
limo
Donnellan, J. E., Jr.; Naga, E. B.; and Levinson, H. S. 1964. Chemically
defined, synthetic media for sporulation and for germination and
growth of Bacillus subtilis. J. Bacteriol. 87 332-336
Fitz-James, P. C. 1955. The phosphorus fractions of Bacillus cereus
and Bacillus Megaterium II. A correlation of the chemical with the
cytological changes occuring during spore germination. Can. J. Microbiol.
1 525-548
Gierer, A. and Schramm, "Q. 1956. Infectivity of ribonucleic acid from
tobacco mosaic virus. Nature 177 702-703
Jacob, F. and Monod, J. 1961. Genetic regulatory mechanism in the
synthesis of protein. J. Mol. Biol. 2 318-356
Keck, K. 1956. An ultramicro technique for the determination of
deoxypentose nucleic acid. Arch• Biochem. Blophys • 63 446-451
Mandels, G. R.; Levinson, H. S. and Hyatt, M. T. 1956. Analysis of respiration
during germination and enlargement of spores of Bacillus megaterium
· and of the fungus Myrothecium verrucaria. J. Gen. Physiol. 32 301-309
Midgley, J. E. M. and McCarthy, B. J. 1962. The synthesis and kinetic
behavior of deoxyribonucleic acid-like ribomucleic acid in bacteria.
Biochim. et Biophis. Acta 61 696-717
Monier, R.; Naono, S.; Hayes, D.; Hayes, F. and Gros, F. 1962. Studies
on the heterogeneity of messenger RNA from E. coli. J. Mol. Biol. 2
301-324
Schneider, W. C. in Colvick, 8. P. and Kaplan, N. 0. editors. Methods
in Enzymology. Academic Press Inc. New York. 1957. Volume III,
p. 680
Sueoka, N. and Cheng, T. 1962. Fractionation of nucleic acids with the
methylated albumin colum. J. Mol. Biol. 4 161-172
volkin, E. 1963. Blosynthesis of RNA in relation to genetic coding
problems. Molecular Genetics, J. H. Taylor, editor. Academic
Press Inc. New York. Part 1, p. 272-289
Woese, C. R. 1961. Unusual ribosome particles occuring diring spore
germination. J. Bacteriol. 82 695-701 •
Woese, C. R. and Porro, J. R. 1960. Correlations between ribonucleic
acid and deoxyribonucleic acid metabolism during spore germination.
J. Bacteriol. 80 812-817
Figure Legends
Figure 1. Turbidity vs. time curve of Bacillus subtilis during germination
postgerminative development. Turbidity determined at 440 mu after
diluting a sample of culture 1:6 in phosphate buffer. Points are
avereges of several determinations.
Figure 2. Germination, emergence, and first division of Bacillus subtilis
spores as determined by scoring, microscopically, slides made at
various times during germination and postgerminative development.
Points are averages of several determinations.
Figure 3. Hution pattern from a methylated album, n-kieselguhr column
of nucleic acids extracted from germinated Bacillus subtilis spores
after 165 minutes growth. The presence of nucleic acids was determined
by measuring the absorbance of the fractions at 255 m.
Figure 4. Total of each type of nucleic acid per mg of spore inoculum
after various periods of growth during germination and postgerminative
development of Bacillus subtilis ·
Figure 5. Ratio of ribosomal RNA to transfer RNA at various times during
germination and postgerminative development of Bacillus subtilis.
Figure 6. Elution pattern from a methylated albumin-kieselguir column of
nucleic acids extracted from Bacillus subt1118 spores after 60
minutes growth. Total nucleic acids determined by their absorption
at 255 mu po incorporated during a 5 minute pulse at the
end of the growth period om determined by counting samples of fractions
in a liquid scintillation counter.
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DATE FILMED
12/9/64







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END