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The isolation of nucleic acids from gram-positive bacteria
ARCHIVES
OF
BIOCHEMISTRY
The Isolation
AND
128,579-582
BIOPHYSICS
of Nucleic
Acids
A. S. JONES The Chemistry
Department, Received
March
from
Gram-positive
Bacteria
R. T. WALKER
AND
University
(1968)
of Birmingham,
29, 1968; accepted
Birmingham
August
16, England
27, 1968
Bentonite has been shown to be an effective inhibitor of enzymes which degrade nucleic acids in certain bacterial extracts. By the use of bentonite and mechanical breakage, DNA and RNA have been isolated from Lactobacillus acidophik and Streptococcus pyogenes. The base compositions have been determined; the GC content The of the DNA for L. acidophilus and S. pyogeneswere 40 and 39.5cfi, respectively. DNA from the two organisms isolat.ed by this procedure each had a molecular weight of 2 X lo6 and the ribosomal RNA in each case was a mixture of components of 20s and 27s.
The procedure most commonly used for the isolation of nucleic acids from micro-organisms is that described by Marmur (1) in 1961. The method appears to be applicable to all Gram-negative micro-organisms and also to a number of Gram-positive ones. The method involves lysis of the cells with 2 5% sodium dodecyl sulphate at 60” for 10 min to extract the nucleic acids followed by their isolation and purification. However, most Gram-positive micro-organisms are resistant to lysis by detergent and only some of these become susceptible after treatment with lysosyme. The nucleic acids can only be obtained from the species which are resistant to both of the mentioned procedures after breaking the cell mechanically, which incurs the danger of mechanical and enzymic degradation of the nucleic acids. One of the most convenient ways of breaking a large number of bacterial cells is in the presence of ballotini glass beads in a rotary ball mill, but precautions have to be taken to prevent enzymic degradation of the nucleic acids and detergents cannot be used because of the resulting loss in breaking efficiency. Previous work (2) has shown that bentonite can be used to inhibit ribonuclease and it appears that it also inhibits deoxyribonuclease (3). The present paper describes the effect of bentonite on the enzymic activity 579
of an extract of broken bacterial cells and a method involving the use of bentonite is described for the isolation of the nucleic acids from two Gram-positive bacteria which are resistant to lysosyme and detergents. MATERIALS
AND
METHODS
Bentonite A suspension of bentonite in 0.01 3~ acetate buffer pH 6.0 was prepared by the method of Brownhill et al. (2). The optical density’ of the final washings of the bentonite was less than 0.2 at 260 mp. Source and Growth
of the Bacteria
Lactobacillus acidophilus, NCIB 8795, was grown in dehydrated whey, 1.3’%, Oxoid peptone, l%, at 37” for 48 hr. The yield of organisms was 120 mg (dry weight)/liter. Streptococcus pyogenes (Group A), was obtained from Dr. P. Pease (Bacteriology Department, Birmingham University, England), and was grown in Oxoid peptone, 1%; sodium chloride, 0.5yo; disodium hydrogen orthophosphate, 1.73%; sodium dihydrogen orthophosphate, 0.27%; glucose, 0.591,. The yield of organisms was 176 mg (dry weight)/liter. Clostridium welchii (NCTC 10578), was grown in Robertson’s cooked meat medium which had been heated to 100” and rapidly cooled to 37” immediately before inoculation. The inoculum was 1 All measurements in l-cm cells.
of optical
density
were made
580
JONES
AND
incubated at 37” for 18 hr and then used to innoculate 500 ml of a medium containing Lablemco beef extract, 0.1%. Oxoid yeast extract, 0.27o, Oxoid peptone, 0.59&, sodium chloride, 0.5’& pH 7.4. The air was expelled from this medium by heating it in screw-capped bottles in an autoclave for 15 min at 15 lb/sq in. The medium was rapidly cooled, 0.570 sterile glucose solution added, followed by 2 ml of inoculum, the bottle filled to the top with more medium from which the air had been expelled and the whole incubated at 37” for 18 hr. Serratia marcescens, NCTC 1377, was grown for 18 hr at 25” in glass trays (12 X 12 inches) containing nutrient agar as previously described (4). Harvesting The cells were harvested by centrifugation of the medium at 0’ at 23,000g for 20 min, except for the cells from S. marcescens which were first scraped from the surface of the agar and washed with 0.14 M sodium chloride solution. Cell Breakage The cells from Cl. we&ii and S. marcescens were broken in aMickle tissue disintegrator (H. Mickle, Mill Works, Gomshall, Surrey, England), in 0.01 M acetat,e buffer (2 ml) pH 6, in the presence of glass beads (1 ml, Junior size, 0.15-mm diameter, Prism0 Safety Corporation, Huntingdon, Pennsylvania, U.S.A.) (5). The cell extract was allowed to stand at 37” for 24 hr to allow the enzymic degradation of the nucleic acids present to take place, the solution dialyzed against 2 M sodium chloride and then exhaustively dialyzed against water to yield a solution which was capable of degrading both DNA and RNA. The optical densities of the solutions at 260 m when diluted to 20 ml (from the cells grown in 500 ml of medium for Cl. we&ii and two plates for S. murcescens) were less than 0.2. To portions of each of the dialyzed solutions (10 ml) a suspension of bentonite (2 ml) was added and the mixtures were shaken at room temperature for 30 min and the bentonite removed by centrifugation at 23,000g. The resulting solution was then tested for its ability to degrade DNA and RNA and was compared with the remainder of the solutions which had not been treated with bentonite. Determination
of the Ability of Solutions DNA and RNA
to Degrade
A solution of DNA or RNA (10 ml), in 0.01 M acetate buffer pH 6, with an optical density of about 1.5 at 260 rng was added to one of the cell extracts (1 ml) isolated as described above from Cl. welchii or S. marcescens, before or after the bentonite treatment, and the solutions incubated at 37”. At intervals, samples (1 ml) were removed,
WALKER 1 M calcium chloride in 75% ethanol (0.5 ml) added and the solutions allowed to stand. The high molecular weight nucleic acids were removed by centrifugation at 20009 and the optical density of the degraded nucleic acids in the supernatant liquids at 260 ml determined. Procedure
for the Isolation of Nucleic Gram-positive Micro-organisms
Acids from
The ribosomal RNA, RNA soluble in M salt and DNA were isolated from S. pyogenes and L. acidophilus in the following manner: Cells from 1 liter of culture medium were obtained by centrifugation at 23,OOOg, washed with 0.14 M sodium chloride and broken in a Mickle tissue disintegrator in 0.01 M acetate buffer pH 6 (2 ml), in the presence of a suspension of bentonite (2 ml) and glass beads (1 ml) for 15 min with cooling (6). The suspension was then centrifuged at 23,OOOgand the optical density of the supernate at 260 ml was determined. The precipitate was reextracted with acetate buffer until no more ultraviolet-absorbing material was extracted, (usually three extractions were required). The supernatant liquids were combined and shaken with chloroform (six times) to remove all traces of protein, ethanol (3 vol) added to the aqueous layer, the precipitate removed by centrifugation, dissolved in 0.2 M sodium chloride (10 ml) and CTAB2 (470 aqueous solution) added until precipitation was complete (about 1 ml required). The precipitate, consisting of the cetyltrimethylammonium salts of the cellular acidic polymeric material, was dissolved in M sodium chloride (10 ml) and the sodium salts of the polymers precipitated by the addition of ethanol (3 vol). The precipitate was dissolved in water (5 ml) which was made M with respect to sodium chloride and left at 0” overnight. The precipitate of ribosomal RNA was removed by centrifugation at 23,OOOg, was reprecipitated from M sodium chloride, dissolved in water, dialyzed and freeze-dried. CTAB was added to the material soluble in M sodium chloride and the concentratiorr of the latter reduced to 0.6 M with a final concentration of CTAB of 1%. The resulting precipitate of the cetyltrimethylammonium salt of DNA was dissolved in M sodium chloride and the sodium salt precipitated by the addition of ethanol (3 ~01). The DNA was dissolved in water, shaken with chloroform to remove traces of CTAB and the incubated with ribonuclease layer aqueous (Armour, crystalline) for 4 hr to remove traces of RNA. The enzyme was removed by shaking the solution six times with chloroform, the aqueous layer was dialyzed against 2 M sodium chloride, 2 Cetyltrimethylammonium
bromide.
NUCLEIC
ACIDS
FROM
GRAM-P0SITIT.E TABLE
THE
EFFECT
OF BENTONITE
ON THE
581
BACTERIA
I
ENZYMIC
ACTIVITY
OF CELL
EXTRACTS
The effect of bentonite on the enzymic activit.y of cell extracts from S. marcescens and CZ. welehii as measured by their ability to render DNA and R?JA nonprecipitable by calcium chloride in ethanol. The figures given are the percentages of nonprecipitable DNA or RNA. Cell extract sauce
With bentonite treatment
Nucleic acid substrate 0 min
yeast RNA S. marcescens RNA Calf thymus DNA S. marcescens DNA
Cl. welchii S. marcescens Cl. welchii S. marcescens
30 min 120 min 24 hr
5 2 0 0
then against water and freeze-dried. Ethanol (3 vol) was added to the supernatant liquid from the CTAB precipitation at 0.6 M sodium chloride and the precipitate thus obtained of the sodium salt of RNA soluble in M sodium chloride was dissolved in water, dialyzed and freeze-dried. Purine and Ppimidine Contents of Nucleic Acids DN.4 samples were hydrolyzed as described by Wyatt and Cohen (7) and the bases det,ermined as described by Wyatt (8). RNA samples were hydrolyzed and the bases determined as described by Markham and Smith (9) after separating the bases in the solvent, system described by Kirby (10)). l’otal
Phosphorus
Total phosphorus was estimated of Jones, Lee and Peacocke (11). RESULTS
AiYD
by t.he method
DISCUSSION
It was found that the cells of S. mwcescew and Cl. welchii could be broken in the P\lickle tissue disintegrator in 0.01 M acetate buffer pH6, in the presence of glass beads. The nucleic acids in the cell extract were allowed to be degraded by the enzymes present and the solutions were dialyzed so that the optieal density of the solution at 260 rnp was less than 0.2. A suspension of bentonite was added to a portion of the solutions and after centrifugation the enzyme activity of the cell extracts was tested against DNA and RNA. The results are shown in Table I. These results show that bentonite is effective in completely removing or inhibiting the action of the enzymes responsible for the degradation of the nucleic acids. The cell extract of Cl. welchii was chosen as representative of a Gram-posit,ive micro-organism and difficulty had been encountered in previous attempts to isoIate the nucleic acids from
3 2 0 0
4 2 0 0
5 2 0 0
Without bent&k 48
hr
0 min
--0 0
5 2 0 0
TABLE NUCLEIC
ACIDS ISOLATED
85 25 0 0
96 98 3 2
48 hr
100 100 6 12 32 80
II FROM
GRAM-POSITIVE
RNA
BACTERIA-SOLUBLE
Wt. obtained/g. dry organism High molecular weight RZVll Wt. obtained/g. dry organism Phosphorus content (s,) Base cont,ent (per 100 nucleotides) Guanine Adenine Cytosine Uracil DNA Wt. obtained/g. dry organism Phosphorus content (%) Base content (per 100 nucleotides) G\lanine Adenille Cytosine Thymine 7; GC
treatment
30 min 120 min 24 hr
Lactobacillus acid@hilus
Streptococcus ~yogenes
13 mg.
33 mg.
43.5 mg.
80 mg.
8.8
8.8
29.0 26.4 23.8 20.8
28.9 27.3 22.7 21.1
G.5 mg.
17 mg.
9.0
8.9
20.4 30.3 19.6 29.7 40.0
19.5 30.5 20.0 30.0 39.5
Cl. welchii due to enzymic degradation. However, the results with the DNA degradation were not cowlusive, as the method chosen to demonstrate degradation was not very sensitive and could probably only detect nucleic acids which had been extensively degraded. The cell extract from S. marcescens was used as it had previously been found that an enzyme causing rapid degradation of DNA was present in this organism (4).
582
JONES
AND
From these results on enzyme inhibition by bentonite, it was decided to attempt to isolate the nucleic acids from two Gram-positive bacteria-Xtreptococcus pyogenes and Lactobacillus acidophilus. These two organisms are quoted by Marmur (1) as being resistant to detergent and lysozyme and thus the nucleic acids cannot be isolated by his method. The nucleic acids were isolated from the organisms which were grown in a fairly small volume of medium (1 liter), but the procedure is such that the amount of cells used could be greatly increased and the cells broken in a rotary ball mill instead of the Rfickle tissue disintegrator. Bentonite prevented any extensive degradation of the ribosomal RNA as shown by the fact that the preparations obtained from both organisms contained components of 20s and 27s. Last traces of RNA were removed from the DNA with ribonuclease because on the small scale used, losses of DNA were large if a second fractionation with CTAB was attempted. The sedimentation coefficient for the samples of DNA from both organisms was 14S, indicating a DNA of molecular weight of about 2 X 106. The samples of freeze-dried DKA showed a hyperchromic effect at 260 rnp of 15% when heated to 100” and then rapidly cooled. The weights obtained and the analyses of the nucleic acids are given in Table II. This method of isolation of nucleic acids from micro-organisms which are resistant to detergent and lysosyme has several advantages over those at present available. NO
WALKER
drastic chemical treatment of the cells is involved and enzymic activity is inhibited. The mechanical treatment, although obviously causing some degradation of the nucleic acids, gives DNA of sufficiently high molecular weight to be of use in cross hybridization experiments with high molecular weight DNA trapped in agar or upon Millipore filters. The method has also been shown to be of use in the isolation of the nucleic acids from CI. welchii and animal tissues, the results of which will be reported elsewhere. ACKNOWLEDGMENT We thankMr.
E. E T. J. Chelton
for assistance.
REFERENCES 1. MARMUR, J., 1. Mol. Biol. 3, 208 (1961). 2. BROWNHILL, T. J., JONES, A. S., AND STACEY, M., Biochem. J. 73, 434 (1959). 3. KRECHETOVA, G. D., CHUDINOVA, I. A., AND SHAPOT, V. S., Biokhimiya, 28, 682 (1963). 4. JONES, A. S., AND WALKER, R. T., J. Gen. Microbial. 31, 187 (1963). 5. KOLBE, J. J., Biochim. Biophys. dcta 38, 373 (1960). 26, 178 6. CHELTON, E. T. J., J. rlppl. Bacterial. (1963). 7. WYATT, G. R.., AND COHEN, S. S., Biochem. J. 66, 734 (1953). 8. WYATT, G. R., Biochem. J. 48, 584 (1951). 9. MARKHAM, R., AND SMITH, J. D., Biochem. J. 49, 401 (1951). 10. KIRBY, K. S., Biochim. Biophys. Acta 18, 575 (1955). 11. JONES, A. S., LEE, W. A., AND PEACOCKE, A. R., J. Chem. Sot. 623 (1951).
OF
BIOCHEMISTRY
The Isolation
AND
128,579-582
BIOPHYSICS
of Nucleic
Acids
A. S. JONES The Chemistry
Department, Received
March
from
Gram-positive
Bacteria
R. T. WALKER
AND
University
(1968)
of Birmingham,
29, 1968; accepted
Birmingham
August
16, England
27, 1968
Bentonite has been shown to be an effective inhibitor of enzymes which degrade nucleic acids in certain bacterial extracts. By the use of bentonite and mechanical breakage, DNA and RNA have been isolated from Lactobacillus acidophik and Streptococcus pyogenes. The base compositions have been determined; the GC content The of the DNA for L. acidophilus and S. pyogeneswere 40 and 39.5cfi, respectively. DNA from the two organisms isolat.ed by this procedure each had a molecular weight of 2 X lo6 and the ribosomal RNA in each case was a mixture of components of 20s and 27s.
The procedure most commonly used for the isolation of nucleic acids from micro-organisms is that described by Marmur (1) in 1961. The method appears to be applicable to all Gram-negative micro-organisms and also to a number of Gram-positive ones. The method involves lysis of the cells with 2 5% sodium dodecyl sulphate at 60” for 10 min to extract the nucleic acids followed by their isolation and purification. However, most Gram-positive micro-organisms are resistant to lysis by detergent and only some of these become susceptible after treatment with lysosyme. The nucleic acids can only be obtained from the species which are resistant to both of the mentioned procedures after breaking the cell mechanically, which incurs the danger of mechanical and enzymic degradation of the nucleic acids. One of the most convenient ways of breaking a large number of bacterial cells is in the presence of ballotini glass beads in a rotary ball mill, but precautions have to be taken to prevent enzymic degradation of the nucleic acids and detergents cannot be used because of the resulting loss in breaking efficiency. Previous work (2) has shown that bentonite can be used to inhibit ribonuclease and it appears that it also inhibits deoxyribonuclease (3). The present paper describes the effect of bentonite on the enzymic activity 579
of an extract of broken bacterial cells and a method involving the use of bentonite is described for the isolation of the nucleic acids from two Gram-positive bacteria which are resistant to lysosyme and detergents. MATERIALS
AND
METHODS
Bentonite A suspension of bentonite in 0.01 3~ acetate buffer pH 6.0 was prepared by the method of Brownhill et al. (2). The optical density’ of the final washings of the bentonite was less than 0.2 at 260 mp. Source and Growth
of the Bacteria
Lactobacillus acidophilus, NCIB 8795, was grown in dehydrated whey, 1.3’%, Oxoid peptone, l%, at 37” for 48 hr. The yield of organisms was 120 mg (dry weight)/liter. Streptococcus pyogenes (Group A), was obtained from Dr. P. Pease (Bacteriology Department, Birmingham University, England), and was grown in Oxoid peptone, 1%; sodium chloride, 0.5yo; disodium hydrogen orthophosphate, 1.73%; sodium dihydrogen orthophosphate, 0.27%; glucose, 0.591,. The yield of organisms was 176 mg (dry weight)/liter. Clostridium welchii (NCTC 10578), was grown in Robertson’s cooked meat medium which had been heated to 100” and rapidly cooled to 37” immediately before inoculation. The inoculum was 1 All measurements in l-cm cells.
of optical
density
were made
580
JONES
AND
incubated at 37” for 18 hr and then used to innoculate 500 ml of a medium containing Lablemco beef extract, 0.1%. Oxoid yeast extract, 0.27o, Oxoid peptone, 0.59&, sodium chloride, 0.5’& pH 7.4. The air was expelled from this medium by heating it in screw-capped bottles in an autoclave for 15 min at 15 lb/sq in. The medium was rapidly cooled, 0.570 sterile glucose solution added, followed by 2 ml of inoculum, the bottle filled to the top with more medium from which the air had been expelled and the whole incubated at 37” for 18 hr. Serratia marcescens, NCTC 1377, was grown for 18 hr at 25” in glass trays (12 X 12 inches) containing nutrient agar as previously described (4). Harvesting The cells were harvested by centrifugation of the medium at 0’ at 23,000g for 20 min, except for the cells from S. marcescens which were first scraped from the surface of the agar and washed with 0.14 M sodium chloride solution. Cell Breakage The cells from Cl. we&ii and S. marcescens were broken in aMickle tissue disintegrator (H. Mickle, Mill Works, Gomshall, Surrey, England), in 0.01 M acetat,e buffer (2 ml) pH 6, in the presence of glass beads (1 ml, Junior size, 0.15-mm diameter, Prism0 Safety Corporation, Huntingdon, Pennsylvania, U.S.A.) (5). The cell extract was allowed to stand at 37” for 24 hr to allow the enzymic degradation of the nucleic acids present to take place, the solution dialyzed against 2 M sodium chloride and then exhaustively dialyzed against water to yield a solution which was capable of degrading both DNA and RNA. The optical densities of the solutions at 260 m when diluted to 20 ml (from the cells grown in 500 ml of medium for Cl. we&ii and two plates for S. murcescens) were less than 0.2. To portions of each of the dialyzed solutions (10 ml) a suspension of bentonite (2 ml) was added and the mixtures were shaken at room temperature for 30 min and the bentonite removed by centrifugation at 23,000g. The resulting solution was then tested for its ability to degrade DNA and RNA and was compared with the remainder of the solutions which had not been treated with bentonite. Determination
of the Ability of Solutions DNA and RNA
to Degrade
A solution of DNA or RNA (10 ml), in 0.01 M acetate buffer pH 6, with an optical density of about 1.5 at 260 rng was added to one of the cell extracts (1 ml) isolated as described above from Cl. welchii or S. marcescens, before or after the bentonite treatment, and the solutions incubated at 37”. At intervals, samples (1 ml) were removed,
WALKER 1 M calcium chloride in 75% ethanol (0.5 ml) added and the solutions allowed to stand. The high molecular weight nucleic acids were removed by centrifugation at 20009 and the optical density of the degraded nucleic acids in the supernatant liquids at 260 ml determined. Procedure
for the Isolation of Nucleic Gram-positive Micro-organisms
Acids from
The ribosomal RNA, RNA soluble in M salt and DNA were isolated from S. pyogenes and L. acidophilus in the following manner: Cells from 1 liter of culture medium were obtained by centrifugation at 23,OOOg, washed with 0.14 M sodium chloride and broken in a Mickle tissue disintegrator in 0.01 M acetate buffer pH 6 (2 ml), in the presence of a suspension of bentonite (2 ml) and glass beads (1 ml) for 15 min with cooling (6). The suspension was then centrifuged at 23,OOOgand the optical density of the supernate at 260 ml was determined. The precipitate was reextracted with acetate buffer until no more ultraviolet-absorbing material was extracted, (usually three extractions were required). The supernatant liquids were combined and shaken with chloroform (six times) to remove all traces of protein, ethanol (3 vol) added to the aqueous layer, the precipitate removed by centrifugation, dissolved in 0.2 M sodium chloride (10 ml) and CTAB2 (470 aqueous solution) added until precipitation was complete (about 1 ml required). The precipitate, consisting of the cetyltrimethylammonium salts of the cellular acidic polymeric material, was dissolved in M sodium chloride (10 ml) and the sodium salts of the polymers precipitated by the addition of ethanol (3 vol). The precipitate was dissolved in water (5 ml) which was made M with respect to sodium chloride and left at 0” overnight. The precipitate of ribosomal RNA was removed by centrifugation at 23,OOOg, was reprecipitated from M sodium chloride, dissolved in water, dialyzed and freeze-dried. CTAB was added to the material soluble in M sodium chloride and the concentratiorr of the latter reduced to 0.6 M with a final concentration of CTAB of 1%. The resulting precipitate of the cetyltrimethylammonium salt of DNA was dissolved in M sodium chloride and the sodium salt precipitated by the addition of ethanol (3 ~01). The DNA was dissolved in water, shaken with chloroform to remove traces of CTAB and the incubated with ribonuclease layer aqueous (Armour, crystalline) for 4 hr to remove traces of RNA. The enzyme was removed by shaking the solution six times with chloroform, the aqueous layer was dialyzed against 2 M sodium chloride, 2 Cetyltrimethylammonium
bromide.
NUCLEIC
ACIDS
FROM
GRAM-P0SITIT.E TABLE
THE
EFFECT
OF BENTONITE
ON THE
581
BACTERIA
I
ENZYMIC
ACTIVITY
OF CELL
EXTRACTS
The effect of bentonite on the enzymic activit.y of cell extracts from S. marcescens and CZ. welehii as measured by their ability to render DNA and R?JA nonprecipitable by calcium chloride in ethanol. The figures given are the percentages of nonprecipitable DNA or RNA. Cell extract sauce
With bentonite treatment
Nucleic acid substrate 0 min
yeast RNA S. marcescens RNA Calf thymus DNA S. marcescens DNA
Cl. welchii S. marcescens Cl. welchii S. marcescens
30 min 120 min 24 hr
5 2 0 0
then against water and freeze-dried. Ethanol (3 vol) was added to the supernatant liquid from the CTAB precipitation at 0.6 M sodium chloride and the precipitate thus obtained of the sodium salt of RNA soluble in M sodium chloride was dissolved in water, dialyzed and freeze-dried. Purine and Ppimidine Contents of Nucleic Acids DN.4 samples were hydrolyzed as described by Wyatt and Cohen (7) and the bases det,ermined as described by Wyatt (8). RNA samples were hydrolyzed and the bases determined as described by Markham and Smith (9) after separating the bases in the solvent, system described by Kirby (10)). l’otal
Phosphorus
Total phosphorus was estimated of Jones, Lee and Peacocke (11). RESULTS
AiYD
by t.he method
DISCUSSION
It was found that the cells of S. mwcescew and Cl. welchii could be broken in the P\lickle tissue disintegrator in 0.01 M acetate buffer pH6, in the presence of glass beads. The nucleic acids in the cell extract were allowed to be degraded by the enzymes present and the solutions were dialyzed so that the optieal density of the solution at 260 rnp was less than 0.2. A suspension of bentonite was added to a portion of the solutions and after centrifugation the enzyme activity of the cell extracts was tested against DNA and RNA. The results are shown in Table I. These results show that bentonite is effective in completely removing or inhibiting the action of the enzymes responsible for the degradation of the nucleic acids. The cell extract of Cl. welchii was chosen as representative of a Gram-posit,ive micro-organism and difficulty had been encountered in previous attempts to isoIate the nucleic acids from
3 2 0 0
4 2 0 0
5 2 0 0
Without bent&k 48
hr
0 min
--0 0
5 2 0 0
TABLE NUCLEIC
ACIDS ISOLATED
85 25 0 0
96 98 3 2
48 hr
100 100 6 12 32 80
II FROM
GRAM-POSITIVE
RNA
BACTERIA-SOLUBLE
Wt. obtained/g. dry organism High molecular weight RZVll Wt. obtained/g. dry organism Phosphorus content (s,) Base cont,ent (per 100 nucleotides) Guanine Adenine Cytosine Uracil DNA Wt. obtained/g. dry organism Phosphorus content (%) Base content (per 100 nucleotides) G\lanine Adenille Cytosine Thymine 7; GC
treatment
30 min 120 min 24 hr
Lactobacillus acid@hilus
Streptococcus ~yogenes
13 mg.
33 mg.
43.5 mg.
80 mg.
8.8
8.8
29.0 26.4 23.8 20.8
28.9 27.3 22.7 21.1
G.5 mg.
17 mg.
9.0
8.9
20.4 30.3 19.6 29.7 40.0
19.5 30.5 20.0 30.0 39.5
Cl. welchii due to enzymic degradation. However, the results with the DNA degradation were not cowlusive, as the method chosen to demonstrate degradation was not very sensitive and could probably only detect nucleic acids which had been extensively degraded. The cell extract from S. marcescens was used as it had previously been found that an enzyme causing rapid degradation of DNA was present in this organism (4).
582
JONES
AND
From these results on enzyme inhibition by bentonite, it was decided to attempt to isolate the nucleic acids from two Gram-positive bacteria-Xtreptococcus pyogenes and Lactobacillus acidophilus. These two organisms are quoted by Marmur (1) as being resistant to detergent and lysozyme and thus the nucleic acids cannot be isolated by his method. The nucleic acids were isolated from the organisms which were grown in a fairly small volume of medium (1 liter), but the procedure is such that the amount of cells used could be greatly increased and the cells broken in a rotary ball mill instead of the Rfickle tissue disintegrator. Bentonite prevented any extensive degradation of the ribosomal RNA as shown by the fact that the preparations obtained from both organisms contained components of 20s and 27s. Last traces of RNA were removed from the DNA with ribonuclease because on the small scale used, losses of DNA were large if a second fractionation with CTAB was attempted. The sedimentation coefficient for the samples of DNA from both organisms was 14S, indicating a DNA of molecular weight of about 2 X 106. The samples of freeze-dried DKA showed a hyperchromic effect at 260 rnp of 15% when heated to 100” and then rapidly cooled. The weights obtained and the analyses of the nucleic acids are given in Table II. This method of isolation of nucleic acids from micro-organisms which are resistant to detergent and lysosyme has several advantages over those at present available. NO
WALKER
drastic chemical treatment of the cells is involved and enzymic activity is inhibited. The mechanical treatment, although obviously causing some degradation of the nucleic acids, gives DNA of sufficiently high molecular weight to be of use in cross hybridization experiments with high molecular weight DNA trapped in agar or upon Millipore filters. The method has also been shown to be of use in the isolation of the nucleic acids from CI. welchii and animal tissues, the results of which will be reported elsewhere. ACKNOWLEDGMENT We thankMr.
E. E T. J. Chelton
for assistance.
REFERENCES 1. MARMUR, J., 1. Mol. Biol. 3, 208 (1961). 2. BROWNHILL, T. J., JONES, A. S., AND STACEY, M., Biochem. J. 73, 434 (1959). 3. KRECHETOVA, G. D., CHUDINOVA, I. A., AND SHAPOT, V. S., Biokhimiya, 28, 682 (1963). 4. JONES, A. S., AND WALKER, R. T., J. Gen. Microbial. 31, 187 (1963). 5. KOLBE, J. J., Biochim. Biophys. dcta 38, 373 (1960). 26, 178 6. CHELTON, E. T. J., J. rlppl. Bacterial. (1963). 7. WYATT, G. R.., AND COHEN, S. S., Biochem. J. 66, 734 (1953). 8. WYATT, G. R., Biochem. J. 48, 584 (1951). 9. MARKHAM, R., AND SMITH, J. D., Biochem. J. 49, 401 (1951). 10. KIRBY, K. S., Biochim. Biophys. Acta 18, 575 (1955). 11. JONES, A. S., LEE, W. A., AND PEACOCKE, A. R., J. Chem. Sot. 623 (1951).