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More on mutants with altered DNA's
Mutation Research
I5
Elsevier P u b l i s h i n g C o m p a n y , A m s t e r d a m P r i n t e d in T h e N e t h e r l a n d s
MORE ON MUTANTS W I T H A L T E R E D DNA's
B E N E D I C T L. W A S I L A U S K A S , H E R B E R T S. R O S E N K R A N Z
J O E E. C O W A R D , P A U L D. E L L N E R
AND
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, N.Y. (U.S.A.) (Received O c t o b e r 26th, 197 o)
SUMMARY
A "DNA-base composition m u t a n t " (No. 17o ) of Bacterium paracoli 5o99 is identified as a member of the genus Bacillus. This suggests that the " m u t a n t " is in reality a contaminant.
The potential significance of bacterial mutants with altered DNA-base compositions has been reviewed recently 3. The existence of mutational events in which major portions of the cellular DNA have been changed are nevertheless still difficult to explain in terms of our current concepts of chemical mutagenesis and the genetic code. I t was to be expected, therefore, that the reported mutants would be closely scrutinized in a number of laboratories. I t was thus found that a " m u t a n t " of Bacterium paracoli appeared to be in reality a FlavobacteriumS, 1° and a " m u t a n t " of Staphylococcus aureus, a Corynebacterium 2. More recently a " m u t a n t " of Agrobacterium tumefaciens was reported to resemble members of the Alcaligenes species n. This led to the suggestion1°, n that the " m u t a n t s " might in reality have been contaminants. More recently, GAUSE e t a l . 4 reported that mutants with altered DNA compositions isolated from a cycloserine-resistant strain of Bacterium paracoli 5099 were still resistant to cycloserine. This was claimed to establish the genetic relationship of the m u t a n t with the parent strain. The present report deals with an analysis and a comparison of the properties of Bacterium paracoli 5099 and such a m u t a n t (No. 17o ) which was reported to possess a changed base composition (42.7% vs. 48% guanine-cytosine for m u t a n t 17o and B. paracoli 5099, respectively) and to exhibit an increased sensitivity to a variety of antibiotics while retaining the cycloserine resistance displayed b y the parent strain. Moreover, m u t a n t 17o was described as being "filamentous". MATERIALS AND METHODS
Bacterial strains. Bacterium paracoli 5o99 and its m u t a n t 17o were obtained from the American Type Culture Collection as strains ATCC 23 280 and ATCC 25 37 ° , respectively. Mutation Res., 12 (1971) 15-19
I0
B . L . WASILAUSKAS, J. E. COWARD, P. I). ELLNER, H. S. ROSENKRANZ
Isolation of DNA. Exponentially growing bacteria were harvested and their DNA was isolated by the procedure described by MARMUR6. Buoyant density centrifugation. Specimens of DNA together with a reference sample (DNA from Micrococcus lysodeikticus, 1.731 g/cm 3) were placed in a cesium chloride solution, density 1.7o g/cm 3, and centrifuged in the Spinco Model E ultracentrifuge at 44740 rev./min for 24 h. The bands formed by the specimens at their equilibrium positions were photographed. Buoyant densities were calculated 9 by comparison with the position of the reference DNA. Antibiotic sensitivity determinations. Portions (o.I Inl) of cultures were spread on the surface of Columbia-base agar (Baltimore Biological Laboratory) plates. When the plates had dried, discs (6 m m diameter) containing various antibiotics (IO/~g) were placed on the plates. Diameters of the zones of inhibition were determined after incubation at 37 ° for 16 h. Biochemical reactions. Standard procedures were used to characterize the metabolic properties of the 2 strains I. Electron microscopy. The bacteria were fixed in 1.5~o glutaraldehyde for 2 h (cacodylate buffered, pH 6.8). After washing in cacodylate buffer, the samples wcre post-fixed in 1% osmium tetroxide for 2 h, washed in maleate buffer (pH 5.2) and dispersed in melted agar. The agar was cut into small pieces, stained for 2 h in 2% uranyl acetate (dissolved in maleate buffer, pH 6.o), dehydrated in graded dilutions of ethyl alcohol, and embedded in epoxy resin (Epon 812). Thin sections were stained with uranyl acetate and lead citrate and viewed in a Philips 200 electron microscope. RESULTS
Buoyant density analyses of the DNA. Analysis of DNA prepared from Bacterium paracoli 5099 and its m u t a n t 17o in gradients of cesium chloride established that they possessed buoyant densities of 1.71o and 1.7o 5 g/cm a, respectively. If it be assumed that these nucleic acids contained no unusual bases or substituents, it can be calculated that their guanine-cytosine contents are 51 and 46%, respectively. Antibiotic sensitivities. GAUSE et al. 4 reported that m u t a n t 17o exhibited an increased sensitivity to all antibiotics tested while retaining the resistance to cycloserine exhibited by its parent B. paracoli 5099. The data of Table I indicate that TABLE I ANTIBIOTIC
SENSITIVITY
TESTS
Antibiotic
Diameter qf zones of inhibition (ram) Bacterium paracoli 5o99 ~l~rutantI7o (A TCC 23 280) (A TCC 25370 )
Chloramphenicol Kanaluycin Erythromycin
24 19 i*~
24 18 18
Tetracycline Streptomycin Hydroxyurea
13 17 24
23 26 o
Actinomyein D Cyclo serine Silver s u l f a d i a z i n e
o 30 8
II 37 8
31ulatio*z l¢cs., 12 (t97 l) 15-1()
MORE ON MUTANTS WITH ALTERED D N A ' s
17
by our criteria, the m u t a n t had a decreased resistance to cycloserine, as well as to erythromycin, tetracycline, streptomycin and actinomycin D. Its susceptibility to chloramphenicol and silver sulfadiazine was identical to that of B. paracoli 5099 while it was more resistant than its parent to hydroxyurea as well as to nalidixic acid (NegGram) and lincomycin (see below). Biochemical and growth properties. B. paracoli 5099 gave rise to smooth white colonies on Columbia-base agar, but m u t a n t 17o yielded dry, wrinkled colonies. The parent strain was Gram-negative while examination of fresh cultures of m u t a n t 17o revealed Gram-positive rods growing in chains. Older cultures (> 6 h) lost their stainability for Gram's stain. Upon prolonged incubation (I week) of m u t a n t 17o on solid medium, spores were detected by appropriate staining procedures. The results of biochemical reactions (Table II) indicate that the 2 strains differ extensively. Bacterium paracoli 5099 (ATCC 23 280) belongs to the coliform group while " m u t a n t " 17o (ATCC 2537 o) must be classified as a member of the genus Bacillus. TABLE II CHARACTERISTICS OF Bacterium paracoli 5099 (ATCC 23280 ) AND ITS "MUTANT" T70 (ATCC 25370) Reaction characteristic
A TCC 23280
A TCC 25 37 °
Primary differences Gram stain Spore formation Reaction in O - F medium a Gas from dextrose Nalidixic acid Lincomycin Motility Growth on EMB agar Growth on MacConkey's agar
negative negative fermenter produced sensitive sensitive motile amber colonies present
positive positive no change none resistant resistant non-motile no growth no growth
Secondary differences Dextrose Maltose Mannitol Lysine decarboxylase Voges-Proskauer
acid and gas acid and gas acid and gas positive positive
negative negative negative negative negative
Similarities Oxidase Pigment Nitrate reduced Phenylalanine deaminase H2S Indole Methyl red Gelatin Lactose Sucrose Catalase Citrate utilized Malonate
negative none positive negative negative negative negative negative negative negative positive negative negative
negative none positive negative negative negative negative negative negative negative positive negative negative
Escherichia sp.
Bacillus sp.
Identification a Oxidation-fermentation medium (Hugh-Leifson). Mutation Res., 12 (i97 I) 15-19
18
B. L. WASILAUSKAS, J. E. COWARD, P. D. ELLNER, H. S. ROSENKRANZ
Fig. I. U l t r a s t r u c t u r a l a p p e a r a n c e of Bacterium paracoli 5099 (ATCC 23 280). Bar represents 0. 5 l*-
Fig. 2. U l t r a s t r u c t u r a l a p p e a r a n c e of " m u t a n t " 17o (ATCC 25 37o). Bar represents o.5/*-
Ultrastructural studies. The appearance under the electron microscope of thin sections of the 2 bacterial strains revealed (Fig. I) t h a t B. paracoli 5099 possesses a cell wall structure characteristic of coliforms 7 while " m u t a n t " 17o displayed structures characteristic 12 of Gram-positive microorganisms (Fig. 2). DISCUSSION
Bacterium paracoli 5o99 and its " m u t a n t " 17o unquestionably differ in the base compositions of their DNA. Our results indicate t h a t this difference is not the Mutation l~es., 12 (i97 I) 15-19
MORE ON MUTANTS WITH ALTERED D N A ' s
19
result of a mutational event but rather it must be due to the fact that " m u t a n t " 17o is in fact a contaminant. Indeed further analyses revealed that " m u t a n t " 17o belonged to the genus Bacillus. It is significant that the base composition of " m u t a n t " 17o (46% guanine+cytosine) falls in the range reported for Bacilli 5. As a result of colony morphology, growth characteristics, staining properties, antibiotic susceptibilities and biochemical reactions, mutant 17o was placed in the Bacillus group while the classification of B. paracoli 5099 as a coliform was confirmed 8. It is interesting that although susceptibility to actinomycin D is not generally used in the identification of microorganisms, this is a property of Gram-positive microorganisms 13; while it has been found that Bacilli are resistant to hydroxyurea (unpublished results). These properties were exhibited by " m u t a n t " 17o. The ultrastructural appearance of mutant 17o also suggested that it was a Gram-positive microorganism. In conclusion, therefore, it must be stated that the existence of mutants with altered DNA compositions remains to be demonstrated. The present report reinforces previous ones1°, n, which suggested that such mutants were in fact contaminants. ACKNOWLEDGEMENTS
This investigation was supported by a grant from the Annie R. Masch Memorial Grant for Cancer Research from the American Cancer Society. One of the authors (B.L.W.) is a post-doctoral trainee, supported by the U.S. Public Health Service (5-ToI-AI-oo245) ; H.S.R. is a Research Career Development Awardee of the National Institute of General Medical Sciences, U.S. Public Health Service (5-K 3GM-29, 024). REFERENCES I BLAIR, J. E., E. H. LENNETTE AND J. P. TRUANT (Eds.), Manual of Clinical Microbiology, A m e r i c a n Society for Microbiology, B e t h e s d a , Md., 197 o. 2 GARRITY, F. L., B. DETRICK AND E. R. KENNEDy, Deoxyribonucleic acid b a s e c o m p o s i t i o n in t h e t a x o n o m y of S t a p h y l o c o c c u s , J. Bacteriol., 97 (1969) 557-560. 3 GAUSE, G. F., A l t e r a t i o n s of D N A base c o m p o s i t i o n in bacteria, Progr. Nucleic Acid Res. Mol. Biol., 8 (1968) 49-71. 4 GAUSE, G. F., A. V. LAIKO, Y. V. DUDNIK, E. M. NETYKSA AND G. V. KOCHETKOVA, R e l a t i o n b e t w e e n t r y p a f l a v i n e s e n s i t i v i t y a n d nucleotide c o m p o s i t i o n of D N A in m u t a n t s of Bacterium paracoli 5o99, Dokl. Akad. Nauk S.S.S.R., 175 (1967) 47o-472. 5 HILL, L. R., A n i n d e x to deoxyribonucleic acid base c o m p o s i t i o n s of bacterial species, J. Gen. Microbiol., 44 (1966) 419-437 . 6 MARMUR, J., A p r o c e d u r e for t h e isolation of deoxyribonucleic acid f r o m m i c r o o r g a n i s m s , J. Mol. Biol., 3 (1961) 2o8-218. 7 MORGAN, C., H. S. ROSENKRANZ AND H. M. ROSE, E l e c t r o n m i c r o s c o p y of c h l o r a m p h e n i c o l t r e a t e d Escherichia coli, J. Bacteriol., 93 (1967) 1987-2oo2. 8 ROSENKRANZ, H. S., AND P. D. ELLNER, M u t a n t of Bacterium paracoli 5099 w i t h a n altered D N A : Identification as a F l a v o b a c t e r i u m , Science, 16o (1968) 893-894. 9 SCHILDKRAUT, C. L., J. MARMUR AND P. DOTY, D e t e r m i n a t i o n of t h e b a s e c o m p o s i t i o n of deoxyribonucleic acid f r o m its b u o y a n t d e n s i t y in CsC1, J. Mol. Biol., 4 (1962) 430-443 . io VAN DER PLAAT, J. B., P. APONTOWEIL AND W. BERENDS, M u t a n t s w i t h altered D N A - b a s e composition, Mutation Res., 7 (1969) 13-18. 11 VAN DER PLAAT, J. B., L. N. VERNIE AND J. DE LEY, S u s p e c t e d m u t a n t of Agrobacterium tumefaciens w i t h a n altered D N A , Mutation Res., 7 (1969) 466-468. 12 VAN ITERSON, W., Bacterial c y t o p l a s m , Bacteriol. Rev., 29 (1965) 299-325 . 13 WAKSMAN, S. A., AND H. B. WOODRUFF, B a c t e r i o s t a t i c a n d bactericidal s u b s t a n c e s p r o d u c e d b y a soil a c t i n o m y c e s , Proc. Soc. Exptl. Biol. Med., 45 (194 °) 6o9-614.
Mutation Res., 12 (1971) 15-19
I5
Elsevier P u b l i s h i n g C o m p a n y , A m s t e r d a m P r i n t e d in T h e N e t h e r l a n d s
MORE ON MUTANTS W I T H A L T E R E D DNA's
B E N E D I C T L. W A S I L A U S K A S , H E R B E R T S. R O S E N K R A N Z
J O E E. C O W A R D , P A U L D. E L L N E R
AND
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, N.Y. (U.S.A.) (Received O c t o b e r 26th, 197 o)
SUMMARY
A "DNA-base composition m u t a n t " (No. 17o ) of Bacterium paracoli 5o99 is identified as a member of the genus Bacillus. This suggests that the " m u t a n t " is in reality a contaminant.
The potential significance of bacterial mutants with altered DNA-base compositions has been reviewed recently 3. The existence of mutational events in which major portions of the cellular DNA have been changed are nevertheless still difficult to explain in terms of our current concepts of chemical mutagenesis and the genetic code. I t was to be expected, therefore, that the reported mutants would be closely scrutinized in a number of laboratories. I t was thus found that a " m u t a n t " of Bacterium paracoli appeared to be in reality a FlavobacteriumS, 1° and a " m u t a n t " of Staphylococcus aureus, a Corynebacterium 2. More recently a " m u t a n t " of Agrobacterium tumefaciens was reported to resemble members of the Alcaligenes species n. This led to the suggestion1°, n that the " m u t a n t s " might in reality have been contaminants. More recently, GAUSE e t a l . 4 reported that mutants with altered DNA compositions isolated from a cycloserine-resistant strain of Bacterium paracoli 5099 were still resistant to cycloserine. This was claimed to establish the genetic relationship of the m u t a n t with the parent strain. The present report deals with an analysis and a comparison of the properties of Bacterium paracoli 5099 and such a m u t a n t (No. 17o ) which was reported to possess a changed base composition (42.7% vs. 48% guanine-cytosine for m u t a n t 17o and B. paracoli 5099, respectively) and to exhibit an increased sensitivity to a variety of antibiotics while retaining the cycloserine resistance displayed b y the parent strain. Moreover, m u t a n t 17o was described as being "filamentous". MATERIALS AND METHODS
Bacterial strains. Bacterium paracoli 5o99 and its m u t a n t 17o were obtained from the American Type Culture Collection as strains ATCC 23 280 and ATCC 25 37 ° , respectively. Mutation Res., 12 (1971) 15-19
I0
B . L . WASILAUSKAS, J. E. COWARD, P. I). ELLNER, H. S. ROSENKRANZ
Isolation of DNA. Exponentially growing bacteria were harvested and their DNA was isolated by the procedure described by MARMUR6. Buoyant density centrifugation. Specimens of DNA together with a reference sample (DNA from Micrococcus lysodeikticus, 1.731 g/cm 3) were placed in a cesium chloride solution, density 1.7o g/cm 3, and centrifuged in the Spinco Model E ultracentrifuge at 44740 rev./min for 24 h. The bands formed by the specimens at their equilibrium positions were photographed. Buoyant densities were calculated 9 by comparison with the position of the reference DNA. Antibiotic sensitivity determinations. Portions (o.I Inl) of cultures were spread on the surface of Columbia-base agar (Baltimore Biological Laboratory) plates. When the plates had dried, discs (6 m m diameter) containing various antibiotics (IO/~g) were placed on the plates. Diameters of the zones of inhibition were determined after incubation at 37 ° for 16 h. Biochemical reactions. Standard procedures were used to characterize the metabolic properties of the 2 strains I. Electron microscopy. The bacteria were fixed in 1.5~o glutaraldehyde for 2 h (cacodylate buffered, pH 6.8). After washing in cacodylate buffer, the samples wcre post-fixed in 1% osmium tetroxide for 2 h, washed in maleate buffer (pH 5.2) and dispersed in melted agar. The agar was cut into small pieces, stained for 2 h in 2% uranyl acetate (dissolved in maleate buffer, pH 6.o), dehydrated in graded dilutions of ethyl alcohol, and embedded in epoxy resin (Epon 812). Thin sections were stained with uranyl acetate and lead citrate and viewed in a Philips 200 electron microscope. RESULTS
Buoyant density analyses of the DNA. Analysis of DNA prepared from Bacterium paracoli 5099 and its m u t a n t 17o in gradients of cesium chloride established that they possessed buoyant densities of 1.71o and 1.7o 5 g/cm a, respectively. If it be assumed that these nucleic acids contained no unusual bases or substituents, it can be calculated that their guanine-cytosine contents are 51 and 46%, respectively. Antibiotic sensitivities. GAUSE et al. 4 reported that m u t a n t 17o exhibited an increased sensitivity to all antibiotics tested while retaining the resistance to cycloserine exhibited by its parent B. paracoli 5099. The data of Table I indicate that TABLE I ANTIBIOTIC
SENSITIVITY
TESTS
Antibiotic
Diameter qf zones of inhibition (ram) Bacterium paracoli 5o99 ~l~rutantI7o (A TCC 23 280) (A TCC 25370 )
Chloramphenicol Kanaluycin Erythromycin
24 19 i*~
24 18 18
Tetracycline Streptomycin Hydroxyurea
13 17 24
23 26 o
Actinomyein D Cyclo serine Silver s u l f a d i a z i n e
o 30 8
II 37 8
31ulatio*z l¢cs., 12 (t97 l) 15-1()
MORE ON MUTANTS WITH ALTERED D N A ' s
17
by our criteria, the m u t a n t had a decreased resistance to cycloserine, as well as to erythromycin, tetracycline, streptomycin and actinomycin D. Its susceptibility to chloramphenicol and silver sulfadiazine was identical to that of B. paracoli 5099 while it was more resistant than its parent to hydroxyurea as well as to nalidixic acid (NegGram) and lincomycin (see below). Biochemical and growth properties. B. paracoli 5099 gave rise to smooth white colonies on Columbia-base agar, but m u t a n t 17o yielded dry, wrinkled colonies. The parent strain was Gram-negative while examination of fresh cultures of m u t a n t 17o revealed Gram-positive rods growing in chains. Older cultures (> 6 h) lost their stainability for Gram's stain. Upon prolonged incubation (I week) of m u t a n t 17o on solid medium, spores were detected by appropriate staining procedures. The results of biochemical reactions (Table II) indicate that the 2 strains differ extensively. Bacterium paracoli 5099 (ATCC 23 280) belongs to the coliform group while " m u t a n t " 17o (ATCC 2537 o) must be classified as a member of the genus Bacillus. TABLE II CHARACTERISTICS OF Bacterium paracoli 5099 (ATCC 23280 ) AND ITS "MUTANT" T70 (ATCC 25370) Reaction characteristic
A TCC 23280
A TCC 25 37 °
Primary differences Gram stain Spore formation Reaction in O - F medium a Gas from dextrose Nalidixic acid Lincomycin Motility Growth on EMB agar Growth on MacConkey's agar
negative negative fermenter produced sensitive sensitive motile amber colonies present
positive positive no change none resistant resistant non-motile no growth no growth
Secondary differences Dextrose Maltose Mannitol Lysine decarboxylase Voges-Proskauer
acid and gas acid and gas acid and gas positive positive
negative negative negative negative negative
Similarities Oxidase Pigment Nitrate reduced Phenylalanine deaminase H2S Indole Methyl red Gelatin Lactose Sucrose Catalase Citrate utilized Malonate
negative none positive negative negative negative negative negative negative negative positive negative negative
negative none positive negative negative negative negative negative negative negative positive negative negative
Escherichia sp.
Bacillus sp.
Identification a Oxidation-fermentation medium (Hugh-Leifson). Mutation Res., 12 (i97 I) 15-19
18
B. L. WASILAUSKAS, J. E. COWARD, P. D. ELLNER, H. S. ROSENKRANZ
Fig. I. U l t r a s t r u c t u r a l a p p e a r a n c e of Bacterium paracoli 5099 (ATCC 23 280). Bar represents 0. 5 l*-
Fig. 2. U l t r a s t r u c t u r a l a p p e a r a n c e of " m u t a n t " 17o (ATCC 25 37o). Bar represents o.5/*-
Ultrastructural studies. The appearance under the electron microscope of thin sections of the 2 bacterial strains revealed (Fig. I) t h a t B. paracoli 5099 possesses a cell wall structure characteristic of coliforms 7 while " m u t a n t " 17o displayed structures characteristic 12 of Gram-positive microorganisms (Fig. 2). DISCUSSION
Bacterium paracoli 5o99 and its " m u t a n t " 17o unquestionably differ in the base compositions of their DNA. Our results indicate t h a t this difference is not the Mutation l~es., 12 (i97 I) 15-19
MORE ON MUTANTS WITH ALTERED D N A ' s
19
result of a mutational event but rather it must be due to the fact that " m u t a n t " 17o is in fact a contaminant. Indeed further analyses revealed that " m u t a n t " 17o belonged to the genus Bacillus. It is significant that the base composition of " m u t a n t " 17o (46% guanine+cytosine) falls in the range reported for Bacilli 5. As a result of colony morphology, growth characteristics, staining properties, antibiotic susceptibilities and biochemical reactions, mutant 17o was placed in the Bacillus group while the classification of B. paracoli 5099 as a coliform was confirmed 8. It is interesting that although susceptibility to actinomycin D is not generally used in the identification of microorganisms, this is a property of Gram-positive microorganisms 13; while it has been found that Bacilli are resistant to hydroxyurea (unpublished results). These properties were exhibited by " m u t a n t " 17o. The ultrastructural appearance of mutant 17o also suggested that it was a Gram-positive microorganism. In conclusion, therefore, it must be stated that the existence of mutants with altered DNA compositions remains to be demonstrated. The present report reinforces previous ones1°, n, which suggested that such mutants were in fact contaminants. ACKNOWLEDGEMENTS
This investigation was supported by a grant from the Annie R. Masch Memorial Grant for Cancer Research from the American Cancer Society. One of the authors (B.L.W.) is a post-doctoral trainee, supported by the U.S. Public Health Service (5-ToI-AI-oo245) ; H.S.R. is a Research Career Development Awardee of the National Institute of General Medical Sciences, U.S. Public Health Service (5-K 3GM-29, 024). REFERENCES I BLAIR, J. E., E. H. LENNETTE AND J. P. TRUANT (Eds.), Manual of Clinical Microbiology, A m e r i c a n Society for Microbiology, B e t h e s d a , Md., 197 o. 2 GARRITY, F. L., B. DETRICK AND E. R. KENNEDy, Deoxyribonucleic acid b a s e c o m p o s i t i o n in t h e t a x o n o m y of S t a p h y l o c o c c u s , J. Bacteriol., 97 (1969) 557-560. 3 GAUSE, G. F., A l t e r a t i o n s of D N A base c o m p o s i t i o n in bacteria, Progr. Nucleic Acid Res. Mol. Biol., 8 (1968) 49-71. 4 GAUSE, G. F., A. V. LAIKO, Y. V. DUDNIK, E. M. NETYKSA AND G. V. KOCHETKOVA, R e l a t i o n b e t w e e n t r y p a f l a v i n e s e n s i t i v i t y a n d nucleotide c o m p o s i t i o n of D N A in m u t a n t s of Bacterium paracoli 5o99, Dokl. Akad. Nauk S.S.S.R., 175 (1967) 47o-472. 5 HILL, L. R., A n i n d e x to deoxyribonucleic acid base c o m p o s i t i o n s of bacterial species, J. Gen. Microbiol., 44 (1966) 419-437 . 6 MARMUR, J., A p r o c e d u r e for t h e isolation of deoxyribonucleic acid f r o m m i c r o o r g a n i s m s , J. Mol. Biol., 3 (1961) 2o8-218. 7 MORGAN, C., H. S. ROSENKRANZ AND H. M. ROSE, E l e c t r o n m i c r o s c o p y of c h l o r a m p h e n i c o l t r e a t e d Escherichia coli, J. Bacteriol., 93 (1967) 1987-2oo2. 8 ROSENKRANZ, H. S., AND P. D. ELLNER, M u t a n t of Bacterium paracoli 5099 w i t h a n altered D N A : Identification as a F l a v o b a c t e r i u m , Science, 16o (1968) 893-894. 9 SCHILDKRAUT, C. L., J. MARMUR AND P. DOTY, D e t e r m i n a t i o n of t h e b a s e c o m p o s i t i o n of deoxyribonucleic acid f r o m its b u o y a n t d e n s i t y in CsC1, J. Mol. Biol., 4 (1962) 430-443 . io VAN DER PLAAT, J. B., P. APONTOWEIL AND W. BERENDS, M u t a n t s w i t h altered D N A - b a s e composition, Mutation Res., 7 (1969) 13-18. 11 VAN DER PLAAT, J. B., L. N. VERNIE AND J. DE LEY, S u s p e c t e d m u t a n t of Agrobacterium tumefaciens w i t h a n altered D N A , Mutation Res., 7 (1969) 466-468. 12 VAN ITERSON, W., Bacterial c y t o p l a s m , Bacteriol. Rev., 29 (1965) 299-325 . 13 WAKSMAN, S. A., AND H. B. WOODRUFF, B a c t e r i o s t a t i c a n d bactericidal s u b s t a n c e s p r o d u c e d b y a soil a c t i n o m y c e s , Proc. Soc. Exptl. Biol. Med., 45 (194 °) 6o9-614.
Mutation Res., 12 (1971) 15-19