PROTOPLASTS ATu'D L-TYPIZ GROWTH OF ESCI~EKICH1il COLZ' JOSHUA LEDERBERG ASD JACQUELINE ST. CL$IR Department of Medical Genetics, School of Medicine and Department of Genctics, College of .Igriculture, University of Wisconsin, Madison, Wisconsin Received for publication August 5, 1957 A4 preceding article (Lcderberg, 1956a) was devoted to the evolution of protoplasts from grooving cells of Escherithia coli treated with penicillin. Further studies have strengthened the correspondence of these protoplasts with the "large bodies" and "L forms" described for many other bacteria. This paper mill give an account of thcsc studies. and an interpretation of others, in support of the hypothesis that L forms are out- growths of protoplasts. Their cell walls may be impaired either by extrinsic inhibition, for cx- ample, wit.h punicillin, or by intrinsic metabolic defects, consequences of genetic mutations. The previous study was motivated mainly bJ the hope of furnishing protoplasts of genetically defined strains of E. coli for physiological and genetic analysis (Spiegelman, 1956; Spooner and Stocker, 1956). It was modeled on the experi- ments of Weibull (1953a) who forestalled the lysis of Bacillus megaferium exposed to lysozyme by maintaining the bacteria in a protective, hypertonic medium. It was found that the lysis of growing E. coli in the presence of penicillin could be forestalled in a medium containing &r/3 sucrose and ~/lo0 Mg*; instead of lysing, t,he rods burgeoned out into osmotically fragile spheres, considered to be protoplasts. Similar effects of penicillin on growing bacteria have been described extensively from an alto- gethcr different viewpoint as an aspect of the development of L forms. For various reasons. species other than E. coli have been preferred for such studies. In general, t'he wall defects have been recognized but not stressed as the essential feat,ure of L forms, and their relationship to protoplasts obtained with lysozyme has been 1 Genetics paper no. 667. This work has been supported by research grants from the National Science Foundation, National Cancer Institute (C-215i !. Public Health Service, Bethesda, Md., Rockefeller Foundation, and the Research Com- mittee of the Graduate School with funds allo- cated by the Wisconsin Alumni Research Founda- tion. obliquely stated. The chief obstacle to a full co- ordination of protoplasts and the "large bodies" of the L form cycle has been the apparent in- viability of lysozyme-produced protoplasts studied by one group of workers in contrast to the continued growth of L forms reported by others. An adcquatc retrospect of the literature on L forms would be a herculean task. Fortunately, we may rely on a number of revien-s for background documentation (Dicnes and lveinbergcr, 1951; Liebermeister and Kellenbergcr, 1956; Kliene- berger-Sobel, 1954; Tulasne, 1951; Kandler and Kandler, 1954). Studies on protoplasts and re- lated aspects of bacterial morphology are in- cluded in a recent symposium (Spooncr and Stocker, 1956). As this paper is in part a restatement of pre- vious knowledge, semantic questions loom large. Protoplast is borrowrd from the botanical vo- cabulary where it serves to distinguish the living content of a plant cell from the lifeless cellulose wall. The walls of bacteria are not so well circum- scribed, either chemically or morphologically, at the present time, and less direct measures help to define the protoplast. For the present, our opera- tional criteria for the absence of a mall are (1) osmotic fragility and (2) loss of rigidity resulting in spherical or amoeboid form. To be sure, these criteria fail to distinguish between t'he total absence of a wall and its functional impairment. L form is a generic term which stems from the cultures labeled L1, LB, etc. (in honor of the Lister Institute) which Klieneberger had isolated from Streptobacillus moniliformis. These isolates were so bizarre in their morphology that their derivation from the bacteria could scarcely be credited, and Klieneberger felt instead that they were a symbiont. However, Dienes subsequently showed the direct, reversible conversion of bacillary into L type growths. Historically, L form refers to one of a specific series of strains. In this paper, however, "L" will bc used more broadly to describe atypical growths resembling 143 144 r,EI)lmlmltC ANI) ST. CIAIR [VOL. $5 figure 3, in contrast to "B" for typical bacil- liform. The time is perhaps nearly ripe for a notation that better reflects our concepts of these structures. It will be convenient to distingnish the scvcral ways in which protoplasts and L growth can he produced. Those achieved by t,he immediate presence of penicillin will be labeled with the prefix PC-; those obtained by withholding diaminopimelic acid (DAP) will be called dap-. "Stabilized" or "fixed" will refer to L forms which have an intrinsic heredkary defect, and therefore display this growth pattern on conventional media; they correspond to L forms in the strict sense advocated by Klienebergcr-Nobel. As this is an extensive rather than intensive report, we have appended much of the discussion t,o the findings as they arc prcscntcd. For the same reason, we will stress, perhaps unduly, the hypothetical status of many infrrenccs. Without question, many of t,hc attendant, details warrant thorough exploration as individnal problems in their own right. Media. The media used were cbvolvcd by trial and error, starting from the fornunlntions of pennassay broth and nutrient broth (Difco) The following medium containing 0.3 M sucrose, M/125 -\rg++, M/GO0 penicillin, plus a nutrient base, was found to be effective in the growth of L colonies of l?. coli. Sucrose broth (per L): cascin digest (Sheffield Chem. Div., Norwich, N. Y.) 10 g; sucrose, 100 g; meat extract (Lemco or Difco), 10 g; EaCl, 3.5 g; glucose, 1 g; ngar, if indicated, 10 g. lifter autoclaving, 10 ml of 20 per cent 11gS04.7H20 was added. If penicillin was indicated, w-e added 1000 LI per ml unless otherwise stated. "Bactwial hydrolyzate" was prepared as follows: 3 g of E. coli mere suspended in 30 ml 2 N sulfuric acid, and nutoclnved in a screwsp vial for 35 min; WC then centrifuged, discarded the sediment, neutralized the supernntant with 10 N NnOH, and sterilized this by reautoclaving. Whcnevcr indicated, t,hc hydrolyznte was used at 1 :lO dilut,ion, giving a final concentration (in terms of the original cells) of about 10 mg per ml. The hydrolyzatc gave an assay wit,h a DAP ausotroph (see below) corresponding to 1 per cent of the dry weight of the original cells as DAP. Since possible stimulation by other factors has not been studied, the assay (annnot be con- sidered quantitatively reliable in absolute terms. However, as IO Kg of J)-iP will permit t,he growth of about 1 mg of bacteria, the correspondence is Pigwe 1. Eschericl~irc coli \r--IO in penicillin sucrose broth. (Abow) Various cells in snccesGve st(ages. 0 to 4 hr. (NrZr,tr!) T,:L((: sl:t#c :L(, Ili&r rn:r~nificat ion. l'h:wc contrnst,. 19581 PROTOPLASTS AND L-TYPE GROWTH OF E. COLI Figure 2. B and L colonies. (Lqft) B gradient, plate of sucrose sgar containing 0 to 700 units per ml of penicillin was seeded with Escherichia coli Y-10 and incubated 2 days. Note zones of B growth (above) and L growth (below) with intermediate zone of no growth. This print was obtained by placing the petri dish directly in an enlarger. (Right) B and L colonies at #i hr, about 10X ; dis- secting microscope, obliqne illumination. Figure 5. (Left) Toung (20 hr) L colony, abc 0.1 mm in diameter. Originally photographed 100X phase contrast. (Ri@t) Squash of L color phase contrast. but at, lY, reasonable. The amount of hydrolyzate used as a supplement is a calculated teufold excess. After preliminary trials with other strains, most of the work reported here involved E. coli strain K-12 and a number of mutant substrains, such as W-G, Y-10, W-1895, and others, for the most part cited in earlier papers (Lederberg et al., 1952). Strain K-12 and its derivatives will be considered collectively as E. coli line 1. Stock cultures were maintained on nutrient agar slants, or in stabs. Routine inocula were grown overnight in penas- say broth and cultures aerated by holding the tubes on a rotator. All cultures were incubated at 37 c. EXPERIMENTS AFiD CONCLUSIONS Formation of protoplmts in penicillin sucrose broth. In the absence of sucrose or other stabilizer, cells of E. coli lysed after an initial swelling, However, as previously described, E. coli cells grown in sucrose-penicillin broth are converted one for one into protoplasts via the stages of figure 1. These protoplasts also lysed rapidly in 146 LEDERBERG AND ST. CLAIR [VOL. 75 water, and mom slowly in broth. In similar ex- periments, hf/5 glucose and M/2000 clinical dextran (kindly furnished by Baster Labora- tories, Inc., Morton Grove, Illinois) had incom- plete protective effects comparable to ~/lo sucrose. Likewise, Weibull (1953a, b) found that ~/50 carbowax protected the protoplasts of Bacillus megaterium. These findings suggest a more complex function for these solutes than sim- ple osmolality, but the disruption by dilution in water will be referred to, for the present, as osmotic fragility. Mg++, replaceable by Ca", is essential for stabilization, perhaps by virtue of a reaction with lipid residues of the plasma membrane (Weibull, 1956). Citrate and Versene bind these cations and thus accentuate the requirement; the presence of citrate in some formulations of penicillin, and in the composition of some mini- mal media, should be kept in mind. The paradox that Verscne is an essential part of one recipe for releasing protoplasts from E. coli (Repaske, 1956) has yet to be resolved. It is well known that lysis by penicillin reaquires active growth of the susceptible bacteria, a principle that has a useful application in methods for the selective isolation of growth factor- dependent mutants (Davis, 1948; Lcdcrberg and Zindcr, 1948). Correspondingly, the cvo- lution of protoplasts also requires growth, and will not occur in non-nutrient media, at low tem- peratures, or in the presence of inhibitory con- centrations of streptomycin, chlortetrncycline or chlornmphenicol. For this reason penicillin is belie\-ed to act by inhibiting new wall synthesis, in contrast to the dissolution of the existing wall by lysozymc. The changes of figure 1 arc there- fore interpreted as the protrusion of an expanding protoplast against a progressively attenuated wall which finally collapses and releases the free protoplast. Owing to intercurrent growth, the penicillin protoplasts are perforce larger than those released by lysozyme, and even more so than the several protoplasts rclcased from a single multiseptate rod of a Bacillus spp. Some fifty and odd penicillin-sensitive strains of E. coli, of various serotypcs, have been treated in penicillin sucrose brot~li with similar results. Protoplast formation has also been secured in a defined medium (Gray and Tat'um, 1944) sup- plemented with sucrose, >Ig*, and penicillin, but was slower and incomplete, presumably be- cause of the lower rate of protoplasmic increase in this medium as compared to broth. The continued growt,h of protoplasts suspended in penicillin sucrose broth is reflected by increases in optical density and induced b-n-galactosidase as well as in the size of the individual protoplast,s. The biosynthetic activity of these protoplasts is being more intensively studied in other lnbora- tories (Spiegelman, 1957) where t,hey have been found to be on a par with the intact bacteria. However, the increase in total mass is not matched by an increase in numbers, nor can any convincing division figures be seen under 6he microscope. On continued incubation? the proto- plasts become very large and highly vacuolate, and they eventually lyse. No better evidence of proliferation was obtained with variations of sucrose broth medium, or by the addition of bovine or equine serum or of other proteins. Freshly prepared protoplasts give a viable count, consisting of B colonies exclusively, amounting to 10 to 50 per cent of the input cells if diluted ,in sucrose broth (without penicillin) and plated in sucrose ngar. This corresponds to the reversion of protoplasts to normal rods which takes place over the course of several hours in sucrose broth as has already been described (Lederbcrg, 1956a). The spheres develop pro- tuberances, one or more of which clongatc into filaments and segment terminally to give typical rods. These changes are taken to represent the resumption of wall-building when the inhibitor is removed. If the protoplasts are diluted in water instead of a protect,ive medium, the viable count drops by a thousandfold or more. The residual viability may be accounted for by dormant "persisters" (Bigger, 1944). Resistant mutants might also be expected among the survivors, but were not found. Likewise, the B reversions from protoplasts maintained in sucrose broth and plated on sucrose agar have behaved like their parent B cultures in their formation of osmotically fragile protoplasts in response to penicillin. Mutants of E. coli line 1 resistant to 1000 u per ml of penicillin were never found in a single step in other experiments involving intense selection, and they are evidently much rarer than the phenotypically deviant persisters. Many features of protoplast st,ructurc are still obscure. .t the protoplasts enlarge, a lune-shaped vacuole appears at one sitlc (figure 1). Older protoplnsts often disnln~- :t narrow crescent of 19581 I'ROTOPLASTS AND L-TYPE GROWTH OF E. COLI 147 phase-dense material to the side of a large, nearly spherical, clear vacuole. The vacuole is bounded by a thin membrane, which presumably invests the entire protoplast, and may correspond to the wrinkled ghost which is seen after lysis. Nothing is known of the contents of this seeming vacuole. India ink preparations show an additional envelope, a transparent capsule, even with strains (like E. coli line 1) which show no capsule in the B form. Since protoplasts of various serotypes are still agglutinable by homologous anti-0 and anti-K serums, the capsule may well represent some disorganized elements of the cell wall. Further immunochemical studies may help to settle this point. When protoplasts are lysed in water, they appear to have dissolved altogether except for the residual ghost and some granular debris. In India ink, however, the lysed proto- plast appears as an enlarged clear space in which the ghost is embedded. Protoplasts are nonmotile, even when prepared from actively motile bacteria. However, they are still extensively flagellated when stained by Leifson's method (1951). This experience cor- responds precisely to Weibull's (1953, a, b). 4s yet, we cannot say whether this paralysis repre- sents a lesion of the flagella or reflects a role of the rigid wall in motility. Preparations stained with Giemsa after HCl hy- drolysis have exhibited a scattering of peripheral nuclear bodies as other workers have described for "large bodies" and L forms. Attempts to release discrete nuclei by controlled lysis with water or with lipase (Spiegclman, 1956) wcrc unsuccessful. Growth of L colonies of E. coli in penicillin sucrose agar. Many authors have stressed the im- portance of the physical texture of the medium in supporting the growth of L forms. In preliminary trials, the recipes given by Diencs (1949) mere effective for Proteus strain 52 (kindly furnished by him). Good yields wcrc also obtained in nu- trient ngar (Difco) with sucrose plus penicillin. It was necessary, as recommended, to limit the agar concent,ration to not more than 0.8 per cent. But comparable experiments with E. coli line 1 were much less successful at first. A long series of trial and error experiments finally uncovered the fol- lowing prescriptions. (1) Shrain specificity:-Different lines and substrains within E. coli line 1 vary in their yield of I, colonies, some cultures being completely un- productive in every medium tried. E. coli strain K-12 was only moderately productive, and was not readily maintained in serial passage of L colonies. Productivity for L colonies was uncor- related with any other recognized genetic marker, isolated clones from the same stock culture sometimes showing wide variations, either in over-all yield, or in the conditions for optimal yield. Strain Y-10 of line 1 was one of the most productive, and has been used routinely in further experiments. It should be stressed that all strains produced protoplasts in penicillin sucrose broth, regardless of whether they went on to L type growth in agar. (2) Agar concentration:-L'nlikc Proteus 52, E. coli required at least 0.8 per cent agar, the threshold varying with the gelling power of the lot. The most uniform results were obtained with 1 per cent Difco agar, which has been routinely adopted. Neither gelatin nor methocel replaced agar as a satisfactory gelling agent. (3) Submerged versus surface growth-We have not succeeded in securing L colonies of E. coli on agar surfaces; all observations in this paper are from pour platings. Agar shake cultures show no special aerobic requirements for L colonies; the largest colonies usually developed in a zone beginning just beneath the surface. (4) Meat extract:-Stimulated the L growth of sonic strains. Yeast extract {\-as, if anything, inhibitory. (5) -1Ig*:-Required just as for the stabi- lization of protoplasts in broth. (6) Sucrose:-Indispensable for L colony for- mation in nutrient agar, but a small number of L colonies of E. coli developed in casein digest-meat extract agar without sucrose. So far as &ted, thcsc did not consist of mutants that would tolerate penicillin in the absence of sucrose. \Vith Proteus, however, sucrose was dispensable in agar media (but improved the yield of protoplasts in broth). (7) pH:-The range from pEI 5.5 to 7.6 was im- posed by the addition of ~/lo phosphate buffer. The optimum was found at about pH 6.3 which approximates the pH of the unbuffered medium. Phosphat,e buffer has therefore been omitted from the recipe to minimize precipibation of mngne- sium phosphates. (8) Growth factors:-In a defined medium (Gray and Tatum, 1944) supplemented with sucrose, penicillin and AIg*, L growth was 148 LEDERBERG AXD ST. CLAIR [VOL. 75 perceptible but very sparse, each colony consist- ing of perhaps a hundred elements after 48 hr. This growth was accentuated by the addition of amino acid mixtures, but no single supplement was uniquely effective. A mixture of B vitamins, including riboflavin, did not stimulate L growth, contrary to the report of Tulasne et al. (1955) for Proteus. (9) Penicillin:-One of the most critical factors for L growth (but not for protoplast formation) was found to be the concentration of penicillin. Although 100 u per ml suffices for the latter, most strains require 1000 u for high yields of L colonies, and may be stimulated even further by 10,000 u. A gradient plate thus shows three zones: B colo- nies at penicillin levels below about 50 u, a zone with virtually no growth, and L colonies at levels over 200 u (figure 2). The zone effect will be of some importance in later discussion. Taking these considerations together, we have adopted the following regime, which gives yields of 10 to 50 per cent of the input cells as L colonies. Strain Y-10 is grown overnight in pennassay broth. The culture is diluted in broth, then mixed with molten penicillin sucrose agar for pour plates about 5 mm deep. (Small petri dishes, 6 cm in diameter, are convenient for many opera- tions.) The plates are allowed to solidify and are incubated. L colonies grow more slowly than B, but can usually be counted after 24 hr, and may reach 1 to 2 mm diameter by 48 hr (figure 2). When viewed under a binocular microscope they can usually be distinguished by their translucent texture; they are also less compact than lens- shaped B colonies, and may show several leaf- like outgrowths. A plate is conveniently surveyed under darkficld illumination at 100 magnifi- cations, at which the individual protoplasts can be made out; for certainty, the colony should be squashed out and examined under phase con- trast at higher power. The unmistakable ap- pearance of such squashes is shown in figure 3. The evolution of L colonies from single bacteria has been followed in agar block preparations. A drop of seeded agar was spread into a thin film on a cover glass, then covered with mineral oil to prevent drying out. With a razor blade, slices were made at right angles to give rectangular blocks with a diameter of about 0.2 mm. Indi- vidual blocks were then spaced out in a regular pattern under the mineral oil. The cover glass was then inverted over an oil chamber. Since the indi- vidual blocks are readily centered under high power lenses, and can be re-located in the pattern, this furnished an efficient method of following several specimens in parallel for serial pho- tography. The formation of pc-protoplasts and their re- version to rods has been followed conveniently in microdroplets of broth preparations (De Fon- brune, 1949; Lederberg, 1954); but L growth did not develop in the liquid medium. Attempts to prepare microdroplets of agar were unsatisfac- tory, owing to the rapid congealing of the agar in the transfer pipette. The development of L colonies from single rods is shown in figure 4. The initial stages are similar to the evolution of protoplasts in broth; however, the cell does not become perfectly spherical. Instead of generating a growing sphere, it forms a number of protuberances, some quite blunt, others very thin and difficult to photo- graph. These enlarge, and after a time, pinch off to give a "daughter protoplast." It has not been possible to follow the continuation of this cycle for very long by means of photomicrographs as the three dimensional aggregate soon becomes too confusing. While L colonies in situ contain more or less irregular elements, presumably due to con- straint by the agar milieu, in squashes these are more uniformly spherical. No L colonies have been found at the surface of the agar, either after surface inoculation or from pour platings. However, an L colony which starts beneath at a lower plane may break through the surface, resulting in a viscous drop or `Lcolony" containing mostly debris and large ghosts. The appearance suggests that the L colony is under considerable pressure, and that its contents may swell, burst and spill out when it reaches a free surface. The subculture of L colonies, especially for quantitative counts, therefore presents some technical difficulties familiar to other practition- ers. It is expedient to cut out a block of agar, transfer it to a small shell vial with one or two volumes of sucrose broth, and to macerate t'he block with a short run of a VirTis Micro-Homog- enizer. The mince can then be taken up in a pipette for dilution and plating. This treatment disperses most of the protoplasts into free sus- pension; some masses remain embedded in agar lumps. The wide range of size of granules and spheres, 19581 PROTOPLASTS AND L-TYPE GROWTH OF E. COLl 149 150 LISDI!XBERG AND ST. CLAIR [\-OL. ij and the prescrlce of agar fragnlt:nt,b int,crfcre with a total count. Single L colonies containing an estimated 10" t.11 10" spherical elements yielded from 10 to 50 L progeny when minced and re- plntccl. This low viability might be inherent in the clcmc~nts of t,hc, colony, or might be ascribrd t,o damage by rc>leaae of ext,ernal pressure or t,o injury from thca maceration. Several strains of E. coli have been subcultured by this m&hod in the L phase for as many as 20 passages bcfort: the experiments were t'erminatcd. The dilution at, I::wI~ passage was about one tenth, so that, th(, cumulative factor of incrcnsc was Io'n, Icaving littlc doubt that the L colonies could have: bc,en propagatccl indefinitely in the prcscnce of pc'nicillin. Samples were also plated into sucrose agar, where they reverted to give approximately the same yield of IZ colonies as of L colonies in penicillin sucrose ngnr. These reversion cultures were indistinguishable from t.he original Y-10 in their growth habit, their scansitivity to penicillin, and their produr*tirity of pc-protoplasts and I, colonirs. Contrary to experience with other bacteria, the prolonged cultivation of E. coli in the L phase in the pr~~s~`n(:(' c\S pcbnicillin gave no stabiliecd L forms. -1 nuniber oj strains, c. g., K-12 and M-6, gave rclativcly low yic>lcls (10m3 or less) of L colonies. Rcvcrsion culturc~ were made of the obtainable 1, growth in t,hr cxpcctation of accumulating mutant.s better adnptcd to form L colonies in thr adopted medium. This expectation was not fu- filled; the L colonies that do develop in t,hrsc circumst,ancc:P nmqt be put down to phenotypic accidents. Like pc-protoplasts, the L growth is osmoticall>- fragile. Minces diluted in mater gave less than 1 per cent of the viable count (B in sucrose agar, L in penicillin sucrose agar) shown by nliquots diluted in sucrose broth. Part of the residual survivorship IIXI~ bc ascribed to prot&cd cle- mcnt,s rmbcdded in bits of agar. These observations may be rationalized into the following conception of L growth as colonies of protoplasts. In broth, the uniformity of extcarnal pressure, the elasticity of the remaining envclopc, and interfacial tc,nsion all tend to conscrv(t the splrcrical shape r~f the protoplast,. Without t,hc norm:11 division mc!chanism, which depends on the wall, the protoplast remains spherical as it grows. Tn R~RT the shnpr of the protoplnst. is im- posed by local stresses in the medium, the net- work of agar fibrils taking the place of the missing rigid mall. At local points of weakness the gro\\-ing protoplast herniates into an adjacent free space, expands, and pinches off a bleb. Many of the blebs may be expcctccl to lack a full complement of vital internal structures, which might account, in part for the low viability. Diaminopimelic acid (DAP). The L growth so far discussed is an effect of external inhibition of wall formation. Vhcn the cells mere placed in a sustaining medium without penicillin, the)- re- covered their normal capucitics and regenerat,ed typical walls. Howcvcr, many workers have re- ported on the occurrence of genetically stabilized L forms which remained defective in the same enviromncnt that sustained normal growth of the parental strains. Two questions concerning these mutants arise: (1) the biochemistry of the defect, and (2) the manner in which the L form arose and survived in place of the parental B form. DAP is an amino acid originall:; discovcrcd in hydrolyzates of Corynebacteriwn diphtheriae and subsequently identified as a component of the cell malls of numerous bacteria (Kork, 1957; Cummins, 1956). Apart from the differential occurrence of the WZC.SO- and t,hc LL-stereoisomers, DAP has been found in the walls of all bacteria examined except for gram-positive cocci and lactobacilli, and including actinomycctcs and myxophyceae, but in no other biological source (except Chlorella). The prcfercntial localization of D:\P in the cell malls of the bacterial species in which it occurs suggests that it has no other struc- tural significance, and t.hc small amounts found in nonwall fractions may represent its role as a metabolic intermediate in wall formation and in the biosynthesis of lysine (Davis, 1952). Davis has isolated an auxotrophic mutant of I?. coli strain IV that required DAP and lysine. Subsequently, Baunmn and Davis (1957) and Meadow et aZ. (195i) observed that cultures grown on limited D;IP undcrwcnt lysis when t,hc DAP supply was exhausted. (Kc arc indcbted to Dr. Davis for initial samples of DAP, for a cult,ure of his DdP-dcpcndrnt mutant, strain 173-25, and for preliminary information on the osmotic fragility of D,U-st,arved cells.) D,ZP is now pro- duced on a commercial scale as a metalrolite accumulated by another mutant (Casidn, 1956) and a generous sample has kindly been furnished by Chne. Pfizer and Co. 19581 PROTOE'L\dTS ANI, T,-TYPE GROWTH OF' E. 1101,l 1.51 Strain 173-25 was grown in broth supplemented with bacterial hydrolyzate or with 10 pg per ml of DXP. The culture was then washed and inocu- lated into sucrose broth! which lacks DAP (having no constituents of bacterial origin). In contrast to the lysis observed in ordinary broth, these cells formed protoplasts by stages similar to figure 1. The same conversion was noted in minimal medium, supplemented with sucrose, w+-, and lysinc, but n-as less complete. As a corollary, the sucrose medium gave a higher turbidity and more &able assay curve for D?rP than the cust.omnry minimal medium wit#hout sucrose. Strain 173-E n-as also plated into sucrose agar, where it grew extensively and exclusively in the form of L colonies similar to, though somcwhat smaller than, t,hose shown in figure 3. Except for occasional (sometimes troublesome) reversc- mutants. no colonies at all were formed in the absence of sucrose. The dap-L colonies grew on serial passage in sucrose sgar in the same fashion as already described for pc-L forms. When DAP was restored, the passage strains promptly rcsumcd B growth. Since D,1P is nb- sent from conventional bacteriological media, the D-U-dependent mut,nnt would have been described as a fixed L form, had it, been isolated prior to these studies. D.\P is not the only tnrgct n-hose impairment would block wall formation. Scithcr DAP nor hydrolyzste was found to reverse inhibition by penicillin, which may thcrcfore antagonize some metabolite missing from the hgdrolyzate, or interfere directly with a wall-building enzyme. Further, several stable L forms of Proteus were received from Dienes (strain Tulasne) and from Kandler (strains 6e, 6f, 5h, and 8~). The strains had been adapted to growth in liquid medium, and they grew quite well as L forms in the casein digest-meat extract agar without sucrose or serum. They showed no response to hydrolyzate or D.\P, and the site of their block, like that of penicillin, remains unspecified. In addition, there was no effect when boiled Prot'eus cells were also added t,o furnish a possible primer of wall formation. Selection of udl-defect mutanfs. The process of stabilization of L grow>-ths in the presence of penicillin has not been syst,cmatically studied, at Icast not, to allow t,hc discrimination of various genrt,ic: hypot,hcscs. :1t 1rw.t four cnmn to mind: (1) that wall-synthwis is subject to apohatwous mutations which may block any of a number of steps and that such mutants hare a selwtive ad- vantage in the circumstances where the>- h:tvc been isolated; (2) that wall formation dtppnds on a self-reproducing cgtoplasmic particle whose: w- production is impaired by penicillin (Sharp et nl., 1957), by analogy with the effects of acriflavinn on yeast (Ephrussi, 1953); (3) that n-all-synthc- sis is itself a self-dependent process, with estrn- sion of the wall depending on t)he integrity of tlw pre-existing structure by analogy with the rolr of polysaccharides as primers for their own axn- thesis; (4) that penicillin has a specific mutagenic effect, so that t,hr same compound produces both phenotypic: and genotypic impairments of wall formation (Briggs et al., 1957). These hypotheses arr not mutally tlxclusivc; howw~ in the present mat~crial the last thrw are dis- couraged by the long-co&nucd propagation of E. coli line 1 as PC-L growth without bwoming a stabilized L form. Hypothesis (1) has bwn par- tially justifird b>- the behavior of the DAI' auxotroph, strain 173-25, which is an example of a wall-defect mutant. However, thv circum- stances of our cxperinients with penicillin cvi- dcntly did not favor selective overgrowth of wall-less mutants, contrary to the caxperiencc of other workers. Tinder what conditions, then, would a wall-less mutant have an advantage, as would be necessary to completr t,lre justifica,tion of the hypothesis (1) ? This question may have several answcra; at least one clue comrs from the zone effects of penicillin mentioned earlier (figure 2). In the presence of "zonal" levels, i. e., 75 to 125 u per ml, L growth of DAP-independent E. COG is quite sparse. On the other hand, in reconstruc- tion experiments, the DAP-dependent strain 173-25 formed L colonies abundantly, regardless of penicillin concentration. But when DAI' was added, L colony formation again failed in the intermediate zone. The component of pharma- ceutical penicillin that is required at higher con- centrations is believed to be the antibiotic itself, since the zone cfftct was also given by a highly purified sample (kindly furnished by Dr. .J. Lein, Bristol Laboratorics, Inc.) and was unaltered bJ the addition of an Excess of heat-inactivated penicillin. We proposc that incomplete inhibi tion accounts in some manner for the zone of poor T, growth, n. ronrlition that can hc relieved either by the addition of mow penicillin 01' by the superimposition of a genetic block. If this reasoning is correct, we should tend t'o find wall- defect mutants among those few L colonies which do form in the intermediate zone of peni- cillin. A number of experimental designs to test this expectation have been tried, none extensively enough to be empirically preferred. The follow ing procedure takes account of some general principles for the isolation of auxotrophic mu- tants which have been detailed elsewhere (Leder- berg, 1950). Cells of Y-10 were harvested from penassay broth and resuspended in mater, then irradiated n,ith ultraviolet to a survivorship of 10-S to 1W2. The irradiated cells were then plated densely into penicillin sucrose agar and incubated for 24 hr. (This corresponds to an interval of intermediate cultivation during which the mutants can come to phenotypic expression in a neutral or perhaps advantageous environ- ment.) A block of agar containing numerous L colonies was then minced and plated at various dilutions into sucrose agar containing zonal penicillin. The yield of L colonies w-as 10-3 or less than that obtained in t'he presence of optimal penicillin. These plates were incubated for 1 week to 10 days, during which t,ime much of the penicillin has been inactivated, in part by the constitutive intracellular penicillinase, which is characteristic of E. coli. As a result,, many non- mutant colonies revert to R form, and if recog- nized need not be picked in further tests. Sus- picious colonies (figure 3) were replated in sucrose agar. If L colonies appeared on replat- ings, they were purified by one or two additional platings, and then tested for growth in hydroly- eate broth and DAP broth. To date, 197 colonies have been replated from sucrose agar with zonal penicillin to sucrose agar, and 4 have proved to be L forms stable enough for further characteri- zation. All these have proven to be DAP auxo- trophs, but a search for other classes of mutants is being extended with this and variant methods. Unless the compounds indicated have other functions important in over-all growth, mutants requiring muramic acid, hexosamines, n-amino acids and their conjugates with uridine-diphos- phoglucose, and other metabolites are among those to be anticipated (Park and Strominger, 1957; Work, 1957). The DAP auxotrophs thus isolated from line 1 have resembled strain 173-25. One isolate may reprcwnt an incomplete block, as it grows substantially normally in broth and minimal liquid medium. However, it forms mainly protoplasts and intermediat,e forms in sucrose broth and L colonies in sucrose agar. &is has already been suggested, hypertonic sucrose may play an active role in t'hc hrrniation of the protoplasts, as well as in helping to maintain them (Zinder and Arndt, 1956). The addition of DAP restored normal morphology in broth or agar, with or without sucrose. The other strains required DAP for growth in media without su- crose. Selection by phage and colic&e. Since the cell walls play an important part in the adsorption and penetration of phages and colicines, these might also serve as selective agents for the isola- tion of wall-defective mutants. PC-L colonies of E. coli line 1 were found to be relatively resistant to phages Tl, T4, T5, and X-2, but still formed plaques when seeded with T3 and T7, and faint plaques with T6, which speaks for differences among the receptors for the various phages. A number of colicines including Fredericq's (1948) type series was also tested for differential effect; most of them inhibited L growth to some degree, though usually less markedly than the corre- sponding B growth. Phages Tl and T5, and colicines E, K, and V have been tested further as aids in the selection of new mutants, analogous to the use of zonal penicillin. This approach has not bwn equally fruitful so far, although recon- struction experiments speak for its validity. The main difficulty has been interference background of transient L colonies which probably result from the wall-dissolving action of the phage itself (Zinder and Arndt, 1956). (The delayed lysis with concurrent L growth of phage-infected cells in sucrose agar warrants study in its own right.) None of the colicincs tried has been com- pletely innocuous to L colonies, and they, therc- fort, do not give perfect discrimination under the conditions used. 1 The only relevant mutant isolated with the r help of phage was resistant to phage Tl, grew nearly normally in broth and in minimal medium, but produced an abundance of protoplasts in sucrose broth. Unlike a partial DAP-auxotroph mentioned previously, this mutant (W3288) was unaffect,ed in its growth pattern and its resistance to Tl by bacterial hydrolyzate. Various chemicals have been used for the 19581 PROTOPLASTS AND L-TYPE GROWTH OF E. COLI 153 selective isolation of plcuropneumonialike organ- isms (Morton and Lecce, 1953); Tulasne and Lavillaureix (1954) further state that thallium acetate differentially inhibits B versus L growth. In exploratory trials, we could find no differential resistance of L growth to graded concentrations of thallium acetate, sodium selenite, sodium tellurite, uranium nitrate, or crystal violet. These compounds were added in penicillin sucrose agar seeded with cells or protoplasts of E. coli Y-10. The scarcity of L form mutants procured in E. coli contrasts with the reported regularity of their occurrence in other material treated with penicillin (e. g., Sharp et al., 1957). We are un- able to judge the relative importance of differ- ences in technical details and in material, or whether other mechanisms (hypotheses (2)) (3)) and (4)) may be involved. However, wall-defect mutants may have a selective advantage in other environments besides zonal penicillin, phage, or colicine or in a genotype less well adapted to PC-L growth than that of Y-10. Further, most workers have used serum in their recipes, and low-titer antibodies against residual wall components might then play some part in the selection of spontaneous mutations. Even before a rationale for selection is perfected, its intensity can be estimated by means of recon- struction experiments. Other observations. The unique role of DAP suggested that antagonistic analogues of this amino acid might have a therapeutic usefulness comparable to that of penicillin. A few com- pounds were tested briefly, against strain K-12 and against strain 173-25 with limiting DAP, but none was inhibitory. They included: n-glu- tamic acid; nn-ol-aminopimelic acid, nn-a-amino- butyric acid (purchased from California Bio- chemical Foundation) and a sulfonic analogue of DAP, 1 , 5diamino-l , 5pentanedisulfonic acid (kindly furnished by Dr. J. Lein, Bristol Labora- tories, Syracuse, N. Y.). The principal incentive for the study of pc- protoplasts in this laboratory was the hope that they might prove to be competent recipients for the transduction of genetically active deoxy- ribonucleic acid (DXA). This hope has so far not been realized in experiments with E. coli. The markers used in these experiments were Lac, M and S, for example M-S' X MfS", with selec- tion for M+S' by platings on minimal medium supplemented with streptomycin. As a rule, the treated recipient protoplasts were allowed to revert to B forms before being plated, the peni- cillin being removed either by the addition of penicillinase, or by washing the suspension with fresh broth. Among the variables systematically tested were: DNA as crude lysates of donor protoplasts, or partially purified after detergent extraction of whole cells; substitution of Ca++ for Mg++; addition of serum albumin; partial osmotic shock in the presence of DSA by dilu- tion of protoplasts in water followed by reconsti- tution with concentrated sucrose. In addition, a large variety of strains was employed for recipi- ents, including some 125 0-serotypes (kindly furnished by Dr. F. jarskov). In some trials the recipients were treated as L colonies in penicillin agar. Attempts to detect the fusion of protoplasts of sexually incompatible (F-) genotypes were equally unrewarding. The design of the experi- ments was similar to that for DKA-transduction, mixtures of protoplasts being evoked and grown together in penicillin agar or broth. We also tried graded osmotic shocks, and spinning a mixed protoplast suspension in 10 per cent sucrose in an air turbine centrifuge at 80,000 X G for 20 min. Whereas the pellet showed evidence of considerable lysis, there was no indication of fusion of protoplasts either from microscopy or tests for recombinants. However, compatible strains (Ft x F-) could recombine if either or both of the parents was introduced as pc-proto- plasts. For example, protoplasts of HfrlMW were mixed with protoplasts of FLaclSr in penicillin sucrose broth, and incubated for 1 hr. They were then agitated to break up residual complexes, di- luted, and plated on EMB lactose streptomycin agar, supplemented with sucrose and Mg++. On this medium, the Hfr parent is suppressed, and La&S recombinants are distinguishable from the F-La&r parent. In protoplast X protoplast tests, the ratio of recombinants to F- parent var- ied from less than 1 to 5 per cent, compared to 5 per cent for the rod x rod controls. In view of the effect of streptomycin even on S' pc-protoplasts (see below) this experimental design is not ideal, and further studies should be based on fuller knowledge of the reversion of protoplasts on various selective media. The mating of HfriW?? protoplasts x E"LacS' rods was consistently 154 LEDERRERG ANU ST. CI,AIL{ fertile (perhaps because this difficulty is not in question)? and a conjugal pair of these was readily observed as a rod with attached proto- p1ast. Hfr and F genotypes retained their character- istic compatibility behavior when reversions were tested after several passages as pc-L colonies. The wall defrct imposed by penicillin thus has no influence on either the genetic continuity or Figure 5. Esckerichiu coli streptomycin-de- phenotypic manifestation of this presumably pendent mlltant; fil:tmentous colony in sucrose surface-related property. agw mit,hout streptomycin. l'hxse contrwt. E. coli 204 is a pleiomorphic strain, briefly described by Klieneberger-Nobel (1949). The replating, the viability of these colonies was very division mechanism of this strain seems to be low; the sucrose medium, therefore, could not intermediate between fission and budding, the compensate for the lesion imposed by lack of units in a growing suspension consisting of de- streptomycin. formed spheres with large protuberances. The Kliencbergcr-Nobel (1954) has listed some culture is resistant to penicillin; hydrolyzate had artifacts in st,crilc media containing lipoid no effect on its morphology. Its mall defect does material which can bc mistaken for the globules not, however? cstend to osmotic fragility, dilu- of L growth. An cqunlly rich source of artcfacts tion from sucrose broth into water having had is nutrient gelatin ngar as used for the selection no effect on viability. 30 sign of the frsgmcnta- of motile bacteria. This mc&m~ tends to con- tion mentioned by Klieneberger-Nobel was tain coacervates which give the appearance of observed in living broth cultures seen under germinating cysts. phase contrast, although Inany cells assumed irregular shapes a.nd comprised an unusual range GENERAL DISCUSSION AIiSD RECAPITUL~4TTION of sizes. It was undecided whether the discrep- Our data support the conception that L growth ancy is dur t,o diffcrcnces of observational OI is a result of a defect in w-311 synthesis. This cultural tJechniquc or to changes that may have defect may be imposed by external inhibition, occurred in t,he behavior of the strain. as by penicillin, or by an internal genetic block. In the course of other experiments, it was found In some cases, the block has been defined bio- that S' (skreptomycin-resistant) mutants of E. chemically, for csamplc, as nnsotrophg for coli lint 1, \vhicli grow wll in 1 mg per ml of dinminopimclic acid. In otlwrs (Proteus 1, forms) streptomycin, wereinhibited by20 pg per ml when it has not been rtparable even by hydrolyzates grown as PC-L forms. This can be viewed either of the wild type bacteria. The irreparable mu- as the negation of the S' effect when the wall is tants might be auxotrophs for metabolit,cs lack- stripped, or a mutual reinforcement of penicillin ing in the hydrolyznte as prepared, or might and streptomycin in respect to another t,arget. represent losses of the w-all-building enzymes. (Jawetz et al., 1954; Linz and Lecocq, 1956). Another hypothesis, t,hat wall-building is self- The former interpretation is favored by the sensi- priming, also warrants further consideration: tivity to st'rcptomycin of dap-L colonies of an the problem may by how to rc-establish the S' strain. primer at a suitable site. dnothcr hint of the relationship of mutation L growth is an nsprct of "unb:danccd growth" at the S locus to alterations of the ccl1 n~~ll was (Cohen and Baruer, 1954) in which one ccl! offered by the reported tendency of strcptomycin- constituent, in this cnsc the cell wall, is selec- dependent mutants to grow as long filaments tively inhibited. The prolonged subculture of and occasional pleiomorphic colonies \\-hen de- pc- and dap-L forms furnished csccllent evidence privcd of streptomycin (Simon, 1955). An $1 that penicillin and diaminopimelic acid play no mutant of E. coli lint 1 was plsted into sucrose essential part in the viability of the bacteria ngar nnd found to give a high yield of colonies other than their influcncc on t'he cell wall, dis- consisting of very long filaments with occasional regarding the role of DAIP as a precursor of sncllin~s (fiQm.c~ 5), mthw than 1, colonies. On lysine which was provided independently-. Since the owr-all inhibition of c&&w mckd~olism, l!cd] PROTl ~I'l.r\tiTS AND L-T`IIPE GROWTH OF E. COLI 1% for example at low temperatures, is no6 lethal to baet'eria, WC should look to home form of un- balanced growt,h as :I general mechanism of bactericidal action whenever a direct structural lesion is not evident. The zonal rffcct, of penicillin reportcad here is reminiscent of t,hc paradoxical cffcct reported by Englc (1951), namely, t'hc greater bactericidal c&et of low(ar than higher concentrations of penicillin. W'r ~"111 only speculate on the source of the protection afforded by higher penicillin lel-cls; as far ni: L forms arc concerned, it is probably still rc,latcd to the wall, since dcpriva- tion for DAP has the same tffcct as augmenting the penicillin level. d puzzling feature of previous rescarchcs, the appearance of genrtically stabilized L forms, which persist as ruch when penicillin is removed from PC-L forms, also has a tentative explanation: that a superimposed genetic block to wall forma- tion confers if selective advantage under the particular circumstances under which pc-L forms arc growing. 11t least one such circumstance has been realized: zonal lercls of penicillin. The development of fixed L forms in other material has not been recorded in sufficient detail to allow a judgment of the g(,ncral applicability of this hypothesis. The proposal that penicillin interferes with synthesis of the 1~x11 wall (Lederberg, 1957; Park and Strominger. 1957) does not imply that pcni- cillin completely prevents the formation of each element of the normal wall. Nor does it specify the pathway of that inhibition. The functional tests-plasticity and osmotic fragility-speak only for the functional impairment of the wall, and there are strong hints that much residual wall material persists on pc-protoplasts. The diversity of wall components in gram-negative bacteria speaks for many potential sit,cs of wall- defect. The reaction of PC-L forms with certain phagcs and colicines, and not others, and the retention of mating capacity, point to the spcci- ficity of the r&dual cllements. It would bc profitable to compare protoplasts made by different methods for these biological specificities. The correlation of protoplasts with L forms is not, novel and various nspccts arc supported by other worker.. (Bonifas, 1954; Vadasz and Juhasz, 1955; and other lf,ork already cited.) ,\part from the morpl~ological similarity between lysoeymr- a.nd penicillin-conditioned protoplasts, perhaps the most direct evidence is the analysis of strept,ococcal L and B forms (Sharp et al.: 1957), the stabilized L forms being found to lack the group A polgsaccharide, both by im- munological test and analysis for rhamnose. Similar analyses would be highly desirable for reversible, penicillin-conditioned L forms of the streptococcus and for the various types of L forms of E. coli2 The mechanistic interpretation of L growth has been confounded by many cyclomorphic schemes of the greatest variety. L forms were, for example, invoked in speculations on mecha- nism of transduction in Salmonella. They were conceived of as "reduced cells" which might somehow persist with an incomplete genetic complement. The chief evidence for this scheme w-as the presence of what we would now call protoplasts in donor cultures treated with phage or with low levels of penicillin for the provoca- tion of lysis. This fanciful speculation (Ledcrberg et al., 1951) is mcntioncd now only to bc con- demned as baseless in the light of further studies (Zinder and Lederberg, 1952; Stocker et al., 1953; Zinder, 1955) which have shown that bacteriophage particles arc the vector in trans- duction. Other roles that have been assigned to L forms are : an aberrant haplophasc (Dienes, 1946) ; sexuality (Dienes and Smith, 1944; Klienebergcr- Nobel, 1950) ; a resistant stage analogous to sports (Tulasnc, 1955) ; a phase of regeneration or rejuvenation, possibly also correlated with sexuality (Klicncberger-Nobel, 1951). There is no indication that L growth is associated with deep-seated changes in genetic makeup; in any case, the vegetative phase of E. coli and probably other bacteria is normally already haploid. When E. co& cells arc exposed to penicillin, division is intcrruptcd, even of cells which have started to constrict, and the protoplast almost always emerges from the middle of the cell at the point of incipient fission. This suggests that the septum and newly formed wall arc the least rigid parts of the wall, or the most susceptible 2 Kandler and Zehender (1967) report that stable L forms of Proteus vulgaris lack DAP while the B and pc-L forms contain it. The stable L forms include strains reported by them, and here- above, not to be restored by the addition of D.4P. Their metabolic defect is therefore presumed to be in the assembly of DAP into a wall component. 156 LEDERBERG AND ST. CLAIR [VOL. 75 to inhibition by penicillin. At lower concentra- tions of penicillin, the cells fail to divide, but form long filaments, again as if the formation of the septum were more readily inhibited than that of the lateral wall. These features have caused some difficulty in the interpretation of fixed and stained preparations. The fusion of protoplasts has been reported by several authors, e. g., Dienes and Smith (1944). Stained preparations can be misleading: for example, stages resembling those of figure 1 have been described as zygospores or fusion figures by Mellon (1925) and Klieneberger-Nobel (1950). Direct continuous observations on living material (Stahelin, 1954; Stempen and Hutchin- son, 1951) leave no doubt that fusion can occur, but is it de novo or refusion? Published photo- graphs generally show adjacent protoplasts that may have coalesced again before the com- pletion of an earlier fission or budding. In our material, nearly all L colonies must arise from single cells (figure 4) and there is no warrant for the interposition of a fusion stage as an obligatory feature of L colony development. Further studies are needed to establish whether protoplasts stemming from different lines of cells can fuse with genetically interesting consequences, and whether this may occur with bacterial strains that do not also mate in their B phases. It should be stressed that the normal conjugal mating process of E. coli involves typical bacillary forms (Lederberg, 19565) and has nothing to do with L forms. However, the morphological de- tails of the mating of protoplasts of compatible genotypes or of protoplasts with rods, though not yet studied, may prove of particular interest, as the two parents can be readily distinguished both by their appearance and their response to osmotic shock. The "resistance" of protoplasts to penicillin is now understood as a corollary of the mechanism of action of this antibiotic; resistance to phages (and according to Dienes, also to complement) gives some credence to t,he adaptive value of protoplasts in special environments. The spontaneous occurrence cf protoplastic outgrowths in old cultures (Sinkovics, 1957) is understandable as an aspect of unbalanced growth whereby the growth of total mass out- strips the synthesis of new wall. The balance of these processes will of course depend on both environmental and genetic determinants. Per- haps the pleiomorphism of the pleuropneumonia organisms is a reflection of the nutritional in- adequacy of the media. As much can be specu- lated for the propensity of Streptobacillus mordi- formis to produce L forms. Other cyclomorphic sequences, without prej- udice to bheir biological interpretation, arc apt subjects for possible correlations with wall synthesis. For example, the cycle of transform- able competence of the pneumococcus (Fox and Hotchkiss, 1957) is patently due to some im- balance of growth of cellular constituents, for which the wall and other envelopes would be the first candidates. For another, the microcysts of Spirilpunl lunutum, as figured by Williams and Rittenberg (1956) show at least a superficial resembIance to the cycle of evolution and rever- sion of protoplasts. The spontaneous occurrence of large bodies and the osmotic fragility of marine luminous bacteria (Johnson and Gray, 1949) also have to be related to the rigidity of the cell wall. While we have not undertaken a comprehen- sive comparative study of Proteus and other bacteria, our limited observations and the pub- lished record would indicate that E. coli is far more delicate and difficult to maintain in the L phase. For example, at suitable stages in the culture cycle, Proteus will give up to 10 per cent of pc-protoplasts in an unprotected medium (Liebermeister and Kellenberger, 1956), in which E. coli would lyse almost completely. Also, Proteus L forms have been successfully adapted to growth in liquid culture (Tulasne et al., 1950; Dienes, 1953), and to some extent on the surface of agar, which we did not succeed in doing with E. coli. The easiest rationalization of such differ- ences is that Proteus has a tougher residual membrane, a protection which may have made this species convenient for earlier investigations, but may also have helped to obscure the essential differences of B and L growth. A liquid-adapted culture of Proteus (strain Tulasne L), grows in massive clumps and the periphery consists of lysed protoplasts and debris. The pressure of the largely inviable mass of growth may take the place of agar in conditioning further outgrowth of the embedded protoplasts. It was noticed that protoplasts subjected to osmotic shock, though leaving little to be seen under phase microscopy, still displaced a definite volume in an India ink suspension. This residual 19581 PROTOPLASTS AND L-TYPE GROWTH OF E. COLZ 157 structure should be looked for and taken into account in evaluating the biosynthetic capacities of disrupted protoplasts, and the association of specific enzymes, e. g., cytochromes (Weibull, 1953b) with membrane (ghost) fractions. Another feature of L growth reported else- where has not been considered here: the occur- rence of viable, filtrable granules (Klieneberger- Nobel, 1951; Kellenberger et al., 1956; Sinkovics, 1957; Vadasz and Juhasz, 1955). The reported efficiency of filtration at low pore diameters is extremely low, which leads to some question as to the reliability of the minimum size estimates. Under these circumstances, there should be more concern for the homogeneity of pore size of filters and for the plasticity of a small proto- plastic bud, which might be squeezed through pores smaller than its characteristic diameter. However, even if taken at face value, the filtrable granules of about 0.3 p diameter are still ample to contain the material necessary for genetic continuity. Lacking L growths that could be propagated in liquid culture, we have made no filtration studies with E. co&. On the same account, we have no basis to com- ment on other reported modes of growth of L forms, e. g., fragmentation of cysts, extrusion of granules, that have not yet been observed in the present material. The recent development of a technique for the production of protoplasts from yeast (Eddy and Williamson, 1957), suggests that further studies of wall-defective organisms will embrace a wide variety of organisms. SUMMARY Escherichia coli growing in the presence of penicillin forms localized stvellings which enlarge to yield spherical protoplasts. The protoplasts lyse in dilute media, but may be maintained in protective media containing &r/3 sucrose plus &r/100 Mg*. In penicillin sucrose broth, the protoplasts continue to enlarge but do not pro- liferate. In the absence of penicillin they revert to form normal rods. An agar medium is described which permits the further growth of protoplasts in the presence of penicillin to produce L colonies. Growth depends on the formation of blebs which enlarge and pinch off. It occurs only in agar medium. In- definite serial passages were made of certain such strains of E. coli K-12 as L growth in penicillin sucrose agar ; these invariably reverted to normal bacillary form when replanted in the absence of penicillin. Optimal L growth requires high concentrations of penicillin, neither L nor bacilliform colonies developing well at intermediate levels. This phenomenon has been applied to the selection of fixed L forms, which grow as L colonies in con- ventional media. So far, these isolates from E. coli have all proved to bc ausotrophic mut'ants requiring diaminopimelic acid, a characteristic constituent of bacterial cell walls. These mutants, and another previously isolated by Davis, grow as L colonies in agnr lacking diaminopimelic acid. They may be passed in series as such, and regenerate bacilliform growth when this metabo- lite is restored. However, other fixed L forms of Proteus responded neither to diaminopimelic acid nor to crude bacterial hydrolyzate. Penicillin-produced L colonies of E. coli were found to have become resistant to certain phages and colicines, but not others; protoplasts of genetically compatible strains proved to retain their ability to undergo sexual recombination. Some but not all the surface receptors of the bacteria are therefore believed to have been lost. Protoplasts suspended in sucrose broth show a transparent capsule, visible in india ink preparations, which may represent remains of the cell wall. Lysed protoplasts, though expanded, are not fully dispersed through the solvent but occupydefinite spaces, again as seen in India ink. Attempts to convey genetic markers to proto- plasts by means of extracts containing deoxy- ribonucleic acid were unsuccessful, as were efforts to promote the fusion of protoplasts of otherwise incompatible E" x F- genotypes. These observations support the proposals that: (1) the mechanism of action of penicillin is to inhibit the synthesis of the bacterial cell wall; (2) that the L forms of Dienes and Klieneberger are colonies of protoplasts, whose aberrant mode of growth is conditioned by the loss of the wall and the failure of the normal division mechanism; (3) the partial or complete defect of the wall may be brought about either by external inhibition (penicillin) or by internal genetic blocks affecting any of various aspects of wall formation. So far, two types of w-all-defect mutants are known: those repaired by diaminopimelic acid, and auxotrophic for it, and those not reparable even by crude bacterial hydrolyzates. The evolution of L fixed growths in the pres- 158 LEDERBERG AND ST. CLAIR [VOL. i5 ence of penicillin may be accounted for by a selec- tive advantage under these conditions of spon- taneous mutants with wall defects. R.EFERENCES Bauixas, N. AND DAVIS, B. D. 1957 Selection of auxotrophic bacterial mutants through the "suicidal" effect of diaminopimelic acid or thymine deprival. Science, 126, 170. BIGGER, J. K. 1944 Treatment of staphylococ- cal infections with penicillin by intermittent sterilization. Lancet, 2, 497-500. BONIFAS, v. 1954 Influence de la pression os- motique sur le maintien, en milieu liquide pbnicillinh, d'une souche de Proteus sous la forme L. Schw. 2. Pathol. Bakteriol., 17, 525-535. BRIGGS, S., CRAWFORD, K., ABRAHAM,E. P., AND GL.&DSTONE,G.P. 1957 Some properties of gram-negative bacilli obtained from a strain of Staphylococcus aureus in the presence of benzylpenicillin. J. Gen. Microbial., 16, 614-627. CASIDA, L. E., JR. 1956 Preparation of diam- inopimelic acid and lysine. U. S. Patent 2,771,396. COHEN, S. S. AND BARNER, H. 1954 Studies on unbalanced growth in Escherichia coli. Proc. Natl. Acad. Sci. U. S., 40, 885-893. CUMYINS, C. s. 1956 The chemical composi- tion of the bacterial cell wall. In Intern&on& review of cytology, Vol. 5, pp. 25-50. .4ca- demic Press, Inc., New York. DAVIS, 13. D. 1948 Isolation of biochemically deficient mutants of bacteria by penicillin. J. Am. Chem. Sot., 70, 4267. DAVIS, B. D. 1952 Diaminopimelic acid and lysine. Nature, 169, 534. DE FONBRUNE, I?. 1949 La technipue de micro- manipulation. Masson Cie., Paris. DIENES, L. 1946 Complex reproductive proc- esses in bacteria. Cold Spring Harbor Sym- posia Quant. Biol., 11, 51-59. DIEBES, L. 1949 The development of Proteus cultures in the presence of penicillin. J. Eacteriol., 67, 529-546. DIEXES, L. 1953 Some new observations on L forms of bacteria. J. Bacterial., 66, 274-279. DIENES, L. AND SWTH, W. E. 1944 The sig- nificance of pleomorphism in Bacteroides strains. J. Bacterial., 48, 125-153. DIEYES, L. AND WEINBERGER, H. J. 1951 The I, forms of bacteria. Bacterial. Revs., 16, 245-288. EIGLE, H. 1951 Further observations of the zone phenomenon in the bactericidal action of penicillin. J. Bacterial., 62, 663-668. EDDY, A. A. AXD WILLIA~MOS, D. H. 1957 A method of isolating protoplasts from yeast. Nature, 179, 1252-1253. EPHRUSSI, B. 1953 Nucleo-cytoplasmic relations in micro-organisms. Clarendon Press, Os- ford. Fox, M. S. AND HOTCHE;ISS, II. D. 1957 Initia- tion of bacterial transformation. Nature, 179, 1322-1325. FREDERICQ, P. 1948 Actions nntibiotiques rkci- proques chez les Enterobacteriaceae. Rev. Belge Pathol. Med. Exp., 19. Suppl. 4, l-107. GRAY, C.H. AND TATUM, E. L. 1944 S-ray in- duced growth factor requirements in bac- teria. Proc. Natl. Acad. Sci. T1. S., 30, 40-l- 410. JAWETZ, E., GUSXISON, J. B., .*NI) COLEMAS, V. R. 1954 Observations 011 the mode of action of antibiotic synergism and ant'ago- nism. J. Gen. Microbial., 10, 191-198. JOHNSOS, F. J. ASD GRAY, 1). H. 1949 Suclei and large bodies of luminous bacteria in rela- tion to salt concentration, osmotic pressure, temperature, and urethane. J. Bacterial., 68, 675-688. KANDLER, 0. AND KANDLER, G. 1954 Unter- suchungen iiber die Morphologie und die Vermehrung der pleuropncumonie-iihnlichen Organismen und der L-Phase der Bakterien. Arch. Mikrobiol., 21, 178-201. KANDLER,~. AND ZEHEXDER, C. 1957 Ubcr das Vorkommen von ~,t-Dialllinopimelins~~~lre bei verschiedenen L-Phasentypen von Pro- teus vulgaris und bei den pleuropneumonie- iihnlichen Organismen. %. Nnturforsch., nc- cepted for publication. KELLESBERGER, E., LIEBERMEI~TER, K., ASD BONIFAS, V. 1956 Studien zur LForm der Bakterien. Z. Naturforsch.. 11. 206215. KLIESEBERGER-NOBEL, E. 1949 Origin, devel- opment and significance of L-forms in bac- terial cultures. J. Gen. 3Iicrobiol., 3, 434- 443. KLIENEBERGER-NOBEL, E. 1950 DO Fusijo~~is necrophorus and Streptobacillus monilifowis show a primitive form of sesuality according to Mellon's views? J. Gen. >licrobiol.? 4, ri. KLJENEBERGER-NOBEL, E. 1951 Filterableforms of bacteria. Bacterial. Revs., 15, 77-103. KLIENEBERGER-NOBEL, E. 1954 \~icroorganisms of the pleuropneumonia group. Biol. Revs. Cambridge Phil. Sot., 29, 154-1334. LEDERBERG, J. 1950 Isolation and characteri- zation of biochemical mutants of bncteri:i. Methods in ~Ietlical Research, 3, 5-22. LEDERBERG, J. 1954 A simple method for iin- lating single microbes. J. Bncteriol., 88, 258-250. 19581 PROTOPLASTS AND L-TYPE GROWTH OF E. COLI 159 LEDERBERG, J. 1956a Bacterial protoplasts in- duced by penicillin. Proc. Natl. Acad. Sci. u. s., 42, 574-577. LEDERBERG, J. 1956b Conjugal pairing in Esch- erichia coli. J. Bacterial., 71,497-498. LEDERBERG, J. 1957 Mechanism of action of penicillin. J. Bacterial., 73, 144. LEDERBERG, J., (24~7~~~1, L.L., AND LEDERBERG, E. M. 1952 Sex compatibility in Escherichia coli. Genetics, 37, 720-730. LEDERBERG, ,J., LEDERBERG, E. M., ZINDER, N. D., AND LIVELY, E. R. 1951 Recombi- nation analysis of bacterial heredity. Cold Spring Harbor Symposia @ant. Biol., 16, 413-443. LEDERBERG, J. AND ZINDER, N. D. 1948 Con- centration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Sot., 70,4267. LEIFSOS, E. 1951 Staining, shape, and arrange- ment of bacterial flagella. J. Bacterial., 62, 377-389. LIEBERMEISTER, K. .~ND KELLENBERGER, E. 1956 Studien zur L-Form der Bakterien. Z. Naturforsch., 11, 20&206. LINZ, R. ASD LECOCQ, E. 1956 Sur l'action bac- tericide de la penicilline, de la streptomycine et d'un melange synergique de pCnicilline et de streptomycine. Ann. inst. Pasteur, 91, 542-563. MEADOW, P., Ho.~RE, D. S., END WORK, E. 1957 Int,errelntionships between lysine and cyc- diaminopimelic acid and their derivatives and nnalogues in mutants of Escherichia coli. Biochem. J. (London), 66, 271-282. MELLON, R. It. 1925 Studies in microbic hered- ity. I. Observations on a primitive form of sexua1it.y (eygospore formation) in the colon- typhoid group. J. Bncteriol., 10,481~501. MORTON, H. Ii:. AND LECCE, J. G. 1953 Selective act.ion of thallium acetate and crystal violet for pleuropneumonialike organisms of human origin. J. 13ncteriol., 66, 646-649. PARK, J. T. ANI) STROMISGF,R, J. L. 1957 Mode of action of penicillin. Science, 126, 99-101. REPA~KE, It. 1956 Lysis of gram-negative bac- teria by lysozymc. Biochim. Biophys. Acts, 22, 189-191. SHARP, J. T., HIJX~SS, IV., .44~1) DIEXES, L. 1957 Examination of the L forms of group A strep- tococci for the group-specific polysaccharide and M protein. J. Exptl. Med., 105, 153-159. SIMON, ELLEN 1955 Bacteriological, immuno- logical and genetical studies with strepto- mycin-dependent bacilli. Ph.D. thesis, Uni- versity of Wisconsin. SIXXOVICS, J. 1957 Pleuropneumonieahnliche Eigenheit,en in alten E. coli-Kulturen. Acta 1Iicrobiologica. Acad. Sci. Hung., 4, 61-i5 SPIEGELMAN, S. 1956 On the nature of the en- zyme-forming system. In Enzymes, units of biological structure and function, pp. 67-89. Edited by 0. H. Gaebler. .\cademic Press. Inc., New York. SPIEGELMAN, S. 1957 Nucleic :icids and the synthesis of proteins. In Symposium on the chemical basis o.f heredity, pp. 232-267. Ed- ited by W. D. McElroy and Ii. Glass. Johns Hopkins Press, Baltimore. SPOONER, E.T.C. AND STOCKER,U.:I.D. (Eds.) 1956 Bacterial anatomy; symposium of the Sot. Gen. Microbial. Cambridge Univ. Press. STSHELIN, H. 1954 Uber osmotisches Verhalten und Fusion nackter Protoplasten von Bacil- lus anthracis. Schw. Z. Pathol. Bakteriol., 17, 296310. STEMPEN, H. AND HUTCHIXSOS, \V. G. 1951 The formation and development of large bodies in Proteus vulgaris 0X-19. J. Bac- teriol., 61, 321-335. STOCKER, B. A. D., ZINDER, N. I>., ANU LEDER- BERG, J. 1953 Transduction of flagellar characters in Salmonella. J. Gen. Microbial., 9, 410-433. TULASNE, R. 1951 Les formes L des bncteries. Revue Immunol., 16, 223-251. TULASNE, R. 1955 Bilan de nos connaissances sur les cycles bactcriens du type L et sur les formes L des batteries. Biol. Med. (Paris), 44,1-33. TULASNE, R. AND LAVILLAUREIS, J. 195-I Con- siderations sur la iransformation experi- mentale des bact&ies en formcs naines. Compt. Rend. Sot. Biol., 148, 58&590. TULASNE, R., TERRANOV~, T., .\NII Lzvr~~aa- RIEX, J. 1955 Obtention de formes L de diverscs batteries b gram negatif sur les milieux sans serum, mnis contGnnnt des vitamines du groupe 1~. Giorn. >Iicrobiol., 1,44-51. TULASNE, R., VENDRELY, It., XIXCB, It., AND MULLER, L. 1950 Technique pour la cul- ture des formes submicroscopiqucs (formes L) du Proteus vulgaris en milien liquide. Compt. rend. acad. sci. Paris, 230, 151-154. V~DASZ, J. AND Ju11.4sz, T. 19% PlaSIII2 glob- ules arising from Salmonella enterilidis under the influence of penicillin and their reversion to the original bacillary form. -Acta Biolo- gica, hcad. Sci. Hung., 6, 171-lY3. WEIBULL, C. 1953~ The isolation of protoplasts from Bacillus megaterium by controlled treat- ment with lysozyme. J. Bacterial., 66, 68% 695. WEIBULL, C. 1953b Characterization of the protoplasmic constituents of Bacillus mega- terium. J. Bncteriol., 66, 696-702. 160 LEDERBERG AND ST. CLAIR [VOL. 75 WEIBULL, C. 1956 The nature of the "ghosts" obtained by lysozyme lysis of Bacillus mega- terium. Exptl. Cell Research, 10, 214-221. WILLIAMS, hf. ASD RITTENBERG, S. C. 1956 Microcyst formation and germination in Spirillum lunatum. J. Gen. Microbial., 15, 20&209. WORK, E. 1957 Biochemistry of the bacterial cell wall. Nature, 179, l-16. ZINDER, N. D. 1955 Bacterial transduction. J. Cellular Comp. Physiol., 46, Suppl. 2,23-49. ZINDER, N. D. AND ARNDT, W. F. 1956 Produc- tion of protoplasts of Escherichia coli by lysozyme treatment. Proc. Natl. Acad. Sci. U. S., 42, 586-590. ZINDER, N. D. AND LEDERBERG, J. 1952 Ge- netic exchange in Salmonella. J. Bacterial., 64, 679-699.