Induction and Propagation of a Bacillus subtilis LForm in Natural and Synthetic Media
H. R. BURMEISTER AND C. W. HESSELTINENorthern Regional Research Laboratory, U.S. Departmenit of Agriculture, Peoria, Illinois 61604
Received for publication 4 March 1968
The L form of Bacillus subtilis NRRL B-3275 was induced in a 7% NaCl brothmedium and subsequently propagated in natural and synthetic media. The L formgrew readily in tryptone broth supplemented with glucose, NaCl, and phosphatebuffer, and in a synthetic medium containing only glucose and biotin, in additionto the required salts. Successive transfers from the bacillus inoculum and subsequentlarge bodies in the tryptone broth with 7% NaCl resulted in gradual selection or
transition from the bacillary form to a stable L form without the addition of anantibiotic. The number of viable granules attained in the broth culture exceeded9 X 107 per ml, and numerous large bodies were always present in rapidly growingcultures.
Protoplasmic granules associated with bacterialcultures were first reported by Klieneberger (10),and their derivation from bacteria was demon-strated by Dienes (7). Since their discovery,numerous L forms have been produced andpropagated, although the L-form growth of mostbacterial species requires complex media andnearly always an agar support. Moreover, success-ful initiation of growth in broth usually requiresthe incorporation of agar blocks containingL-form colonies. However, mutants of Proteusthat lack the subsurface components of typical3B type L-form colonies on agar have been shownto grow readily on direct transfer to broth (2).
Development of synthetic media for the studyof bacterial L forms other than those of Proteushas evolved slowly. A semidefined broth mediumwas compounded by Abrams (1), and a com-pletely synthetic broth was described by Medilland O'Kane (14) for the Proteus L forms. Inaddition, a Staphylococcus aureus L form (5) andthe 3B type L form of Bacillus subtilis (15) havebeen cultured on semidefined and defined media,respectively. The B. subtilis L form required anagar support.
Transition of bacteria to L forms previouslyhas been observed only on media solidified withagar (9), although commitment can occur inliquid penicillin media in the absence of celldivision (11).
B. subtilis NRRL B-3275 did not require agarfor L-form induction, transfer, or propagation.The L form was induced with 7% NaCl in atryptone broth. Several transfers of the bacillus
and subsequent large bodies provided L-formcultures that could be readily propagated in brothor on agar media. This report describes themethods of induction, propagation, growth rates,and some morphological features of large bodiesof this L form.
MATERIALS AND METHODSCulture identification. A bacillus was isolated from
moist corn and became the subject of study duringcharacterization because large, spheroplast-likebodies, as well as bacilli, were always observed intest media containing 7% NaCI. The motile, gram-positive bacillus averaged 0.7 A in diameter and 2.0 ,uin length. Endospores of the organism were formedin abundance on Trypticase Soy Agar (BBL). Thespores were about 0.6 ,u in diameter and 1.5 A inlength. Colonies from heat-shocked spores grewreadily on glucose agar, soybean agar, and in tryp-tone broth containing 7% NaCI. Starch, casein, andgelatin were hydrolyzed, and the pH of 0.3% tryp-tone broth declined from 7.2 to about 6.0 after 7days of incubation at 28 C. Reduction of nitrateto nitrite, however, was not shown. The isolation wastentatively identified as B. subtilis and was given thenumber NRRL B-3275. Additional characteristics ofthis bacterium will be given in another section of thispaper.
Media. The L form was propagated in three media,each medium containing the listed ingredients perliter, and resulted in pH values from 6.8 to 7.0. T-medium consisted of tryptone, 3.0 g; glucose, 1.0 g;KH2PO4, 1.0 g; K2HPO4, 3.0 g; and NaCl, 17.0 g.The glucose-salts medium (GS) contained glu-cose, 5.0 g; KH2PO4, 2.0 g; K2HPO4, 4.0 g;(NH4)S04, 0.5 g; NaCI, 17.0 g; MgCI2*6H20,0.25 g; CaCl2, 50,g; CuS04. 5H20, 40 ,ug; FeCl3-
6H0, 100 jug; MnSO4 -H20, 100 Mg; ZnSO4-7H20,400 ,ug; and biotin, 20 MAg. The third medium wasYeast Nitrogen Base (YNB; Difco) supplemented withK2HPO4, 3.0 g; glucose, 5.0 g; and NaCI, 17.0 g. Theglucose and the MgCl2 were autoclaved separately,and YNB required filtering to obtain a clear broth.
Induction of the L form. Colonies that were to beinduced to the L form were grown on Nutrient Agar(Difco) inoculated with spores heat-shocked for 30min at 70 C. A portion of a 36-hr colony was trans-ferred to 10 ml of T-medium with 7% NaCI. Trans-fers (0.5 ml) were continued in this medium untilonly viscid strands of large, spherical bodies weremicroscopically visible; four to eight transfers weregenerally required. L forms so induced were succes-sively transferred to T-medium and T-medium with1.2% agar (15) a minimum of five times to observetheir stability in the reduced salt concentration. Inaddition, the cultures were plated onto T-mediumwith the NaCl deleted to test their osmotic fragility.Transfers were made at 2-day intervals with an incu-bation temperature of 28 C.
Proofofosmotic fragility. In this study, the criteriafor functional impairment or absence of a cell wallwere based on those of Lederberg and St. Clair (13),and they include (i) osmotic fragility, and (ii) lossof rigidity resulting in spherical or amoeboid forms.The inability of the organism to grow on transfer tomedia with less than 0.1 M NaCl but growing in thesame medium with 0.3 M NaCl was considered proofof osmotic fragility. Our working definition of astable L form was taken from Landman and Halle(12) and refers to L forms induced by a mass conver-sion process, but they continued to multiply in theL stage for successive generations after the inducingagent had been withdrawn.
Physiological tests. Carbon assimilation tests weremade in GS medium with the carbon source to betested replacing glucose, and a biotin requirementwas shown by successively transferring 0.1 ml ofculture in GS medium from which the biotin wasomitted. The remainder of the tests utilized T-mediumand T-medium with 1.2% agar as the basal substrate.
Growth measurements. Three flasks of each growthmedium, 50 ml in a 300-ml Erlenmeyer flask, wereinoculated with 0.5 ml of a culture grown in thesame medium and of the following ages. TheT-medium inoculum was from a culture transferredat 2-day intervals; it was incubated in stationaryculture at 28 C; and inoculum for GS and YNB wasincubated for 3 days. Optical densities of L-formgrowth were determined at a wavelength setting of450 m,u in a Bausch & Lomb Spectronic-20 colorime-ter. Direct counts of large bodies were made in aPetroff-Hausser bacteria counting chamber; L-formnumbers were also estimated by most probablenumber (MPN) in T-medium with a three-tubemethod (3).
RESULTS AND DIscussIoN
Induction and stability of the L form. Readyinduction of the L form from B-3275 and thesimplicity of propagation allow one freely toinitiate L-form cultures. In a composite of two
tests, 14 of 22 colonies were induced to multiplyin a stable, spherical form, after eight successivetransfers in T-medium with 7% NaCl. Several ofthe L forms were cultured in T-medium for morethan 40 transfers without reversion. The ease ofinduction and propagation of Proteus L formshave been attributed to their comparatively toughmembranes (2), and a similar featuremay accountfor the stability of this B. subtilis strain in the L-form state. Unlike L forms described previously,agar support is not essential for induction andpropagation of this B. subtilis L form. It isapparent that a high salt concentration providesspecific inhibitory effects concerning cell-wallsynthesis and the physical condition for reproduc-tion of this osmotically fragile cell.
Evidence for the derivation of the L form fromB. subtilis B-3275. The L form has a high degree ofsimilarity to B. subtilis B-3275 in its physiologicalcharacters (Table 1). Eleven of the thirteen com-pounds tested as sole carbon sources resulted insimilar growth responses for the L form and thebacillus. Rhamnose and sodium citrate were notsatisfactory carbon sources for the L form, but thebacillus grew adequately upon repeated transferin the salts of GS medium with these carbonsources. The bacillus differed from the L form inits ability to hydrolyze gelatin. In addition, starchwas readily hydrolyzed by the bacillus, whereasonly a faint zone of hydrolysis was evidentbeneath the smear of L-form growth.A biotin requirement was discovered in the
bacillus, and the induced L form, likewise,required only this vitamin. Both bacterial formsare sensitive to aflatoxin. The significance of thistest is questionable, but only species of the genusBacillus (6) and members of the Actinomycetales(4) have been shown to be inhibited by 30 jig perml of this mycotoxin. When one considers thephysiological similarities of the two bacterialforms and the initiation of the L form from heat-shocked spores, the probability for the derivationof the spherical form from the bacillus isextremely good.
Growth and growth measurements. In stationarybroth cultures of the L form, an opaquenessdeveloped throughout the medium and a film ofcells collected on the bottom of the culture me-dium. When swirled, the growth could be observedas a mixture of white and transparent, viscidstrands. On a moist agar surface of T-medium,the L-form colonies grew to a diameter of 2 to 3mm in 4 days; the white viscid growth becamedull yellow in about 1 week. A drop of broth on acover slip allows it to be placed on the surface,with only slight disruption of the colony, to bemicroscopically examined. Large, pliable cellscould be observed at the edges of large colonies
(Fig. 1). Large bodies in rapidly growing cultureswere generally phase dark with a light centralarea in one orientation (Fig. 2); these featureswere also observed in lysozyme protoplasts. Asthe large body developed, the lighter area near the
cell center increased in size and smaller phasedark bodies became evident at the periphery (Fig.3). A typical log-phase culture containednumerous phase dark large bodies some joined byviscid connecting strands (Fig. 4). Growth rates
p~~ ~~~~~~P
SMO~~~~~~~~~~
FIG. 1. Periphery of a large L-form colony of Bacillus subtilis propagated on T-medium agar. This picture was
taken with an incandescent light source passing through a layer of T-medium agar and a plastic petri dish bottom.
FIG. 2. Phiase micrograph of a large body growing in T-medium for 48 hr at 28 C. The large bodies are mor-
phologically similar to protoplasts.FIG. 3. Large body ofmaximal size with disvtinct peripheral bodies. Phase-contrast micrograph.FIG. 4. Large bodiesfrom a 24-hr culture ofthe Lform growing in T-medium at 28 C. Phase-contrast micrograph.
Other testsBiotin ................... Required RequiredCatalase................. + +Acetylmethylcarbinol. . . . + +Growth in 7% NaCl..... + +Growth at pH 6.0........ + +Growth anaerobic .......
Growth at 55 C..........
Growth at 45 C.......... + +Casein hydrolysis........ + +Gelatin hydrolysis ....... +Starch hydrolysis ........ + +Nitrite from nitrate ......Acid from xylose........ + +Aflatoxin sensitivity, 30,Pg/ml.+ +
* Symbols: +, growth or positive test; -, nogrowth or negative reaction.
of this organism were compared in three media(Fig. 5). Most rapid reproduction occurred in theT-medium, a maximum was obtained within 30hr; growth in the synthetic media reached amaximum in about 3 days. The number of largebodies and the number of viable units in T-me-dium remained at a maximum for about 3 days.After the third day, there was a marked decreasein viable numbers and in the large body count.Thenumber of viable units as determined by MPNexceeded the large body count by almost 1,000-fold at each sampling period (Table 2); thisobservation suggests that the L-form cultures arecomposed of particles not detectable by lightmicroscopy or that the large bodies give rise tonumerous viable granules. The latter observationis consistent with that ofDienes (8), who observedthe development of viable granules inside largebodies of L forms derived from streptococci andand staphylococci.
This B. subtilis L form is unlike those derivedfrom most other bacteria in the following ways.
Time, hr.
FiG. 5. Comparative growth of Bacillus subtilisL form in a natural (T-medium) and two syntheticmedia (GS, YNB) in a stationary culture at 28 C.
TABLE 2. MPN, number oflarge bodies, and opticaldensity (OD) ofL form cultured in stationary
T-medium broth at 28 C
Time MPN Large bodies OD
days
0 9.3 X 105 0.021 9.3 X 107 1.8 X 106 0.262 9.3 X 107 1.3 X 106 0.253 9.3 X 107 1.0 X 105 0.244 2.3 X 106 3.3 X 104 0.215 2.7 X 104 4.0 X 104 0.23
(i) Agar or penicillin is not required for the transi-tion of bacilli to L-colony forming cells, and astable growth can be maintained in the absence ofcompounds known to inhibit cell-wall synthesis;and (ii) it can be propagated in synthetic mediaand grows readily in broth and on agar surfaces.
LITERATURE CiTED1. ABRAMS, R. 1955. A method for the cultivation of
L forms in liquid media. J. Bacteriol. 70:251.2. ALTENBERN, R. A., AND 0. E. LANDMAN. 1960.
Growth of L-forms of Proteus mirabilis inliquid media. J. Bacteriol. 79:510-518.
3. AMERICAN PUBLIc HEALTH AssOCIATION. 1960.Standard methods for the examination of waterand wastewater, 11th ed. American PublicHealth Association, Inc., New York.
4. ARAm, T., T. ITO, AND Y. KoYAMA. 1967. Anti-microbial activity of aflatoxins. J. Bacteriol.93:59-64.
5. BANVILLE, R. R. 1964. Factors affecting growthof Staphylococcus aureus L forms on semi-defined medium. J. Bacteriol. 87:1192-1197.
6. BuRMJErER, H. R., AND C. W. HFS5ELTINE. 1966.Survey of the sensitivity of microorganisms toaflatoxin. Appl. Microbiol. 14:403-404.
7. DmENE, L. 1939. "1" organisms of Klienebergerand Streptobacillus moniliformis. J. Infect.Diseases 65:24-42.
8. DIENES, L. 1967. Morphology and reproductiveprocesses of the L forms of bacteria. I. Strep-tococci and staphylococci. J. Bacteriol. 93:693-702.
9. DIENES, L., AND J. T. SHARP. 1956. The role ofhigh electrolyte concentration in the productionand growth of L forms of bacteria. J. Bacteriol.71:208-213.
KLIENEBERGER, E. 1935. The natural occurrence
of pleuropneumonia-like organisms in ap-parent symbiosis with Streptobacillus monili-formis and other bacteria. J. Pathol. Bacteriol.40:93-105.
11. LANDMAN, 0. E., AND H. S. GINOZA. 1961.Genetic nature of stable L forms of Salmonellaparatyphi. J. Bacteriol. 81:875-886.
12. LANDMAN, 0. E., AND S. HALLE. 1963. Enzy-mically and physically induced inheritancechanges in Bacillus subtilis. J. Mol. Biol.7:721-738.
13. LEDERBERG, J., AND J. ST. CLAIR. 1958. Proto-plasts and L-type growth of Escherichia coli.J. Bacteriol. 75:143-160.
14. MEDILL, M. A., AND D. J. O'KANE. 1954. Asynthetic medium for the L type colonies ofProteus. J. Bacteriol. 68:530-533.
15. RYTER, A., AND 0. E. LANDMAN. 1964. Electronmicroscope study of the relationship betweenmesosome loss and the stable L state (or proto-plast state) in Bacillus subtilis. J. Bacteriol.88:457-467.
