Vol. 37, No. 4
INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Oct. 1987, p. 456457
0020-7713/87/040456-02$02.OO/O
Copyright 0 1987, International Union of Microbiological Societies
Polynucleobacter necessarius gen. nov. sp. nov. an Obligately
Endosymbiotic Bacterium Living in the Cytoplasm of Euplotes
aediculatus
KLAUS HECKMANN" AND HELMUT J . SCHMIDT
Zoologisches Institut der Universitat Miinster, 04400 Munster, Federal Republic of Germany
Obligately endosymbiotic bacteria living in the cytoplasm of ciliates of the genus Euplotes constitute the new
genus Polynucleobacter gen. nov. These endosymbionts are commonly known as omicron and omicronlike
particles. The best-studied form, as well as the type species, is omicron from stock 15 of Euplotes aediculatus.
We propose the name Polynucleobacter necessarius gen. nov., sp. nov., for this bacterium.
particle, which is equivalent to 3.5 X lo9 daltons. Taking the
average number of nucleoids per particle into account, a
DNA content of 0.5 x lo9 daltons per nucleoid is calculated.
This value is in close agreement with the value for the kinetic
complexity of the DNA, which was determined to be 0.57 X
lo9 daltons when corrected for G+C content (5). It was
therefore concluded that P. necessarius has a small genome,
about one-fourth the size found in Escherichia coli, and that
each symbiont contains multiple copies of the genome,
apparently one genome copy per nucleoid.
It is possible to remove the symbionts from E. aediculatus
by treating a rapidly growing culture with 100 to 500 U of
penicillin per ml for 5 to 6 days. Aposymbiotic hosts may
undergo one or two fissions but then stop multiplying and
eventually die about 15 to 20 days after their last fission. The
same results were obtained with several other antibiotics or
sufficiently high doses of X rays (2). Reinfection and rescue
of E. aediculatus have been achieved either by adding a cell
homogenate from symbiont bearers or by injecting the
symbiont-containingcytoplasm (1,2). On the other hand, P .
necessarius appears to be totally dependent on its host as
well. Numerous attempts to grow them outside their hosts
have failed (2; Schmidt, unpublished data).
Endosymbiotic bacteria often are seen in protozoa. However, only a few of them have been studied extensively, and
several of these are still referred to by Greek letters, as is
customary for cytoplasmic elements. Among the endosymbionts which are well characterized but do not have binary
names are omicron and the omicronlike endosymbionts. The
purpose of the present paper is to name this group of
symbionts in accordance with the rules of the Bacteriological
Code. The name Polynucleobacter was selected to indicate a
characteristic feature of this genus: all members show multiple nucleoids easily visible in these endosymbionts after
staining with acetocarmin, acetoorcein, or deoxyribonucleic
acid (DNA)-fluorescent dyes. The epithet necessarius was
chosen to indicate the relationship of omicron and its host
Euplotes aediculatus, which depend upon each other (3).
Omicron. The characteristic features of omicron, originally described by Heckmann (2), are summarized as follows. Approximately 900 to 1,000 cells of P. necessarius
inhabit the cytoplasm of their host. They have a length of 2.5
to 7.5 pm (most commonly 5 pm) and a width of about 0.3
pm, and are gram negative. If P. necessarius is stained with
DNA-specific dyes, 3 to 9 and in some cases up to 12
intensely stained and regularly spaced dots become visible
(Fig. 1).They are considered to be nucleoids. When studied
with the electron microscope, these nucleoids differ from
those of most free living bacteria by exhibiting an electrondense central core, which resembles the chromatin of eucaryotes (Fig. 2). Whether this core is formed by proteins
associated with DNA or some other material is not clear.
The symbionts are contained in vesicles to which ribosomes
are often attached. P. necessarius reproduces by transverse
binary fission in a typical bacterial manner. The fission
products were, however, often found to differ from each
other in size.
It is possible to isolate P. necessarius from E. aediculatus
homogenates and to obtain pure preparations of the
symbionts in quantities large enough for an extraction and
characterization of their DNA. The guanine-plus-cytosine
(G+C) content was computed to be 48 mol% by thermal
denaturation (T,J. A value 2.8% lower was found when the
G+C content was calculated from the buoyant density of the
DNA, which was 1.7036 g/cm3 (5). Similar differences reported by others are explained as caused by rare bases, but
no DNA chemistry is available for P . necessarius. The
average DNA content of this symbiont is 5.8 x lop3pg per
FIG. 1. Fluorescence micrograph of P. necessarius in the cytoplasm of a slightly crushed E. aediculatus cell after staining with
N,N'-diethylpseudoisocyanin chloride. The symbionts are revealed
by the DNA-specific yellow fluorescence of their nucleoids. Ma,
Macronucleus; Mi, micronucleus, x 1,200.
-
* Corresponding author.
456
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 22:18:35
VOL. 37, 1987
NOTES
457
FIG. 2. Electron micrograph of P. necessarius (reprinted from reference 2, by permission). Longitudinal section. The ultrastructure of this
symbiont resembles that of gram-negative bacteria. x 60,000.
Omicronlike endosymbionts. Symbionts that look similar
to omicron were found in six other freshwater Euplotes spp.
( E . eurystomus, E . plurnipes, E . daidaleos, E . octocarinatus, E. patella, and E. woodrufi). These species, like E .
aediculatus, have a 9 type 1fronto-ventral cirrus pattern and
are therefore considered to be closely related (4). The
symbionts may differ from omicron in size, shape, and other
features, but they always share with omicron the feature that
they possess multiple nucleoids. The omicronlike symbionts
have not yet been studied in enough detail to propose species
names for them. Stocks of Euplotes species with omicronlike
symbionts were subjected to a penicillin treatment of the
kind that had earlier been found to remove omicron (4). It
was found that their cells stopped dividing and died, as had
E . aediculatus cells, once they were deprived of their
symbionts. This suggests that all Euplotes spp. with a 9 type
1 cirrus pattern suffer from a common deficiency which
arose in a common ancestor of this group of species. This
ancestor must have lived in symbiosis with a procaryote
which compensated for the acquired deficiency.
Descriptionof Polynucleobacter gen. nov. Polynucleobacter
(pol.y.nuc’leo.bac.ter.;Gr. p o f y s , numerous; masc. L. nucleus, nut, kernel; bacter, the masc. equivalent of the Gr.
neut. n. bactrum, a rod). Bacterial endosymbionts. Multiple
nucleoids. Inhabit the cytoplasm of the following closely
related Euplotes spp.: E . aediculatus, E. eurystomus, E .
plumipes, E . daiduleos, E. octocarinatus, E. patella, and E .
woodrufi. Essential for their host species. Nonmotile. The
type species is Polynucleobacter necessarius.
Description of Polynucleobacter necessarius sp. nov. Polynucleobacter necessarius (nec.es.sar i.us.; L. adj. necessarius, indispensable, necessary). Bacterial endosymbionts;
900 to 1,000 symbionts inhabit the cytoplasm of E. aediculatus and are essential for this ciliate. Three to twelve
nucleoids. Gram negative. Dimensions, 0.3 pm wide by 2.5
to 7.5 bm long. Nonmotile. Cell division is by simple binary
fission. DNA G+C composition, 47.7 mol% (melting point
profile) and 44.9 mol% (buoyant density). Analytical complexity, 3.5 x lo9 daltons. Kinetic complexity, 0.57 x lo9
daltons. Usually 3 to 9 and sometimes up to 12 genomes per
cell. The type strain of P. necessarius is living in stock 15 of
E. aediculatus, which is deposited at the American Type
Culture Collection, Rockville, Md. (ATCC 30859). The
strain is also available from the Heckmann culture collection, Munster, Federal Republic of Germany.
We thank N. Staubach (Institut fur Altertumsforschung der
Universitat Miinster) for his helpful suggestions regarding the proposed scientific names, and H . - W . Kuhlmann and R . L.
Quackenbush for reading the manuscript.
LITERATURE CITED
1. Fujishima, M. and K. Heckmann. 1984. Intra- and interspecies
transfer of endosymbionts in Euplotes. J. Exp. Zool. 230:339345.
2. Heckmann, K. 1975. Omikron, ein essentieller Endosymbiont
von Euplotes aediculatus. J . Protozool. 22:97-104.
3 . Heckmann, K. 1983. Endosymbionts of Euplotes. Int. Rev.
Cytol. SUppl. 14:111-144.
4. Heckmann, K., R. ten Hagen, and H.-D. Gortz. 1983. Freshwater
Euplotes species with a 9 type 1 cirrus pattern depend upon
endosymbionts. J. Protozool. 30:284-289.
5 . Schmidt, H. J. 1982. Isolatian of omikron-endosymbionts from
mass cultures of Euplotes aediculatus and characterization of
their DNA. Exp. Cell Res. 140:417-425.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 22:18:35