JOURNAL OF BACTERIOLOGY, Apr. 1973, p. 378-389 Vol. 114, No.

1
Copyright © 1973 American Society for Microbiology Printed in U.S.A.

Gram Characteristics and Wall Ultrastructure of

Arthrobacter crystallopoietes During
Coccus-Rod Morphogenesis
C. M. WARD, JR.,' AND G. W. CLAUS
Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 3 January 1973

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Arthrobacter crystallopoietes growing exponentially as cocci were changed to
rods by adding succinate to the medium. Cells were sampled before, during, and
after this transition for Gram-staining and ultrastructural studies. Cells were
Gram stained by the standardized method of Bartholomew, and all samples
were fixed and prepared for thin sectioning in an identical manner. Cocci were
gram positive, and thin sections demonstrated a gram-positive type of cell wall
having an average thickness of 31 nm. Cells sampled during morphogenesis
appeared as cocci with most having a single rodlike projection. The coccus
portion of these transition cells was gram positive and bound by a gram-positive
type of wall having an average thickness of 29 nm. The rodlike projection of the
transition cells appeared to be gram negative; it was also surrounded by a
gram-positive type of wall, but its average thickness was only 22 nm.
Gram-negative rods of the type species, Arthrobacter globiformis, were also
examined and found to produce a gram-positive type of wall with a 19-nm
average thickness. Evidence for the trilaminar region, characteristic of most
gram-negative bacterial cell walls, was totally lacking in both species. These
results suggest that variations in cell wall thickness may be an important
contributing factor to the variable Gram-staining characteristics of this genus.

Thin sections of gram-positive bacterial cell
walls usually reveal a thick, electron-dense,
rather homogenous structure immediately
overlaying the plasma membrane (12). Al-
though the gram-negative wall is depicted as
several layers, the most distinctive layer ap-
pears as a unit membrane on'the outer portion
of the wall (7, 12). These structural differences
closely parallel large differences in cell wall
chemistry. One might assume that these gross
structural and chemical differences are reflec-
tions of distinct and complex genetic dissimi-
larities; therefore, changes from one Gram-
staining characteristic to another during
growth of a pure culture seems very unlikely.
On the other hand, certain bacteria show
marked changes in Gram-staining characteris-
tics during growth. Gram-staining variability is
characteristic of the genus Arthrobacter (6, 31),
and these variations are often accompanied by
changes in cell shape. The type species, Ar-
throbacter globiformis, is characterized as
I
Present address: Department of Cell Biology, Roche
Institute, Nutley, N.J. 07110.
378
gram-positive cocci that change to gram-nega-
tive rods during exponential growth and then
back to gram-positive cocci when exponential
growth ceases (8). Ensign and Wolfe (10) dem-
onstrated that morphogenesis in A.
crystallopoietes parallels that of the type spe-
cies and can be experimentally controlled by
nutritional manipulation. A. crystallopoietes
cocci are described as gram-variable cocci that
shift to gram-positive rods during exponential
growth (9).
This report examines both the Gram-stain-
ing characteristics and the cell wall ultrastruc-
ture of A. crystallopioetes before, during, and
after coccus-to-rod morphogenesis. These re-
sults are compared with an examination of the
Arthrobacter type species and contrasted with
the generalization concerning the ultrastruc-
ture of gram-positive and gram-negative cell
walls.
MATERIALS AND METHODS
Cultures. A. crystallopoietes ATCC 15481 was
received from the American Type Culture Collection
VOL. 114, 1973 ARTHROBACTER CELL WALLS
in 1970 and maintained on slants containing 0.5%
peptone and 1.5% agar. A. globiformis ATCC 4336
was received from the Department of Microbiology,
Pennsylvania State University (PSU) culture collec-
tion in 1970 and also maintained on peptone-agar
slants. Both Arthrobacter stock cultures were stored
at 4 C and transferred every 3 months. Acetobacter
suboxydans ATCC 621 was obtained from the Ameri-
can Type Culture Collection in 1968, maintained at
-20 C in 66% glycerol (13), and transferred every 4 to
6 months. Staphylococcus aureus ATCC 10390 was
received from the Department of Microbiology, PSU
culture collection in 1970 and maintained at 4 C on
nutrient agar slants. Bacillus megaterium PSU 41
and Pseudomonas aeruginosa PSU 191 were received
in 1971 from the PSU culture collection and main-
tained at 4 C on brain-heart infusion agar slants.
Growth. Subcultures of A. crystallopoietes or A.
globiformis were grown at 28 C in 500-ml Nephelo
culture flasks (Bellco Glass Inc., Vineland, N.J.)
containing 50 ml of 1% peptone (Difco Laboratories,
Detroit, Mich.) reciprocating at 200 strokes per min
with a 3.8-cm amplitude (standard shaking). Cells
from 2 ml of late exponential- or early stationary-
phase cultures were centrifuged and washed three
times with 0.9% saline buffered at pH 7.2 with 0.01 M
potassium phosphate. Cells were then suspended in
1.0 ml of buffered saline, and the entire volume was
used to inoculate 500-ml Nephelo flasks containing
50 ml of a glucose-salts medium containing nitrilo-
triacetic acid (10). Cells were grown in nitrilotriacetic
acid-glucose-salts (NGS) medium at 28 C with stan-
dard shaking. In order to initiate morphogenesis of A.
crystallopoietes, a sterile solution of sodium succi-
nate was added 28 h after inoculation to achieve a 1%
(wt/vol) concentration in the NGS medium.
A. suboxydans was grown at 28 C on the basal
complex medium (3) containing 5% glycerol, and S.
aureus was grown on 0.8% nutrient broth. When
these cultures reached mid-exponential growth, they
were fixed by the addition of 20% (wt/vol) paraform-
aldehyde. After 30 min, 10% (vol/vol) formaldehyde
was used to wash each culture and to store the cells at
4 C. These formaldehyde-fixed cultures were used as
standards for the Gram reaction.
B. megaterium and P. aeruginosa were grown in a
medium containing 0.05% glucose, 0.5% tryptone,
0.5% yeast extract, and 0.5% potassium phosphate
(22). Cultures were harvested by centrifugation after
10 h of growth and immediately were used as
standards for the lysozyme-EDTA experiment.
Gram staining and light microscopy. Each mi-
croscope slide contained the experimental
Arthrobacter cultures and separate smears of the
formaldehyde-fixed gram-positive and gram-nega-
tive standards. The Gram-staining procedure was
carefully standardized according to the procedure of
Bartholomew (1). Reaction times were, in seconds: 60
for crystal violet, 60 with Lugol iodine, 30 with 95%
ethanol, and 30 with safranine. Observations were
made on Arthrobacter cultures only if 98 to 100% of
the formaldehyde-fixed standards yielded the proper
appearance. A Zeiss model WL microscope was
adjusted for Kohler illumination, and all photo-
379
graphic records were made by using a 0.5 neutral
density filter, a 350- to 600-nm band-pass filter, a 1.4
numerical aperture achromatic condenser with the
iris adjusted to the same position for each observa-
tion, a 100x, 1.3 numerical aperture planapochro-
matic objective, and a 1.0 Optovar setting. Kodak
high-contrast copy film was used to record all images,
and this was developed in Kodak D-76.
Electron microscopy. All Arthrobacter samples
were prepared for electron microscopy observation in
an identical manner. One milliliter of 1% OSO4 was
allowed to react with 9 ml of culture for 30 min. Cells
were centrifuged at 2,200 x g for 10 min, suspended
in 1 ml of 1% OSO4 and 0.1 ml of a solution containing
1% tryptone with 0.5% NaCl (18), and then were
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allowed to stand at 24 C for about 16 h. The fixed
cells were removed by centrifugation, mixed with
warm 4% Noble agar, allowed to solidify, and then
were cut into two mm3 blocks. The blocks were
placed into a pH 6.1 solution containing 0.5% uranyl
acetate, 0.4% sodium acetate, 0.6% sodium barbitol,
0.7% sodium chloride and 0.01% calcium dichloride,
were allowed to remain in the 0.5% uranyl acetate for
2 h, and then were dehydrated for 30 min in each of
the following percentages of ethanol: 20, 40, 60, 95,
and 100. Cells were infiltrated for 30 min each in
50:50 and 25:75 mixtures of ethanol-Spurr epoxy
(35) and then for 16 h with 100% Spurr epoxy, and
this was polymerized for 24 h at 70 C. Sections
prepared with the LKB Ultrotome III were stained
with lead citrate (37) and viewed with a Phillips
EM300 by using a 300-gm condenser aperture and a
60-kV accelerating potential. Specimen images were
exposed on Kodak electron microscope film (Estar
thick base) and were developed in D-19.
RESULTS
Growth and controlled morphogenesis. On
unsupplemented NGS medium, cells grew ex-
ponentially with about a 13-h doubling time,
and all cells were observed as cocci during each
growth phase (Fig. 1). On the other hand, when
succinate was added to a 28-h culture of cocci
growing on the NGS medium, the doubling
time was reduced to about 5 h, and the cocci
began to form rods. Ten hours after succinate
addition, all cells in the culture appeared as
long rods (Fig. 1). These results are consistent
with those of Ensign and Wolfe (10) and
confirm that succinate experimentally initiates
the coccus-to-rod morphogenesis.
Characteristics of cocci and rods. Under
carefully controlled staining conditions, A.
crystallopoietes appeared as gram-positive
cocci through all phases of growth on unsupple-
mented NGS medium. A. crystallopoietes rods,
formed after addition of succinate to the NGS
medium, also stained gram positively.
Thin sections of A. crystallopoietes harvested
during exponential growth as cocci or rods
consistently exhibited a cell wall characteristic
380 WARD AND CLAUS J. BACTERIOL.

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1.0 rods---
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02 0.5
transition
stage -w-
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0.3 -
/-- cocci

,i
As I 0.2

do;- U .1, 10 20
-
30 40
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50 60 70
1
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1
90 100
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FIG. 1. Growth and morphology of A. crystallopoietes on nitroltriacetic acid-glucose-salts (NGS) medium.

During exponential and stationary phases of growth on unsupplemented NGS medium (1), cells appeared as
gram-positive cocci (Fig. la). After addition of succinate to the NGS medium, growth was more rapid (x).
Ten hours after succinate addition, the entire population appeared as gram-positive rods (Fig. Ib). A con-
trolled Gram-staining procedure similar to that of Bartholomew (1) was used for all observations. Bar markers
represent 1 gm.
of gram-positive bacteria (Fig. 2 and 3). The were identical to those normally observed only
only ultrastructural difference detected be- in gram-positive bacteria. The trilaminar por-
tween the walls of cocci and rods was a differ- tion (outer membrane) so characteristic of
ence in thickness. The mean thickness of the other gram-negative bacteria was never ob-
coccus wall (Fig. 2a) was 31.1 ± 0.8 nm, and served. In comparing their cell walls with those
the mean thickness of the rod wall (Fig. 3a) was of A. crystallopoietes, two distinct differences
22.7 ± 0.9 nm. These observations indicate were noted. First, the mean cell wall thickness
that the rod cell wall is 27% thinner than that of the gram-negative A. globiformis rod was
surrounding the cocci. only 18.7 ± 0.4 nm: this is about 20% thinner
The distinctly gram-positive nature of A. than the cell walls of A. crystallopoietes rods
crystallopoietes rods conflicts with the reported and about 40% thinner than the cell walls of A.
characteristics of the type species A. crystallopoietes cocci. The second noted differ-
globiformis. For this reason, A. globiformis rods ence was in the affinity of the cell walls for
and cocci were also subjected to the basic electron-dense stains. A. crystallopoietes cell
Bartholomew Gram-staining procedure. Before walls were readily stained by applying 0.4%
morphogenesis, A. globiformis appeared as lead citrate to silver-grey sections for 5 min. On
gram-positive cocci; after morphogenesis, they the other hand, when A. globiformis sections
appeared entirely as gram-negative rods. Thus, were stained in this manner, the cells appeared
A. crystallopoietes rods were distinctly differ- to be surrounded only by a plasma membrane.
ent from A. globiformis in their gram-staining A. globiformis cell walls were detected only
properties, and this suggested a possible differ- after increasing the lead citrate staining time
ence in the chemistry or structure, or both, of to 10 min and following this with 2 h on 2%
their cell walls. uranyl acetate.
Gram-negative cultures of A. globiformis Effect of lysozyme and ethylenedia-
rods were fixed and embedded in a manner minetetrnacetate on cocci and rods.
identical to that used with A. crystallopoietes. Arthrobacter rods and cocci were compared
The ultrastructural appearance of the cell wall with known gram-positive and gram-negative
(Fig. 4), the appearance of the septum in standards in their susceptibility to lysozyme
dividing cells, and the mesosome structure and ethylenediaminetetraacetate (EDTA).
 :a~~_P
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1. S't V " . 'I ., =
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4~~~~S

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membrane (PM), appears to be typical of walls normally found surrounding gram-positive cel.-oizna
bar marker represents 0.1 ,im. That part of the cell enclosed by brackets is enlarged (Fig. 2a), and this
enlargement includes a 31-nm bar marker representing the mean thickness of the coccus cell wall.
FIG. 3. Longitudinally sectioned A. crystallopoietes rod representative of a population harvested from
nitrilotriacetic acid-glucose-salts medium 10 h after the addition of succinate. The cell wall immediately
surrounds the plasma membrane (PM) and has characteristics typical of gram-positive cells. Horizontal bar
marker represents 0.1 ,um. That part of this rod enclosed by brackets is enlarged (Fig. 3a) and is typical of the
23-nm mean wall thickness surrounding the rod-shaped cells. Fig. 3a also includes the 31-nm bar marker
representing the mean thickness of the coccus cell wall.
381
382 WARD AND CLAUS J. BACTERIOL.

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FIG. 4. Longitudinally sectioned A. globiformis rod representative of a gram-negative, mid-exponential-
phase culture growing in nitrilotriacetic acid-glucose-salts medium. Sections had to be stained with 0.4% lead
citrate for 10 min and by 2% uranyl acetate for 2 h before the wall material could be visualized. Cells contained
numerous examples of gram-positive type mesosomes (M). Horizontal bar marker represents 0.1 MAm. The
inset enlarges that part of the rod enclosed in brackets showing the mean 19-nm wall thickness, and it includes
the 31-nm bar marker representing the mean wall thickness for A. crystallopoietes cocci. Although these rods
exhibited a gram-negative staining reaction, a trilaminar region typical of sectioned gram-negative walls was
never observed.
Table 1 shows that EDTA had no effect on the Six hours after succinate addition, both the
extent of lysis of gram-positive A. coccus and the attached, fully formed rods
crystallopoietes rods or cocci or the gram-posi- routinely gave a gram-positive staining reac-
tive standard, but EDTA did enhance lysis of tion. The consistency with which the newly
the gram-negative standard. The gram-nega- forming projections yielded a gram-negative
tive A. globiformis rods were not lysed more appearance suggests that the newly forming
readily in the presence of EDTA. These data cell wall was different from that of the parental
support the ultrastructural observations show- coccus.
ing a gram-positive type of cell wall surround- Thin sections of cells harvested 3 h after
ing the rods of both Arthrobacter species. succinate addition (transition cells) frequently
Characteristics of A. crystallopoietes dur- revealed a shape identical to cells observed by
ing morphogenesis. Although fully formed light microscopy (Fig. 6 and 7). The entire cell
cocci or rods of A. crystallopoietes stained gram wall surrounding the transition cells always
positively, this characteristic was not main- appeared to be the gram-positive type: rather
tained during the transition from cocci to rods. homogeneous, electron dense, and lacking a
Cells sampled 3 h after addition of succinate trilaminar layer (outer membrane). However,
stained as gram-positive cocci with predomi- differences in cell wall thickness were always
nant gram-negative projections (Fig. 5), and apparent when the transition cell coccus was
this appearance continued for the next 2 h, compared with its rodlike projection. The mean
during which time the projections elongated. thickness of cell wall surrounding the coccus
 TABLE 1. Cell wall degradation of Arthrobacter after additions of lysozyme or ethylenediaminetetraacetate,
or both
Effect of additions on culture turbidity
Cells Culture media
and agea
Gram reaction
and morphologyLyome
(AOD)b ET
Lysozyme +yszyeDTA T
A.
crstalopoites
NS 28h Grm (+)cocc 0.5 0.5 0.06
A. crystallopoietes NGS/ 33h Gram c+odsi 0.59 0.60 0.06
A. globiformis NGS 12 h Gram ()rods 0.27 0.23 0.11
Bacillus megaterium TYPG 10 h Gram (+ r6ds 0.23 0.23 0.13
Pseudomonas aeruginosa TYPG 10 h Gram (-) rods +0.09 1.27 0.41
aCultural conditions are described in Materials and Methods. Media used: glucose-salts medium
containing nitrilotriacetic acid (NGS); NGS plus the addition of sodium succinate 28 h after inoculation
(NGS/S); and 0.5% tryptone, 0.5% yeast extract, 0.5% potassium 'phosphate, and 0.05% glucose medium

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(TYPG).
bChanges in optical density values (A GD) represent decreases, except where indicated, in the turbidity of
reaction mixtures. All reaction mixtures contained 0.5 ml of 0.1 M tris(hydroxymethyl)aminomethane buffer,
pH 7.5; 0.5 ml of cell suspension so that the total reaction mixture would initially be about 1.0 GD, and water
to bring each mixture to a volume of 3.0 ml. Additions were 0.4 ml of 0.25 mg of lysozyme/ml and 0.2 ml of 8 mg
of ethylenediaminetetraacetate (EDTA) per ml adjusted to pH 7.5.

dr '4

Gn)Standard-
I
0 w
.9
Sb

*0
*

V 0
FIG. 5. Gram-stained populations of A. crystallopoietes during transition between cocci and rods 3 h after
succinate addition to nitrilotriacetic acid-glucose-salts medium. The appearance of transition cells is
compared with the gram-positive and gram-negative standards. All microscopy and photography procedures
used both here and in Fig. I were identical. The coccus portion of transition cells gave a strong gram-positive
reaction and appears in these photographs to be very dark. In contrast, the rod portion appeared colored only
by the counter stain and appears to be much lighter here than the gram-positive portion of the cell. Horizontal
bar markers represent 1 rim.
383
384 WARD AND CLAUS J. BACTERIOL.

a b

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A

'r .
; ,c
I
-t
'i
- . ". t

I
ftN.

FIG. 6. Longitudinally sectioned A. crystallopoietes sampled during transition between cocci and rods 3 h
after succinate addition to nitrilotriacetic acid-glucose-salts medium. The cell appears more rounded at one
end and more elongated at the other. The cell wall, at both ends of the transition cells appears to be the
gram-positive type, and they appear to differ only in thickness. Horizontal bar marker represents 0.1 um. The
enlargement of the wall surrounding the rod portion (Fig. 6a) and of that surrounding the coccus portion (Fig.
6b) are compared with the 31-nm bar marker.
portion was 28.8 + 0.5 nm, whereas that than those around the fully formed rod and the
surrounding the rodlike projection was 21.5 + transition cell rod projection. Although the wall
0.3 nm. ultrastructure appeared similar, the walls sur-
Cell wall thickness data is summarized in rounding the gram-negative cells were signifi-
Table 2. All wall measurements were subjected cantly thinner than those surrounding the
to Student's t test (33) and found to be sig- gram-positive cells.
nificantly different with a 99.5% certainty at Longitudinal sections through transition
the 0.01 level of significance. The walls sur- cells during an early and late stage of division
rounding the fully formed coccus and the are depicted in Fig. 8. The apparent method of
transition cell coccus were considerably thicker septum ingrowth (Fig. 8) and the structure of
VOL. 114, 1973 ARTHROBACTER CELL WALLS 385

-
-
-
-

r
F

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FIG. 7. Longitudinally sectioned A. crystallopoietes sampled during transition from cocci to rods. Both
cells demonstrate the extent of cell elongation possible before cell division and the compact chromatin (C). In
addition to the cell wall appearance, the mesosome (M) also is typical of gram-positive cells. Horizonta' bar
marker represents 0.1 gim.

mesosomes (Fig. 7 and 8) in A. crystallopoietes DISCUSSION

are both typical of gram-positive bacteria. The It is generally agreed that Arthrobacter spe-
asymmetrical position of the newly forming cies can exist as either rods or cocci, yet there
division septum was noted only in cultures appears to be no agreement on the Typical
undergoing morphogenesis. Gram characteristics of these forms. The rods
386 WARD AND CLAUS J. BACTERIOL.
TABLE 2. Cell-wall thickness and Gram reaction of Arthrobacter crystallopoietes and A. globiformis
Medium (culture No. of No. of Average wall
Cells M Morphology cells measure- thickness Gram
e )a
measured ments (nm)b rreactiont

A. crystallopoietes NGS (28 h) Cocci 12 66 31.1 ± 0.8 +

NGS/S (31 h) Coccus portion of tran- 48 297 28.8 ± 0.5 +
sition cells
NGS/S (31 h) Projection portion of tran- 48 350 21.5 ± 0.3
sition cells
Peptone (8 h) Rods 29 121 22.7 ±0.9 +
A. globiformis NGS (12 h) Rods 50 226 18.7 ± 0.4

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a Cultural conditions are described in Materials and Methods. Media used: glucose-salts medium
containing nitrolotriacetic acid (NGS); NGS plus the addition of sodium succinate 28 h after inoculation
(NGS/S); and 1% peptone.
bMeasurements on enlarged prints of thin-sectioned cells were taken only where the plasma membrane was
slightly pulled away from the inside surface of the cell wall. Transition cells were divided in thirds
perpendicular to their long axis, and measurements were taken randomly along the wall and along all but the
middle third of the cell. By using Student's t test (33), it was found that each measurement listed above was
significantly different from all others with a 99.5% certainty at the 0.01 level of significance.
' Gram reaction method was the carefully controlled procedure of Bartholomew (1).
of various species have been described as gram typical of gram-positive bacteria.
negative (8, 23), gram negative with gram-posi- The evidence presented here demonstrates
tive granules (21, 29), gram negative to gram that Arthrobacter is surrounded by an ultra-
variable (5), "predominately" gram positive structurally typical, gram-positive cell wall.
(29), and gram positive (9, 16, 17). Cocci have Nevertheless, carefully controlled Gram-stain-
been described as "generally" gram negative ing procedures show that certain forms of
(23), gram variable (9), gram variable to gram Arthrobacter are gram negative. In order to
positive (5), gram positive to gram negative explain these results, one must consider what is
with positive granules (17), "predominately" known about the chemistry and anatomy of the
gram positive (29), and gram positive (8, 16, bacterial cell wall and their relation to the
21). On the basis of this literature, it seems Gram reaction.
that the Gram reaction does not reflect a stable In contrast to gram-positive walls, typical
characteristic of this genus, nor does it justify gram-negative walls contain a lipoprotein
assumption of a set of ultrastructural charac- membrane and much less peptidoglycan (27).
teristics for Arthrobacter that is normally as- Treatment with 95% ethanol presumably
sociated with either gram-positive or gram- removes the wall lipid and concentrates the
negative bacteria. wall peptidoglycan through dehydration. The
Four observations presented here suggest altered wall that remains does not prevent
that A. globiformis, the type species, and A. rapid outward diffusion of the solubilized crys-
crystallopoietes are cytologically typical of the tal violet-iodine complex (CV-I), and therefore
gram-positive bacteria. First, the appearance the CV-I is rapidly lost to the surrounding etha-
of sectioned walls is like that of other gram- nol. Any alterations in the cell's peripheral
positive bacteria in that it consistently lacks structure that would slow or prevent this out-
the distinct outer membrane that is typical of ward diffusion might allow part or all of the cell
gram-negative bacteria (7, 12). Secondly, cross to appear gram positive. For example, Cardio-
walls formed during division of both species bacterium hominis (32) stains as a gram-nega-
resemble those found only in gram-positive tive rod with gram-positive regions occurring
bacteria (15, 36). Thirdly, lysozyme easily particularly at the cell poles. Ultrastructural
degrades the wall and allows lysis in the studies show C. hominis surrounded by a typi-
absence of EDTA. Finally, the mesosomes cal gram-negative wall, but these cells also con-
found within these species are structurally tain peripheral layers of membrane inside the
similar to the mesosomes of gram-positive plasma membrane, especially at the poles (24).
bacteria (26, 36). The only other known fine- Most experimental results confirm that the
structural study on Arthrobacter is that of Gram reaction is dependent upon the relative
Stevenson (35) who demonstrated that A. rates of CV-I diffusion from the cell in ethanol
pascens also exhibits cytological characteristics (2, 28, 30); therefore, the additional mem-
 ~ ~ ~b
VOL. 114, 1973 ARTHROBACTER CELL WALLS 387

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_
~~~~a

~
~ ~ ~ ~ ~ ~ ~ ~ ~ W

FIG. 8. Sections of A. crystallopoietes sampled during morphogenesis on nitrilotriacetic acid-glucose-salts

medium 3 h after succinate addition and demonstrating two stages in the division process. The asymmetrical
position of the newly forming septum is typical of dividing cells during the transition stage. The appearance of
the newly forming septum and the completed septum are characteristic of gram-positive cells. Horizontal bar
markers represent 0.1 pm.
branes stacked along the C. hominis periphery bly more wall material to be concentrated by
at the poles could retard CV-I diffusion and dehydration in ethanol, the thick gram-positive
make these areas appear gram positive. wall is thought to present a more formidable
In comparison with the gram-negative wall, barrier to CV-I diffusion. However, the thick-
the wall of gram-positive bacteria contains no ness of the gram-positive cell wall is reported to
lipid and more amino sugars (peptidoglycan), vary from 15 to 80 nm (12, 27), and evidence
and it is usually much thicker (27). Because available to date suggests that cells having
there is no lipid to be removed and considera- walls at the low end of this spectrum often stain
388 WARD AND CLAUS
gram negative. For example, Haemophilus
vaginalis has an ultrastructurally typical gram-
positive wall that is only 15-nm thick, and
these cells stain gram negative to gram varia-
ble (25). Our data show a similarly appearing
19-nm wall around A. globiformis, and these
cells consistently stain gram negative. On the
other hand, our studies on the structurally
typical gram-positive wall of A.
crystallopoietes rods and cocci demonstrate a
23- and 31-nm wall thickness, and these cells
consistently stain gram positive (Table 2).
These data suggest that the thickness of the
typical gram-positive cell wall will influence its
ability to retard CV-I diffusion during the
Gramr-staining procedure.
The rate of CV-I diffusion from the bacterial
cell is not an all-or-none phenomenon, but
relatively fast or slow depending upon the type
of cell being stained (2, 28, 30). This rate is
usually constant for all cells in a pure culture,
but certain bacteria will exhibit both gram-posi-
tive and gram-negative cells on the same slide,
and these cultures are called "gram variable."
This characteristic is often given to older cul-
tures that were uniformly gram positive during
their exponential growth. Young cultures of
certain bacteria have also been described as
gram variable, but there are only a few of these
cases where we also have information on their
wall ultrastructure or chemistry. A. globiformis
rods and H. vaginalis are described as gram
negative to gram variable (5, 11, 25), and both
are surrounded by very thin walls of the gram-
positive type (Table 2, 25). Our results sug-
gest that these wall thicknesses may be near
or below that necessary to consistantly yield
a gram-positive appearance. At this lower
limit, slight variations in wall thickness from
cell to cell may be enough to account for a
gram-variable appearance in young cultures.
Our data showing a 7-nm (25%) difference in
wall thickness between the coccus and rodlike
projection of transition cells, and a correspond-
ing difference in gram reaction, support this
possibility. Nevertheless, there is a question of
whether Arthrobacter species are gram variable
(4, 5, 8, 9, 31). Our results show that A.
globiformis rods stain uniformly gram nega-
tive, that A. crystallopietes rods and cocci are
consistently gram positive, and that the stand-
ard deviation for the mean wall thickness of
each cell population is very small (Table 2).
In addition to its effect on the gram reaction,
cell wall thickness in Arthrobacter may be
correlated with growth rate. Ensign and Wolfe
(10) demonstrated that growth of A.
crystallopoietes rods is faster than their growth
J. BACTERIOL.
as cocci. Our results confirm this (Fig. 1) and
also show that rods maintaining this faster
growth rate are surrounded by a thinner wall
than that which surrounds the cocci (Table 2;
Fig. 2, 3). Other Arthrobacter species form
rods only during periods of exponential growth,
and this report demonstrates that rods of the
actively growing type-species are very thin.
Since less wall material surrounds the faster
growing rods, it appears that cell wall synthesis
is not directly proportional to growth rate.
Others have reported that nongrowing gram-
positive bacteria have thicker walls than those
Downloaded from http://jb.asm.org/ on September 12, 2015 by guest
growing exponentially (12).
It is generally concluded that the wall
strength needed to maintain a rod-shaped cell
is greater than that necessary for maintenance
of a sphere within an environment of low
osmolarity. Yet in this study we found that
Arthrobacter rods are surrounded by thinner
walls than those surrounding their cocci. If we
assume that the observed decrease in thickness
during coccus-to-rod morphogenesis is also ac-
companied by a corresponding decrease in
peptidoglycan concentration, then either the
peptidoglycan remaining around the thinner
walls has sufficient strength to maintain the
rod shape or the rod peptidoglycan may be
chemically changed to compensate in strength
for the reduced quantity. Krulwich et al. (19,
20) have found chemical differences between
the rod and coccus peptidoglycan of A.
crystallopoietes. Although the interpeptide
cross-linking within the polymer from each cell
type was similar, they found that the polysac-
charide backbone of the peptidoglycan isolated
from the rod wall was three to four times longer
than that isolated from the coccus wall. They
suggested that this chemical difference could
make the rod walls more rigid than those of the
cocci. If this chemical change accompanies
morphogenesis, then it could account for the
extra strength needed to maintain the thinner
rod wall.
Cultures sampled shortly after initiating
morphogenesis showed many transition cells
that lacked a division septum (Fig. 6, 7). Thus,
it appears that the beginning of coccus-to-rod
morphogenesis precedes the initiation of cell
division. When a division septum is evident
(Fig. 8), the shape of the transition cell sug-
gests that the newly forming rod is a linear
extension of the coccus perpendicular to the
plane of the newly forming septum. The lead-
ing edge of this rodlike projection is surrounded
by a wall that is thinner than that around other
areas of the transition cell. Higgins and Shock-
man (14, 15) demonstrated that the newly
VOL. 114, 1973 ARTHROBACTER CELL WALLS
forming wall in dividing Streptococcus is adja-
cent to the invaginating septum, that it is 18.
thinner than the older wall, and that its linear
extension is followed by wall thickening. It will
be interesting for future investigators to deter-
mine whether the leading edge of Arthrobacter
transition cells is also a region active in wall 19.
biosynthesis.
ACKNOWLEDGMENTS
This investigation was supported in part by grant 20.
GB-17493 from the National Science Foundation.
We gratefully acknowledge Carol Ann Baker for her
technical assistance in preparing this manuscript.
21.
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