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DNA uptake by Streptomyces species
117
Biochimica et Biophysica Acta, 442 (1976) 117--122 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 91432
DNA UPTAKE BY STREPTOMYCES SPECIES
P. R O E L A N T S a and V. K O N V A L I N K O V A L. LEDOUX b
b, M. M E R G E A Y
b, P.F. L U R Q U I N b and
aReeherche et Industrie Th$rapeutique (R.L T.), Bacteriology D e p a r t m e n t - Research Division, B-1330 Rixensart and b Department of Radiobiology, Centre d'Etude de l'Energie Nucl$aire (C.E.N./S.C.K.), B-2400 Mol (Belgium) (Received May 24th 1976)
Summary Uptake of homologous and heterologous radioactively labelled DNA was studied in ten 8treptornyces species. Among these, S. kasugaensis and S. virginiae were shown to take up 0.5--2.0% of the supplied donor DNA at a well-defined point in their growth cycle. The heterologous donor DNA taken up retained its original buoyant density in CsC1 gradients. Other strains either extensively degraded the donor DNA or showed poor uptake efficiency.
Previous attempts at demonstrating genetic transformation among
Streptornyces were based on the transfer of information for antibiotic production [1--5]. These reports were not supported by extensive biochemical and genetic data and are still awaiting confirmation [6,7]. An effective transformation system has been clearly established by Hopwood and Wright [8] in Thermoactinomyces vulgaris. However, this organism differs significantly (and notably by its low G + C content, sporulation physiology and temperature optimum) from the other Actinomycetales and looks to be not~ representative of this genus regarding transformation. As a first step in evaluating the possibility of transformation among some Streptomycetaceae of industrial interest, we have measured the ability of various strains to take up added exogenous DNA. These results are reported here. The organisms used were Streptornyces coelicolor A3 [2], S. fradiae (ATCC 10745), S. kanarnyceticus (ATCC 12853), S. kasugaensis (ATCC 15714), S. rimosus (ATCC 14827), S. tenebrarius (ATCC 17920), S. griseus (our isolate SG279), S. virginiae (ATCC 13161) and its derived mutant Nb. 10 and Micromonospora purpurea (ATCC 15835). Spores of each strain were inoculated in 125-ml flasks with 15 ml of the following medium convenient for Streptomyces: K2HPO4, 1 g; NaC1, 1 g;
118 MgSO4" 7H20, 1 g; L-asparagine, 1 g; casamino acids Difco, 1 g; glycerol, 10 g; 1000 ml distilled water and for Micromonospora: K2HPO4, 1 g; NaC1, 1 g; MgSO4" 7H20, 1 g; (NH4)2SO4, 1 g; yeast extract Difco, 1 g; glucose 10 g; 1000 ml distilled water. The flasks were incubated on a reciprocal shaker at 28°C and growth, measured as increase in absorbance at 660 nm, reached a maximum after about 20 h. Samples (1 ml) were taken at regular intervals. Mycelia were spun down, washed and resuspended in 1 ml fresh medium supplemented with tritiumlabelled DNA extracted from S. virginiae or Bacillus subtilis uracil-requiring mutants. (see legend of Fig. 1). The preparation of these DNAs involved lysis of the cells with lysozyme followed by 2% sodium dodecyl sulfate, incubation with pronase and a final purification step in preparative CsC1 density gradients as previously described
[9,15]. After 30 minutes of incubation with donor [3H] DNA, mycelia were treated with pancreatic deoxyribonuclease and processed for acid-precipitable radioactivity determination as described in the legend of Fig. 1. (a) Uptake kinetics. Preliminary results showed that the amount of donor DNA bound by the Streptomyces cells did not increase significantly at concentrations higher than 10--12 #g/ml (concentration range = 0.5--100 ~g/ml
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Fig. 1. U p t a k e of [ 3 H ] D N A b y S t r e p t o m y c e t e cells as a f u n c t i o n o f t h e g r o w t h cycle. Cells w e r e i n c u b a t e d w i t h 10 # g / m l [ 3 H ] D N A . A f t e r 30 rain i n c u b a t i o n a t 2S°C a D N A a s e s o l u t i o n ( 1 0 0 ~g in 1 m l fresh m e d i u m w i t h o u t c a r b o h y d r a t e s o u r c e ) was a d d e d a n d t h e i n c u b a t i o n was c o n t i n u e d f o r 10 rain at 37°C. M y c e l i a w e r e c e n t r i f u g e d , w a s h e d t w i c e w i t h c a r b o h y d r a t e - f r e e m e d i u m , r e s u s p e n d e d in 2 m l 5% t r i c h l o r o a c e t i c acid a n d h e l d f o r S0 rain a t 0°C. M y c e l t a w e r e finally r e c o v e r e d o n a Whatm a n glass fiber filter ( G F / C ) a n d w a s h e d several t i m e s w i t h c o l d t r i c h l o r o a c e t i e acid. T h e filters w e r e d r i e d , p l a c e d in s c i n t i l l a t i o n fluid ( t o l u e n e w i t h 0 . 2 5 g/1 2,2 ' - p - p h e n y l - b i s - ( 5 - p h e n y l o x a z o l ) a n d 5 g/1 2 , 5 - d i p h e n y l o x a z o l ) a n d r a d i o a c t i v i t y w a s m e a s u r e d in a P a c k a r d s c i n t i l l a t i o n s p e c t r o m e t e r , o - - ~ , g r o w t h m e a s u r e d b y A600 nm" ( A ) U p t a k e of [ 3 H ] D N A f r o m B. s u b t i l i s b y S. kasugaensis; o - - 4 , filterb o u n d c p m . (B) U p t a k e o f [ 3H] D N A f r o m B. subtilis b y S. virginiae; ~ - 4 , f i l t e r - b o u n d c p m . (C) UP o t a k e of 3H-labelled h o m o l o g o u s D N A b y S. virginiae a n d D N A s y n t h e s i s as f o l l o w e d b y [ 1 4 C ] t h y m i d i n e i n c o r p o r a t i o n in acid p r e c i p i t a b l e m a t e r i a l ; o - - o , u p t a k e of p o l y m e r i z e d h o m o l o g o u s D N A ; ~--~, d o n o r D N A e x p o s e d t o D N A a s e p r i o r to i n c u b a t i o n w i t h cells; D--~, [ t 4 C ] t h y m i d i n e i n c o r p o r a t e d a f t e r 3 0 rain i n c u b a t i o n in 1 m l s a m p l e s t o w h i c h 9 0 0 0 c p m o f [2 - 1 4 C ] t h y m i d i n e w e r e a d d e d (spec. act. 48 C i / m o l ) . T h e specific r a d i o a c t i v i t y of B. subtilis [ 3 H ] D N A was 1 × 106 cpm/~tg a n d t h a t o f S. uirginiae [ 3 H ] D N A w a s 3 • l 0 s c p m # t g . B o t h p r e p a r a t i o n s w e r e o b t a i n e d f r o m cells l a b e l e d w i t h 50 ~ C i / m l [ 3 H ] u r i d i n e .
119
DNA). Subsequently, all experiments were carried o u t with 10 pg/ml donor DNA. Table I reports the maximum uptake of B. subtilis DNA estimated as acid-precipitable radioactivity. The amount of DNA b o u n d b y the S. virginiae mutant strain Nb. 10 was found to be higher than for the other strains: around 1--2% of the labelled donor DNA was found in mycelia. As a first approximation, this represents a b o u t 2% of the endogenous DNA content. The ATCC 13161 strain retained only 0.3% of the supplied radioactivity. S. kasugaensis b o u n d 0.5% of the donor DNA; for S. fradiae, S. kanamyceticus, So rirnosus and S. tenebrarius uptake was less than 0.1% and for S. coelicolor and Micromonospora, less than 0.1%. TABLE I
B. S U B T I L I S [ S H ] D N A U P T A K E E F F I C I E N C Y MONOSPORA STRAINS
IN VARIOUS STREPTOMYCES
AND MICRO-
1 0 ~ g o f D N A w e r e given t o e a c h 1-rrfl s a m p l e and r e p r e s e n t e d 1 0 ~ c p m . T h e A 6 6 o n m o f t h e c u l t w t e s ranged f r o m 1 . 0 t o 1.5. I n c u b a t i o n w i t h d o n o r D N A w a s for 3 0 r a i n a t 2 8 ° C . A c i d - i n s o l u b l e radioa c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d u n d e r F i g . 1.
R e c i p i e n t strains
B. subtilis D N A u p t a k e in c p m
8. c o e l i c o l o r A 3 [ 2 ] 8. fradiae A T C C 1 0 . 7 4 5 S. griseus S G 2 7 9 8. k a n a m y c e t i c u 8 A T C C 1 2 . 8 5 3 S. kasugaensis A T C C 1 5 . 7 1 4 S. tenebrarius A T C C 1 7 . 9 2 0 S. rirnosus A T C C 1 4 . 8 2 7 S. pirginiae A T C C 1 3 . 1 6 1 S. pirginiae m u t a n t N b 1 0 M. p u r p u r e a A T C C 1 5 . 8 3 5
1 0 2, 1 0 2 2 " 1 0 3, 1 . 2 " 1 0 3 10 3 10 3 2 . 5 " 1 0 4, 2 . 4 • 1 0 4 , 4 • 1 0 4 7 10 2 2 • 1 0 3, 5 • 1 0 3 8-10 3 8 " 10 4, 2 • 1 0 s 10 2 "
In the case of S. coelicolor, it was found that the DNA of B. subtilis or S. coelicolor was extensively degraded exocellularly which corrobated a previous observation of H o p w o o d and Wright [7]. The DNA-binding pattern by the t w o best recipients S. virginiae and S. kasugaensis is shown in Fig. 1. It can be seen that uptake of DNAaseresistant radioactivity occurred at a defined m o m e n t of the growth cycle, strongly evoking a competence phase as described for a number of bacterial species [ 10]. A similar DNA absorption peak was also reported for S. griseus
[11]. Furthermore, the uptake p h e n o m e n o n seems to be independent of the origin (homologous or heterologous) of the donor DNA. Indeed, Figs. 1B and C describe the uptake of B. subtilis and S. virginiae [3H]DNA respectively by S. virginiae mycelia. It is shown that DNAase-resistant binding occurs at the same time of the growth cycle and it was found that the extent of binding was similar with both DNAs. The same observation holds true for S. coelicolor [3H]DNA as a donor (data not shown). Similarly, S. kasugaensis b o u n d S. coelicolor and B. subtilis [3H]DNA to the same extent and according to the same time-based pattern. Fig. 1C also demonstrates that a comparable a m o u n t of S. virginiae [ 3H] DNA, previously degraded upon a 10-min incubation at 37°C with 100 pg/ml pancreatic deoxyribonuclease, is very poorly utilized for endogenous
120 DNA synthesis. Thus, it turns out that the uptake p h e n o m e n o n is linked to the polymerized nature of the donor DNA. Moreover, DNA synthesis as measured by the incorporation of ['4C] thymidine into acid-precipitable material is found to drop drastically at the time of m a x i m u m DNA uptake as is shown in Fig. IC. Apparently there is a correlation between donor DNA uptake and the decline of endogenous DNA synthesis. The peak positions of DNA binding oscillate somewhat among different uptake experiments. Peaking was found to occur from the late exponential phase to the first hours of the stationary phase. Using S. virginiae as a recipient, peaks observed in t w e n t y DNA-uptake experiments arose at an average time of 22 h 30 min + 2 h 30 min after the inoculation of the culture. An average value of 22 + 2 h was observed using S. kasugaensis as a recipient in seven experiments. The kinetics of DNA binding were linear during the first 7 min of incubation but went on for at least another 23 min with a much shallower slope using a saturating concentration of 10 pg/ml donor DNA. (b) Nature of the intracellular radioactivity. No tool to look accurately for the genetic expression of homologous DNA taken up was available at the time of these experiments. Among the existing difficulties one notes the necessity of a sporulation step on solid medium before testing donor DNA expression. Suitable experimental conditions are currently being worked out. We found it of interest to get some insight into the fate of the donor DNA inside and outside the cells at a time when m a x i m u m uptake was shown to occur (see Fig. 1). To serve this purpose B. subtilis [3H]DNA was used as a donor since its lower G+C content (43%, CsC1 b u o y a n t density = 1.703 g. cm -3) makes it readily distinguishable from the host DNA having a G+C content of 71% (CsC1 b u o y a n t density = 1.730 g. cm-3). The use of heteropycnic ~ionor DNA is further justified on the basis of the similarity between homologous and heterologous DNA uptake curves {Figs. 1B and C}. The mycelia of S. kasugaensis or S. virginiae were lysed by treatment with lysozyme (200 pg/ml in saline/EDTA solution) immediately after incubation with [3H]DNA from B. subtilis and DNAase treatment. The DNA was extracted from the mycelium according to Marmur [12] and analyzed in CsC1 density gradients. Figs. 2A and B clearly show that most of the intracellular radioactivity bands at the position of the donor DNA (d = 1.703 g. cm-3). The shoulder located to the left of the main radioactive band is most probably a result of donor DNA breakdown and reutilization. Indeed, breakdown of donor DNA must have occurred intracellularly since about 30% of the radioactivity found associated with cells incubated in the presence of [3H] DNA was acid-soluble. This portion of the intracellular radioactivity did not show up in CsC1 gradients as a result of our DNA extraction procedure which does not allow recovery of low molecular weight compounds. The incubation medium still contained 95% acid-precipitable radioactive compounds after the 30-min incubation with mycelia. That extracellular degradation was very limited was confirmed by sedimentation velocity analysis of the donor DNA before and after incubation. Fig. 2C shows that the rate of sedimentation of the incubated donor [3H] DNA in a 5--20% alkaline sucrose gradient was somewhat decreased but that no radioactivity was floating at the top of the gradient.
121
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Fig. 2. ( A ) CsCI d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f D N A e x t r a c t e d f r o m S. kasugaensis i n c u b a t e d f o r 3 0 m i n w i t h B. subtflis [ S H ] D N A 2 2 h a f t e r i n o c u l a t i o n (see Fig. 1 A ) . (B) CsC1 g r a d i e n t c e n t r i f u g a t i o n o f D N A e x t r a c t e d f r o m S. virginiae i n c u b a t e d f o r 3 0 r a i n w i t h B. subtiits [ S H ] D N A 2 0 h a f t e r i n o c u l a t i o n (see Fig. 1B). D N A w a s e x t r a c t e d f r o m m y c e H a ( a c e t o n i c p o w d e r ) b y g r i n d i n g w i t h solid C O 2 a n d i n c u b a t i n g t h e r e s u l t i n g e x t r a c t w i t h l y s o z y m e , p r o n a s e a n d r i b o n u c l e a s e s u c c e s s i v e l y as d e s c r i b e d for the isolation of other bacterial DNAs [9,15]. The DNA solutions were then brought to a refractive i n d e x o f 1 . 3 9 9 5 w i t h CIC! ( 8 u p r a p t t r , M e r c k ) , a d j u s t e d t o 3 m l w i t h a CsCl s o l u t i o n o f t h e s a m e d e n s i t y a n d c e n t r i f u g e d a t 2 0 ° C f o r 6 3 h a t a 0 0 0 0 r e v , / m i n in a M a r t i n C h r i s t S W 6 0 r o t o r . The slope o f t h e g r a d i e n t w a s d e t e r m i n e d b y a d d i n g u n l a b e l l e d 8. eoc|lcolor D N A (pffi 1 . 7 3 0 g / c m 3) and B. subtills D N A (p ,, 1 . 7 0 8 g/oreS). The absorbance a t 2 6 0 n m c o r r e s p o n d i n g t o t h e s e m a r k e r D N A I w a s m e a s t t r e d i n a C a r y 1 4 r p e c t t o p h o t o m e t e r , l ~ a d i o a c t i v i t y d e t e r m i n a t i o n s w e r e d o n e b y l i q u i d scintillat i o n i n the presence o f l n s t a g e l ( P a c k a r d ) . e - e , A260 n m ; ~ ' ~ , 3H c p m . (C) S e d i m e n t a t i o n v e l o c i t y a n a l y s i s o f B. subtllis [ S H ] D N A u s e d in t h e u p t a k e e x p e r i m e n t s . T h e D N A s a m p l e s (1150/~1) w e r e l a y e r e d o n t o p o f 5 m l 5 - 2 0 % a l k a l i n e s u c r o s e g r a d i e n t s a n d s p u n f o r 3 h a t 4 6 0 0 0 r e v . / m i n in a n SW 5 2 M a r t i n C h r i s t r o t o r a t 2 0 ° C . T h e g r a d i e n t s w e r e f r a c t i o n a t e d f r o m t h e b o t t o m a n d t h e r a d i o a c t i v i t y o f t h e f r a c t i o n s w a s d e t e r m i n e d as a b o v e , e - - e , d o n o r D N A b e f o r e i n c u b a t i o n w i t h cells; c - - ~ , d o n o r D N A r e c o v e r e d f r o m t h e s u p e r n a t a n t a f t e r 3 0 r a i n i n c u b a t i o n w i t h S. virginiae g r o w i n g f o r 2 0 h. S e d i m e n t a t i o n is f r o m r i g h t t o left.
These results would indicate that the exogenous D N A could remain relatively undegraded and retained its original buoyant density inside the recipient cells. However, the mean size of the donor D N A molecules recovered from the incubated cells is reduced as compared to that of the original donor DNA. This is indicated by the band-broadening observed in Figs. 2A and B where it can be seen that the banding pattern of the exogenous D N A is wider than that of the reference B. subtilis DNA. This phenomenon may be attributed in part to intracellular degradation of donor D N A and in part to shearing introduced during the extraction of D N A from the treated Streptomyces cells which involves grinding in dry ice. In conclusion, these findings are unexpected with regard to the usual observations made on the fate of heterospecific D N A taken up by bacteria [ 13,14]. However, taken together, these experiments present reasonably sound analogies with a competence phase as described in other bacteria, and allow us to select S. virginiae and to a lesser extent S. kasugaensis as recipients to look intensively for genetic expression and the metabolic fate of the donor DNA.
122
Thanks are due to Dr. B. B o o n for encouragement during this work and to Dr. D.A. H o p w o o d for generous gift of strains. This work was supported by grants of IRSIA and FRFC. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
H a r a d a , Y. ( 1 9 5 9 ) J. Agric. C h e m . Soc. J a P . 33, 7 4 4 - - 7 5 2 . H o r v a t h , J. ( 1 9 5 9 ) A c t a M i k r o b i o l . 6, 2 2 7 . M a t s e l y u k , B.P. ( 1 9 6 4 ) M i k r o b i o l . Zh. N a u k . U k r . R S R 2 6 , 8 - - 1 5 . R a m a c h a n d r a n , S., S u k a p u r e , R.S. a n d T h i r u m a l a c h a r , M.J. ( 1 9 6 5 ) H i n d u s t a n A n t i b i o t i c s Bull. 7, 197--202. Biswas, G . D . a n d S e n , S.P. ( 1 9 7 1 ) J. A p p l . B a c t e r i o l . 3 4 , 2 8 7 - - 2 9 3 . S e r m o n t i , G. a n d H o p w o o d , D.A. ( 1 9 6 4 ) in T h e B a c t e r i a ( G u n s a l u s , I.C. a n d S t a n i e r , R . Y . , eds.), Vol. 5, A c a d e m i c Press, N e w Y o r k . H o p w o o d , D . A . ( 1 9 7 3 ) in A c t i n o m y c e t a l e s ( S y k e s , G. a n d S k i n n e r , F . A . , eds.), p p . 1 3 1 - - 1 5 3 , A c a d e m i c Press, N e w Y o r k . H o p w o o d , D . A . a n d W r i g h t , H.M. ( 1 9 7 2 ) J. G e n . M i c r o b i o l . 7 1 , 3 8 3 - - 3 9 8 . L u r q u i n , P.F. a n d B e h k i , R . M . ( 1 9 7 5 ) M u t a t . Res. 29, 3 5 - - 5 1 . B o t t , K . F . a n d Wilson, G . A . ( 1 9 6 7 ) J. B a e t e r i o l . 9 4 , 5 6 2 - - 5 7 0 , G e r m a i n e , G . R . a n d A n d e r s o n , D.L. ( 1 9 6 6 ) J. B a c t e r i o l . 9 2 , 6 6 2 - - 6 6 7 . M a r m u r , J. ( 1 9 6 1 ) J. Mol. Biol. 3, 2 0 8 - - 2 1 8 . M o r r i s o n , D. a n d G u i l d , W. ( 1 9 6 7 ) J. B a c t e r i o l . 1 1 2 , 1 1 5 7 - - 1 1 6 8 . P i e c h o w s k a , M., S o l t y k , A. a n d S h u g a r , D. ( 1 9 7 5 ) J. B a c t e r i o l . 1 2 2 , 6 1 0 - - 6 2 2 . Cha.rles, P. ( 1 9 7 2 ) in U p t a k e o f I n f o r m a t i v e M o l e c u l e s b y Living Cells ( L e d o u x , L., e d . ) , p p . 1 0 - - 2 8 , North-Holland, Amsterdam.
Biochimica et Biophysica Acta, 442 (1976) 117--122 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 91432
DNA UPTAKE BY STREPTOMYCES SPECIES
P. R O E L A N T S a and V. K O N V A L I N K O V A L. LEDOUX b
b, M. M E R G E A Y
b, P.F. L U R Q U I N b and
aReeherche et Industrie Th$rapeutique (R.L T.), Bacteriology D e p a r t m e n t - Research Division, B-1330 Rixensart and b Department of Radiobiology, Centre d'Etude de l'Energie Nucl$aire (C.E.N./S.C.K.), B-2400 Mol (Belgium) (Received May 24th 1976)
Summary Uptake of homologous and heterologous radioactively labelled DNA was studied in ten 8treptornyces species. Among these, S. kasugaensis and S. virginiae were shown to take up 0.5--2.0% of the supplied donor DNA at a well-defined point in their growth cycle. The heterologous donor DNA taken up retained its original buoyant density in CsC1 gradients. Other strains either extensively degraded the donor DNA or showed poor uptake efficiency.
Previous attempts at demonstrating genetic transformation among
Streptornyces were based on the transfer of information for antibiotic production [1--5]. These reports were not supported by extensive biochemical and genetic data and are still awaiting confirmation [6,7]. An effective transformation system has been clearly established by Hopwood and Wright [8] in Thermoactinomyces vulgaris. However, this organism differs significantly (and notably by its low G + C content, sporulation physiology and temperature optimum) from the other Actinomycetales and looks to be not~ representative of this genus regarding transformation. As a first step in evaluating the possibility of transformation among some Streptomycetaceae of industrial interest, we have measured the ability of various strains to take up added exogenous DNA. These results are reported here. The organisms used were Streptornyces coelicolor A3 [2], S. fradiae (ATCC 10745), S. kanarnyceticus (ATCC 12853), S. kasugaensis (ATCC 15714), S. rimosus (ATCC 14827), S. tenebrarius (ATCC 17920), S. griseus (our isolate SG279), S. virginiae (ATCC 13161) and its derived mutant Nb. 10 and Micromonospora purpurea (ATCC 15835). Spores of each strain were inoculated in 125-ml flasks with 15 ml of the following medium convenient for Streptomyces: K2HPO4, 1 g; NaC1, 1 g;
118 MgSO4" 7H20, 1 g; L-asparagine, 1 g; casamino acids Difco, 1 g; glycerol, 10 g; 1000 ml distilled water and for Micromonospora: K2HPO4, 1 g; NaC1, 1 g; MgSO4" 7H20, 1 g; (NH4)2SO4, 1 g; yeast extract Difco, 1 g; glucose 10 g; 1000 ml distilled water. The flasks were incubated on a reciprocal shaker at 28°C and growth, measured as increase in absorbance at 660 nm, reached a maximum after about 20 h. Samples (1 ml) were taken at regular intervals. Mycelia were spun down, washed and resuspended in 1 ml fresh medium supplemented with tritiumlabelled DNA extracted from S. virginiae or Bacillus subtilis uracil-requiring mutants. (see legend of Fig. 1). The preparation of these DNAs involved lysis of the cells with lysozyme followed by 2% sodium dodecyl sulfate, incubation with pronase and a final purification step in preparative CsC1 density gradients as previously described
[9,15]. After 30 minutes of incubation with donor [3H] DNA, mycelia were treated with pancreatic deoxyribonuclease and processed for acid-precipitable radioactivity determination as described in the legend of Fig. 1. (a) Uptake kinetics. Preliminary results showed that the amount of donor DNA bound by the Streptomyces cells did not increase significantly at concentrations higher than 10--12 #g/ml (concentration range = 0.5--100 ~g/ml
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Fig. 1. U p t a k e of [ 3 H ] D N A b y S t r e p t o m y c e t e cells as a f u n c t i o n o f t h e g r o w t h cycle. Cells w e r e i n c u b a t e d w i t h 10 # g / m l [ 3 H ] D N A . A f t e r 30 rain i n c u b a t i o n a t 2S°C a D N A a s e s o l u t i o n ( 1 0 0 ~g in 1 m l fresh m e d i u m w i t h o u t c a r b o h y d r a t e s o u r c e ) was a d d e d a n d t h e i n c u b a t i o n was c o n t i n u e d f o r 10 rain at 37°C. M y c e l i a w e r e c e n t r i f u g e d , w a s h e d t w i c e w i t h c a r b o h y d r a t e - f r e e m e d i u m , r e s u s p e n d e d in 2 m l 5% t r i c h l o r o a c e t i c acid a n d h e l d f o r S0 rain a t 0°C. M y c e l t a w e r e finally r e c o v e r e d o n a Whatm a n glass fiber filter ( G F / C ) a n d w a s h e d several t i m e s w i t h c o l d t r i c h l o r o a c e t i e acid. T h e filters w e r e d r i e d , p l a c e d in s c i n t i l l a t i o n fluid ( t o l u e n e w i t h 0 . 2 5 g/1 2,2 ' - p - p h e n y l - b i s - ( 5 - p h e n y l o x a z o l ) a n d 5 g/1 2 , 5 - d i p h e n y l o x a z o l ) a n d r a d i o a c t i v i t y w a s m e a s u r e d in a P a c k a r d s c i n t i l l a t i o n s p e c t r o m e t e r , o - - ~ , g r o w t h m e a s u r e d b y A600 nm" ( A ) U p t a k e of [ 3 H ] D N A f r o m B. s u b t i l i s b y S. kasugaensis; o - - 4 , filterb o u n d c p m . (B) U p t a k e o f [ 3H] D N A f r o m B. subtilis b y S. virginiae; ~ - 4 , f i l t e r - b o u n d c p m . (C) UP o t a k e of 3H-labelled h o m o l o g o u s D N A b y S. virginiae a n d D N A s y n t h e s i s as f o l l o w e d b y [ 1 4 C ] t h y m i d i n e i n c o r p o r a t i o n in acid p r e c i p i t a b l e m a t e r i a l ; o - - o , u p t a k e of p o l y m e r i z e d h o m o l o g o u s D N A ; ~--~, d o n o r D N A e x p o s e d t o D N A a s e p r i o r to i n c u b a t i o n w i t h cells; D--~, [ t 4 C ] t h y m i d i n e i n c o r p o r a t e d a f t e r 3 0 rain i n c u b a t i o n in 1 m l s a m p l e s t o w h i c h 9 0 0 0 c p m o f [2 - 1 4 C ] t h y m i d i n e w e r e a d d e d (spec. act. 48 C i / m o l ) . T h e specific r a d i o a c t i v i t y of B. subtilis [ 3 H ] D N A was 1 × 106 cpm/~tg a n d t h a t o f S. uirginiae [ 3 H ] D N A w a s 3 • l 0 s c p m # t g . B o t h p r e p a r a t i o n s w e r e o b t a i n e d f r o m cells l a b e l e d w i t h 50 ~ C i / m l [ 3 H ] u r i d i n e .
119
DNA). Subsequently, all experiments were carried o u t with 10 pg/ml donor DNA. Table I reports the maximum uptake of B. subtilis DNA estimated as acid-precipitable radioactivity. The amount of DNA b o u n d b y the S. virginiae mutant strain Nb. 10 was found to be higher than for the other strains: around 1--2% of the labelled donor DNA was found in mycelia. As a first approximation, this represents a b o u t 2% of the endogenous DNA content. The ATCC 13161 strain retained only 0.3% of the supplied radioactivity. S. kasugaensis b o u n d 0.5% of the donor DNA; for S. fradiae, S. kanamyceticus, So rirnosus and S. tenebrarius uptake was less than 0.1% and for S. coelicolor and Micromonospora, less than 0.1%. TABLE I
B. S U B T I L I S [ S H ] D N A U P T A K E E F F I C I E N C Y MONOSPORA STRAINS
IN VARIOUS STREPTOMYCES
AND MICRO-
1 0 ~ g o f D N A w e r e given t o e a c h 1-rrfl s a m p l e and r e p r e s e n t e d 1 0 ~ c p m . T h e A 6 6 o n m o f t h e c u l t w t e s ranged f r o m 1 . 0 t o 1.5. I n c u b a t i o n w i t h d o n o r D N A w a s for 3 0 r a i n a t 2 8 ° C . A c i d - i n s o l u b l e radioa c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d u n d e r F i g . 1.
R e c i p i e n t strains
B. subtilis D N A u p t a k e in c p m
8. c o e l i c o l o r A 3 [ 2 ] 8. fradiae A T C C 1 0 . 7 4 5 S. griseus S G 2 7 9 8. k a n a m y c e t i c u 8 A T C C 1 2 . 8 5 3 S. kasugaensis A T C C 1 5 . 7 1 4 S. tenebrarius A T C C 1 7 . 9 2 0 S. rirnosus A T C C 1 4 . 8 2 7 S. pirginiae A T C C 1 3 . 1 6 1 S. pirginiae m u t a n t N b 1 0 M. p u r p u r e a A T C C 1 5 . 8 3 5
1 0 2, 1 0 2 2 " 1 0 3, 1 . 2 " 1 0 3 10 3 10 3 2 . 5 " 1 0 4, 2 . 4 • 1 0 4 , 4 • 1 0 4 7 10 2 2 • 1 0 3, 5 • 1 0 3 8-10 3 8 " 10 4, 2 • 1 0 s 10 2 "
In the case of S. coelicolor, it was found that the DNA of B. subtilis or S. coelicolor was extensively degraded exocellularly which corrobated a previous observation of H o p w o o d and Wright [7]. The DNA-binding pattern by the t w o best recipients S. virginiae and S. kasugaensis is shown in Fig. 1. It can be seen that uptake of DNAaseresistant radioactivity occurred at a defined m o m e n t of the growth cycle, strongly evoking a competence phase as described for a number of bacterial species [ 10]. A similar DNA absorption peak was also reported for S. griseus
[11]. Furthermore, the uptake p h e n o m e n o n seems to be independent of the origin (homologous or heterologous) of the donor DNA. Indeed, Figs. 1B and C describe the uptake of B. subtilis and S. virginiae [3H]DNA respectively by S. virginiae mycelia. It is shown that DNAase-resistant binding occurs at the same time of the growth cycle and it was found that the extent of binding was similar with both DNAs. The same observation holds true for S. coelicolor [3H]DNA as a donor (data not shown). Similarly, S. kasugaensis b o u n d S. coelicolor and B. subtilis [3H]DNA to the same extent and according to the same time-based pattern. Fig. 1C also demonstrates that a comparable a m o u n t of S. virginiae [ 3H] DNA, previously degraded upon a 10-min incubation at 37°C with 100 pg/ml pancreatic deoxyribonuclease, is very poorly utilized for endogenous
120 DNA synthesis. Thus, it turns out that the uptake p h e n o m e n o n is linked to the polymerized nature of the donor DNA. Moreover, DNA synthesis as measured by the incorporation of ['4C] thymidine into acid-precipitable material is found to drop drastically at the time of m a x i m u m DNA uptake as is shown in Fig. IC. Apparently there is a correlation between donor DNA uptake and the decline of endogenous DNA synthesis. The peak positions of DNA binding oscillate somewhat among different uptake experiments. Peaking was found to occur from the late exponential phase to the first hours of the stationary phase. Using S. virginiae as a recipient, peaks observed in t w e n t y DNA-uptake experiments arose at an average time of 22 h 30 min + 2 h 30 min after the inoculation of the culture. An average value of 22 + 2 h was observed using S. kasugaensis as a recipient in seven experiments. The kinetics of DNA binding were linear during the first 7 min of incubation but went on for at least another 23 min with a much shallower slope using a saturating concentration of 10 pg/ml donor DNA. (b) Nature of the intracellular radioactivity. No tool to look accurately for the genetic expression of homologous DNA taken up was available at the time of these experiments. Among the existing difficulties one notes the necessity of a sporulation step on solid medium before testing donor DNA expression. Suitable experimental conditions are currently being worked out. We found it of interest to get some insight into the fate of the donor DNA inside and outside the cells at a time when m a x i m u m uptake was shown to occur (see Fig. 1). To serve this purpose B. subtilis [3H]DNA was used as a donor since its lower G+C content (43%, CsC1 b u o y a n t density = 1.703 g. cm -3) makes it readily distinguishable from the host DNA having a G+C content of 71% (CsC1 b u o y a n t density = 1.730 g. cm-3). The use of heteropycnic ~ionor DNA is further justified on the basis of the similarity between homologous and heterologous DNA uptake curves {Figs. 1B and C}. The mycelia of S. kasugaensis or S. virginiae were lysed by treatment with lysozyme (200 pg/ml in saline/EDTA solution) immediately after incubation with [3H]DNA from B. subtilis and DNAase treatment. The DNA was extracted from the mycelium according to Marmur [12] and analyzed in CsC1 density gradients. Figs. 2A and B clearly show that most of the intracellular radioactivity bands at the position of the donor DNA (d = 1.703 g. cm-3). The shoulder located to the left of the main radioactive band is most probably a result of donor DNA breakdown and reutilization. Indeed, breakdown of donor DNA must have occurred intracellularly since about 30% of the radioactivity found associated with cells incubated in the presence of [3H] DNA was acid-soluble. This portion of the intracellular radioactivity did not show up in CsC1 gradients as a result of our DNA extraction procedure which does not allow recovery of low molecular weight compounds. The incubation medium still contained 95% acid-precipitable radioactive compounds after the 30-min incubation with mycelia. That extracellular degradation was very limited was confirmed by sedimentation velocity analysis of the donor DNA before and after incubation. Fig. 2C shows that the rate of sedimentation of the incubated donor [3H] DNA in a 5--20% alkaline sucrose gradient was somewhat decreased but that no radioactivity was floating at the top of the gradient.
121
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Fig. 2. ( A ) CsCI d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f D N A e x t r a c t e d f r o m S. kasugaensis i n c u b a t e d f o r 3 0 m i n w i t h B. subtflis [ S H ] D N A 2 2 h a f t e r i n o c u l a t i o n (see Fig. 1 A ) . (B) CsC1 g r a d i e n t c e n t r i f u g a t i o n o f D N A e x t r a c t e d f r o m S. virginiae i n c u b a t e d f o r 3 0 r a i n w i t h B. subtiits [ S H ] D N A 2 0 h a f t e r i n o c u l a t i o n (see Fig. 1B). D N A w a s e x t r a c t e d f r o m m y c e H a ( a c e t o n i c p o w d e r ) b y g r i n d i n g w i t h solid C O 2 a n d i n c u b a t i n g t h e r e s u l t i n g e x t r a c t w i t h l y s o z y m e , p r o n a s e a n d r i b o n u c l e a s e s u c c e s s i v e l y as d e s c r i b e d for the isolation of other bacterial DNAs [9,15]. The DNA solutions were then brought to a refractive i n d e x o f 1 . 3 9 9 5 w i t h CIC! ( 8 u p r a p t t r , M e r c k ) , a d j u s t e d t o 3 m l w i t h a CsCl s o l u t i o n o f t h e s a m e d e n s i t y a n d c e n t r i f u g e d a t 2 0 ° C f o r 6 3 h a t a 0 0 0 0 r e v , / m i n in a M a r t i n C h r i s t S W 6 0 r o t o r . The slope o f t h e g r a d i e n t w a s d e t e r m i n e d b y a d d i n g u n l a b e l l e d 8. eoc|lcolor D N A (pffi 1 . 7 3 0 g / c m 3) and B. subtills D N A (p ,, 1 . 7 0 8 g/oreS). The absorbance a t 2 6 0 n m c o r r e s p o n d i n g t o t h e s e m a r k e r D N A I w a s m e a s t t r e d i n a C a r y 1 4 r p e c t t o p h o t o m e t e r , l ~ a d i o a c t i v i t y d e t e r m i n a t i o n s w e r e d o n e b y l i q u i d scintillat i o n i n the presence o f l n s t a g e l ( P a c k a r d ) . e - e , A260 n m ; ~ ' ~ , 3H c p m . (C) S e d i m e n t a t i o n v e l o c i t y a n a l y s i s o f B. subtllis [ S H ] D N A u s e d in t h e u p t a k e e x p e r i m e n t s . T h e D N A s a m p l e s (1150/~1) w e r e l a y e r e d o n t o p o f 5 m l 5 - 2 0 % a l k a l i n e s u c r o s e g r a d i e n t s a n d s p u n f o r 3 h a t 4 6 0 0 0 r e v . / m i n in a n SW 5 2 M a r t i n C h r i s t r o t o r a t 2 0 ° C . T h e g r a d i e n t s w e r e f r a c t i o n a t e d f r o m t h e b o t t o m a n d t h e r a d i o a c t i v i t y o f t h e f r a c t i o n s w a s d e t e r m i n e d as a b o v e , e - - e , d o n o r D N A b e f o r e i n c u b a t i o n w i t h cells; c - - ~ , d o n o r D N A r e c o v e r e d f r o m t h e s u p e r n a t a n t a f t e r 3 0 r a i n i n c u b a t i o n w i t h S. virginiae g r o w i n g f o r 2 0 h. S e d i m e n t a t i o n is f r o m r i g h t t o left.
These results would indicate that the exogenous D N A could remain relatively undegraded and retained its original buoyant density inside the recipient cells. However, the mean size of the donor D N A molecules recovered from the incubated cells is reduced as compared to that of the original donor DNA. This is indicated by the band-broadening observed in Figs. 2A and B where it can be seen that the banding pattern of the exogenous D N A is wider than that of the reference B. subtilis DNA. This phenomenon may be attributed in part to intracellular degradation of donor D N A and in part to shearing introduced during the extraction of D N A from the treated Streptomyces cells which involves grinding in dry ice. In conclusion, these findings are unexpected with regard to the usual observations made on the fate of heterospecific D N A taken up by bacteria [ 13,14]. However, taken together, these experiments present reasonably sound analogies with a competence phase as described in other bacteria, and allow us to select S. virginiae and to a lesser extent S. kasugaensis as recipients to look intensively for genetic expression and the metabolic fate of the donor DNA.
122
Thanks are due to Dr. B. B o o n for encouragement during this work and to Dr. D.A. H o p w o o d for generous gift of strains. This work was supported by grants of IRSIA and FRFC. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
H a r a d a , Y. ( 1 9 5 9 ) J. Agric. C h e m . Soc. J a P . 33, 7 4 4 - - 7 5 2 . H o r v a t h , J. ( 1 9 5 9 ) A c t a M i k r o b i o l . 6, 2 2 7 . M a t s e l y u k , B.P. ( 1 9 6 4 ) M i k r o b i o l . Zh. N a u k . U k r . R S R 2 6 , 8 - - 1 5 . R a m a c h a n d r a n , S., S u k a p u r e , R.S. a n d T h i r u m a l a c h a r , M.J. ( 1 9 6 5 ) H i n d u s t a n A n t i b i o t i c s Bull. 7, 197--202. Biswas, G . D . a n d S e n , S.P. ( 1 9 7 1 ) J. A p p l . B a c t e r i o l . 3 4 , 2 8 7 - - 2 9 3 . S e r m o n t i , G. a n d H o p w o o d , D.A. ( 1 9 6 4 ) in T h e B a c t e r i a ( G u n s a l u s , I.C. a n d S t a n i e r , R . Y . , eds.), Vol. 5, A c a d e m i c Press, N e w Y o r k . H o p w o o d , D . A . ( 1 9 7 3 ) in A c t i n o m y c e t a l e s ( S y k e s , G. a n d S k i n n e r , F . A . , eds.), p p . 1 3 1 - - 1 5 3 , A c a d e m i c Press, N e w Y o r k . H o p w o o d , D . A . a n d W r i g h t , H.M. ( 1 9 7 2 ) J. G e n . M i c r o b i o l . 7 1 , 3 8 3 - - 3 9 8 . L u r q u i n , P.F. a n d B e h k i , R . M . ( 1 9 7 5 ) M u t a t . Res. 29, 3 5 - - 5 1 . B o t t , K . F . a n d Wilson, G . A . ( 1 9 6 7 ) J. B a e t e r i o l . 9 4 , 5 6 2 - - 5 7 0 , G e r m a i n e , G . R . a n d A n d e r s o n , D.L. ( 1 9 6 6 ) J. B a c t e r i o l . 9 2 , 6 6 2 - - 6 6 7 . M a r m u r , J. ( 1 9 6 1 ) J. Mol. Biol. 3, 2 0 8 - - 2 1 8 . M o r r i s o n , D. a n d G u i l d , W. ( 1 9 6 7 ) J. B a c t e r i o l . 1 1 2 , 1 1 5 7 - - 1 1 6 8 . P i e c h o w s k a , M., S o l t y k , A. a n d S h u g a r , D. ( 1 9 7 5 ) J. B a c t e r i o l . 1 2 2 , 6 1 0 - - 6 2 2 . Cha.rles, P. ( 1 9 7 2 ) in U p t a k e o f I n f o r m a t i v e M o l e c u l e s b y Living Cells ( L e d o u x , L., e d . ) , p p . 1 0 - - 2 8 , North-Holland, Amsterdam.