JOURNAL OF Vol. Biochemical Effects Novobiocin ... · WISHNOWETAL. 20 w 16 4-0o t 2 2 zo O O 8 00...
JOURNAL OF BACTERIOLOGY, Apr., 1965 Copyright a 1965 American Society for Microbiology Vol. 89, No. 4 Printed in U.S.A. Biochemical Effects of Novobiocin on Staphylococcus aureus RODNEY M. WISHNOW,' JACK L. STROMINGER,2 CLAIRE H. BIRGE, AND ROBERT H. THRENN Departments of Pharmacology, Washington University School of Medicine, St. Louis, Missouri, and University of Wisconsin Medical School, Madison, Wisconsin Received for publication 12 November 1964 ABSTRACT WISHNOW, RODNEY M. (Washington University School of Medicine, St. Louis, Mo.), JACK L. STROMINGER, CLAIRE H. BIRGE, AND ROBERT H. THRENN. Biochemical effects of novobiocin on Staphylococcus aureus. J. Bacteriol. 89:1117-1123. 1965.-Novobiocin in- duced accumulation in Staphylococcus aureus of uridine nucleotide precursors of the cell-wall glycopeptide, of a cytidine nucleotide precursor of the teichoic acid in the cell wall, and of a large number of other ultraviolet-absorbing materials. These substances accumulated promptly on addition of the antibiotic at levels near the growth-inhibitory concentration. Isotopic experiments showed that, in addition to inhibiting cell-wall synthesis, novobiocin also inhibited both protein and nucleic acid synthesis. The lack of selectivity in the action of novobiocin is similar to that previously observed with gen- tian violet, and is quite different from the selective inhibition of cell-wall synthesis ob- served with penicillin or with D-cycloserine. Although inhibition of cell-wall synthesis is not excluded as one of the primary lethal effects of novobiocin, it is also possible that all of the observed phenomena are secondary to some other primary metabolic lesion. In recent years, many attempts have been made to elucidate the mechanism of action of various antibiotics. A striking feature of this work has been the increasing recognition of the importance of the cell wall and membrane as targets for cyto- toxic agents. Novobiocin has an antibacterial spectrum similar to that of erythromycin, and has provea useful in the treatment of resistant staphylococcal infections. It has a complex struc- ture consisting of a substituted phenol, a substi- tuted coumarin, and the sugar moiety, noviose (the carbamate ester of a derivative of L-rham- nose). It has been reported briefly that novobiocin causes accumulation of cell-wall precursors in Staphylococcus aureus (Strominger and Threnn, 1959). Details of novobiocin-induced nucleotide accumulation and its specificity are reported in this paper. MATERIALS AND METHODS Microorganism. S. aureus strain Copenhagen is a hemolytic, coagulase-positive, penicillin-sensi- tive strain, phage type 3B,3C,55,71,29,80, em- ployed in previous studies in this laboratory. 1 Present address: Department of Medicine, New York University School of Medicine, Bellevue Hospital, New York. 2 Present address: Department of Pharma- cology, University of Wisconsin Medical School, Madison. Uridine nucleotide accumulation. Measurement of accumulation of derivatives of uridine diphos- phate (UDP)-acetylmuramic acid, which are cell-wall precursors, was carried out as described previously (Strominger, 1957) with 100-ml cul- tures. As a matter of convenience, extraction of cells with 1 ml of water in a boiling-water bath for 10 min is now preferred to extraction with cold trichloroacetic acid. Acetylamino sugar-contain- ing nucleotides in the hot-water extract were measured colorimetrically. Accumulated nucleotides were recovered from the extracts by charcoal adsorption and elution, as described by Strominger (1962a). On a small scale (100-ml cultures), the nucleotide eluates were subjected to two-dimensional paper chroma- tography in solvent A (isobutyric acid-0.5 N NH40H, 5:3) and solvent B [ethanol-1 N ammo- nium acetate (pH 7), 7.5:3]. Nucleotides were then visualized by printing the chromatograms in ultraviolet light with reflex document copying paper. On a larger scale (9 liters) nucleotides in tri- chloroacetic acid extracts were separated on Dowex 1-chloride, essentially as described by Strominger (1957), and then recovered from the column eluates by charcoal adsorption and elution and purified by paper chromatography in solvents A and B. Measurement of cell-wall, protein, and nucleic acid synthesis with isotopes. Experiments to deter- mine cell-wall, protein, and nucleic acid synthesis were carried out as described previously (Nathen- 1117 on June 25, 2020 by guest http://jb.asm.org/ Downloaded from
JOURNAL OF Vol. Biochemical Effects Novobiocin ... · WISHNOWETAL. 20 w 16 4-0o t 2 2 zo O O 8 00 0-t E-1 4 0 01 ' 0 10 20 30 40 50 150 Novobiocin Conc. (,uEg/mI) FIG. 1. Accumulation
Text of JOURNAL OF Vol. Biochemical Effects Novobiocin ... · WISHNOWETAL. 20 w 16 4-0o t 2 2 zo O O 8 00...
JOURNAL OF BACTERIOLOGY, Apr., 1965Copyright a 1965 American Society for Microbiology
Vol. 89, No. 4Printed in U.S.A.
Biochemical Effects of Novobiocin on
RODNEY M. WISHNOW,' JACK L. STROMINGER,2 CLAIRE H. BIRGE,AND ROBERT H. THRENN
Departments of Pharmacology, Washington University School of Medicine, St. Louis, Missouri, andUniversity of Wisconsin Medical School, Madison, Wisconsin
Received for publication 12 November 1964
ABSTRACTWISHNOW, RODNEY M. (Washington University School of Medicine, St. Louis, Mo.),
JACK L. STROMINGER, CLAIRE H. BIRGE, AND ROBERT H. THRENN. Biochemical effects ofnovobiocin on Staphylococcus aureus. J. Bacteriol. 89:1117-1123. 1965.-Novobiocin in-duced accumulation in Staphylococcus aureus of uridine nucleotide precursors of thecell-wall glycopeptide, of a cytidine nucleotide precursor of the teichoic acid in the cellwall, and of a large number of other ultraviolet-absorbing materials. These substancesaccumulated promptly on addition of the antibiotic at levels near the growth-inhibitoryconcentration. Isotopic experiments showed that, in addition to inhibiting cell-wallsynthesis, novobiocin also inhibited both protein and nucleic acid synthesis. The lack ofselectivity in the action of novobiocin is similar to that previously observed with gen-
tian violet, and is quite different from the selective inhibition of cell-wall synthesis ob-served with penicillin or with D-cycloserine. Although inhibition of cell-wall synthesisis not excluded as one of the primary lethal effects of novobiocin, it is also possible thatall of the observed phenomena are secondary to some other primary metabolic lesion.
In recent years, many attempts have been madeto elucidate the mechanism of action of variousantibiotics. A striking feature of this work hasbeen the increasing recognition of the importanceof the cell wall and membrane as targets for cyto-toxic agents. Novobiocin has an antibacterialspectrum similar to that of erythromycin, andhas provea useful in the treatment of resistantstaphylococcal infections. It has a complex struc-ture consisting of a substituted phenol, a substi-tuted coumarin, and the sugar moiety, noviose(the carbamate ester of a derivative of L-rham-nose). It has been reported briefly that novobiocincauses accumulation of cell-wall precursors inStaphylococcus aureus (Strominger and Threnn,1959). Details of novobiocin-induced nucleotideaccumulation and its specificity are reported inthis paper.
MATERIALS AND METHODS
Microorganism. S. aureus strain Copenhagen isa hemolytic, coagulase-positive, penicillin-sensi-tive strain, phage type 3B,3C,55,71,29,80, em-ployed in previous studies in this laboratory.
1 Present address: Department of Medicine,New York University School of Medicine, BellevueHospital, New York.
2 Present address: Department of Pharma-cology, University of Wisconsin Medical School,Madison.
Uridine nucleotide accumulation. Measurementof accumulation of derivatives of uridine diphos-phate (UDP)-acetylmuramic acid, which arecell-wall precursors, was carried out as describedpreviously (Strominger, 1957) with 100-ml cul-tures. As a matter of convenience, extraction ofcells with 1 ml of water in a boiling-water bath for10 min is now preferred to extraction with coldtrichloroacetic acid. Acetylamino sugar-contain-ing nucleotides in the hot-water extract weremeasured colorimetrically.
Accumulated nucleotides were recovered fromthe extracts by charcoal adsorption and elution,as described by Strominger (1962a). On a smallscale (100-ml cultures), the nucleotide eluateswere subjected to two-dimensional paper chroma-tography in solvent A (isobutyric acid-0.5 NNH40H, 5:3) and solvent B [ethanol-1 N ammo-nium acetate (pH 7), 7.5:3]. Nucleotides were thenvisualized by printing the chromatograms inultraviolet light with reflex document copyingpaper.On a larger scale (9 liters) nucleotides in tri-
chloroacetic acid extracts were separated onDowex 1-chloride, essentially as described byStrominger (1957), and then recovered from thecolumn eluates by charcoal adsorption and elutionand purified by paper chromatography in solventsA and B.Measurement of cell-wall, protein, and nucleic
acid synthesis with isotopes. Experiments to deter-mine cell-wall, protein, and nucleic acid synthesiswere carried out as described previously (Nathen-
Novobiocin Conc. (,uEg/mI)FIG. 1. Accumulation of uridine diphosphate
acetylamino sugar compounds in Staphylococcusaureus as a function of novobiocin concentration.Measurements were made after 90 min of incubation.
son and Strominger, 1961) with minor modifica-tions. Logarithmic-phase cells (400 ml of culture)were harvested and suspended in nutrient broth,diluted 1:10 with water. After 5 min of incubationat 37 C in the presence of novobiocin, either p32_inorganic phosphate (100lc) or C'4-L-lysine (3,c)was added to both experimental and control flasks.After 15 min of further incubation, the cells wererapidly centrifuged and washed twice with water.The cells were ruptured in a Nossal disintegratorwith Ballotini no. 12 glass beads, and cell wallsand the cold trichloroacetic acid precipitates ofthe soluble cell contents (which contains bothprotein and nucleic acid) were then prepared.Radioactivity measurements were made in aNuclear-Chicago windowless gas-flow counter.Protein was measured by the modified Folinphenol procedure (Lowry et al., 1951).
Uridine nucleotide accumulation as a function oftime and novobiocin concentration. Novobiocin in-duced the accumulation in S. aureus Copenhagenof UDP-acetylamino sugar compounds, which arecell-wall precursors. The lowest concentration ofnovobiocin which induced uridine nucleotide ac-cumulation was 1.5 ,g/ml. Maximal accumula-tion occurred at 15 jg/ml (Fig. 1). M1easurementof antibiotic sensitivity by tube dilution with aninoculum of 1 %O of a fully grown culture showedthat the minimal growth-inhibitory concentra-tion of novobiocin was about 4 mig/ml; total in-hibition occurred at 16 jig/ml.
Nucleotide accumulation began promptly afterthe addition of novobiocin. The time for half-maximal accumulation was about 5 min. Themaximal accumulation of nucleotide was reachedin 60 min, and amounted to 17 ,imoles per liter ofculture (Fig. 2).
Observation of accumulated nucleotides by two-dimensional paper chromatography. A 100-mlamount of culture at half-maximal growth wastreated with novobiocin (50,g/ml) for 90 min.The cells were collected by centrifugation,washed, and extracted with hot water. Cold tri-chloroacetic acid (final concentration, 5%) wasadded, and the insoluble material was removedby centrifugation. Nucleotides were adsorbd ontocharcoal and recovered by elution with ammoni-acal ethyl alcohol. About 90% of the ultraviolet-absorbing material was recovered by this proce-dure, and was subjected to two-dimensional paperchromatography (Fig. 3). The pattern of nucleo-tides obtained can be contrasted with the nucleo-tide accumulation observed with "gentian vio-let" (G. T. Gurr, Ltd., lot 635, 100 ,ug/ml), peni-cillin (200,ug/ml), and D-cycloserine (Oxamycin,100 ,ug/ml) prepared at the same time andin a similar manner (Fig. 4 to 6). [As previouslyreported (Strominger, 1959), most of bottles ofgentian violet do not induce uridine nucleotideaccumulation in S. aureus. The phenomenonappears to be due to an impurity in the par-ticular lot of dye employed; hence, "gentianviolet" is written in quotation marks.] Theamount of ultraviolet-absorbing compoundspresent in the extract of a control culture (Fig. 7)is too small to be seen by this technique.
r0 I I1 1, , , ,0 20 40 60
Time (Minutes)FIG. 2. Accumulation of uridine diphosphate
acetylamino sugar compounds in Staphylococcusaureus induced by novobiocin, as a function of time.
B IFIG. 3. Nucleotides accumulated in cells treated for 90 min with novobiocin (50 ug/ml). (A) Two-dimen-
sional chromatogram of the cell extract photographed in ultraviolet light. The first dimension (solvent A) wasrun in isobutyric acid-i N NH40H (5:3), and the second dimension (solvent B), in ethanol-i M ammoniumacetate, pH 7 (7.5:3). (B) Schematic drawing of A. The numbers are the same as those used in Table i.
FIG. 4. Nutcleotides accumulated in cells treatedfor 90 min with "gentian violet" (100 ,ug/ml). Com- FIG. 5. Nucleotides accumulated in cells treatedpounds i, 2, 3, 5, 6, 8, 9, 10, ii, and i3 were the same for 90 min with penicillin. The major materialsas those found in novobiocin-treated cells. Compound present are compound 4 (a mixture of UDP-acetyl-4 in this case was a mixture of UDP-acetylglucosa- muramic acid and UDP-acetylmuramyl-L-ala inmine, UDP-acetylglucosamine-pyruvate enol ether, this case) and compound 7 (UDP-acetylmuramyl-and UDP-acetylmuramic acid. Compound 7 (UDP- L-ala-D-glu-L-lys-D-ala-D-ala). No cytidine nucleo-acetylmuramyl-L-ala-D-glu-L-lys-D-ala-D-ala) and tides accumulate, and compound 5 contains onlycompound i2 (nicotinamide adenine dinucleotide UMP. Small amounts of compounds i, 2, 5, and 8phosphate) were absent. Small amounts of several are also seen.unidentified compounds not present in novobiocin-treated cells can be seen.
FIG. 6. Nucleotides accumulated in cells treatedfor 90 min with D-cycloserine (100 pg/ml). Themajor compound present (near the position of com-pound 9) is UDP-acetylmuramyl-L-ala-D-glu-L-lys,and is not seen in any of the other figures. Compound4 (UDP-acetylmuramic acid plus UDP-acetylmur-amyl-L-ala in this case) had also accumulatedTraces of compounds 1, 2, and 3 can be seen.
Novobiocin, like "gentian violet," induced mas-sive accumulation of a large number of differentultra -iolet-absorbing materials. The patterns ob-served with these two substances were very simi-lar. In contrast, a much more specific accumula-tion of only a few compounds occurred withpenicillin and D-cycloserine.
Analysis of nucleotides from novobiocin-treatedcells. The ultraviolet-absorbing compounds wereeluted from the chromatogram with water. Thebase present was identified by its ultraviolet-ab-sorption spectrum in acid and alkali. Nicotin-amide adenine dinucleotide and nicotinamideadenine dinucleotide phosphate were identified inpart from the secondary absorption maximum ofthese compounds at 320 m,u in 0.1 M NaCN.Phosphate, acetylamino sugar, and amino acidanalyses were also carried out. By these tech-niques, as well as by the position on the two-di-mensional chromatogram, a provisional identifica-tion of some of the compounds was obtained(Table 1). Two of the ultraviolet-absorbing areascontained UDP-acetylamino sugar compounds.One of these areas contained mainly UDP-
acetylmuramic acid. This area would also havecontained UDP-acetylmuramyl-L-ala. This lattercompound is present in penicillin-treated andcycloserine-treated cells but is absent from bothnovobiocin-treated and "gentian violet"-treated
cells. The other area contained a peptide deriva-tive of UDP-acetylmuramic acid and is describedbelow.As in the case of "gentian violet"-inhibited
cells, cytidine diphosphate (CDP)-ribitol alsoaccumulated in the presence of novobiocin. Asmall amount of CDP-glycerol was also found.
Detailed analysis of the peptide-containing UDP-acetylamino sugar compound. Analysis of the pep-tide-containing UDP-acetylamino sugar com-pound was carried out to compare this nucleotidewith the peptide-containing compound obtainedfrom S. aureus strain H treated with penicillin.The samples employed were obtained from large-scale preparations on Dowex 1-chloride. By themethods employed, the two nucleotides wereidentical (Table 2), and are represented by thestructure UDP-acetylmuramyl-L-ala-D-glu-L-lys-D-ala-D-ala.
Effects of novobiocin on the incorporation of C14-lysine and P32-inorganic phosphate into cell wall,protein, and nucleic acid. To investigate thespecificity of the effect of novobiocin on cell-wallsynthesis, the incorporation of C14-lysine andP32-inorganic phosphate into cell wall and into theprotein and nucleic acid fraction was measured(Tables 3 and 4). At a novobiocin concentrationof 15 ,ug/ml, the incorporation of C'4-lsine intocell walls was inhibited by 72%. The incorpora-tion of label into the protein and nucleic acidfraction, however, was also inhibited by 44%. Ata novobiocin concentration of 100 ,ug/ml, slightlygreater effects were observed.
FIG. 7. Nucleotides present in an untreated cul-ture. Only comnpound 1 (AMP) is present in anamount sufficient to be detected under these condi-tions.
TABLE 1. Identification of compounds from novobiocin-treated Staphylococcus aureus
Ratio of Ratio ofCom- organic acetyl-pound Base phosphate amino Other data Provisional identificationeno.a to sugar to
1 A 0.9 AMP2 C 1.3 CMP3 A 1.9 Secondary absorbancy maximum at NAD
325 m,u in 0.1 M KCN4 U 2.1 1.1 No amino acids after acid hydrolysisf UDP-acetylmuramic acid5 U + C 1.4 0 Glycerol after enzymatic hydrolysisg UMP + CDP-glycerol6 C 2.0 Ribitol after enzymatic hydrolysisg CDP-ribitol7 U 2.1 1.2 Amino acids present, see Table 3f UDP-acetylmuramyl-ala-
glu-lys-ala-ala8 G 0.9 GMP9 U 2.7 0 UDP?
10 C 2.0 CDP11 A 2.1 ADP12 A 2.8 Secondary absorbancy maximum at NADP
325 min in 0.1 M KCN13 G 1.8 GDP
a Numbers of the compounds refer to those in Fig. 3.b A = adenosine; C = cytosine; G = guanosine; U = uracil. The base present was determined from
its characteristic ultraviolet-absorption spectrum between 220 and 310 m,u in 0.01 N HCl and 0.05 NNaOH, measured in a Cary recording spectrophotometer, model 14. The spectrum of compound 5 couldnot be identified. This compound (0.02 /hmole) was hydrolyzed in 40% perchloric acid at 100 C for 2 hr,and was then chromatographed in isopropanol-HCl (Markham and Smith, 1952). Two ultraviolet-absorbing areas were seen in the positions of uracil and cytosine. Ultraviolet-absorption spectra ofthese two compounds (measured after elution from the paper) were identical to those of uracil andcytosine.
c Organic phosphate was measured by the method of Lowry et al. (1951).d Acetylamino sugar was measured, after hydrolysis in 0.1 N HCl, by the modified Morgan-Elson reac-
tion (Ressig, Strominger, and Leloir, 1955; Strominger, 1957).e In addition to the data presented, all of the compounds mentioned were in the same position on the
f Qualitative examination of amino acids was carried out by hydrolyzing 0.1 to 0.2,umole in 6 N HClat 100 C overnight. After removal of HCl in vacuo, the sample was subjected to paper chromatographyin n-butanol-acetic acid-water (3:1:1). The paper was developed with ninhydrin reagent.
" Samples (0.1 ,umole) were hydrolyzed by snake venom phosphodiesterase (Worthington Biochemi-cals Corp., Freehold, N.J.) and Escherichia coli phosphomonoesterase (Sigma Chemical Co., St. Louis,Mo.). The polyol present in a sample of the hydrolysate was then identified by thin-layer chromatogra-phy followed by detection with the periodate-Schiff reagent as described by Saukkonen et al. (1964). Weare grateful to J. J. Saukkonen for providing us with this procedure prior to publication.
Incorporation of P32-inorganic phosphate intocell wall at a novobiocin concentration of 15,ug/ml was inhibited by 31%. At this concentra-tion, the incorporation of P32-inorganic phosphateinto cell protein and nucleic acid was inhibitedby 75%. Similar effects were observed at a novo-biocin concentration of 100 ,ug/ml.
DISCUSSIONIt is apparent that novobiocin induces accumiiu-
lation of the uridine nucleotide precursors of theglycopeptide polymer in the cell wall of S. aureus
as well as of CDP-ribitol, a precursor of theteichoic acid polymer in the cell wall. Like "gen-tian violet," however, a large number of otherultraviolet-absorbing compounds accumulate,most of which have not been completely identi-fied. This massive accumulation induced by thesetwo substances stands in contrast to the more se-lective accumulation of the uridine nucleotideprecursors of the glycopeptide which is inducedby penicillin or by D-cycloserine (Strominger,1962b). Isotope experiments, moreover, clearlydemonstrate that the effect of novobiocin is not
a Results are expressed as micromoles permicromole of uridine.bFor methods of measurement, see Table 1.c A sample (0.2 to 0.3,umole) was hydrolyzed in
6 N HCI at 100 C overnight, and then subjectedto chromatography on Dowex 50 with HCI as theeluent, as used previously (Strominger, Park, andThompson, 1959).
d Measured on samples eluted from the Dowex50 column. The specificity of D-amino acid oxidaseand L-alanine-a-ketoglutarate transaminase wasemployed. The pyruvate formed in each case wasmeasured with reduced nicotinamide adeninedinucleotide and lactic dehydrogenase.
TABLE 3. Incorporation of C54-lysine into cell walland into cell protein and nucleic acid in
Flask condition Cell wall Inhibition protein Inhibitionand nucleic
Control 10,800 1,970Novobiocin, 3,450 72 1,100 44
15 ,ug/mlNovobiocin, 1,050 90 970 51
* Results are expressed as counts per minuteper milligram of protein. For details of the methodemployed, see Nathenson and Strominger (1961).
restricted to its effect on cell-wall synthesis, butincludes inhibition of both protein and nucleicacid synthesis. Again, these data are strikinglysimilar to data obtained with "gentian violet"(Nathenson and Strominger, 1961) and are incontrast to the selective effect of penicillin or
D-cycloserine on cell-wall synthesis.The effect of novobiocin on cell-wall synthesis
is, however, a prompt effect of addition of theantibiotic, and occurs at concentrations which are
TABLE 4. Incorporation of P32-inorganic phosphateinto cell wall and into cell protein and nucleic
acid in Staphylococcus aureus
CellFlask condition Cell wall Inhibition protein Inhibition
Control 19,100 6,720Novobiocin, 13,150 31 1,650 75
15 ,ug/mlNovobiocin, 11,100 42 1,200 82
* Results are expressed as counts per minuteper milligram of protein. For details of the methodemployed, see Nathenson and Strominger (1961).
similar to the minimal growth-inhibitory concen-tration of this antibiotic. At the present time itis impossible, therefore, to state whether inhibi-tion of cell-wall synthesis is the primary lethaleffect, whether it is one of several lethal effects,or whether it is secondary to some other primaryaction of novobiocin. Other investigators workingwith Escherichia coli have suggested that it causesdamage to the membranes of growing cells or thatall of its effects may be due to intracellular mag-nesium binding (Brock and Brock, 1959; Brock,1962a, b). More recently, it has been suggestedthat novobiocin damages membrane integrity byinducing an intracellular magnesium deficiency,magnesium ions being necessary to stabilize cellmembranes (Brock, 1962b). Novobiocin is acoumarin derivative, and it has been shown, also,to inhibit oxidative phosphorylation in Myco-bacterium phlei (Weber and Rosso, 1963). A vita-min K compound is involved in electron transportin this organism, so that inhibition of bothoxidative phosphorylation and of cell growth bynovobiocin was reversed by vitamin K. It is diffi-cult to find a common denominator between theseresults and the data reported here. The synthesisof the accumulated nucleotides in S. aureus re-quires both divalent cation and a large amountof adenosine triphosphate. It is, of course, notcertain that the effect of novobiocin on differentbacteria is due to the same mechanism of action.
Other investigators (Shockman and Lampen,1962) have shown that novobiocin inhibitsgrowth of protoplasts of Streptococcus faecalis atthe same concentration at which it inhibits intactcells. Growth of these protoplasts was not in-hibited by penicillin or by D-cycloserine. Baci-tracin, vancomycin, and ristocetin, which haveall been implicated as inhibitors of cell-wall syn-thesis, caused inhibition of protoplast growth aswell as of cells, although the concentration of
ristocetin required to inhibit protoplasts was fivetimes that required to inhibit cells. Isotopic ex-
periments with ristocetin suggest that its effecton cell-wall synthesis is a specific one (Wallas andStrominger, 1963). The inhibition of protoplastgrowth by novobiocin is in agreement with thelarge number of effects of this antibiotic in S.aureus. Moreover, it explains the failure of thisantibiotic to induce spheroplast formation inEscherichia coli (unpublished data), since con-
tinued growth of the protoplast after cell-wallsynthesis ceases is a requirement for emergence ofthe spherical form of E. coli.
This investigation was supported by PublicHealth Service research grant AI-06247 from theNational Institute of Allergy and Infectious Dis-eases, by National Science Foundation grantGB-1823, and by a grant from The Upjohn Co.,Kalamazoo, Mich. Novobiocin (Albamycin) was
generously supplied by The Upjohn Co.
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