GROWTH OF CELLULAR FORMS IN CULTURES OF CHROMIATIN BODIES

GROWTH OF CELLULAR FORMS IN CULTURES OF CHROMIATIN BODIES ISOLATED FROM BACILLUS MEGATERIUM1 B. R. CHATTERJEE AND ROBERT P. WILLIAMS Departnment of Microbiology, Baylor University College of Medicine, Houston, Texas Received for publication 15 October 1962 ABSTRACT CHATTERJEE, B. R. (Baylor University College of Medicine, Houston, Texas) AND ROBERT P. WILLIAMS. Growth of cellular forms in cultures of chromatin bodies isolated from Bacillus megaterium. J. Bacteriol. 85:623-627. 1963.- Chromatin bodies isolated from old cultures of Bacillus megaterium were capable of growing into protoplastlike cells when cultured in broth enriched with horse serum, yeast extract, adenosine triphosphate, and penicillin. A tendency toward formation of rod forms of bacteria was observed in such cultures. Omission of penicillin from the medium resulted in development of short bacterial forms. In 3 of 29 experiments, actual bacillary forms indistinguishable from the parent B. megaterium organism were recovered. Culture of the chromatin bodies in plain nutrient broth did not produce any growth. Inoculation on serum-enriched agar medium of a culture of chromatin bodies, after they had begun multiplication in serum-enriched broth, resulted in development of large bodies characteristic of L forms. Ability of chromatin bodies to grow was not affected by heating for 2 hr at 80 C or by sonic treatment for up to 25 min. The possible role of such resistant chromatin bodies in the latency and persistence of infectious diseases was discussed. the isolated chromatin bodies, rather than whole cells, could be cultured in a suitable medium and developed into protoplasts, L forms, or even mature bacilli. MATERIALS AND M\IETHODS The asporogenous strain KM of B. megaterium was used. Growth of the bacteria, their microscopic examination and photography, and isolation of the chromatin bodies were carried out by the methods of Chatterjee and Williams (1962a). To obtain completely cell-free preparations of chromatin bodies, the concentration of lysozyme was raised to 5 mg/ml. Addition of sodium lauryl sulfate (final concentration, 0.1 %), 1 hr after lysozyme treatment, effected clarification of the suspensions, and this procedure was used whenever necessary. Liquid cultures of isolated chromatin bodies were grown in Penassay Broth (Difco). Solidmedium cultures were grown on Brain Heart Infusion Agar (Difco). Both media were enriched with 1 % yeast extract (Difco), 20% PPLO Serum Fractions (Difco), 5% NaCl, and 10-3 M adenosine triphosphate (ATP; Schwarz Laboratories, Inc., Mt. Vernon, N.Y.). Where indicated, 1,000 IU/ml of crystalline penicillin (Chas. Pfizer & Co., Inc., Brooklyn, N.Y.) were added. All cultures were incubated at 37 C. A Raytheon sonic oscillator (250 w; 10 kc) was used for sonic treatment of the chromatin bodies. We reported the development of chromatin bodies within aging cells of Bacillus megaterium RESULTS and B. anthracis, and by suitable techniques the Growth of protoplastlike cells from chromatin bodies were isolated from old cultures (Chatterjee bodies. NVhen an arbitrary quantity of isolated and Williams, 1962a). Chromatin bodies with chromatin bodies was inoculated into nutrient similar morphological and staining characteristics broth, no growth ensued. However, if an equal were also observed in developing L cultures of B. quantity of the bodies was inoculated into the anthracis (Chatterjee and Williams, 1962b). serum-enriched liquid medium containing 1,000 These observations suggested that development IU/ml of penicillin, a considerable number of of a chromatin body might be a phase in the protoplastlike bodies appeared after 2 to 3 days bacterial transition into L forms. The following of incubation. Within 48 hr, the serum broth experiments were planned to determine whether became turbid, and the culture, when examined 623

624 CHATTERJEE AND WILLIAMS J. BACTERIOL. under a microscope, showed an increase in chromatin bodies. They were no longer in small constellations of two or more spherical bodies, as they were after isolation (Fig. 1), but had multiplied; large clusters of the bodies, which seemed interconnected by thin strands of material, had developed from the constellations (Fig. 2). From the third day onward, increasing numbers of small and large protoplastlike cells appeared in the cultures (Fig. 3). Quite often, these protoplastlike cells occurred in large clusters with some spherical chromatin bodies inside. After many hours, these protoplastlike cells broke down, either into shapeless masses of granular material (Fig. 4), or into small spherical bodies, some of which had a homogenous appearance like large cocci, whereas others with a refractile center and dark periphery looked much like the chromatin bodies. These changes are illustrated in Fig. 5 to 10. Other cell types in addition to the protoplastlike cells also appeared in serum broth cultures. Thin-walled cylindrical forms containing chromatinlike structures sometimes developed (Fig. 11). As shown in Fig. 12, cylindroid forms were evident which were neither spherical nor cylindrical, but presumably arose from a spherical element. Completely spherical cells with the appearance of true protoplasts (Fig. 13) were also evident. Serum was essential for the growth observed. Enrichment of nutrient broth with yeast extract or ATP did not promote growth of the chromatin bodies. Yeast extract and ATP could be omitted from the serum broth, but their omission resulted in a considerable slowing down of the growth and a decrease in the number of protoplastlike cells. Transfer from serum broth to nutrient broth of a developing culture of chromatin bodies resulted in complete disappearance of the protoplastlike cells. Growth of bacilli from chromatin-body cultures. After growth for 4 to 5 days in a serum broth culture containing no penicillin, a small, pleomorphic form of Bacillus appeared in a large proportion of the cultures. Within another 2 to 3 days of growth, these pleomorphic bacteria almost completely replaced the chromatin bodies that had resulted from breakdown of the protoplastlike cells. During their emergence, some of these bacterial forms still appeared to be attached to, or arising from, clusters of chromatin bodies or protoplastlike cells (Fig. 14 and 15). The rod forms varied in length from 1 to 7,t. As shown in Fig. 16, the majority of these cells were club-shaped, with one extremity wider than the other. The presence of penicillin in the medium completely inhibited development of the bacillary forms. Once the bacilli developed in the culture, they could be grown in nutrient broth FIG. 1. Chromatin bodies of Bacillus megaterium after isolation. (All pictures were taken through a phase-contrast microscope.) X 1,000. FIG. 2. Multiplication of chromatin bodies into large clusters in serum broth. X 2,000. FIG. 3. Protoplastlike cells grown from chromatin bodies in a serum broth culture. X 2,000. FIG. 4. Breakdown of protoplastlike cells into small, granular elements. X 2,000. FIG. 5 to 10. Sequence of pictures taken over a 17-hr period showing ultimate breakdown of a cluster of protoplastlike cells into small coccoid or chromatinlike elements. The arrow shows emergence of a rodlike element from a hyphalike projection of the cluster. In Fig. 10, only the shadow of the rod is visible because the rod lies in a different focal plane from the rest of the picture. The protoplastlike cells were grown in a serum broth culture from chromatin bodies. X 2,000. FIG. 11. Cylindrical form with a chromatinlike structure (arrow) at one end grown from chromatin bodies in a serum broth culture. X 2,000. FIG. 12. Tendency toward cylindroid form of a presumably spherical element. Same culture as shown in Fig. 11. X 2,000. FIG. 13. Large protoplastlike cell in the same culture as shown in Fig. 11. X 2,000. FIG. 14. Emergence of a rod form of bacterium from a cluster of chromatin bodies in a serum broth culture. X 2,000. FIG. 15. Another mode of development of rod forms from protoplastlike cells. X 2,000. FIG. 16. Appearance of the short, pleomorphic bacillary forms as they developed in a serum broth culture of the chromatin bodies. X 2,000. FIG. 17. Growth on nutrient agar of the short bacterial forms obtained by culture in serum broth of heated (80 C for 2 hr) chromatin bodies. X 2,000. FIG. 18. Large body of Dienes and Weinberger (1951) developing on serum agar. The inoculum was obtained from a culture of chromatin bodies grown in serum broth. Arrow shows the envelope of the cell. X 2,500.

FIGS. 1-18 625

626 CHATTERJEE AND WILLIAMS J. BACTERIOL. or nutrient agar, and maintained through an indefinite number of transfers. On agar, the organisms gave rise to pinhead- or pinpoint-sized, opaque, slightly raised colonies. Regeneration of the parent Bacillus form occurred in 3 of 29 experiments. The biochemical, morphological, and staining characteristics of these forms were identical to those of the parent B. megaterium organism from which the chromatin bodies were isolated. Growth of L forms from chromatin bodies. If an inoculum were taken from a serum broth culture of the chromatin bodies before appearance of protoplastlike cells and spread over the surface of a serum agar plate, the cellular elements illustrated in Fig. 18 would appear after 3 to 4 days of incubation. These large spherical bodies contained 10 to 50 small, spherical, chromatinlike elements encased within a common envelope. Similar cellular elements are found in developing L forms and have been termed by Dienes and Weinberger (1951) as large bodies. Attempts to transfer the spherical bodies from serum agar plates to fresh medium to keep them viable were unsuccessful. Inoculation of isolated chromatin bodies directly into serum agar did not produce growth of any kind. Resistance of chromatin bodies to physical treatments. No growth could be obtained from the asporogenous strain KM of B. megaterium if thick suspensions of the cells were heated at 80 C for 1 hr. However, isolated chromatin bodies would produce growth similar to that described above even after the suspensions were heated at 80 C for as long as 2 hr. No regeneration of the parent bacillary form could be obtained from heated samples. Figure 17 shows the growth of the pleomorphic bacterial forms on nutrient agar after heat treatment. Sonic treatment of the chromatin bodies for as long as 25 min did not alter their appearance or viability. We previously demonstrated that the chromatin bodies cannot be identified with bacterial spores (Chatterjee and Williams, 1962b). DISCUSSION As reported previously (Chatterjee and Williams, 1962c), the chromatin bodies are the nuclear equivalents of these bacteria. When stained by acid-giemsa or Feulgen methods, the isolated bodies show the same type of peripheral staining as when inside the cells. Staining with acridine orange demonstrates the characteristic yellowish-green fluorescence of deoxyribonucleic acid. Retention of physical integrity and viability by these elements through the rigors of the isolation procedure, heating, and sonic treatment suggests that, during the later phases of the growth cycle, the bacterial chromatin is surrounded by a protective membrane. Ability of the chromatin bodies to organize and multiply into the pleomorphic rod forms, or at times to regenerate parent bacillary organisms, suggests that the bodies are not merely nuclear material but perhaps are a specialized cellular entity. The chromatin bodies might be the starting material for formation of L forms in bacterial cultures. Also, resistance of the chromatin. bodies to adverse physical conditions indicates that they, as well as bacterial spores, represent a means for survival of bacteria. If regeneration from chromatin bodies of the parent bacillary forms can be demonstrated in other organisms, particularly pathogens, the phenomenon could lead to speculation about problems in epidemiology and chronic, resistant infections. It is frequently observed in endemic areas that the causative organisms disappear from their common natural sources between outbreaks of disease. Perhaps the infectious agent of nonsporeforming organisms could persist during these intervals through development of a resistant chromatin-body stage. In recurrent and resistant infections, causative organisms also frequently disappear in the animal host during administration of chemotherapeutic drugs. After specific treatment ceases, the organisms appear again. Wittler et al. (1960) recovered some unusual bacterial forms from such cases. Some of these forms are similar to the protoplastlike cells and granular elements described in this paper. Latency or persistence of a pathogen in the diseased host might occur through development of a chromatin-body stage. Subsequent regeneration of the parent-type bacterium would complete the cycle, and bring about reappearance of the causative organism. ACKNOWLEDGMENT This investigation was supported by research grant A1-01535-05 from the National Institutes of Health, U.S. Public Health Service.

VOL. 85, 1963 CELLULAR FORMS FROM CHROMATIN BODIES 627 LITERATURE CITED CHATTERJEE, B. R., AND R. P. WILLIAMS. 1962a. Cytological observations on the chromatin bodies of two Bacillus species. J. Bacteriol. 83:1112-1118. CHATTERJEE, B. R., AND R. P. WILLIAMS. 1962b. Cytological changes in aging bacterial cultures. J. Bacteriol. 84:340-344. CHATTERJEE, B. R., AND R. P. WILLIAMS. 1962c. Growth and organizing ability of the nuclear material isolated from Bacillus megaterium. Intern. Congr. Microbiol., 8th, Montreal, 1962, p. 39. DIENES, L., AND H. J. WEINBERGER. 1951. The L forms of bacteria. Bacteriol. Rev. 15:245-288. WITTLER, R. G., W. F. MALIZA, P. E. KRAMER, J. D. TUCKETT, H. N. PRICHARD, AND H. J. BAKER. 1960. Isolation of Corynebacterium and its transitional forms from a case of subacute bacterial endocarditis treated with antibiotics. J. Gen. Microbiol. 23:315-333.