Reprinted from JouRNAL oF BACTERIOLOGY
Vol. 75, No. 2, pp. 148-160, February, 1958
Printed in U.S.A.

PROTOPLASTS AND L-TYPE GROWTH OF ESCHERICHIA COLP

JOSHUA LEDERBERG axp JACQUELINE 8ST. CLAIR

Department of Medical Genetics, School of Medicine and Department of Genetics, College of
Agriculture, University of Wisconsin, Madison, Wisconsin

Received for publication August 5, 1957

A preceding article (Lederberg, 1956a) was
devoted to the evolution of protoplasts from
growing cells of Escherichia 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 will give an account of
these 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 ex-
ample, with penicillin, or by intrinsic metabolic
defects, consequences of genetic mutations,

The previous study was motivated mainly by
the hope of furnishing protoplasts of genetically
defined strains of FE. coli for physiological and
genctic analysis (Spiegelman, 1956; Spooner and
Stocker, 1956). It was modeled on the experi-
ments of Weibull (1953a) who forestalled the
lysis of Bacillus megaterium exposed to lysozyme
by maintaining the bacteria in a protective,
hypertonic medium. It was found that the lysis
of growing £. coli in the presence of penicillin
could be forestalled in a medium containing
M/3 sucrose and M/100 Mgt"; instead of lysing,
the 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-
gether different viewpoint as an aspect of the
development of L forms. For various reasons,
species other than £. colt have been preferred for
such studies. In general, the wall defects have
been recognized but not stressed as the essential
feature 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-2157). 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.

148

obliquely stated. The chicf 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 adequate retrospect of the literature on L
forms would be a herculean task. Fortunately, we
may rely on a number of reviews for background
documentation (Dienes and Weinberger, 1951;
Liebermeister and Kellenberger, 1956; Kliene-
berger-Nobel, 1954; Tulasne, 1951; Kandler and
Kandler, 1954). Studies on protoplasts and re-
lated aspects of bacterial morphology are in-
cluded in a recent symposium (Spooner and
Stocker, 1956).

As this paper is in part a restatement of pre-
vious knowledge, semantic questions loom large.
Protoplast is borrowed 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 cireum-
scribed, either chemically or morphologieally, 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 wall are (1)
osmotic fragility and (2) loss of rigidity resulting
in spherical or amoeboid form. To be sure, these
criteria fail to distinguish between the total
absence of a wall and its functional impairment.
L form is a generic term which stems from the
cultures labeled Li, Le, ete. (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 direet, 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 be used more
broadly to describe atypical growths resembling
144

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 distinguish the several
ways in which protoplasts and L growth can be
- produced. Those achieved by the immediate
presence of penicillin will be labeled with the
prefix pce-; those obtained by withholding
diaminopimelic acid (DAP) will be called dap-.
“Stabilized” or “fixed” will refer to L forms which
have an intrinsic hereditary defect, and therefore
display this growth pattern on conventional
media; they correspond to L forms in the strict
sense advocated by Klieneberger-Nobel.

As this is an extensive rather than intensive
report, we have appended much of the discussion
to the findings as they are presented. For the
same reason, we will stress, perhaps unduly, the
hypothetical status of many inferences. Without
question, many of the attendant details warrant
thorough exploration as individual problems in
their own right.

MATERIAL AND METHODS

Media. The media used were evolved by trial
and error, starting from the formulations of
pennassay broth and nutrient broth (Difco).

LEDERBERG AND ST. CLAIR

[vou. 75

The following medium containing 0.3 M sucrose,
m/125 Me**, 1/600 penicillin, plus a nutrient
base, was found to be effective in the growth
of L colonies of F. colt.

Sucrose broth (per L): casein digest (Sheffield
Chem. Div., Norwich, N. Y.) 10 g; sucrose, 100
g; meat extract (Lemco or Difco), 10 g; NaCl,
3.5 g; glucose, 1 g; agar, if indicated, 10 g. After
autoclaving, 10 ml of 20 per cent MgSO,-7H.O
was added. If penicillin was indicated, we added
1000 u per ml unless otherwise stated.

“Bacterial hydrolyzate’ was prepared as
follows: 3 g of #. coli were suspended in 30 ml
2 .N sulfuric acid, and autoclaved in a screweap
vial for 35 min; we then centrifuged, discarded
the sediment, neutralized the supernatant with
10 n NaOH, and sterilized this by reautoclaving.
Whenever indicated, the hydrolyzate was used
at 1:10 dilution, giving a final concentration (in
terms of the original cells) of about 10 mg per ml.
The hydrolyzate gave an assay with a DAP
auxotroph (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 cannot be con-
sidered quantitatively reliable in absolute terms.
However, as 10 pg of DAP will permit the growth
of about 1 mg of bacteria, the correspondence is

 

(0 a.

Figure {. Escherichia colt Y-10 in penicillin sucrose broth. (Above) Various cells in successive stages.
0to4 hr. (Below) Late stage at higher magnification, Phase contrast.
1958]

PROTOPLASTS AND L-TYPE GROWTH OF E. COLI

145

 

Figure 2. B and L colonies. (Left) A gradient plate of sucrose agar 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 48 hr, about 10x; dis-

secting microscope, oblique illumination.

 

Figure 3. (Left) Young (20 hr) L colony, about
0.1 mm in diameter. Originally photographed at
100X phase contrast. (Right) Squash of L colony,
phase contrast.

reasonable. The amount of hydrolyzate used as a
supplement is a calculated tenfold excess.

After preliminary trials with other strains, most
of the work reported here involved £. coli strain
K-12 and a number of mutant substrains, such as
W-6, Y-10, W-1895, and others, for the most part
cited in earlier papers (Lederberg e¢ al., 1952).
Strain K-12 and its derivatives will be considered
collectively as #. 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 AND CONCLUSIONS

Formation of protoplasts in penicillin sucrose
broth. In the absence of sucrose or other stabilizer,
cells of H. coli lysed after an initial swelling.
However, as previously described, #. colt 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

water, and more slowly in broth. In similar ex-
periments, M/5 glucose and m/2000 clinical
dextran (kindly furnished by Baxter Labora-
tories, Inc., Morton Grove, Illinois) had incom-
plete protective effects comparable to m/10
sucrose. Likewise, Weibull (1958a, }) found that
m/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.

Mgt, 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 Versene is an essential part of one recipe for
releasing protoplasts from #. coli (Repaske,
1956) has yet to be resolved.

It is well known that lysis by penicillin requires
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; Lederberg
and Zinder, 1948). Correspondingly, the evo-
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, chlortetracycline or
chloramphenicol. For this reason penicillin is
believed to act by inhibiting new wall synthesis,
in contrast to the dissolution of the existing wall
by lysozyme. The changes of figure | are 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 released from a
single multiseptate rod of a Bacillus spp.

Some fifty and odd penicillin-sensitive strains
of F. coli, of various serotypes, have been treated
in penicillin sucrose broth with similar results.
Protoplast formation has also been secured in a
defined medium (Gray and Tatum, 1944) sup-
plemented with sucrose, Mgt, and penicillin,
but was slower and incomplete, presumably be-

LEDERBERG AND ST. CLAIR

[vou. 75

cause of the lower rate of protoplasmic increase
in this medium as compared to broth.

The continued growth of protoplasts suspended
in penicillin sucrose broth is reflected by increases
in optical density and induced 8-p-galactosidase
as well as in the size of the individual protoplasts.
The biosynthetic activity of these protoplasts is
being more intensively studied in other labora-
tories (Spiegelman, 1957) where they 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 the
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 im sucrose agar. 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
(Lederberg, 1956a). The spheres develop pro-
tuberances, one or more of which clongate 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 protective 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 Z. 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 structure are still
obscure. As the protoplasts enlarge, a lune-shaped
vacuole appears at one side (figure 1). Older
protoplasts often display a narrow crescent of
1958]

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 #. coli line 1) which show no capsule in the
B form. Since protoplasts of various serotypes
are still agglutinable by homologous anti-O 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, 6). As
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 (Spiegelman, 1956) were
unsuccessful.

Growth of L colonies of EH. colt 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 Dicncs (1949) were
effective for Proteus strain 52 (kindly furnished
by him). Good yields were also obtained in nu-
trient agar (Difco) with sucrose plus penicillin. It
was necessary, as recommended, to limit the agar
concentration to not more than 0.8 per cent. But
comparable experiments with H. coli line 1 were
much less successful at first. A long series of trial
and error experiments finally uncovered the fol-
lowing prescriptions.

(1) Strain specificity:—Different lines and
substrains within #. coli line 1 vary in their yield
of L colonies, some cultures being completely un-

PROTOPLASTS AND L-TYPE GROWTH OF E£. COLI

147

productive in every medium tried. HE, cold 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 infurther
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:—Unlike 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 F.
colt 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 bencath the surface.

(4) Meat cxtract:—Stimulated the L growth of
some strains. Yeast extract was, if anything,
inhibitory.

(5) Mg**+:—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 #. coli developed in casein digest-meat
extract agar without sucrose. So far as tested,
these did not consist of mutants that would
tolerate penicillin in the absence of sucrose. With
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 m/10 phosphate buffer.
The optimum was found at about pH 6.3 which
approximates the pH of the unbuffered medium.
Phosphate buffer has therefore been omitted from
the recipe to minimize precipitation of magne-
sium phosphates.

(8) Growth factors:—In a defined medium
(Gray and Tatum, 1944) supplemented with
sucrose, penicillin and Mgtt, L growth was
148

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-

LEDERBERG AND ST. CLAIR

[vou. 75

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 ‘‘colony”
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 subeulture 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 the
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,
149

COLI

GROWTH OF £.

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PROTOPLASTS AND L-TY

1958]

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150

and the presence of agar fragments interfere with
a total count. Single L colonies containing an
estimated 10% to 10+ spherical elements yielded
from 10 to 50 L progeny when minced and re-
plated. This low viability might be inherent in the
elements of the colony, or might be ascribed to
damage by release of external pressure or to
injury from the maceration.

Several strains of #. coli have been subcultured
by this method in the L phase for as many as 20
passages before the experiments were terminated.
The dilution at each passage was about one tenth,
so that the cumulative factor of increase was
10”, leaving little doubt that the L colonies could
have been propagated indefinitely in the presence
of penicillin. Samples were also plated into sucrose
agar, where they reverted to give approximately
the same yield of B colonies as of L colonies in
penicillin sucrose agar. These reversion cultures
were indistinguishable from the original Y-10 in
their growth habit, their sensitivity to penicillin,
and their productivity of pe-protoplasts and L
colonics.

Contrary to experience with other bacteria, the
prolonged cultivation of #. coli in the L phase in
the presence of penicillin gave no stabilized L
forms.

A number of strains, e. g., K-12 and W-6, gave
relatively low yields (10 or less) of L colonies.
Reversion cultures were made of the obtainable
L growth im the expectation of accumulating
mutants better adapted to form L colonies in the
adopted medium. This expectation was not ful-
filled; the L colonics that do develop in these
circumstances must be put down to phenotypic
accidents.

Like pe-protoplasts, the L growth is osmotically
fragile. Minces diluted in water gave less than 1
per cent of the viable count (B in sucrose agar, L
in penicillin sucrose agar) shown by aliquots
diluted in sucrose broth. Part of the residual
survivorship may be ascribed to protected cle-
ments embedded 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 external
pressure, the elasticity of the remaining envelope,
and interfacial tension all tend to conserve the
spherical shape of the protoplast. Without the
normal division mechanism, which depends on the
wall, the protoplast remains spherical as it
grows. In agar the shape of the protoplast is im-

LEDERBERG AND 8T. CLAIR

[woL. 75
posed by local stresses in the medium, the net-
work of agar fibrils taking the place of the missing
rigid wall. At local points of weakness the growing
protoplast herniates into an adjacent free space,
expands, and pinches off a bleb. Many of the blebs
may be expected 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. When the cells were placed in a
sustaining medium without penicillin, they re-
covered their normal capacities and regenerated
typical walls. However, many workers have re-
ported on the occurrence of genctically stabilized
L forms which remained defective in the same
environment 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 originally discovered in
hydrolyzates of Corynebacterium diphtheriae and
subsequently identified as a component of the cell
walls of numerous bacteria (Work, 1957;
Cummins, 1956). Apart from the differential
occurrence of the meso- and the Lu-stereoisomers,
DAP has been found in the walls of all bacteria
examined except for gram-positive cocci and
lactobacilli, and including actinomycetes and
myxophyceac, but in no other biological source
(except Chlorella). The preferential localization
of DAP in the cell walls of the bacterial species in
which it occurs suggests that it has no other struc-
tural significance, and the 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
FE. colt strain W that required DAP and lysine.
Subsequently, Bauman and Davis (1957) and
Meadow ef al. (1957) observed that cultures
grown on limited DAP underwent lysis when the
DAP supply was exhausted. (We are indebted to
Dr. Davis for initial samples of DAP, for a culture
of his DAP-dependent mutant, strain 173-25,
and for preliminary information on the osmotic
fragility of DAP-starved cells.) DAP is now pro-
duced on a commercial scale as a metabolite
accumulated by another mutant (Casida, 1956)
and a generous sample has kindly been furnished
by Chas. Pfizer and Co.
1958]

Strain 173-25 was grown in broth supplemented
with bacterial hydrolyzate or with 10 ug per ml
of DAP. 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,
Mg**, and lysine, but was less complete. As a
corollary, the sucrose medium gave a higher
turbidity and more stable assay curve for DAP
than the customary minimal medium without
sucrose.

Strain 173-25 was also plated into sucrose agar,
where it grew extensively and exclusively in the
form of L colonies similar to, though somewhat
smaller than, those shown in figure 3. Except
for occasional (sometimes troublesome) reverse-
mutants, no colonies at all were formed in the
absence of sucrose. The dap-L colonies grew on
serial passage in sucrose agar in the same fashion
as already described for pe-L forms.

When DAP was restored, the passage strains
promptly resumed B growth. Since DAP is ab-
sent from conventional bacteriological media,
the DAP-dependent mutant would have been
described as a fixed L form, had it been isolated
prior to these studies.

DAP is not the only target whose impairment
would block wall formation. Neither DAP nor
hydrolyzate was found to reverse inhibition by
penicillin, which may therefore antagonize some
metabolite missing from the hydrolyzate, 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 Sy). 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 DAP, and the site of their block, like that of
penicillin, remains unspecified. In addition, there
was no effect when boiled Proteus cclls were
also added to furnish a possible primer of wall
formation.

Selection of wail-defect mutants. The process of
stabilization of L growths in the presence of
penicillin has not been systematically studied, at
least not to allow the discrimination of various
genetic hypotheses. At least four come to mind:

PROTOPLASTS AND L-TYPE GROWTH OF EF. COLI

fol

(1) that wall-synthesis is subject to spontancous
mutations which may block any of a number of
steps and that such mutants have a selective ad-
vantage in the circumstances where they have
been isolated; (2) that wall formation depends on
a self-reproducing cytoplasmic particle whose re-
production is impaired by penicillin (Sharp et al.,
1957), by analogy with the effects of acriflavine
on yeast (Ephrussi, 1953); (3) that wall-synthe-
sis is itself a self-dependent process, with exten-
sion of the wall depending on the integrity of the
pre-existing structure by analogy with the role
of polysaccharides as primers for their own syn-
thesis; (4) that penicillin has a specific mutagenic
effect, so that the same compound produces
both phenotypic and genotypic impairments of
wall formation (Briggs ef al., 1957). These
hypotheses arc not mutally exclusive; however
in the present material the last three are dis-
couraged by the long-continued propagation of
&., colt line 1 as pe-L growth without becoming a
stabilized L form. Hypothesis (1) has been par-
tially justified by the behavior of the DAP
auxotroph, strain 173-25, which is an example of
a wall-defect mutant. However, the circum-
stances of our experiments with penicillin evi-
dently did not favor selective overgrowth of
wall-less mutants, contrary to the experience of
other workers. Under what conditions, then,
would a wall-less mutant have an advantage, as
would be necessary to complete the justification
of the hypothesis (1)?

This question may have several answers; at
least one clue comes from the zone effects of
penicillin mentioned earlier (figure 2). In the
presence of “zonal” Icvels, i. e., 75 to 125 u per
ml, L growth of DAP-independent F. coli 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 DAP 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 effect was also given by a highly
purified sample (kindly furnished by Dr. J. Lein,
Bristol Laboratories, Inc.) and was unaltered by
the addition of an excess of heat-inactivated
penicillin. We propose that incomplete inhibi-
tion accounts in some manner for the zone of
poor IL growth, a condition that ean be relieved
152

either by the addition of more penicillin or by
the superimposition of a genetic block. If this
reasoning is correct, we should tend to 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 water, then
irradiated with ultraviolet to a survivorship of
10-3 to 10-*%. 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 was 10~ or
less than that obtained in the presence of optima]
penicillin. These plates were incubated for 1 week
to 10 days, during which time much of the
penicillin has been inactivated, in part by the
constitutive intracellular penicillinase, which is
characteristic of #. colt. As a result, many non-
mutant colonies revert to B 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-
zate 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, p-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.

LEDERBERG AND ST. CLAIR

[vou. 75

One isolate may represent an incomplete block,
as it grows substantially normally mm broth and
minimal liquid medium. However, it forms mainly
protoplasts and intermediate forms in sucrose
broth and L colonies in sucrose agar. As has
already been suggested, hypertonic sucrose may
play an active role in the herniation 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 colicine. 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. Pe-L colonies of
E. colt line 1 were found to be relatively resistant
to phages T1, T4, T5, and A-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 T1 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 been 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 colicines tried has been com-
pletely innocuous to L colonies, and they, therc-
fore, do not give perfect discrimination under the
conditions used.

The only relevant mutant isolated with the
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
unaffected in its growth pattern and its resistance
to Ti by bacterial hydrolyzate.

Various chemicals have been used for the
1958] PROTOPLASTS AND L-TYPE GROWTH OF E. COLI 153

selective isolation of pleuropneumonialike 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 FE. coli
Y-10.

The scarcity of L form mutants procured in
£. 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 pe-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: p-glu-
tamic acid; pL-a-aminopimelic acid, DL~a-amino-
butyric acid (purchased from California Bio-
chemical Foundation) and a sulfonic analogue of
DAP, 1,5-diamino-1,5-pentanedisulfonic 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 (DNA). This hope has so far
not been realized in experiments with £. coli.
The markers used in these experiments were Lac,
M and §, for example M-S" X M785, with selec-
tion for MtS" 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 Catt
for Mg**; addition of serum albumin; partial
osmotic shock in the presence of DNA 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 O-serotypes (kindly
furnished by Dr. F. @rskov). In some trials the
recipients were treated as L colonics 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 DN A-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 cither or
both of the parents was introduced as pc-proto-
plasts. For example, protoplasts of Hfr,M~ were
mixed with protoplasts of F-Lac;-S' in penicillin
sucrose broth, and incubated for | hr. They were
then agitated to break up residual complexes, di-
luted, and plated on EMB lactose streptomycin
agar, supplemented with sucrose and Mgtt. On
this medium, the Hfr parent is suppressed, and
LactS' recombinants are distinguishable from the
F-Lac-S" 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 percent fortherod x rod controls. In view of the
effect of streptomycin even on S*™ pe-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 HfrM~S*
protoplasts & F Lac S' rods was consistently
154

fertile (perhaps because this difhculty is not in
question), and a conjugal pair of these was
readily observed as a rod with attached proto-
plast.

Hfr and F~ genotypes retained their character-
istic compatibility behavior when reversions were
tested after several passages as pc-L colonies.
The wall defeet imposed by penicillin thus has no
influence on cither the genetic continuity or
phenotypic manifestation of this presumably
surface-related property.

E. coli 204 is a pleiomorphic strain, briefly
described by Klieneberger-Nobel (1949). The
division mechanism of this strain seems to be
intermediate between fission and budding, the
units in a growing suspension consisting of de-
formed spheres with large protuberances. The
culture is resistant to penicillin; hydrolyzate had
no effect on its morphology. Its wall defect does
not, however, extend to osmotic fragility, dilu-
tion from sucrose broth into water having had
no effect on viability. No sign of the fragmenta-
tion mentioned by Klieneberger-Nobel was
observed in living broth cultures seen under
phase contrast, although many cells assumed
irregular shapes and comprised an unusual range
of sizes. It was undecided whether the discrep-
ancy is due to differences of observational or
cultural technique or to changes that may have
occurred in the behavior of the strain.

In the course of other experiments, it was found
that S* (streptomycin-resistant) mutants of £.
coli line.1, which grow well in 1 mg per ml of
streptomycin, were inhibited by 20 ug per ml when
grown as pc-L forms. This can be viewed either
as the negation of the S* effect when the wall is
stripped, or a mutual reinforcement of penicillin
and streptomycin in respect to another target.
(Jawetz et al., 1954; Linz and Lecocq, 1956).
The former interpretation is favored by the sensi-
tivity to streptomycin of dap-L colonies of an
S* strain.

Another hint of the relationship of mutation
at the § locus to alterations of the cell wall was
offered by the reported tendency of streptomycin-
dependent mutants to grow as long filaments
and oceasional pleiomorphic colonies when de-
prived of streptomycin (Simon, 1955). An S@
mutant of E. coli Jine 1 was plated into sucrose
agar and found to give a high yield of colonies
consisting of very long filaments with occasional
swellings (figure 5), rather than TL colonics. On

LEDERBERG AND ST. CLAIR

 

colt
pendent mutant; filamentous colony in sucrose
agar without streptomycin. Phase contrast.

Figure §. Escherichia streptomycin-de-

replating, the viability of these colonies was very
low; the sucrose medium, therefore, could not
compensate for the lesion imposed by lack of
streptomycin.

Klieneberger-Nobel (1954) has listed some
artifacts in sterile media containing lipoid
material which can be mistaken for the globules
of L growth. An equally rich souree of artefacts
is nutrient gelatin agar as used for the selection
of motile bacteria. This medium tends to con-
tain coacervates which give the appearance of
germinating cysts.

GENERAL DISCUSSION AND RECAPITULATION

Our data support the conception that L growth
is a result of a defect in wall synthesis. This
defect may be imposed by external inhibition,
as by penicillin, or by an internal genetic block.
In some eases, the block has been defined bio-
chemically, for cxample, as auxotrophy for
diaminopimelic acid. In others (Proteus L forms)
it has not been reparable even by hydrolyzates
of the wild type bacteria. The irreparable mu-
tants might be auxotrophs for metabolites lack-
ing in the hydrolyzate as prepared, or might
represent losses of the wall-building enzymes.
Another hypothesis, that wall-building is self-
priming, also warrants further consideration:
the problem may be how to re-establish the
primer at a suitable site.

L growth is an aspect of ‘unbalanced growth”
(Cohen and Barner, 1954) in which one cell
constituent, in this case the cell wall, is selec-
tively inhibited. The prolonged subculture of
pc- and dap-L forms furnished execllent evidence
that penicillin and diaminopimelic acid play no
essential part in the viability of the bacteria
other than their influence on the cell wall, dis-
regarding the role of DAP as a precursor of
lysine which was provided independently. Since
the over-all inhibition of cellular metabolism,
1953]

for example at low temperatures. is not lethal to
bacteria, we should look to some form of un-
balanced growth as a general mechanism of
bactericidal action whenever a direct structural
lesion is not evident.

The zonal effect of penicillin reported here is
reminiscent of the paradoxical effect reported by
Kagle (1951), namcly, the greater bactericidal
effect of lower than higher concentrations of
penicillin. We can only speculate on the source
of the protection afforded by higher penicillin
levels; as far as L forms are concerned, it is
probably still related to the wall, since depriva-
tion for DAP has the same effect as augmenting
the penicillin level.

A puzzling feature of previous researches, the
appearance of genctically stabilized L forms,
which persist as such when penicillin is removed
from pe-L forms, also has a tentative explanation:
that a superimposed genetic block to wall forma-
tion confers a selective advantage under the
particular circumstances under which pc-L forms
are growing. At least one such circumstance has
been realized: zonal levels of penicillin. The
development of fixed L forms in other material
has not been recorded in sufficient detail to
allow a judgment of the general applicability of
this hypothesis.

The proposal that penicillin interferes with
svnthesis of the cell wall (Lederberg, 1957; Park
and Strominger. 1957) does not imply that peni-
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 pe-protoplasts. The
diversity of wall components in gram-negative
bacteria speaks for many potential sites of wall-
defect. The reaction of pe-L forms with certain
phages and colicines, and not others, and the
retention of mating capacity, point to the speci-
ficity of the residual elements. It would be
profitable to compare protoplasts made by
different methods for these biological specificities.

The correlation of protoplasts with L forms is
not novel and various aspects are supported by
other workers (Bonifas, 1954; Vadasz and
Juhasz, 1955; and other work already cited.)
Apart from the morphological similarity between
\vsozyme- and penicillin-conditioned protoplasts,

PROTOPLASTS AND L-TYPE GROWTH OF &. COLI

155

perhaps the most direct evidence is the analysis
of streptococcal L and B forms (Sharp eé al.,
1957), the stabilized L forms being found to
lack the group A polysaccharide, 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. coli?

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
was 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 (Lederberg
et al., 1951) is mentioned now only to be 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 are the vector in trans-
duction.

Other roles that have been assigned to L forms
are: an aberrant haplophase (Dienes, 1946);
sexuality (Dienes and Smith, 1944; Klieneberger-
Nobel, 1950); a resistant stage analogous to
spores (Tulasne, 1955); a phase of regeneration
or rejuvenation, possibly also correlated with
sexuality (Klieneberger-Nobel, 1951). There is
no indication that L growth is associated with
decp-scated changes in genetic makeup; in any
case, the vegetative phase of Z. colt and probably
other bacteria is normally already haploid.

When E. colt cells are exposed to penicillin,
division is interrupted, 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 are the least
rigid parts of the wall, or the most susceptible

2 Kandler and Zehender (1957) report that
stable L forms of Proteus vulgaris lack DAP while
the B and pe-L forms contain it. The stable L
forms include strains reported by them, and here-
above, not to be restored by the addition of DAP.
Their metabolic defect is therefore presumed to
be in the assembly of DAP into a wall component.
156

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 #. coli involves typical bacillary
forms (Lederberg, 1956b) 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 the 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-

LEDERBERG AND ST. CLAIR

[vox. 75

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 monili-
formis to produce L forms.

Other cyclomorphic sequences, without prej-
udice to their biological interpretation, are 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
Spirillum lunatwm, as figured by Williams and
Rittenberg (1956) show at least a superficial
resemblance to the cycle of evolution and rever-
sion of protoplasts. The spontaneous occurrence
of large bodies and the osmotic fragility of marine
juminous 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 #. 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 pe-protoplasts in an unprotected medium
(Liebermeister and Kellenberger, 1956), in which
E. colt would lyse almost completely. Also,
Proteus L forms have been successfully adapted
to growth in liquid culture (Tulasne ef 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
1958]

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; Sinkovies,
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 » 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 EF. colt.

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 colt growing in the presence of
penicillin forms localized swellings which enlarge
to yield spherical protoplasts. The protoplasts
lyse in dilute media, but may be maintained in
protective media containing M/3 sucrose plus
M/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 £. colt K-12 as L growth in penicillin
sucrose agar; these invariably reverted to normal

PROTOPLASTS AND L-TYPE GROWTH OF #. COLI

157

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 F.
colt have all proved to be auxotrophic mutants
requiring diaminopimelic acid, a characteristic
constituent of bacterial cell walls. These mutants,
and another previously isolated by Davis, grow
as L colonies in agar 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. colt 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
occupy definite 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 F~ 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 wall-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

ence of penicillin may be accounted for by a selec-
tive advantage under these conditions of spon-
taneous mutants with wall defects.

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