Vol. 129, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Jan. 1977, p. 550-553 Copyright © 1977 American Society for Microbiology

Incorporation of 3,4-Dihydroxybutyl-1-Phosphonate, a Glycerol 3-Phosphate Analogue, into the Cell Wall of Bacillus

subtilis1 DAVID A. KLEIN, ROBERT ENGEL, AND BURTON E. TROPP* Department of Chemistry, Queens College of the City University of New York, Flushing, New York 11367

Received for publication 4 August 1976

3,4-Dihydroxybutyl-l-phosphonate, a bacteriostatic agent toward Bacillus subtilus 168 and a bactericidal agent toward strain W 23, is incorporated into cell walls and phospholipids of each strain. The present study was initiated to examine the effects of 3,4-dihydroxybutyl-1-phosphonate, dilithium salt (Li203PCH2CH2CHOHCH2OH) (7) upon Bacillus subtilis BD 170 (thr trp) and 1005 (met). The former, a derivative of strain 168 (Marburg), contains polyglycerol phosphate cell wall teichoic acid (2). The latter, a derivative of strain W 23, contains polyribitol phosphate cell wall teichoic acid (2). As indicated in Fig. 1, 2.5 mM analogue is bacteriostatic toward strain BD 170 and bactericidal toward strain 1005. In view of the differences in the cell wall teichoic acid composition of the two strains, one might expect the analogue to affect teichoic acid metabolism in each strain in a different fashion. To determine whether the analogue is incorporated into cell walls, 80-ml cultures were incubated in 500-ml Erlenmeyer flasks, which were shaken in a water bath (New Brunswick Metabolyte G77), set at 200 rpm and 370C. When the cultures reached 20 Klett units, 3,4-dihydroxy[3-3H]butyl-1-phosphonate (7) (33 mCi/mmol) was added to a final concentration of 30 uM. After a 1-h incubation, cells were collected by centrifugation at 5,000 x g for 10 min, washed once with cold 0.9% NaCl, and resuspended in 13 ml of cold distilled water. The suspension was disrupted by three 30-s bursts of sonic oscillation. A 2-ml sample was removed for lipid extraction, and the remainder was centrifuged for 10 min at 5,000 x g. Cell walls were isolated from the supernatant by centrifugation at 24,000 x g for 15 min. The pellet was suspended in 2.5% sodium dodecyl sulfate and heated for 30 min at 80 to 90°C to I Taken in part from a dissertation to be submitted to the Faculty of Biochemistry of the City University of New York in partial fulfillment of the requirements for the Ph.D. degree (D.A.K.). A preliminary account of this research was presented at the Seventh American Chemical Society Northeast Regional Meeting.

destroy autolysin activity, and the detergent was removed from the walls by five water washings. Because of difficulty in quantitatively recovering cell walls, we considered it desirable to relate incorporation of labeled analogue into cell wall to hexosamine content, which was determined by the method of Levvy and McAllan (11). On this basis, 4.2 mol of 3,4-dihydroxybutyl-1-phosphonate per 100 mol of hexosamine were incorporated into the walls of strain BD 170. The corresponding value for strain 1005 is 0.83 mol of analogue per 100 mol of hexosamine. The fact that the intact analogue was incorporated was established by the following step. The wall.suspensions were hydrolyzed in 2 N HCl for 24 h at 1000C in sealed tubes, lyophilized, redissolved, and spotted on Whatman no. 1 filter paper. The chromatograms were developed in n-propanol-concentrated ammonia-water (6:3:1). Greater than 90% of the label migrated with an Rf of 0.23, identical to that for 3,4-dihydroxybutyl-1-phosphonate. The remainder had an Rf of 0.52 and was not studied further. The radioactive material in the cell wall of each strain was completely solubilized by lysozyme or Pronase, but not by chymotrypsin, trypsin, deoxyribonuclease, or ribonuclease (Table 1). Pronase treatment causes cell wall hexosamines to be released into the soluble fraction. The label in the wall could not be removed by chloroform-methanol extraction. The data are consistent with intact analogue being covalently attached to the cell wall in each strain. The incorporation of analogue into phospholipids was monitored as previously described (13, 15). Under the growth conditions described above, strain BD 170 incorporated 0.12 nmol of intact analogue into phospholipids per ml of culture. The corresponding value for strain 1005 is 0.17 nmol/ml of culture. The lipid ex-

550

NOTES

VOL. 129, 1977

TABLE

551

1. Percentage of cell wall-associated tritium

solubilizeda % 3H solubilized in

Enzyme

200

strain

1005 BD 170 100 100 Lysozyme 100 100 5~~~~~a Pronase 2 4 a-Chymotrypsin 4 5 Trypsin 7 9 DNase and RNaseb a Enzymatic release of label from the cell walls of strain BD 170 and 1005. Cell walls were subjected to enzymatic digestion for 5 h at 37°C at the concentrations and conditions indicated: trypsin at 0.5 mg/ml in 100 mM potassium phosphate buffer, pH 7.6; ribonuclease and deoxyribonuclease, each at 10 ,ug/ml in 50 mM tris(hydroxymethyl)aminomethane, pH 8.0, with 10 mM MgCl2; Pronase at 2 mg/ml in 50 mM potassium phosphate, pH 7.0; and a-chymotrypsin at 0.5 mg/ml in 100 mM potassium phosphate, pH 7.6. All enzymes were purchased from the Sigma Chemical Co., St. Louis, Mo. At the end of the incubation period, the mixture was centrifuged at la 23,000 x g, and the pellet and supernatant were counted in a Beckman LS 200 liquid scintillation counter. b DNase, Deoxyribonuclease, RNase, ribonuclease. 0

Htours 0 ~~

~

~

~

~

~

~

0

tracts were chromatographed on a diethylaminoethyl-cellulose column as previously described (15). The results presented in Fig. 2 are for lipids isolated from strain 1005. The same chromatographic pattern was obtained for strain BD 170. The material in the major peak, containing 90 to 95% of the radioactivity found in the unfractionated lipid extract, had the chromatographic properties expected of (1,2diacyl)-sn-glyceryl D-4'-phosphoryloxybutyl-1phosphonate, the phosphonate analogue of phosphatidylglycerophosphate (15). Phospholipase C treatment of the pooled phospholipid converted the label from chloroform- to watersoluble material. Chromatography of the water-soluble material indicated that 10% of the radioactivity migrated as 4-phospho-3-hydroxybutyl-l-phosphonate. Due to the presence

growth turbidimetrically in a Klett-Summerson colorimeter with a 660-nm filter. Viable cell counts were FIG. 1. Effect of 3,4-dihydroxybutyl-1-phospho- determined by diluting samples from the culture into nate upon growth and viability. The culture medium a minimal salts medium (1) and counting colonies on consisted of10 g of casein hydrolysate (pH 7.0) and 5 Eugon agar (Difco Laboratories, Detroit, Mich.). g of NaCl per liter. Cells were cultured in 20 ml of Symbols: 5 mM lithium chloride: (0) Klett units medium in 250ml Erlenmeyer fla,ka fitted with side and (0) viable cell number; 2.5 mM dilithium salt of arms. Incubations were in a water bath shaker (New 3,4-dihydroxybutyl-a-phosphonate: (0) A'left- units Brunswick Metabolyte G 77) set at 160 rpm and and (U) viable cell number. (A) Strain BD 170; (B) Hours

37°C. Cell growth

was

determined by monitoring

strain 1005.

552

NOTES

J. BACTERIOL.

3

2

E

0

10

20 Fraction Number

30

40

FIG. 2. Fractionation of lipid extracts obtained from strain 1005 on a diethylaminoethyl-cellulose column. The procedure was as previously described (15).

of phosphatase activity in the preparations of Bacillus cereus phospholipase C (15), these results are consistent with the identification of the major peak as the phosphonate analogue of phosphatidylglycerophosphate. The bacteriostatic effect toward strain BD 170 is quite similar to that reported for Escherichia coli (12). The bactericidal effect on strain 1005 was unexpected. At this time we suspect that the analogue inhibits the growth of E. coli by interacting with cytidine 5'-diphos-

phate-diglyceride:sn-glycerol 3-phosphate phosphatidyltransferase (13, 14, 15). The in vivo data indicate that the analogue serves as a substrate for the B. subtilis enzyme and, as with E. coli, the phosphonate does not appear to replace glycerol 3-phosphate in the acylcoenzyme A:sn-glycerol 3-phosphate acyltransferase-catalyzed reaction. Interaction with phosphatidyltransferase may explain growth inhibition in B. subtilis, but fails to explain why the analogue is bactericidal to strain 1005 but not strain BD 170 or E. coli (12). The possibility that strain BD 170 partially detoxifies the drug by incorporating large amounts into cell wall must be viewed with caution, since similar degrees of growth inhibition were observed at various concentrations of the drug in each of the B. subtilis strains (data not shown). Recent publications concerning the presence of an oligomer of glycerol 3-phosphate serving as a linker between the peptidoglycan and the teichoic acid donated by lipoteichoic acid carrier (3, 5, 9, 10) offer a reasonable explanation for our observations concerning the incorporation of analogue into cell wall. In each strain,

analogue may replace glycerol 3-phosphate in the linker region. In strain BD 170 it may also be able to replace glycerol 3-phosphate in the cell wall teichoic acid itself. This is consistent with the observation that 5.4 times as much analogue is incorporated into the wall of BD 170 than 1005. Preliminary experiments (R. Deutsch, R. Engel, and B. E. Tropp) indicate that the analogue inhibits phosphoglyceride synthesis in B. subtilis. One might predict that an alteration of phosphatidylglycerol metabolism would have a marked effect upon lipoteichoic acid fonnation (6, 8). A better understanding of the effects of 3,4-dihydroxybutyl-1phosphonate must await further in vivo studies and the initiation of in vitro investigations. We wish to thank A. Tomasz of the Rockefeller University for helpful discussions and advice and L. Mindich and D. Dubnau of the Public Health Research Institute of the City of New York for providing the strains of Bacillus subtilis used in the present study. This investigation was supported by Public Health Service grant GM 21400-03 from the National Institute of General Medical Sciences, National Science Foundation grant PCM76-07326, and a grant from the Faculty Research Award Program of the City University of New York.

LITERATURE CITED 1. Anagnostopulis, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. 2. Baddiley, J. 1972. Teichoic acids in cell walls and mem-

branes of bacteria. Essays Biochem. 8:35-79. 3. Bracha, R., and L. Glaser. 1976. In vitro synthesis of teichoic acid linked to peptidoglycan. J. Bacteriol. 125:872-879. 4. Cheng, P-J., W. D. Nunn, R. J. Tyhach, S. L. Goldstein, R. Engel, and B. E. Tropp. 1975. Investigations concerning the mode of action of 3,4-dihydroxybutyl-1-phosphonate on Escherichia coli. J. Biol. Chem. 250:1633-1639. 5. Coley, J., A. R. Archibald, and J. Baddiley. 1976. A linkage unit joining peptidoglycan to teichoic acid in Staphylococcus aureus H. FEBS Lett. 61:240-242. 6. Emdur, L. I., and T. H. Chiu. 1974. Turnover of phosphatidylglycerol in Streptococcus sanguis. Biochem. Biophys. Res. Commun. 59:1137-1144. 7. Goldstein, S. L., D. Braksmayer, B. E. Tropp, and R. Engel. 1974. Isosteres of natural phosphates. 2. Synthesis of the monosodium salt of 4-hydroxy-3-oxobutyl-1-phosphonic acid, an isostere of dihydroxyacetone phosphate. J. Med. Chem. 17:363-364. 8. Glaser, L., and B. Lindsay. 1974. The synthesis of lipoteichoic acid carrier. Biochem. Biophys. Res. Commun. 59:1131-1136. 9. Hancock, I., and J. Baddiley. 1976. In vitro synthesis of the unit that links teichoic acid to peptidoglycan. J. Bacteriol. 128:880-886. 10. Heckels, J. E., A. R. Archibald, and J. Baddiley. 1975. Studies on the linkage between teichoic acid and peptidoglycan in a bacteriophage-resistant mutant of Staphylococcus aureus H. Biochem. J. 149:637-647. 11. Lewy, G. A., and A. McAllan. 1959. The N-acylation and estimation of hexosamines. Biochem. J. 73:127132.

VOL. 129, 1977 12. Shopsis, C. S., R. Engel, and B. E. Tropp. 1972. Effects of phosphonic acid analogues of glycerol 3-phosphate on the growth of Escherichia coli. J. Bacteriol. 112:408-412. 13. Shopsis, C. S., R. Engel, and B. E. Tropp. 1974. The inhibition of phosphatidylglycerol synthesis in Escherichia cohi by 3,4-dihydroxybutyl-1-phosphonate. J. Biol. Chem. 249:2473-2477. 14. Shopsis, C. S., W. D. Nunn, R. Engel, and B. E. Tropp.

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1973. Effects of phosphonic acid analogues of glycerol 3-phosphate on the growth of Escherichia coli: phospholipid metabolism. Antimicrob. Agents Chemother. 4:467473. 15. Tyhach, R. J., R. Engel, and B. E. Tropp. 1976. The metabolic fate of 3,4-dihydroxybutyl-1-phosphonate in Escherichia coli: formation of a novel lipid, the phosphonic acid analogue of phosphatidylglycerophosphate. J. Biol. Chem. 251:6717-6723.