Effect of carbon source during growth on sensitivity of Pseudomonasfluorescens to actinomycin Dl
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CYNTHIAA. WALKER A N D NORMAN N. DURHAM Departrnent of Microbiology, Oklahotnn Stote University, Still\voter, Oklohotno 74074 Accepted September 6, 1974
WALKER, C. A., and DURHAM, N. N . 1975. Effect of carbon source during growth on sensitivity of Pserrdomonasjirorescens to actinomycin D. Can. J. Microbiol. 21: 69-74. Sensitivity to actinomycin D(AD) varies in P s e r t J o t n o t ~ ~ s ~ r t o r r ~cells ~ c egrown t ~ . ~ in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity inglucose-grown cells. Succinate-grown cells bound 2-3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lip0 olysaccharide masks the binding sites and, therefore, is responsible for the difference in blngng of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic. WALKER,C. A., e t DURHAM, N . N. 1975. Effect of carbon source duringgrowth on sensitivity of Pse~rdomotzosjuorescensto actinomycin D. Can. J. Microbiol. 21: 69-74. Chez Pseirdomonos jirorescens, la sensibilite B I'actinomycine D (AD) varie selon que la culture est faite dans un milieu minimal contenant des sels e t du glucose ou du succinate. Dans le milieu minimal avec succinate la croissance est inhibee par des concentrations plus faibles d'AD que dans le milieu minimal avec glucose. Dans le cas des cellules cultivees pendant 2 h dans le milieu avec succinate additionnk d'AD, la captation de metabolites radioactifs est inhibee par I'AD alors que les cellules cultivees avec glucose sont insensibles. Le traitement par I'EDTA augmente la sensibilitk B I'AD des cellules cultivees avec le succinate e t n'affecte pas celle des cellules cultivees avec glucose. Les cellules cultivees avec succinate fixent 2 B 3 fois plus de 3H-ADque les cellules cultivees avec glucose. Par rapport aux cellules cultivees avec succinate, les cellules cultivkes en presence de glucose ont une enveloppe plus riche en lipopolysaccharides. I1 est possible que ce materiel lipopolysaccharidique masque les sites de fixation et qu'il soit ainsi responsable de la difference dans la quantite d'AD fixee par les cellules cultivkes avec glucose e t succinate. La disponibilitt des sites de fixation est aussi impliquke dans la sensibilite des cellules B I'antibiotique. [Traduit par le journal]
Introduction The pseudomonads, a highly adaptive group of microorganisms, are notoriously insensitive to antibiotics. Actinomycin D (AD), which prevents protein synthesis by inhibiting deoxyribonucleic acid (DNA) - directed ribonucleic acid (RNA) synthesis (7, 9, 15), is partially active against these gram-negative organisms. AD inhibits uptake and incorporation of selected amino acids and uracil, a; well as the synthesis of certain inducible enzymes in Pseudomonas jiuorescens (4). The primary mechanism of inhibition is thought to be the physical blockage of RNA polymerase (5). Thus, for the antibiotic to reach its effective site of action, the molecule must first penetrate the cell permeability barrier. Walker and Durham (17) demonstrated that 'Received June 17, 1974.
the sensitivity of P. fluorescens NND to AD is partially dependent -on the carbon source and concentration in which the organism is growing. Growth of the organism in either glucose or succinate as the sole source of carbon and energy effectively alters the inhibition pattern of the organism. This investigation characterizes the physiological differences in cells grown in these two carbon sources.
Materials and Methods Test Organism The organism used in this study was Pseudomonas flrorescens N N D (1 1 ) . Stock cultures were maintained on 0.2% (w/v) succinate-salts agar slants stored at 4C. Medium The minimal salts mcdium had the following composition (w/v): 0.42% ;:&PO4, 0.32% K H Z P 0 4 , 0.2% NH4CI, and 0.2% NaCI. The carbon source was added to a final concentration of 2.78 x lo-' M and the p H adjusted to 7.0 with K O H before sterilization by auto-
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CAN. 1. MICROBI(3L. VOL. 21, 1975
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claving for 15 min at 121C and 15 Ib pressure. Glucose was sterilized separately by autoclaving for 10 min and added aseptically to the sterile minimal salts medium just before use. A sterile trace minimal salts solution (0.1 ml) was added to each 100 ml of synthetic medium (3). Atztibiotics Actinomycin D (Merck, Sharp, and Dohme) was dissolved in sterile, glass-distilled water (300 pg/ml) and stored at 4C until used. Groivt/~Studies Growth studies were conducted in 18 x 150 mm tubes, 250-nil sidearm flasks, or a mass fermenter, depending on the cell mass needed for analysis. Tubes and flasks were incubated at 37C on a reciprocal shaker, and growth was followed by measuring the increase in absorbance at 540 nni using a Coleman Junior I1 spectrophotometer. Radioisotope Studies Uptake of radioactive carbon sources was examined by growing the cells in 26.0 ml of medium in a 250-ml sidearm flask. The cells were incubated at 37C with shaking for 3 h at which time either 3.0 ml of A D (300 ~ g / m l or ) 3.0 ml of potassium phosphate buffer (pH 7.0) was added, and the cells incubated 2 additional h. The cells were harvested, suspended to an absorbance at 540 nm, equivalent to 0.35 mglml dry cell weight, in minimal salts niedii~mcontaining no carbon source, and equilibrated to 37C. Five milliliters of this cell suspension was added t o tubes containing the appropriate carrier (0.5 mg/ml), labeled carbon source (0.5 pCi/nll), and either A D (30 pg/ml) or potassium phosphate buffer. Samples (0.5 ml) were removed, filtered through Millipore filters (0.45micron (p) pore size), and washed twice with minimal salts buffer. The filters were placed in a counting vial, Aqi~asol(10 ml) was added, and the radioactivity determined in a liquid scintillation spectrometer (Nuclear Chicago) which has an efficiency of 40% under these conditions.
Ef/~ylenediatnit~etetr.ancetic Acid (EDTA) Treatmetzt of Cells Cells were suspended in Tris (2-amino-2(hydroxymethyl)-1,3-propanediol) buffer (0.12 M, p H 8.0) t o an absorbance at 540 nm, equivalent to 0.45 mg/ml dry cell weight, and treated with EDTA (pH 7.0, final concentraM) for 2 min at 37C. The action of EDTA tion I x was stopped by making a 1: 10 dilution into fresh medium. The cells were inoculated into culture tubes and growth in the presence and absence of A D was followed spectrophotometrically. Plate counts established that cell viability was not altered by the EDTA treatment.
Preparatiotz of Cell Envelopes Cells were grown in 5.0-liter volumes on a mass fermenter or in 0.5-liter volumes in Fernbach flasks. The cells were harvested, suspended in minimal volumes of physiological saline, and frozen in 10-ml volumes. The cells were broken as a frozen pellet using an X-press (AB BIOTEC, Stockholm, Sweden), placed in 100 ml of saline, and centrifuged at 1935 x g a t 4C. Thelarger debris was removed, envelope material was sedimented by centrifugation at 27 000 x g for 20 min, and purified by repeated washing. The envelope material was frozen in saline until used. Phase-contrast microscopy revealed that less than 5% whole cells remained.
Results Eflect of A D on Growth AD readily inhibits cells growing in a succinate-salts medium and the inhibition is concentration dependent (Fig. 1). An inhibition of 50% or more was commonly observed at the lower concentrations while complete inhibition was evident at higher concentration. Microscopic examination of the inhibited cells revealed the presence of filaments. In contrast, glucose-grown cells exhibited a decreased sensitivity to AD (Fig. 1). Less than a 50% inhibition of the total cell mass was noted even at the higher concentrations of the antibiotic (30 pg/ml) as compared to total inhibition of growth in the succinate-grown cells at the same concentrations. Microscopically, the glucose-grown cells showed only a few aberrant cells (about 10%) after growth in AD containing medium.
Eflect of A D on Uptake of Radioactive Metabolites AD blocks uptake of selected amino acids in P. fl~roresceris (4). Experiments were performed to measure the uptake of several metabolites by cells grown on the two carbon sources. Labeled substrates included 14C-glucose (specific activity 3.0 mcilrnmol), 14C-succinate (specific activity 1.8 mCilmmol), 14C-alanine (specific activity 5.0 mCilmmol), 14C-phenylalanine (specific activity 10.3 mCilmmol), and 14C-uracil. The reBinding of 3H-AD Binding was carried out in a 5.5-ml total liquid volume sults for 14C-phenylalanine are presented in Fig. at room temperature. The cells were exposed to 5.0 2. The uptake of metabolites was not influenced pCi 3H-AD (Schwarz Bioresearch, 5.5 mCi/mg) for 15 min, centrifuged at 17 300 x g at 4C, and washed twice by the presence or absence of AD in the uptake with physiological saline. The cell pellets were digested solutions for both succinate- and glucose-grown overnight in 0.5 ml of NCS reagent (Nuclear-Chicago) at cells, but growth in the presence of the antibiotic room temperature, and the digested material was added in the succinate medium for 2 h did produce an to 10 ml of scintillation fluid (2.0 g of 2,5-diphenyloxazole inhibition. With all radioactive substrates, prior (PPO), 0.025 g 1,4-bis-[2-(5-phenyloxazolyl)]-benzene (POPOP) in 237 ml dioxane). Radioactivity was deter- incubation of the cells in the succinate medium containing AD for 2 h inhibited active uptake in mined by liquid scintillation spectrophotometry.
WALKER AND DURHAM: SENSITIVITY OF P. FLUORESCENS TO A D I
SUCCINATE MINIMAL MEDIUM
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GLUCOSE MINIMAL MEDIUM
.9 -
.9 -
.5 -
.5 -
E
0
cr"
.I.05 -
.05
.Oil . 0
2
4 6 8 TIME (HOURS)
1
0
0
2
0
1 4 6 8 T I M E ( HOURS)
0
l
FIG1. Effect of AD on cells growing in succinate or glucose minimal salts medium. ( a ) , Control; (A), 1 0 pg/ml AD ; (m),20 pg/ml AD ; ( e l , 3 0 wg/ml AD.
tially linear in both sets of cells. The break in the adsorption curves suggests that two separate parameters may be participating in the measurable phenomenon-binding and uptake. T o determine if the difference in 3H-AD accumulation was a property of the cell envelope, similar studies
TlME (MIN)
Fig. 2. Uptake of 14C-phenylalanine by succinate- and glucose-grown cells. ( a ) , Succinate-grown cells with and without AD added to the uptakesystem; ( e ) , succinate AD grown cells with and without AD added to the uptake system; (D), glucose-grown cells with and without AD added to the uptake system, arzd glucose + AD grown cells with and without AD added t o the uptake system. AD, when present, was at a concentration of 3 0 pg/ml.
+
succinate-grown cells, but similar treatment did not inhibit uptake of the substrates in the glucosegrown cells. Binding of 3H-AD to Glucose- and Succinategrown Cells Experiments were performed to measure binding of 3H-AD to cells grown in the different carbon sources. Cells grown in succinate bind 4-5 times as much 3H-AD as the same cell mass of glucose-grown cells (Fig. 3). Adsorption is par-
M g Dry Cell Weight
FIG.3 . Binding of 3H-AD to glucose (A) and succinate ( 0 ) grown cells. Cells were grown for 12 h, harvested, and suspended in saline to the indicated dry cell weight. Five milliliters of cell suspension was added to 0.5 ml of 3H-AD (specific activity 8.4 Ci/mmol, 5.0 pCi/ml).
CAN: J. MICROBIOL.
VOL.
21, 1975
.9 .5
-
CONTROL
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 11/10/14 For personal use only.
L
.o1
0
2
4
6
8
1
TIME ( H O U R S
0
TIME ( H O U R S )
FIG.4. Effect of EDTA treatment on AD sensitivity of succinate-grown cells. (e), Control; (A) 5 Pg/ml A D ; (m), 10 pg/ml AD ; (0120 ,~ g / m AD. l
were conducted with isolated cell envelope material. Results indicate that envelopes (adjusted to same absorbancy level and showing similar protein levels) prepared from cells grown in either nutritional condition adsorb equivalent levels of activity when exposed to the labeled antibiotic. Extensive washing of whole cells increases the AD-binding capacity and the sensitivity of the cells during growth in A D by 10-15% in succinate-grown cells, and 30-40% in glucose-grown cells. These data suggest there may be an equivalent number of binding sites per cell and, when the envelope is isolated or when cells are washed extensively, the sites are exposed and made available for binding with AD. Thus, the accessibility of the sites for binding with A D is influenced by and varies with the composition of the envelope. Cellular Setuitivit.y to A D after E D T A Treatment Leive (12, 13) reported that the permeability of Escherichia coli was increased by treatment with EDTA, and EDTA treatment caused the releaseof lipopolysaccharide from the cell surface. EDTA is reported to have a more extensive effect on Pseudotnonas aer~t~qinosn cells and envelope, solubilizing not only lipopolysaccharide, but also protein and phospholipid (8, 16). A modified procedure (14) was used with cells grown under the two nutritional conditions to determine whether the sensitivity of cells to A D would be influenced by EDTA treatment. EDTA treatment of succinate-grown cells caused a pronounced increase in sensitivity to AD (Fig. 4). A complete inhibition of growth in
the EDTA-treated cells was noted at the lowest concentration of antibiotic tested (5 pg/ml). A similar EDTA treatment of glucose-grown cells increased the lag time, but the level of inhibition was not significantly different from the growth response exhibited by the control cells (Fig. 5). These results augment the observations with the binding studies that succinate-grown cells have a different envelope coinposition and (or) conformation than the glucose-grown cells. Tliiobarbituric Acid Detertnination of Envelope Lipopolysacclzaride Lipopolysaccharide (LPS) is thought to be involved with cellular permeability (18). The difference in A D sensitivity exhibited by the cells grown in the two different nutritional sources after EDTA treatment as well as the difference in their AD-binding capacities implied the possibility of LPS involvement. LPS was determined by measuring ketodeoxyoctosonic acid (KDO) (2) in stationary-phase glucose- and succinategrown cells. Results were as follows: glucosegrown P. j~rorescenscells contained about 80 pg thiobarbituric acid positive material per inilligram dry cell weight, while succinate-grown P. jltorescens cells contained about 40 pg thiobarbituric acid positive material per milligram dry cell weight. The succinate-grown cells have only about 50% as much thiobarbituric acid positive material as glucose-grown cells suggesting a greatly reduced level of LPS in the envelope of cells grown with succinate as the carbon and energy source.
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WALKER AND DURHAM: SENSITIVITY O F P. FLUORESCENS TO AD
,011 0
2
8
4
I
TlME (
6
8
1
I
0
HOURS )
,011 0
2
4
I
TlME
6
8
1
I
0
(HOURS)
FIG. 5. Effect of EDTA treatment o n A D sensitivity of glucose-grown cells. (a), Control; (A), 5 pg/ml A D ; 10 ~lglmlA D ; (O), 20 ~ g / m AD. l
(m),
Discussion The degree of sensitivity of P. ji'uorescens to A D varies significantly depending on the carbon source in which the organism is growing. Cells grown in succinate exhibited pronounced sensitivity to A D when compared with glucosegrown cells. Uptake studies suggested that incubation of cells in succinate medium containing A D for 2 h significantly reduced the ability of the cells to take up selected substrates. The succinate-grown cells bound and (or) accumulated large amounts of 3H-AD, and EDTA treatment greatly increased AD-promoted growth inhibition. In contrast, the glucose-grown cells were much less sensitive. A D did not alter uptake of selected metabolites even when the cells were grown in the Dresence of the antibiotic. The glucose-grown cells bind about one-fourth as much 3H-AD as did the succinate-grown cells, and EDTA treatment did not promote increased A D sensitivity. The experimental data indicate that a pronounced difference exists in the outer cell surface of cells grown in the two different carbon sources. 3H-AD binding studies with whole cells and cell envelopes support the hypothesis that an equivalent number of binding sites exist on the cell surface for the antibiotic but the sites are masked by cell components accumulating during growth on selected carbon and energy sources. Extensive washing of whole cells or during preparation of envelopes made the "buried" sites available. The conclusions relating to chemical composi-
tion of the cell and growth medium are augmented by reports that E. coli cells grown on succinate had "leakier" membranes than cells grown on glucose (lo), an increase in the amount of phospholipid and the ratio of fatty acids in the phospholipid of the envelope were associated with a mutation to antibiotic resistance in P. aeruginosa (I), and the ultrastructural and chemical composition in P. aeruginosa was altered when grown in Mg2+-deficient medium (6). Results from this study establish that glucosegrown cells have a higher concentration of lipopolysaccharide than succinate-grown cells, which influences binding of A D to the cell surface, and in this sense, may function in the cells exhibiting different permeability traits which are reflected in sensitivity to the antibiotic.
Acknowledgment This work was supported-by American Cancer Society Grant IN-91 and U.S. Public Health Service, National Cancer Institute Grant No. CA-14343. 1. ANDERES, E . A., W. E . S A N D I N EP. , ~R.~ELLIKER. ~ 1971. Lipids of antibiotic-sensitive and -resistant
strains of Pseudomonas aeruginosa. Can. J. Microbiol. 17: 1357-1365. 2. CYNKIN,A,, and G. ASHWELL.1960. Estimation of 3-deoxysugars by means of the malonaldehydethiobarbituric acid reaction. Nature (Lond.), 186: 155-156. 3. DURHAM,N. N. 1958. Studies on the metabolism of p-nitrobenzoic acid. Can. J. Microbiol. 4: 141-148. N. N., and K. C. KEUDELL.1%9. Synthe4. DURHAM,
sis of protocatechuate oxygenase by Pseudomonas j7~rorescens in the presence of actinomycin D. Antimicrob. Agents Chemother. pp. 67-72.
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CAN. J. MICROBIOL. VOL. 21, 1975
tion of carbohydrate catabolism and synthesis of mac5. GELLERT,M., C. E. SMITH,D. NEVILLE,and G. romolecules during enzyme synthesis in PseudoFELSENFELD.1965. Actinomycin binding to DNA: monnsjuorescens. J . Bacteriol. 90: 23-28. Mechanism and specificity. J. Mol. Biol. 11: 4 4 5 4 5 7 . 12. LEIVE, L. 1965. Actinomycin sensitivity in 6. GILLELAND, H . E., JR., J. D. STINNETT,and R. G. Eschericl~ia coli produced by EDTA. Biochem. EAGON.1974. Ultrastructural and chemical alteration Biophys. Res. Commun. 18: 13-17. of the cell envelope of Pserrdonzonns aeruginosn, as13. LEIVE,L. 1965. A non-specific increase in permeabilsociated with resistance to ethylenediaminetetraity in Esclzerichin coli produced by EDTA. Proc. Natl. acetate resulting from growth in a Mg2+-deficient Acad. Sci., U.S.A. 53: 745-750. medium. J. Bacteriol. 117: 302-31 1. I. H., M. RABINOVITZ, and E . REICH. 14. LEIVE,L. 1968. Studies on the permeability change 7. GOLDBERG, produced in coliform bacteria by ethylenediaminetet1962. Basis of actinomycin action. I. DNA binding and raacetate. J. Biol. Chem. 243: 2373. inhibition of RNA polymerase synthetic reactions by 1968. Studies of 15. MULLER,W., and D. M. CROTHERS. actinomycin. Proc. Natl. Acad. Sci. U.S.A. 48: the binding of actinomycin and related compounds to 2094-2101. DNA. J. Mol. Biol. 35: 251-290. 8. GRAY,G. W., and S. G. WILKINSON. 1965. The action JR., and R. G. of ethylenediaminetetraacetic acid on Pse~tdonzotzcrs 16. ROGERS,S. W., H. E. GILLELAND, EAGON. 1969. Characterization of a protein-liponerlryitzosa. J. Appl. Bacteriol. 28: 153-164. polysaccharide complex released from cell walls of L . D., W. FULLER, and E. REICH.1963. 9. HAMILTON, Pse~rdomotzrrs aerlrginosa by ethylenediaminetetraX-ray diffraction and molecular model building acetic acid. Can. J. Microbiol. 15: 743-748. studies of the interaction of actinomycin with nucleic C. A.. and N. N. DURHAM. 1972. Effect of 17. WALKER, acids. Nature (Lond.), 198: 538-540. carbon sources on the envelope lipopolysaccharide 10. KABACK, H . R . 1970. The transport of sugars across component of Pse~tdomonas j~rorescetzs. Abstr. isolated bacterial membranes. III Current topics in Annu. Meet. Am. Soc. Microbiol. p. 43. membranes and transport. Vol. 1. Edited by F. Bron1971. Molecular 18. WRIGHT,A., and S. KANEGASAKI. ner and A. Kleinzeller. Academic Press, New York aspects of lipopolysaccharides. Physiol. Rev. 51: and Lond. p. 36. 748-784. 1 1 . KIRKLAND, J. J., and N. N. DURHAM. 1965. Correla-
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CYNTHIAA. WALKER A N D NORMAN N. DURHAM Departrnent of Microbiology, Oklahotnn Stote University, Still\voter, Oklohotno 74074 Accepted September 6, 1974
WALKER, C. A., and DURHAM, N. N . 1975. Effect of carbon source during growth on sensitivity of Pserrdomonasjirorescens to actinomycin D. Can. J. Microbiol. 21: 69-74. Sensitivity to actinomycin D(AD) varies in P s e r t J o t n o t ~ ~ s ~ r t o r r ~cells ~ c egrown t ~ . ~ in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity inglucose-grown cells. Succinate-grown cells bound 2-3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lip0 olysaccharide masks the binding sites and, therefore, is responsible for the difference in blngng of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic. WALKER,C. A., e t DURHAM, N . N. 1975. Effect of carbon source duringgrowth on sensitivity of Pse~rdomotzosjuorescensto actinomycin D. Can. J. Microbiol. 21: 69-74. Chez Pseirdomonos jirorescens, la sensibilite B I'actinomycine D (AD) varie selon que la culture est faite dans un milieu minimal contenant des sels e t du glucose ou du succinate. Dans le milieu minimal avec succinate la croissance est inhibee par des concentrations plus faibles d'AD que dans le milieu minimal avec glucose. Dans le cas des cellules cultivees pendant 2 h dans le milieu avec succinate additionnk d'AD, la captation de metabolites radioactifs est inhibee par I'AD alors que les cellules cultivees avec glucose sont insensibles. Le traitement par I'EDTA augmente la sensibilitk B I'AD des cellules cultivees avec le succinate e t n'affecte pas celle des cellules cultivees avec glucose. Les cellules cultivees avec succinate fixent 2 B 3 fois plus de 3H-ADque les cellules cultivees avec glucose. Par rapport aux cellules cultivees avec succinate, les cellules cultivkes en presence de glucose ont une enveloppe plus riche en lipopolysaccharides. I1 est possible que ce materiel lipopolysaccharidique masque les sites de fixation et qu'il soit ainsi responsable de la difference dans la quantite d'AD fixee par les cellules cultivkes avec glucose e t succinate. La disponibilitt des sites de fixation est aussi impliquke dans la sensibilite des cellules B I'antibiotique. [Traduit par le journal]
Introduction The pseudomonads, a highly adaptive group of microorganisms, are notoriously insensitive to antibiotics. Actinomycin D (AD), which prevents protein synthesis by inhibiting deoxyribonucleic acid (DNA) - directed ribonucleic acid (RNA) synthesis (7, 9, 15), is partially active against these gram-negative organisms. AD inhibits uptake and incorporation of selected amino acids and uracil, a; well as the synthesis of certain inducible enzymes in Pseudomonas jiuorescens (4). The primary mechanism of inhibition is thought to be the physical blockage of RNA polymerase (5). Thus, for the antibiotic to reach its effective site of action, the molecule must first penetrate the cell permeability barrier. Walker and Durham (17) demonstrated that 'Received June 17, 1974.
the sensitivity of P. fluorescens NND to AD is partially dependent -on the carbon source and concentration in which the organism is growing. Growth of the organism in either glucose or succinate as the sole source of carbon and energy effectively alters the inhibition pattern of the organism. This investigation characterizes the physiological differences in cells grown in these two carbon sources.
Materials and Methods Test Organism The organism used in this study was Pseudomonas flrorescens N N D (1 1 ) . Stock cultures were maintained on 0.2% (w/v) succinate-salts agar slants stored at 4C. Medium The minimal salts mcdium had the following composition (w/v): 0.42% ;:&PO4, 0.32% K H Z P 0 4 , 0.2% NH4CI, and 0.2% NaCI. The carbon source was added to a final concentration of 2.78 x lo-' M and the p H adjusted to 7.0 with K O H before sterilization by auto-
70
CAN. 1. MICROBI(3L. VOL. 21, 1975
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claving for 15 min at 121C and 15 Ib pressure. Glucose was sterilized separately by autoclaving for 10 min and added aseptically to the sterile minimal salts medium just before use. A sterile trace minimal salts solution (0.1 ml) was added to each 100 ml of synthetic medium (3). Atztibiotics Actinomycin D (Merck, Sharp, and Dohme) was dissolved in sterile, glass-distilled water (300 pg/ml) and stored at 4C until used. Groivt/~Studies Growth studies were conducted in 18 x 150 mm tubes, 250-nil sidearm flasks, or a mass fermenter, depending on the cell mass needed for analysis. Tubes and flasks were incubated at 37C on a reciprocal shaker, and growth was followed by measuring the increase in absorbance at 540 nni using a Coleman Junior I1 spectrophotometer. Radioisotope Studies Uptake of radioactive carbon sources was examined by growing the cells in 26.0 ml of medium in a 250-ml sidearm flask. The cells were incubated at 37C with shaking for 3 h at which time either 3.0 ml of A D (300 ~ g / m l or ) 3.0 ml of potassium phosphate buffer (pH 7.0) was added, and the cells incubated 2 additional h. The cells were harvested, suspended to an absorbance at 540 nm, equivalent to 0.35 mglml dry cell weight, in minimal salts niedii~mcontaining no carbon source, and equilibrated to 37C. Five milliliters of this cell suspension was added t o tubes containing the appropriate carrier (0.5 mg/ml), labeled carbon source (0.5 pCi/nll), and either A D (30 pg/ml) or potassium phosphate buffer. Samples (0.5 ml) were removed, filtered through Millipore filters (0.45micron (p) pore size), and washed twice with minimal salts buffer. The filters were placed in a counting vial, Aqi~asol(10 ml) was added, and the radioactivity determined in a liquid scintillation spectrometer (Nuclear Chicago) which has an efficiency of 40% under these conditions.
Ef/~ylenediatnit~etetr.ancetic Acid (EDTA) Treatmetzt of Cells Cells were suspended in Tris (2-amino-2(hydroxymethyl)-1,3-propanediol) buffer (0.12 M, p H 8.0) t o an absorbance at 540 nm, equivalent to 0.45 mg/ml dry cell weight, and treated with EDTA (pH 7.0, final concentraM) for 2 min at 37C. The action of EDTA tion I x was stopped by making a 1: 10 dilution into fresh medium. The cells were inoculated into culture tubes and growth in the presence and absence of A D was followed spectrophotometrically. Plate counts established that cell viability was not altered by the EDTA treatment.
Preparatiotz of Cell Envelopes Cells were grown in 5.0-liter volumes on a mass fermenter or in 0.5-liter volumes in Fernbach flasks. The cells were harvested, suspended in minimal volumes of physiological saline, and frozen in 10-ml volumes. The cells were broken as a frozen pellet using an X-press (AB BIOTEC, Stockholm, Sweden), placed in 100 ml of saline, and centrifuged at 1935 x g a t 4C. Thelarger debris was removed, envelope material was sedimented by centrifugation at 27 000 x g for 20 min, and purified by repeated washing. The envelope material was frozen in saline until used. Phase-contrast microscopy revealed that less than 5% whole cells remained.
Results Eflect of A D on Growth AD readily inhibits cells growing in a succinate-salts medium and the inhibition is concentration dependent (Fig. 1). An inhibition of 50% or more was commonly observed at the lower concentrations while complete inhibition was evident at higher concentration. Microscopic examination of the inhibited cells revealed the presence of filaments. In contrast, glucose-grown cells exhibited a decreased sensitivity to AD (Fig. 1). Less than a 50% inhibition of the total cell mass was noted even at the higher concentrations of the antibiotic (30 pg/ml) as compared to total inhibition of growth in the succinate-grown cells at the same concentrations. Microscopically, the glucose-grown cells showed only a few aberrant cells (about 10%) after growth in AD containing medium.
Eflect of A D on Uptake of Radioactive Metabolites AD blocks uptake of selected amino acids in P. fl~roresceris (4). Experiments were performed to measure the uptake of several metabolites by cells grown on the two carbon sources. Labeled substrates included 14C-glucose (specific activity 3.0 mcilrnmol), 14C-succinate (specific activity 1.8 mCilmmol), 14C-alanine (specific activity 5.0 mCilmmol), 14C-phenylalanine (specific activity 10.3 mCilmmol), and 14C-uracil. The reBinding of 3H-AD Binding was carried out in a 5.5-ml total liquid volume sults for 14C-phenylalanine are presented in Fig. at room temperature. The cells were exposed to 5.0 2. The uptake of metabolites was not influenced pCi 3H-AD (Schwarz Bioresearch, 5.5 mCi/mg) for 15 min, centrifuged at 17 300 x g at 4C, and washed twice by the presence or absence of AD in the uptake with physiological saline. The cell pellets were digested solutions for both succinate- and glucose-grown overnight in 0.5 ml of NCS reagent (Nuclear-Chicago) at cells, but growth in the presence of the antibiotic room temperature, and the digested material was added in the succinate medium for 2 h did produce an to 10 ml of scintillation fluid (2.0 g of 2,5-diphenyloxazole inhibition. With all radioactive substrates, prior (PPO), 0.025 g 1,4-bis-[2-(5-phenyloxazolyl)]-benzene (POPOP) in 237 ml dioxane). Radioactivity was deter- incubation of the cells in the succinate medium containing AD for 2 h inhibited active uptake in mined by liquid scintillation spectrophotometry.
WALKER AND DURHAM: SENSITIVITY OF P. FLUORESCENS TO A D I
SUCCINATE MINIMAL MEDIUM
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GLUCOSE MINIMAL MEDIUM
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.9 -
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FIG1. Effect of AD on cells growing in succinate or glucose minimal salts medium. ( a ) , Control; (A), 1 0 pg/ml AD ; (m),20 pg/ml AD ; ( e l , 3 0 wg/ml AD.
tially linear in both sets of cells. The break in the adsorption curves suggests that two separate parameters may be participating in the measurable phenomenon-binding and uptake. T o determine if the difference in 3H-AD accumulation was a property of the cell envelope, similar studies
TlME (MIN)
Fig. 2. Uptake of 14C-phenylalanine by succinate- and glucose-grown cells. ( a ) , Succinate-grown cells with and without AD added to the uptakesystem; ( e ) , succinate AD grown cells with and without AD added to the uptake system; (D), glucose-grown cells with and without AD added to the uptake system, arzd glucose + AD grown cells with and without AD added t o the uptake system. AD, when present, was at a concentration of 3 0 pg/ml.
+
succinate-grown cells, but similar treatment did not inhibit uptake of the substrates in the glucosegrown cells. Binding of 3H-AD to Glucose- and Succinategrown Cells Experiments were performed to measure binding of 3H-AD to cells grown in the different carbon sources. Cells grown in succinate bind 4-5 times as much 3H-AD as the same cell mass of glucose-grown cells (Fig. 3). Adsorption is par-
M g Dry Cell Weight
FIG.3 . Binding of 3H-AD to glucose (A) and succinate ( 0 ) grown cells. Cells were grown for 12 h, harvested, and suspended in saline to the indicated dry cell weight. Five milliliters of cell suspension was added to 0.5 ml of 3H-AD (specific activity 8.4 Ci/mmol, 5.0 pCi/ml).
CAN: J. MICROBIOL.
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Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 11/10/14 For personal use only.
L
.o1
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FIG.4. Effect of EDTA treatment on AD sensitivity of succinate-grown cells. (e), Control; (A) 5 Pg/ml A D ; (m), 10 pg/ml AD ; (0120 ,~ g / m AD. l
were conducted with isolated cell envelope material. Results indicate that envelopes (adjusted to same absorbancy level and showing similar protein levels) prepared from cells grown in either nutritional condition adsorb equivalent levels of activity when exposed to the labeled antibiotic. Extensive washing of whole cells increases the AD-binding capacity and the sensitivity of the cells during growth in A D by 10-15% in succinate-grown cells, and 30-40% in glucose-grown cells. These data suggest there may be an equivalent number of binding sites per cell and, when the envelope is isolated or when cells are washed extensively, the sites are exposed and made available for binding with AD. Thus, the accessibility of the sites for binding with A D is influenced by and varies with the composition of the envelope. Cellular Setuitivit.y to A D after E D T A Treatment Leive (12, 13) reported that the permeability of Escherichia coli was increased by treatment with EDTA, and EDTA treatment caused the releaseof lipopolysaccharide from the cell surface. EDTA is reported to have a more extensive effect on Pseudotnonas aer~t~qinosn cells and envelope, solubilizing not only lipopolysaccharide, but also protein and phospholipid (8, 16). A modified procedure (14) was used with cells grown under the two nutritional conditions to determine whether the sensitivity of cells to A D would be influenced by EDTA treatment. EDTA treatment of succinate-grown cells caused a pronounced increase in sensitivity to AD (Fig. 4). A complete inhibition of growth in
the EDTA-treated cells was noted at the lowest concentration of antibiotic tested (5 pg/ml). A similar EDTA treatment of glucose-grown cells increased the lag time, but the level of inhibition was not significantly different from the growth response exhibited by the control cells (Fig. 5). These results augment the observations with the binding studies that succinate-grown cells have a different envelope coinposition and (or) conformation than the glucose-grown cells. Tliiobarbituric Acid Detertnination of Envelope Lipopolysacclzaride Lipopolysaccharide (LPS) is thought to be involved with cellular permeability (18). The difference in A D sensitivity exhibited by the cells grown in the two different nutritional sources after EDTA treatment as well as the difference in their AD-binding capacities implied the possibility of LPS involvement. LPS was determined by measuring ketodeoxyoctosonic acid (KDO) (2) in stationary-phase glucose- and succinategrown cells. Results were as follows: glucosegrown P. j~rorescenscells contained about 80 pg thiobarbituric acid positive material per inilligram dry cell weight, while succinate-grown P. jltorescens cells contained about 40 pg thiobarbituric acid positive material per milligram dry cell weight. The succinate-grown cells have only about 50% as much thiobarbituric acid positive material as glucose-grown cells suggesting a greatly reduced level of LPS in the envelope of cells grown with succinate as the carbon and energy source.
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WALKER AND DURHAM: SENSITIVITY O F P. FLUORESCENS TO AD
,011 0
2
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I
TlME (
6
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I
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HOURS )
,011 0
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4
I
TlME
6
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(HOURS)
FIG. 5. Effect of EDTA treatment o n A D sensitivity of glucose-grown cells. (a), Control; (A), 5 pg/ml A D ; 10 ~lglmlA D ; (O), 20 ~ g / m AD. l
(m),
Discussion The degree of sensitivity of P. ji'uorescens to A D varies significantly depending on the carbon source in which the organism is growing. Cells grown in succinate exhibited pronounced sensitivity to A D when compared with glucosegrown cells. Uptake studies suggested that incubation of cells in succinate medium containing A D for 2 h significantly reduced the ability of the cells to take up selected substrates. The succinate-grown cells bound and (or) accumulated large amounts of 3H-AD, and EDTA treatment greatly increased AD-promoted growth inhibition. In contrast, the glucose-grown cells were much less sensitive. A D did not alter uptake of selected metabolites even when the cells were grown in the Dresence of the antibiotic. The glucose-grown cells bind about one-fourth as much 3H-AD as did the succinate-grown cells, and EDTA treatment did not promote increased A D sensitivity. The experimental data indicate that a pronounced difference exists in the outer cell surface of cells grown in the two different carbon sources. 3H-AD binding studies with whole cells and cell envelopes support the hypothesis that an equivalent number of binding sites exist on the cell surface for the antibiotic but the sites are masked by cell components accumulating during growth on selected carbon and energy sources. Extensive washing of whole cells or during preparation of envelopes made the "buried" sites available. The conclusions relating to chemical composi-
tion of the cell and growth medium are augmented by reports that E. coli cells grown on succinate had "leakier" membranes than cells grown on glucose (lo), an increase in the amount of phospholipid and the ratio of fatty acids in the phospholipid of the envelope were associated with a mutation to antibiotic resistance in P. aeruginosa (I), and the ultrastructural and chemical composition in P. aeruginosa was altered when grown in Mg2+-deficient medium (6). Results from this study establish that glucosegrown cells have a higher concentration of lipopolysaccharide than succinate-grown cells, which influences binding of A D to the cell surface, and in this sense, may function in the cells exhibiting different permeability traits which are reflected in sensitivity to the antibiotic.
Acknowledgment This work was supported-by American Cancer Society Grant IN-91 and U.S. Public Health Service, National Cancer Institute Grant No. CA-14343. 1. ANDERES, E . A., W. E . S A N D I N EP. , ~R.~ELLIKER. ~ 1971. Lipids of antibiotic-sensitive and -resistant
strains of Pseudomonas aeruginosa. Can. J. Microbiol. 17: 1357-1365. 2. CYNKIN,A,, and G. ASHWELL.1960. Estimation of 3-deoxysugars by means of the malonaldehydethiobarbituric acid reaction. Nature (Lond.), 186: 155-156. 3. DURHAM,N. N. 1958. Studies on the metabolism of p-nitrobenzoic acid. Can. J. Microbiol. 4: 141-148. N. N., and K. C. KEUDELL.1%9. Synthe4. DURHAM,
sis of protocatechuate oxygenase by Pseudomonas j7~rorescens in the presence of actinomycin D. Antimicrob. Agents Chemother. pp. 67-72.
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CAN. J. MICROBIOL. VOL. 21, 1975
tion of carbohydrate catabolism and synthesis of mac5. GELLERT,M., C. E. SMITH,D. NEVILLE,and G. romolecules during enzyme synthesis in PseudoFELSENFELD.1965. Actinomycin binding to DNA: monnsjuorescens. J . Bacteriol. 90: 23-28. Mechanism and specificity. J. Mol. Biol. 11: 4 4 5 4 5 7 . 12. LEIVE, L. 1965. Actinomycin sensitivity in 6. GILLELAND, H . E., JR., J. D. STINNETT,and R. G. Eschericl~ia coli produced by EDTA. Biochem. EAGON.1974. Ultrastructural and chemical alteration Biophys. Res. Commun. 18: 13-17. of the cell envelope of Pserrdonzonns aeruginosn, as13. LEIVE,L. 1965. A non-specific increase in permeabilsociated with resistance to ethylenediaminetetraity in Esclzerichin coli produced by EDTA. Proc. Natl. acetate resulting from growth in a Mg2+-deficient Acad. Sci., U.S.A. 53: 745-750. medium. J. Bacteriol. 117: 302-31 1. I. H., M. RABINOVITZ, and E . REICH. 14. LEIVE,L. 1968. Studies on the permeability change 7. GOLDBERG, produced in coliform bacteria by ethylenediaminetet1962. Basis of actinomycin action. I. DNA binding and raacetate. J. Biol. Chem. 243: 2373. inhibition of RNA polymerase synthetic reactions by 1968. Studies of 15. MULLER,W., and D. M. CROTHERS. actinomycin. Proc. Natl. Acad. Sci. U.S.A. 48: the binding of actinomycin and related compounds to 2094-2101. DNA. J. Mol. Biol. 35: 251-290. 8. GRAY,G. W., and S. G. WILKINSON. 1965. The action JR., and R. G. of ethylenediaminetetraacetic acid on Pse~tdonzotzcrs 16. ROGERS,S. W., H. E. GILLELAND, EAGON. 1969. Characterization of a protein-liponerlryitzosa. J. Appl. Bacteriol. 28: 153-164. polysaccharide complex released from cell walls of L . D., W. FULLER, and E. REICH.1963. 9. HAMILTON, Pse~rdomotzrrs aerlrginosa by ethylenediaminetetraX-ray diffraction and molecular model building acetic acid. Can. J. Microbiol. 15: 743-748. studies of the interaction of actinomycin with nucleic C. A.. and N. N. DURHAM. 1972. Effect of 17. WALKER, acids. Nature (Lond.), 198: 538-540. carbon sources on the envelope lipopolysaccharide 10. KABACK, H . R . 1970. The transport of sugars across component of Pse~tdomonas j~rorescetzs. Abstr. isolated bacterial membranes. III Current topics in Annu. Meet. Am. Soc. Microbiol. p. 43. membranes and transport. Vol. 1. Edited by F. Bron1971. Molecular 18. WRIGHT,A., and S. KANEGASAKI. ner and A. Kleinzeller. Academic Press, New York aspects of lipopolysaccharides. Physiol. Rev. 51: and Lond. p. 36. 748-784. 1 1 . KIRKLAND, J. J., and N. N. DURHAM. 1965. Correla-
