F o l i a Microbiol. 24, 4 4 5 - - 4 4 8 (1979)
Aeration Sensitizes to Hydroxyurea
Streptococcus faecalis
R. LAlZTI and J. H~INON~ Departraent of Biochemistry, University of Turku, S~.20500 Turku gO, Finland t~eceived October 24, 1978
A B S T R A C T . H y d r o x y u r e a in u p to 60 m ~ c o n c e n t r a t i o n d i d n o t i n h i b i t g r o w t h or D N A s y n t h e s i s in n o n a e r a t e d c u l t u r e s of Streptococcus ]ascalis A T C C 8043. I n c o n t r a s t , in c u l t u r e s a ~ r a t e d b y s h a k i n g a l r e a d y 1 mM h y d r o x y u r e a d e c r e a s e d t h e r a t e o f n e t D N A s y n t h e s i s a n d in h i g h e r c o n c e n t r a t i o n s of t h e d r u g t h e g r o w t h o f t h e t o t a l cell m a s s also slowed d o w n a n d t h e n u m b e r of cells per c h a i n i n c r e a s e d f r o m 1 -- 2 to 10. T h e differential rato o f D N A s y n t h e s i s , b u t n o t t h o g r o w t h of t h o t o t a l cell m a s s , c o u l d be r e s t o r e d a l m o s t to t h e c o n t r o l level b y a d d i n g t h y m i d i n e t o t h e m e d i u m . T h u s t h e r e a r e a t least t w o t a r g e t s for h y d r o x y u r e a in t h e cells o f S. faecalis grown in a e r a t e d c u l t u r e s .
Hydroxyurea prevents DNA synthesis by inhibiting the reduction of ribonucleotides by ribonucleotide reductase (Reichard 1972). In our studies concerning the role of inorganic pyrophosphatase (EC 3.6.1.1) in DNA synthesis in bacteria we observed that cultures of Streptococcus faecalis were normally resistant to hydroxyurea, but became sensitive to it when aerated by shaking. Our results also suggest that, in addition to ribonucleotide reductase, there is an other target for hydroxyurea in the cells of S. faecalis growing in aerated cultures.
MATERIALS AND METHODS
Organism and cultivation. Streptococcus faecalis ATCC 8043 was used. The growth medium contained in 1 litre of distilled water: yeast extract 15 g, Bacto-tryptone (Difco) 5 g, K~HPOa 2 g. Glucose (10 g) was added as sterile solution after the medium had been sterilized in the autoclave (15 min, 120 ~ Growth took place at 37 ~ in Erlenmeyer flasks stoppered with cotton plugs. Nonaerated cultures were allowed to stand still except for the gentle manual shaking immediately before sampling. Aerated cultures were shaken in a rotary shaker (4.2 Hz). The initial volume of the culture in 500 ml flask was 150 ml. The growth of the total cell mass was followed by measuring the turbidity of the culture with a Klett-Summerson colorimeter equipped with filter 62 (590--650 nm). The ratio turbidity/dry weight was observed to change by at most 10 %, when
446 R . L A H T I a n d J . H E I N O N E N
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F~G. 1. E f f e c t o f h y d r o x y u r e a o n g r o w t h a n d D N A s y n t h e s i s in a e r a t e d c u l t u r e s of S. faecalis; left: g r o w t h o f t h e t o t a l cell m a s s ( t u r b i d i t y , K U ) ; right: differential r a t e o f D N A s y n t h e s i s ( a r b i t r a r y unit.s, u); t h e : d r u g c o n c e n t r a t i o n s 0, l, 5, 20, 40, a n d 60 raM.
the long chains of cells were formed. Because this change does not alter our results significantly, t u r b i d i t y is used as a measure of total cell mass in this paper. In the beginning of an experiment an overnight culture (in the stationary phase of growth) was diluted with prewarmed medium to reach the turbidity of 10 K l e t t units. The culture was immediately divided into several portions. One served as a control, to the others h y d r o x y u r e a (Sigma) was added. Determination of net D N A synthesis. Samples (1--5 ml, depending on the t u r b i d i t y of the culture) were withdrawn from the cultures and the cells were separated b y centrifugation in the cold. The amounts of D~qA in the samples were determined b y measuring the enhancement of the fluorescence of ethidium ions in the presence of the alkali-treated samples (Donkersloot et al. 1972). The amount of D]~A is given in the figures as arbitrary units (the fluorometer readings referred to the sample volume of 1 ml). RESULTS AND DISCUSSION
In normal nonaerated cultures of S. faecaHs h y d r o x y u r e a had practically no effect either on growth or on net D N A synthesis even in 60 m ~ concentration. However, rather unexpectedly aeration b y shaking made the cultures of S. faecalis sensitive to the drug. Already 1 m ~ h y d r o x y u r e a decreased the differential rate of ])NA synthesis, although the growth of the total cell mass was unaffected. At higher drug concentrations the relative rate of DNA synthesis was further decreased and the growth of the total cell mass also slowed down (Fig. 1). In contrast to Escherichia coli, the cell of S. faeealis did not t u r n into long filaments in the presence of h y d r o x y u r e a b u t the n u m b e r of ceils per chain increased from 1--2 up to 10, as observed b y phasecontrast microscope.
1979
SENSITIVITY OF S. faecalis TO t I Y D R O X Y U R E A I "
|
J
20.0
125
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I
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447
--
U
KU
100
100 75
50 50
20 25
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I
0
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50
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I
100
150 KU
FIG. 2. Thymidine prevents the inhibition of DNA synthesis by hydroxyurea; left: growth of the total cell mass (turbidity, KU); right: differential rate of DNA synthesis (arbitrary units, u); C control, H 12 m E hydroxyurea, H -~ T 12 mM hydroxyurea + 0.7 m ~ thymidine.
The sensitization of S. faecalis to hydroxyurea by aeration is a puzzling phenomenon. Aeration per se is not deleterious to the cells. On the contrary, the final yield of total cell mass was increased 30--40 ~o by aeration. This has also been observed by Dolin (1955). The oxidation of hydroxyurea to the real effector, the change in the metabolism to hydroxyurea-sensitive enzyme systems, or the stimulation of the uptake of the drug by aeration are some possible explanations. Thymidine, but not thymine, almost completely abolished the specific retardation of net DNA synthesis caused by hydroxyurea. The inhibition of growth could not be relieved by thymidine, however (Fig. 2). This suggests that there are at least two independent targets of hydroxyurea in the cells of S. faecalis growing in aerated cultures. The first target, responsible for the inhibition of DNA synthesis by the drug, is evidently ribonucleotide reductase. The autolytic enzyme(s) required in the synthesis of the cell envelope and in the separation of the cells after cell division might be the other target, because the mean number of cells in one chain increased drastically in the presence of hydroxyurea and this effect could not be reversed by tbymidine. Feiner et al. (1973) have earlier proposed that autolysins of Staphylococcus aureus and Micrococcus lysodeil~ticus are inhibited by the drug. This work was supported by the Finnish National ]~eseareh Council for Science.
448
R. L A H T I and J. H E I N O N E N
Vol. 24
REFERENCES
DoLI~ M. I.: The DPNH-oxidizing enzymes of Streptococcus faecalis. II. The enzyme utilizing oxygen, eytochrome c, peroxide and 2,6-dichlorophenol-indophenol or ferricyanide as oxidants. Arch.B;~ochem. Biophys. 55, 415 (1955). DOI~'~KERSLOOT J. A., ROBRISH S. A., KRICHEVSKY I. M.: Fluorometrie determination of deoxyribonucleic acid in bacteria with ethidium bromide. Appl.Microbiol. 24, 179 (1972). FEINER 1~,. R., (~OWARDJ. E., ROSEIVKRANZH. S.: Effects of hydroxyurea on Staphylococcus epidvrmi~ and ~licrococcus lysodeikticus. Antimicrob.Agents Chemother. 3, 432 (1973). REICH~D P.: Control of deoxyribonueleotide synthesis in vivo and in vitro, p. 3 in Advances in Enzyme tCegulation (G. Weber, Ed.), Vol. 10. Pergamon Press, Oxford 1972.
Aeration Sensitizes to Hydroxyurea
Streptococcus faecalis
R. LAlZTI and J. H~INON~ Departraent of Biochemistry, University of Turku, S~.20500 Turku gO, Finland t~eceived October 24, 1978
A B S T R A C T . H y d r o x y u r e a in u p to 60 m ~ c o n c e n t r a t i o n d i d n o t i n h i b i t g r o w t h or D N A s y n t h e s i s in n o n a e r a t e d c u l t u r e s of Streptococcus ]ascalis A T C C 8043. I n c o n t r a s t , in c u l t u r e s a ~ r a t e d b y s h a k i n g a l r e a d y 1 mM h y d r o x y u r e a d e c r e a s e d t h e r a t e o f n e t D N A s y n t h e s i s a n d in h i g h e r c o n c e n t r a t i o n s of t h e d r u g t h e g r o w t h o f t h e t o t a l cell m a s s also slowed d o w n a n d t h e n u m b e r of cells per c h a i n i n c r e a s e d f r o m 1 -- 2 to 10. T h e differential rato o f D N A s y n t h e s i s , b u t n o t t h o g r o w t h of t h o t o t a l cell m a s s , c o u l d be r e s t o r e d a l m o s t to t h e c o n t r o l level b y a d d i n g t h y m i d i n e t o t h e m e d i u m . T h u s t h e r e a r e a t least t w o t a r g e t s for h y d r o x y u r e a in t h e cells o f S. faecalis grown in a e r a t e d c u l t u r e s .
Hydroxyurea prevents DNA synthesis by inhibiting the reduction of ribonucleotides by ribonucleotide reductase (Reichard 1972). In our studies concerning the role of inorganic pyrophosphatase (EC 3.6.1.1) in DNA synthesis in bacteria we observed that cultures of Streptococcus faecalis were normally resistant to hydroxyurea, but became sensitive to it when aerated by shaking. Our results also suggest that, in addition to ribonucleotide reductase, there is an other target for hydroxyurea in the cells of S. faecalis growing in aerated cultures.
MATERIALS AND METHODS
Organism and cultivation. Streptococcus faecalis ATCC 8043 was used. The growth medium contained in 1 litre of distilled water: yeast extract 15 g, Bacto-tryptone (Difco) 5 g, K~HPOa 2 g. Glucose (10 g) was added as sterile solution after the medium had been sterilized in the autoclave (15 min, 120 ~ Growth took place at 37 ~ in Erlenmeyer flasks stoppered with cotton plugs. Nonaerated cultures were allowed to stand still except for the gentle manual shaking immediately before sampling. Aerated cultures were shaken in a rotary shaker (4.2 Hz). The initial volume of the culture in 500 ml flask was 150 ml. The growth of the total cell mass was followed by measuring the turbidity of the culture with a Klett-Summerson colorimeter equipped with filter 62 (590--650 nm). The ratio turbidity/dry weight was observed to change by at most 10 %, when
446 R . L A H T I a n d J . H E I N O N E N
200
|
J
"'
'1
'"
':
Vol. 24
.
100
t
5:~.
KU 100
-
!
"
I'
U
,
75 1
50
50
2O
9
5"
20
25
h0
"
10 !
|
0
2
t
0 h
9.
!
!
I
50
100
150
.
,
200 KU
F~G. 1. E f f e c t o f h y d r o x y u r e a o n g r o w t h a n d D N A s y n t h e s i s in a e r a t e d c u l t u r e s of S. faecalis; left: g r o w t h o f t h e t o t a l cell m a s s ( t u r b i d i t y , K U ) ; right: differential r a t e o f D N A s y n t h e s i s ( a r b i t r a r y unit.s, u); t h e : d r u g c o n c e n t r a t i o n s 0, l, 5, 20, 40, a n d 60 raM.
the long chains of cells were formed. Because this change does not alter our results significantly, t u r b i d i t y is used as a measure of total cell mass in this paper. In the beginning of an experiment an overnight culture (in the stationary phase of growth) was diluted with prewarmed medium to reach the turbidity of 10 K l e t t units. The culture was immediately divided into several portions. One served as a control, to the others h y d r o x y u r e a (Sigma) was added. Determination of net D N A synthesis. Samples (1--5 ml, depending on the t u r b i d i t y of the culture) were withdrawn from the cultures and the cells were separated b y centrifugation in the cold. The amounts of D~qA in the samples were determined b y measuring the enhancement of the fluorescence of ethidium ions in the presence of the alkali-treated samples (Donkersloot et al. 1972). The amount of D]~A is given in the figures as arbitrary units (the fluorometer readings referred to the sample volume of 1 ml). RESULTS AND DISCUSSION
In normal nonaerated cultures of S. faecaHs h y d r o x y u r e a had practically no effect either on growth or on net D N A synthesis even in 60 m ~ concentration. However, rather unexpectedly aeration b y shaking made the cultures of S. faecalis sensitive to the drug. Already 1 m ~ h y d r o x y u r e a decreased the differential rate of ])NA synthesis, although the growth of the total cell mass was unaffected. At higher drug concentrations the relative rate of DNA synthesis was further decreased and the growth of the total cell mass also slowed down (Fig. 1). In contrast to Escherichia coli, the cell of S. faeealis did not t u r n into long filaments in the presence of h y d r o x y u r e a b u t the n u m b e r of ceils per chain increased from 1--2 up to 10, as observed b y phasecontrast microscope.
1979
SENSITIVITY OF S. faecalis TO t I Y D R O X Y U R E A I "
|
J
20.0
125
I
I
I
447
--
U
KU
100
100 75
50 50
20 25
10 I
I
0
2
"I
~. h
I
50
I
I
100
150 KU
FIG. 2. Thymidine prevents the inhibition of DNA synthesis by hydroxyurea; left: growth of the total cell mass (turbidity, KU); right: differential rate of DNA synthesis (arbitrary units, u); C control, H 12 m E hydroxyurea, H -~ T 12 mM hydroxyurea + 0.7 m ~ thymidine.
The sensitization of S. faecalis to hydroxyurea by aeration is a puzzling phenomenon. Aeration per se is not deleterious to the cells. On the contrary, the final yield of total cell mass was increased 30--40 ~o by aeration. This has also been observed by Dolin (1955). The oxidation of hydroxyurea to the real effector, the change in the metabolism to hydroxyurea-sensitive enzyme systems, or the stimulation of the uptake of the drug by aeration are some possible explanations. Thymidine, but not thymine, almost completely abolished the specific retardation of net DNA synthesis caused by hydroxyurea. The inhibition of growth could not be relieved by thymidine, however (Fig. 2). This suggests that there are at least two independent targets of hydroxyurea in the cells of S. faecalis growing in aerated cultures. The first target, responsible for the inhibition of DNA synthesis by the drug, is evidently ribonucleotide reductase. The autolytic enzyme(s) required in the synthesis of the cell envelope and in the separation of the cells after cell division might be the other target, because the mean number of cells in one chain increased drastically in the presence of hydroxyurea and this effect could not be reversed by tbymidine. Feiner et al. (1973) have earlier proposed that autolysins of Staphylococcus aureus and Micrococcus lysodeil~ticus are inhibited by the drug. This work was supported by the Finnish National ]~eseareh Council for Science.
448
R. L A H T I and J. H E I N O N E N
Vol. 24
REFERENCES
DoLI~ M. I.: The DPNH-oxidizing enzymes of Streptococcus faecalis. II. The enzyme utilizing oxygen, eytochrome c, peroxide and 2,6-dichlorophenol-indophenol or ferricyanide as oxidants. Arch.B;~ochem. Biophys. 55, 415 (1955). DOI~'~KERSLOOT J. A., ROBRISH S. A., KRICHEVSKY I. M.: Fluorometrie determination of deoxyribonucleic acid in bacteria with ethidium bromide. Appl.Microbiol. 24, 179 (1972). FEINER 1~,. R., (~OWARDJ. E., ROSEIVKRANZH. S.: Effects of hydroxyurea on Staphylococcus epidvrmi~ and ~licrococcus lysodeikticus. Antimicrob.Agents Chemother. 3, 432 (1973). REICH~D P.: Control of deoxyribonueleotide synthesis in vivo and in vitro, p. 3 in Advances in Enzyme tCegulation (G. Weber, Ed.), Vol. 10. Pergamon Press, Oxford 1972.
