Microbiol. Immunol. Vol. 28 (9), 1057-1064, 1984

The Effect of Pipemidic Acid on the Growth of a Stable

L-Form of •ôNH•ôStaphylococcus aureus•ôNS•ô

Kunihiko YABU,*,1 Hiromi ToMizu,1 and Yayoi KANDA2

1 Department of Biology, Hokuriku University School of Pharmacy, Kanazawa-machi, Kanazawa, Ishikawa ,920-11, and 2Department of Microbiology, Teikyo University School of Medicine, Kaga 2-chome, Ilabashi-ku, Tokyo 173

(Accepted for publication, June 12, 1984)

Bacterial L-forms usually display spherical forms in an osmotically protective

medium and seem to lack the typical binary fission process of cellular division ob servedin most bacteria (7). Although various modes of replication, such as budding binary fission, and release of elementary bodies from large bodies have been

observed by light and electron microscopy (2, 5, 16), little is known about the processes involved in replication of L-forms. In the course of an experiment designed to test the effect of DNA synthesis inhibitors on the growth of a stable L-form of •ôNH•ôStaphylococcus•ôNS•ôaureus which grows in liquid medium, it was found that pipemidic acid, a synthetic antibacterial agent

structurally related to nalidixic acid (13), induced a marked morphological altera tionat growth inhibitory concentrations. This study was initiated in an attempt

to clarify the mechanism of replication of stable L-forms by analyzing the mor

phologicalalteration caused by pipemidic acid. The stable L-form used was isolated as follows. S. •ôNH•ôaureus•ôNS•ôFDA 209P was

grown in 10ml of Brain Heart Infusion broth (Difco) at 37C. The culture grown at 5•~105 colony-forming units (CFU) per ml was washed with saline by filtration and suspended in saline containing 100ƒÊg of N-methyl-N'-nitro-N-nitrosoguanidine

per nil. The suspension was incubated for 30min at 37C. The cells were col lectedand resuspended in liquid L-form medium containing 3 units of lysostaphin

(Sigma) per ml. After three weeks of incubation at 30C, the surviving clone that formed typical L-form colonies on solid medium was selected and transferred to

liquid medium.

The L-form was maintained and cultured in a modified semidefined liquid

medium (1). The basal medium contained the following ingredients per 100ml:

5.0g of NaCl, 0.5g of K2HPO4, 40mg of MgSO4.7H2O, 2.0g of sodium lactate,

1.0g of galactose, 0.5g of vitamin free casamino acid (Difco), 5.0mg of uracil, 0.5mg of nicotinic acid, 0.1mg of thiamine hydrochloride, and 0.1ƒÊg of biotin.

The pH of the basal medium was adjusted to 6.8 with HCI. This basal medium was supplemented with 10 ml of 10% bovine serum albumin (Sigma). Agar me diumwas prepared by the addition of 1.2% (wt/vol) agar (Difco) to the liquid medium.

1057 1058 K. YABU ET AL

A 0.2-ml sample of 16-hr culture was inoculated into test tubes containing

5ml of the liquid medium. In experiments, pipemidic acid (obtained from Dai nipponPharmaceutical Co.), nalidixic acid, oxolinie acid, novobiocin (purchased

from Sigma Chemical Co.), or AB206 (obtained from Sumitomo Chemical Co.)

was added at the indicated concentrations. Cultures were grown at 30 C. Sam

plesfrom cultures were taken at the time of inoculation and at I-day intervals for viable count determination. Each sample was diluted in the liquid medium, and

duplicate samples of the culture were processed by gentle blending for 5 see on a

Vortex mixer to obtain accurate viable counts. A 0.1-ml portion of the appropriate dilution was then plated in triplicate. Agar plates were incubated for 5 days at

30C.

The growth rate of the L-form was determined by viable counts in the liquid

medium containing varying amounts of pipemidic acid. Growth was inhibited at

a constant level at a concentration of 100ƒÊg per ml, but decreased gradually with incubation time at 200ƒÊg per ml (Fig. 1). The metamorphosis of the L-form was

followed by phase-contrast microscopy during one week of incubation. The L- form cells were quite small in size in the exponential growth phase and became

swollen in prolonged incubation (Fig. 2, A and C). In the presence of 100ƒÊg

per ml of pipemidic acid, large spherical forms with phase-bright vacuoles were seen from the second day of incubation. In a period of two or three days, vacuoles within large bodies increased gradually in size and attained sizes of 5 to 10ƒÊm or

Fig. 1. Effect of pipemidic acid on the

growth of Staphylococcus aureus L-form in liquid medium. An exponential culture was used to inoculate test

tubes containing 5ml of liquid me

diumwithout any drug (•›) or with

pipemidic acid at a concentration of 50ƒÊg per ml (•£), 100ƒÊg per ml

(•œ), or 200ƒÊg per ml (•¡). Test tubes were incubated at 30C and

samples were removed at the times

indicated for viable counts.

Fig. 2. Phase-contrast micrographs of L-forms grown in the presence and absence of 100ƒÊg

of pipemidic acid per ml. The culture conditions were the same as shown in Fig . 1. A and C show control cultures grown for 2 and 6 days, respectively. B and D show cultures grown for

2 and 6 days in the presence of the drug, respectively. Bars represent 10ƒÊm. NOTES 1059

Fig. 2 1060 K . YABU ET AL

Fig. 3 NOTES 1061 more at the end of incubation (Fig. 2, B and D). Electron microscopic observations of the cultures fixed with glutaraldehyde-osmium (6) revealed that the large bodies contained numerous small vesicles, some of which had abnormally thickened mem branesand were empty or void of obvious cytoplasm and threads of DNA (Fig . 3B). In the control culture, such vesicles were not observed but the nuclei were usually polydispersed (Fig. 3A). With the addition of other synthetic DNA synthesis inhibitors , nalidixic acid and oxolinic acid, at the growth inhibitory concentration (100ƒÊg per ml) , numerous L-form cells irregularly shaped like a comma or bean-sprout were found by phase contrastmicroscopic examination (data not shown). The addition of AB206, a quinoline derivative similar to oxolinic acid, at a concentration of 25ƒÊg per ml produced a morphological alteration similar to that induced by pipemidic acid. However, this drug seemed to lead the L-form to lysis in an early stage of growth

(data not shown). All these synthetic antibacterial agents were capable of inducing morphological changes in the L-form at growth inhibitory concentrations although they had no effect on the morphology of the parent coccal cell. In contrast, in the presence of novobiocin at the growth inhibitory concentration (0.3ƒÊg per ml), no apparent morphological change was detected in the L-form by phase-contrast microscopy, but the growth was greatly reduced. It is well known that nalidixic acid inhibits DNA synthesis with little effect

Fig. 4. Effect of pipemidic acid on the incorporation of thymidine (A), uracil (B), and L-

leucine (C) into DNA, RNA, and protein of the L-form. To each I ml of a growing L-form

culture at 5•~106 CFU per ml was added [14C]thymidine (IƒÊCi/ml), [14C]uracil (1ƒÊCi/ml), or L-[14C]leucine (1ƒÊCi/ml) at a final concentration of 0.2mM. Radioactive compounds

a nd drugs were added simultaneously at the beginning of the incubation at 30C. At the times indicated, the reaction was stopped by the addition of I ml of 100% trichloroacetic

acid. The precipitates were collected on a glass fiber filter and washed three times with 5ml

of cold 5% trichloroacetic acid. The filters were dried and counted in a liquid scintilation

counter. Control (•›), pipemidic acid: 50ƒÊg per ml (• ); 100ƒÊg per ml (• ).

Fig. 3. Electron micrographs of thin sections of L-forms grown for 6 days in the absence (A)

and presence of 100ƒÊg of pipemidic acid per ml (B). Bars represent 1.0ƒÊm. 1062 K. YABU ET AL

Fig. 5. Densitometric scan of the autoradiogram of 14C-labeled membrane proteins prepared

from the L-forms grown in the absence (A) and presence (B) of 100ƒÊg of pipemidic acid per ml. An exponential culture was allowed to grow for 15 hr at 30C in the liquid medium

with or without the drug. [14C]Amino acid mixture (1ƒÊCi/ml) was added and the incu

bationwas continued for 1hr. The cultures were harvested by centrifugation, suspended in 0.067M phosphate buffer (pH 7.2), and washed twice with the same buffer. Isolated mem

branepreparations were solubilized and separated by SDS-polyacrylamide gel electro

phoresisbased on the method described by Kubak and Yotis (10). After fluorography according to the method of Bonner and Laskey (3), gels were exposed to Kodak X-Omat

XRP-5 film for two weeks at-80C. The intensity of the bands was determined with a Shi madzuCS910 TLC scanner. Arrows indicate peaks C1, C2, and C3, which were greatly

increased or decreased in the presence of pipemidic acid. Their apparent molecular weights were 14K, 44K, and 70K, respectively. on RNA and protein synthesis (8, 12, 15). Oxolinic acid and AB206 also inhibit DNA synthesis in Escherichia coli (11, 14). To examine whether pipemidic acid inhibits DNA synthesis of the L-form, the incorporation of radioactive thymidine, uracil, and L-leucine into trichloroacetic acid-insoluble material was measured. Pipemidic acid inhibited the incorporation of thymidine, but showed no inhibitory effect on the L-form RNA and protein synthesis (Fig. 4). It has been reported that nalidixic acid alters cellular protein patterns by causing the appearance of some and the disappearance of other pulse-labeled pep tidesin E. colt (4). Figure 5 shows the effect of pipemidic acid on the synthesis of membrane proteins of the L-forms. When the membrane fraction of the L-forms labeled with [14C]amino acid mixture was analyzed by SDS-polyacrylamide gel elec NOTES 1063

trophoresis, visualized by autoradiography, and traced by the densitometric scan of autoradiograms, 15 or more peaks could be separated, as seen in Fig. 5A. In the

presence of 100ƒÊg of pipemidic acid per ml, the synthesis of proteins with mole cularweights of 14K and 70K was depressed whereas the synthesis of a protein of

44K was markedly stimulated (Fig. 5B). Pipemidic acid altered membrane protein

patterns although it was not clear how these membrane proteins were associated with the drug-induced morphological alteration of the L-form.

Pipemidic acid not only inhibited DNA synthesis but also caused morphological

alteration of the L-form cell which was devoid of an organized cell wall. Nalidixic

acid, oxolinic acid, and AB206 also produced an extensive irregular shape of L-form

cells in osmotically protective medium. Electrophoretic analysis and electron

microscopic observations suggested alterations in protein of the membrane of the

L-form grown in the presence of pipemidic acid.

Some studies on modes of L-form replication indicated that large bodies re

leasednumerous elementary bodies which propagated by the usual process of binary

fission or budding (5, 9). Although the mechanism by which elementary bodies

are formed inside large bodies has not been explained, Wyrick and Rogers (16)

assumed that elementary bodies may be formed through partition of a large body

by internal membranes surrounding and enclosing DNA and cytoplasm. The

morphological alteration of the L-form of S. aureus induced by pipemidic acid may

reflect interruption of the formation of elementary bodies within a large body by

blocking DNA synthesis, and consequently the formation of small vesicles and

abnormal vacuoles without involving DNA. Further investigations on membrane

protein synthesis interference by pipemidic acid will help to analyze the process

of formation of vesicles inside large bodies.

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coccus aureus membrane proteins. Biochim. Biophys. Acta 687: 238-246. 11) Nagate, T., Komatsu, T., Izawa, A., Ohmura, S., Namiki, S., and Mitsuhashi, S. 1980. Mode of action of a new nalidixic acid derivative, AB206. Antimicrob. Agents Chemother. 17: 763 - 769. 12) Pedrino, A.M., Geroldi, D., Siccardi, A., and Falaschi, A. 1972. Studies on the mode of action of nalidixic acid. Eur. J. Biochem. 25: 359-365. 13) Shimizu, M., Takase, Y., Nakamura, S., Katae, H., Minami, A., Nakata, K., Inoue, S., Ishi yama,M., and Kubo, Y. 1975. Pipemidic acid, a new antibacterial agent active against Pseu domonasaeruginosa: In vitro properties. Antimicrob. Agent Chemother. 8: 132-138. 14) Staudenbauer, W.L. 1976. Replication of Escherichia coli DNA in vitro: Inhibition by oxolinic acid. Eur. J. Biochem. 62: 491-497. 15) Sugino, A., Peebles, C.L., Kreuzer, K.N., and Cozzarelli, N.R. 1977. Mechanism of action of nalidixic acid: Purification of Escherichia coli nal gene product and its relationship to DNA gyrase and a novel Nicking-closing enzyme. Proc. Natl. Acad. Sci. U.S.A. 74: 4767-4771. 16) Wyrick, P.B., and Rogers, H.J. 1973. Isolation and characterization of cell wall-defective vari antsof Bacillus subtilis and Bacillus licheniformis. J. Bacteriol. 116: 456-465.

( Received for publication, February 20, 1984)