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Sarcobium Zyticum gen. nov., sp. nov., an Obligate Intracellular
Bacterial Parasite of Small Free-Living Amoebae?
WINCENTY J. DROZANSKI
Department of General Microbiology, Maria Curie-Skiodowska University, 20-033 Lublin, Poland
A new genus and species of obligate intracellular bacterial parasite of small free-living amoebae is described.
This bacterium causes fatal infections in amoebae belonging to the Acanthamoeba-NaegZeria group. It does not
grow on any artificial substrate deprived of living amoeba cells. The entry of the bacterium into a host occurs
by phagocytosis, but growth occurs in the cytoplasm, not in phagosomes. This parasite is readily distinguished
from other kinds of previously recognized bacteria that live within amoeba cells on the basis of its host cell lytic
activity. The bacterium is a gram-negative, short rod with tapered ends. It multiplies intracellularly by a
transverse central pinching-off process. Cells are motile by means of a polar tuft of flagella. The bacterium is
surrounded by a distinct multilayered cell envelope with a chemtype A,y peptidoglycan. The peptidoglycan is
unique in its high content of glucosamine residues with free amino groups. The DNA base composition of this
organism is 43 mol% guanine plus cytosine. The name Sarcobium Zyticum gen. nov., sp. nov. is proposed for
this bacterium. The type strain of S. Zyticum is strain L2 (= PCM 2298).
More than 30 years ago a new obligate intracellular
bacterial parasite (OIBP) which causes fatal infections of
hosts was found in a raw culture of amoebae that were
freshly isolated from soil (7). This OIBP was isolated from
soil from the Lublin area, Poland. In previous reports I have
described the infection processes for Acanthamoeba castellanii, Hartmannella rhysodes, Hartmannella astronyxis,
Mayorella palestinensis, Didasculus thorntoni, Schizopyrenus ruselli, and Naegleria gruberii, as well as 41 other
unclassified amoebae isolated from soil and water reservoirs
(8, 10). The OIBP did not grow on any medium when living
amoebae were not present. Even a thick suspension of
disrupted amoeba cells was ineffective. It has been shown
that the OIBP cannot multiply if after infection the mixed
culture is heated at 43°C for 10 min; this treatment is
sufficient to kill trophic forms of the amoeba, but the
bacterium survives. Microscopy has shown (8, 11).that the
parasites are readily phagocytized by amoebae and that their
immediate place of residence is within phagosomes. The
neighboring phagosomes fuse extensively with parasite-containing vacuoles; however, even dead bacteria evade lysosoma1 attack because the cell wall is resistant to the host’s
bacteriolytic enzymes. Later, living bacteria escape from
phagosomes into the cytoplasm, where multiplication occurs. The final effect of the infection is total lysis of the host
cells. Although the morphology of the OIBP and the presence of peptidoglycan in its cell wall suggest that this
organism is a bacterium, it has not been possible to classify
it previously because of a lack of nutritional tests; the OIBP
does not correspond to any bacterium described in Bergey’s
Manual of Determinative Bacteriology (3). Most of the many
kinds of previously recognized bacteria that live within
amoeba cells behave as temperate endosymbionts, and some
appear to provide benefit to the hosts (1,4,15-18,23,25). To
my knowledge, the only exception is the OIBP, which
causes lysis of the amoeba cells. This organism was recog-
nized as being unusual, and therefore I decided to attempt to
classify it.
MATERIALS AND METHODS
Organisms and isolation procedure. The OIBP, the causative organism of fatal infections of small free-living amoebae belonging to the Acanthamoeba-Naegleria group, was
found in a raw culture of amoebae that were isolated from
soil from the Lublin area, Poland (7)’ by using the enrichment method. Crumbs of soil were placed onto the surface of
a non-nutrient agar plate covered with a thin film of a thick
suspension of Aerobacter aerogenes and incubated at room
temperature for 8 to 10 days. In this way the growth of all
microorganisms except those that feed on bacteria or parasitize bacterium feeders was checked. When this procedure
was used, several different isolates which could infect amoebae were obtained. The isolate described in this paper was
designated strain L2T (T = type strain). Strain L2T was
purified from contaminating bacteria by allowing infected
amoebae to migrate on agar surface as described previously
(8). A two-member (host-parasite) culture was established
by growing infected amoebae in PYG medium (8).
Acanthamoeba casteltanii, which was obtained from W.
Balamuth, Department of Zoology, University of California,
was used as a host for maintenance of the OIBP.
Propagation of the OIBP. The OIBP was propagated by
transferring a small amount of lysate from an ipfected culture
into a 2-day-old culture of amoebae grown on PYG medium
as described previously (8, 24). Bacteria were also propagated on amoeba cells harvested from an axenic culture at
the exponential phase of growth and suspended in saline
prepared as described by Band (2). The OIBP propagated on
amoebae suspended in saline produced more uniform cells
and survived for a longer period of time than the OIBP
propagated on amoebae in PYG medium (9).
Microscopic observations. Observations on the course of
infection were made with living materials by using phasecontrast microscopy and fixed preparations stained by the
methods of Macchiavello (5) and Gimenez (15). The stains
used for bacteria liberated from their hosts were the stains
t Dedicated to W. J. H. Kunicki-Goldfinger on the occasion of his
70th birthday in friendship and admiration.
82
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FIG. 1. Phase-contrast micrograph of Acantharnoeba castellanii
2 h after infection with the OIBP, showing enlarged parasitophorous
vacuoles containing a few parasites. Bar = 10 pm.
described by Norris and Swain (22). For transmission electron microscopy, infected amoebae were fixed in amoeba
saline supplemented with 1.5% glutaraldehyde and postfixed
in Veronal-acetate-buffered OsO,. Following dehydration in
ethanol, pellets packed by centrifugation were embedded in
FIG. 3. Electron micrograph of a section through an AcanVestopal W (Serva). Thin sections of embedded specimens
tharnoeba c a s t e l l h i cell infected with the OIBP, showing an
were cut with a Tesla model BS490A ultramicrotome and
vacuole containing a few bacteria and
~ lead citrate.
~
sam~
~enlargedl parasitophorous
d
stained with uranyl acetate and ~
structures. Bar = pm.
ples for negative staining with sodium phosphotungstate
FIG. 2. Phase-contrast micrograph of Acantharnoeba castellanii
8 h after infection, showing one large lacuna (long arrow) filled with
the bacteria and several enlarged parasitophorous vacuoles (short
arrows). Bar = 10 pm.
were prepared as described previously (12). Grids were
examined with Zeiss model DZ and Tesla model BS613
electron microscopes that were operated at 50 or 80 kV.
Oxygen consumption measurements. A conventional Warburg respirometer was used for oxygen consumption measurements. Each flask usually received 4 x lo1' OIBP cells
suspended in saline and substrate to bring the total volume to
2.0 ml. The bacteria were prepared by the procedure described below. A 2-day-old axenic culture of Acanthamoeba
castellanii, which had reached a concentration of 3 X lo6
cells per ml, was infected with the OIBP. The ratio of
bacteria to amoebae at the time of infection was approximately 7:l. The infected amoebae were incubated on a
shaker at 28°C. Bacteria liberated from the infected amoebae
were pelleted and freed from cysts and unbroken host cells
by fractional centrifugation at 120 and 3,000 x g, resuspended in saline, and starved for 2 h on a shaking water bath.
Respiration measurements were carried out at pH 7.4 and
25°C for 2 h with a gas phase of air. CO, was trapped with 0.2
ml of 20% KOH.
DNA extraction and estimation of base composition. DNA
was extracted from bacteria frozen in saline (0.15 M NaC1,
0.1 M EDTA, pH 8.0) after disruption of the cells in 2%
sodium dodecyl sulfate at 60°C and then purified by using the
method Of Marmur (20)*The purified DNA
were
dissolved in ssc (0.15 M NaCl Plus 0.015 M sodium citrate)
and stored at 2°C Over chloroform. Base ComPosition was
determined by performing a melting point analysis, using the
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INT. J. SYST.BACTERIOL.
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FIG. 5 . Micrograph of Acantharnoeba castellanii, showing bacteria surrounded by electron-transparent zones. Bar = 1 pm.
FIG. 4. Micrograph of Acanthamoeba castellanii, showing a
parasitophorous vacuole with a stream of cytoplasm inside (arrowheads). Bar = 1 pm.
method of Marmur and Doty (21) and DNA from Escherichia
coli (51 mol% guanine plus cytosine) as a reference.
RESULTS
Microscopic observations. (i) Microscopy of infected amoebae. Typical Acanthamoeba castellanii cells which were
infecied with the OIBP are shown in Fig. 1and 2. At 2 h after
infection the cells remained remarkably intact. When phasecontrast microscopy was used, differentiation of the cytoplasm into ecto- and endoplasmic layers was clearly observed. Also, the nuclei and other cell structures appeared to
be physically undamaged. However, the food vacuoles bearing bacteria became enlarged, yet they were not filled with
OIBP cells. Within 6 to 9 h after infection, although the
bacteria were present in almost all of the food vacuoles, no
distinct morbid changes in host cells were observed. The
differentiation of ectoplasm and endoplasm was still maintained, and the host nuclei were prominent and appeared to
be physiologically intact. The amoebae moved normally,
collected food, and even reproduced; however, the invading
bacteria had increased in numbers and occupied a large area
of the host endoplasm. In the later stages of infection some
bacteria remained in the parasitophorous vacuoles, but most
of them were found in the cytoplasm, where lacunae filled
with parasites were formed. In heavily infected cells the
organelles were displaced by bacteria. In the last stage of
infection the host cells took on a ball-shaped form and were
filled with rapidly moving bacteria. The rupture of each
plasma membrane liberated about 2 x lo3 bacteria into the
environment.
An examination of ultrathin sections of Acanthamoeba
castellanii by transmission electron microscopy revealed
that soon after infection bacteria engulfed by host cells were
tightly surrounded by membranes of endocytic vacuoles.
Later in infection the parasitophorous vacuoles increased to
abnormal sizes because of the fusion of neighboring endocytic vacuoles containing bacteria with each other and
with lysosomes (Fig. 3). At this stage of infection no division
figures of bacteria were observed, and parasitophorous
vacuoles were filled with membranelike structures scattered
in an electron-translucent background, as well as with a few
bacteria. Later in infection the membranes of bacteriumbearing parasitophorous vacuoles underwent lysis, and parasites escaped into the cytoplasm (Fig. 4 and 5 ) . The
electron-translucent space surrounding bacteria gradually
underwent resorption, and as a consequence bacteria came
into direct contact with the host cytoplasm, where they were
clearly distinquished from other subcellular components
(Fig. 6). Once the bacteria were inside the cytoplasm,
multiplication started. In the last stage of infection lacunae
filled with dividing bacteria increased in size, and the proliferating parasites disarranged the host cell structure, causing
autolysis of mitochondria and disruption of the plasma
membrane (Fig. 7). Division of the parasites was not observed outside the amoebae cells; however, the liberated
bacteria remained alive for several months after being released from their hosts.
(ii) Morphology and fine structure of the OIBP. The bacteria liberated from host cells were straight rods with tapered
ends. The mean size of the OIBP was calculated from
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85
FIG. 6 . Micrograph of Acanthamoeba castellanii, showing bacteria scattered in the host cytoplasm (arrows). Bar = 1 km.
FIG. 7. Micrograph of Acanthamoeba castellanii, showing lacunae filled with bacteria. Bar = 2 Fm.
organisms stained with phosphotungstic acid, was 0.6 pm
wide by 1.9 pm long. Cells more than 5.0 pm long generally
occurred in poorly aerated cultures. The surfaces of negatively stained bacteria were electron opaque and smooth. On
cells immediately liberated from their hosts tufts of flagella
on one pole were observed (Fig. 8). The maximum number
of flagella observed at a single tuft was five. Phase-contrast
microscopic observations of bacteria freshly liberated from
hosts revealed the presence of motile rods. Motility occurred
within the hosts and decreased rapidly after the bacteria
were liberated into the ambient medium. The bacterium
were gram negative. Neither capsules nor spores were
observed. Thin sections showed that the OIBP was essentially a gram-negative bacterium with a trilaminar outer
envelope and a cytoplasmic membrane (Fig. 9). With the
technique used no definite structures were observed in the
cytoplasm that might be interpreted as spores, beta-hydroxybutyric acid, or starch granules. The cytoplasm contained an
obvious nucleoplasm which appeared to be a filamentous
skein distributed along the entire length of the bacterium.
The part of the cytoplasm that was not occupied by the
nucleoplasm contained numerous small electron-dense granules that probably represented ribosomes.
Peptidoglycan. The procaryotic nature of this organism
was established by the discovery in its cell wall of compounds that are regarded as specific to bacteria (e.g., the
bag-shaped peptidoglycan composed of glucosamine, muramic acid, alanine, glutamic acid, and diaminopimelic acid
in a molar ratio of 0.4:0.4:1.4:1:1)(12).The peptidoglycan
was completely insensitive to lysozyme and bacteriolytic
N,O-diacetylmuramidase of amoeba1 origin. The resistance
to the bacteriolytic enzymes was presumably due to the large
content of glucosamine residues with free amino groups in
the glucan strands of the peptidoglycan (13).
Oxygen consumption. Purified suspensions of the OIBP
exhibited metabolic activity, as shown by consumption of
oxygen and production of carbon dioxide in the presence of
disrupted amoeba cells or PYG medium. Freshly collected
bacteria in buffered saline had an endogenous Qo, of 24 k 3
Fl per lolo cells ( Q ~ ? ( lolo
~ cells) = pl of O2 taken up per 1
x lo1' cells per h). A homogenate of Acanthamoeba casteflanii prepared from a suspension containing 2 x lo7 cells per
ml did not consume 0, but stimulated respiration of the
OIBP fivefold compared with endogenous respiration. Glucose and related sugars, as well as glutamate and amino
acids synthesized by Acanthamoeba castellanii or included
in the synthetic medium to support the growth of amoebae
(6), were not metabolized by the OIBP. KCN at a final
M invariably inhibited all respiraconcentration of 5 X
tory activities of the OIBP.
DNA base composition. The DNA of the OIBP contained
43 mol% guanine plus cytosine as determined by thermal
melting point analysis.
DISCUSSION
Certain species of free-living amoebae harbor microorganisms. Many of these microorganisms are the same size as
ordinary bacteria, and some have been identified as archaeobacteria. Unfortunately, because of their specialized ecological niche, none of these organisms can be cultivated apart
from the eucaryotic cells which they inhabit. Consequently,
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INT. J. SYST.BACTERIOL.
DROZANSKI
FIG. 8. Electron micrograph of negatively stained OIBP with a
tuft of flagella at one pole. Bar = 1 pm.
little information that permits assessment of the taxonomic
affinities of these organisms has been obtained. However,
the assignment of names to organisms which have not been
cultivated in vitro is feasible partly because of our increasing
knowledge of the bacteria living inside eucaryotic cells and
partly because of the fact that the most reliable taxonomic
characteristics are DNA base ratio and the chemical cornposition of cell walls. Most of the previously described
bacteria which inhabit amoeba cells have one feature in
common; namely, they behave as temperate parasites. Some
even provide benefit to the host. The only exception to my
knowledge is the OIBP described in this paper. The uncontrolled growth pattern of this organism in the cytoplasm of
amoebae belonging to Acanthamoeba-Naegleria group, its
DNA base composition, and the chemical makeup of its cell
wall peptidoglycan are sufficient to warrant placing it in a
new genus. The proposed new genus Sarcobium does not
appear to belong to any of the currently recognized families
of bacteria.
Description of Sarcobium gen. nov. Sarcobium (Sar. co’ bi.
urn. Gr. n. sarcos, flesh; Gr. n. bios, life; M. L. neut. n.
Sarcobium, that which lives in the sarcode or flesh [cytoplasm]). Rods that are 0.6 by 1.9 pm. Gram negative. Motile
by means of three to five polar flagella. Cell division occurs
by a transverse central pinching-off process. The DNA base
composition is 43 mol% guanine plus cytosine, as determined by the thermal denaturation method. Chemotype A,?
peptidoglycan with a free amino group on the glucosamine
residues. Obligate intracellular parasite of amoebae belonging to the Acanthamoeba-Naegferiugroup and related protozoa (e.g., Dictiostelium discoideum). The bacterium is
able to escape from parasitophorous vacuoles into the cytoplasm, where it proliferates.
FIG. 9. Electron micrograph of a section through cells of the
OIBP. The ultrastructure of this bacterium resembles that of gramnegative bacteria. Bar = 1 pm.
Description of Sarcobium lyticum sp. nov. Sarcobium lyticum (ly’ ti. cum. M. L. neut. adj. lyticum, that which causes
lysis). Description as for the genus. Type species of the
genus Sarcobium. The type strain of Sarcobium lyticum is
strain L2,T which has been deposited in a two member
culture with the Polish Culture Collection of Microorganisms
as culture PCM 2298. The description of the type strain is the
same as that given above for the genus and species, and, in
the absence of cultivation in pure culture, this description
forms the nomenclatural type Rule 18a of the International
Code of Nomenclature of Bacteria [19]).
ACKNOWLEDGMENT
This work was supported in part by a grant from the Committee of
Microbiology of the Polish Academy of Sciences.
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