A M . ZOOLOCIST, 9:759-764 (1969).
Interference with Deposition of Minerals in a Coccolithophorid Protozoon
HENRY D. ISENBERG AND LEROY S. LAVINE
The Long Island Jeiuish Hospital and State University of
Neiu York Downstate Medical Center
SYNOPSIS. Laboratory studies with axenic culture of the coccolithophorid, Hymenomonas MP 156, in chemically defined media have established that this primitive protist deposits its minerals in essentially the same manner as multicellular organisms. An
appreciable number of inhibitors and natural compounds prevent deposition of minerals in laboratory experiments. Among these cations other than Ca+2, certain amino
acids, select intermediates of carbohydrate metabolism, and inhibitors of enzymes
occupy a prominent place. Interference with deposition of minerals does not necessarily
abolish proliferation of the coccolithophorid. However, electron-microscopic, autoradiographic, histological, and analytical studies of the various modes of interference
reveal a profound alteration of organelles and physiological activities. The ability of
the protist to use its own extracellular, mineralized, calcareous structures as a source
of Ca+2 for growth under conditions of reduced cations in the environment suggests a
possible role for these microorganisms in cation equilibria of natural waters.
The theme of this symposium is the penetration of CaCO3 substrates by organisms.
The topic of this paper suggests preoccupation with a biological activity which has
the opposite effect, namely deposition of
mineral or, more specifically, CaCO3.
While it would be incorrect to equate or
compare these contrary functions, they do
share obvious features. The understanding
of certain broad aspects of biological activities leading in either direction should
contain information applicable to the other, especially when one admits that our
present state of knowledge concerning the
interactions between living forms and the
mineral world is, at best, scanty. Our laboratory studies have been directed primarily
at an appreciation of the intimate physics
and chemistry attending the interaction
between an organic matrix and minerals in
a mineralizing microorganism. As part of
this investigation we have been concerned
with interfering with the process of mineral deposition by various means and have
also challenged this particular organism
with its own mineralized extracellular
structures as a source of Ca+2 for growth
and other activities. This report is a summary of these phases of our study and a
Supported by Crant DE 01662 from the National
Institute of Dental Research.
discussion
findings.
of
the
pertinence
of
the
METHODS
The test object of our studies is the
phytofiagellate, Hymenomonas MP 156
(Cricosphacra
carterae) .(Paasche, 1968).
Hymenomonas is a supralittoral, euryhaline coccolithophorid, possessed of two
flagella; it produces microscopic calcareous pearls or coccoliths. It is grown in
axenic culture in chemically defined media
and under conditions described earlier
(Isenberg, et al, 1963, 1965, 1967). The
phytoflagellate is incubated at 18°C in a
cold incubator constantly illuminated with
eight 40-watt, cold-white, fluorescent
bulbs. The details of harvesting, and analytical, histological, autoradiographic, and
electron-microscopic procedures have been
published in detail (Isenberg, el al., 1965
a, b; 1966, 1967).
REVIEW OF PREVIOUSLY PUBLISHED MATERIAL
Moss (1964) has defined biological deposition of minerals as a biphasic process
in which the synthesis of an organic matrix
precedes impregnation with mineral.
Studies with Hymenomonas MP 156
demonstrate these criteria but also permit
an analytical approach hitherto unattaina-
759
760
HENRY D. ISENBERG AND LEROY S. LEVINE
ble with other higher organisms. This organism, like all other coccolithophores, extrudes its mineralized structures into the
environment. It thus provides the investigator with natural, mineralized, organic
structures which are cell-free and not subject to the influences of the various anabolic and catabolic processes which obscure
and distort deposition of mineral in most
other biological structures. TJymenomonas,
under standard controlled conditions of
culture, usually presents itself as an ovoid
to round, biflagellated, chloroplast-bearing
organism, 10-12 JX in size; also seen with
ease are structures which suggest intracellular coccoliths. These latter subcellular
elements are seen in cells in the late stages
of logarithmic growth and throughout the
stationary phase, i.e., only after the first
10-12 days of incubation. Autoradiographic and analytic studies with Ca45 (Isenberg, et al., 1964) demonstrate that this
cation is localized at specific centers within
the cell. Further evidence for this opinion can be gleaned from histochemical
studies employing tinctorial techniques to
demonstrate active calcification in mammalian tissues. S35O4-enriched cultures
studied autoradiographically and with special staining procedures (Douglas, et al.,
1967) showed copious amounts of intracellular material with the staining properties
of mammalian sulfated acid-mucopolysaccharides under conditions which favor
deposition of mineral. Peripheral and extracellular coccoliths were also stained by these
procedures. Obviously, some clues to the
mechanism of mineral deposition could be
derived from an analysis of these structures.
Crystallographic examination of extracellular coccoliths (Isenberg, et al., 1963) revealed not only calckic CaCO :i but the dark
central core so suggestive of an organic
component.
Initial failure to separate the organic
from the inorganic phases constituting the
coccolith was finally overcome by employing distilled water as the solvent. While
final solution of the calcareous structures
required more than two months, an organic component, Fraction 1 (Fx), was released
along with some CaCO 3 during the first
three days (Isenberg, et al., 1966, 1967).
Fraction 1 has a molecular weight of
40-50,000 and along with several carbohydrate and amino acid moieties contains appreciable quantities of hydroxyproline, unusual among microbial proteins or peptides (Isenberg, et al., 1966). We feel that
the chemical data suggest that coccolithogenesis contains the blueprint to biological mineral deposition.
Electron-microscopy of
Hymenornonns
calcifying under constant illumination in
the laboratory was most revealing. Such
cells do not show a nucleus displaying
heterochromatin clumping, mitochondria,
nor Golgi cisternae. Instead structures of
dictiosomal origin abound and are modified into a variety of subcellular organelles which finally lead to structures we
have termed intracellular coccolith precursor (Isenberg, et al., 1966, 1967). Another striking feature are the numerous
intra- and extracellular coccolith-associated,
as well as free, structures we have referred
to as fibers. The fibers appear identical with
structures called scales in other species
(Paasche, 1968); however, the striking periodicity (of ca. 200 A) displayed is obviously
imposed by the presence of the imino acids,
proline and hydroxyproline (Glimcher,
I960). In addition, this periodicity of 200 A
is reminiscent of the periodicity by fibers
produced in tissue cultures of fibroblasts or
the protofibrils of ichthyocol when reconstituted in a mineral environment similar
to the total solids of the culture medium
used in these studies (Gross, 1963). It must
be made clear that these fibers by their
periodicity suggest that they must contain
imino acids; that they are identical with
the fraction F x has not been demonstrated.
There is also no implication that F t or
the scale fibers contain a microbial form of
collagen. What is emphasized here is that
structures of primitive protists actively engaged in capture of minerals contain hydroxyproline. That hydroxyproline in active capture of CaCO 3 is not restricted to
the coccolithophorids has been demon-
INTERFERENCE WITH COCCOLITHOGENESIS
strated with totally unrelated Protista in
our laboratory.
Additional structures are involved in
coccolithogenesis; however, actual capture
of mineral, i.e., interaction of organic and
inorganic phases, occurs in organelles we
have dubbed "mineral reservoir" and
"fingerprint" (Isenberg, et al., 1966, 1967).
It is here that the organization of the
mineral phase takes place, followed by
transport to specific areas in the intracellular coccolith precursor. When all of the
"hard" segments are firmly bound in place
the mature coccolith is extruded. This
highly speculative scheme has been described in detail (Isenberg, et al., 1966).
We can, however, assert that the two cytoplasmic organelles, the "mineral reservoir"
and "fingerprint", are restricted to the
mineral-depositing Hymenomonas and are
apparently essential for this process, for
they are invariably absent in cells which
for various reasons cannot deposit minerals.
INTERFERENCE STUDIES
Interference with deposition of mineral
in this particular coccolithophorid can be
achieved in a variety of ways. It is made
possible primarily by the ability of this
protist to continue to grow in laboratory
experiments even when mineral deposition
is abolished; the converse can also be accomplished and has been very helpful in
delineating certain conditions of Ca+2-capture. Coccolithogenesis is not usually observed in the artificial medium during the
initial and logarithmic growth phases
(Isenberg, et al., 1965); it appears at
about the time that the culture enters the
stationary growth phase and proceeds until
almost all of the added cation is incorporated into the coccoliths. Minimal growth
without coccolithogenesis is observed when
Ca+2 is present at 10~6 M; growth and
coccolithogenesis increase in proportion
with increasing concentrations of Ca+2 to
an optimal yield of cells and coccoliths
at lO- 2 M (Isenberg, et al., 1963). Mg+2
plays no role in coccolithogenesis but stimu-
761
lates growth. It cannot substitute for Ca+2
in the inorganic phase of the coccolith nor
for the proliferative requirements. Ba+2
behaves similarly except that it is toxic for
growth at levels above 10~4 M. It was
surprising to find Sr+2 a very adequate
Ca+2 substitute for the growth of the organisms. However, Hymenomonas failed to
include this cation in its coccoliths (Isenberg, et al., 1963). Tinctorial studies with
the histological stains enumerated above
showed that none of the active cell sites
developed during Sr+2 replacement; S35autoradiography demonstrated the absence
of sulfated polysaccharides in Sr+2-grown
cells (Douglas, et al, 1967). Electronmicroscopy of Sr+2-nurtured cells showed
the presence of mitochondria and chloroplasts. However, the major portion of
the cell was invariably occupied by vacuolated incomplete ICP's filled with
fibrous structures lacking the striking periodicity of the mineralizing organism in
most instances. Several Golgi vesicles were
present. Some apparently opened to give
rise to the fibrous elements. Obvious fat
inclusions were also discerned. Teleologically speaking, the Sr+2-grown cell
does not surrender so easily to the abolition of coccolithogenesis. Other electronmicrographs (Isenberg, et al., 1966) revealed besides a very obvious Golgi apparatus and several small mitochondria as
well as chloroplasts, that the protist almost
succeeded in forming coccoliths with this
cation, for very small coccolith segments
were seen in one of the incomplete ICP's,
segments sufficiently hard to be removed
from the cell mechanically during preparation of undecalcified cells. The degeneration of these attempts into fat bodies was
also demonstrated. However, some of the
fibers showed the typical periodicity. Totally lacking in all of the preparations
were the "mineral reservoir" and "fingerprint" (Isenberg, el al., 1966, 1967).
Since the mineral component of Hymenomonas is calcitic CaCO3 (Isenberg, et
al., 1963) the effect of the carbonic anhydrase inhibitor, 2-acetyl-l, 3, 4-thiodiazole5-sulfonamide-sodium (Diamox), on growth
762
HENRY D. ISENBERG AND LEROY S. LEVINE
and deposition of mineral by the protist permitted the organism to tolerate an apwas evaluated. Varying amounts of the preciable number of organic compounds
inhibitor ranging from lO" 8 to 10- 2 M (Isenberg, et al., 1965); since the fate of
were added to media which differed from these various additions was not followed in
one another only by the presence of lactate detail, the actual role of several organic
in one, thus providing a somewhat more intermediates of carbohydrate metabolism
heterotrophic milieu in one experiment. and of amino acids can only be surmised
The anhydrase inhibitor halted cocco- on the basis of detailed analyses which inlithogenesis completely at 10- 3 M; growth volved nitrogen-, anthrone-, Lowry-, and
of the organism on the other hand was Ca+2-determinations in cells and coccoimpaired but minimally even at the 10~2 liths. However, Crenshaw (1964), using
M level of the inhibitor. The presence of different ratios of cations, was unable to
the lactate did not interfere with inhibit- substitute other organic compounds for
ion at the 10- 3 M concentration. At 10"* lactate in short-term experiments. While
M Diamox, lactate-grown cells were hardly the actual utilization of these organic cominhibited, as measured in terms of total pounds has not yet been confirmed, some
cellular nitrogen. Cells without added or- exerted such a dramatic effect on the
ganic nutrient were inhibited to 50% ot chemical composition of various coccoyield by the same amount of inhibitor. lithophorid cellular elements and products,
The presence of lactate also resulted in a on the colonial and cellular morphology
consistent, detectable level of intracellular of the organism, on its tinctorial and autoCa+2. The data obtained in these experi- radiographic behavior, and on finements support the Sr+2 studies in demon- structural anatomy, as if to underline the
strating the separation of growth and minadvantages of protistal illiteracy (Isenberg,
eral-capture. They certainly support the
view that intracellular carbonate levels are et al., 1965, 1966, 1967). In general, the
determinative for deposition of mineral, substitution of organic acids for lactate1
and suggest that Ca+2 is but a very suitable and of amino acids for lactate and NOs"
cation for removal of anions (Isenberg, et led to either increased proliferation of cells
al., 1963-1966). Histological studies of Dia- with decreased or inhibited coccomox-grown cells presented no tinctorial lithogenesis or to a diminution of proliferaevidence of mineralizing activities. Elec- tive activities with an enhanced productron-microscopy yielded cells very reminis- tion of coccoliths. A suitable means of
cent of those in which coccolithogenesis comparing the effects of these additives or
of mineralization,
had been interrupted with Sr+2. Autoradi- substitutes is the index
2
45
35
deposited/^g
cellular
the
ratio
of
jug
Ca+
ography with Ca or S indicated that
nitrogen
(Isenberg,
et
al.,
1965,
1967).
Diamox-inhibition did not permit formaThe
other
chemical
fractions,
i.e.,
antion of adequate mineralizing sites within
cells. The fine-structural studies showed throne and Lowry-reacting materials of
feeble attempts at the production of ICP's. cellular and coccolithic origin reflected this
Occasional fibers were seen in electro- ratio. The role of carbonate is especially
micrographs (Isenberg, et al., 1966, 1967). important with acetate, where the anion
But the cells showed numerous well de- effected a reduction of the index of minerveloped mitochondria, well formed dic- alization, and with propionate and to a
tiosomes, and vacuoles with adielectronic lesser degree citrate. With these commaterial. The ICP's seen with Diamox-inhi- pounds the presence of CO3~2 in small
bited mineralization revealed less resem- amounts and without an artificially conblance to active mineral deposition than trolled atmosphere led to an appreciable
those prevented from effective calcification increase in coccolith Ca+2 per unit cellular
with Sr+2.
nitrogen. Among the amino acids, proline, hydroxyproline, and tyrosine led to
Adjustment of the Mg/Ca ratio to 1 increased deposition of mineral. With
INTERFERENCE WITH COCCOLITHOGENESIS
these amino acids, proliferative activities
were curtailed markedly, while coccolithogenesis per cell appeared vastly increased. Chemical data obtained with other amino acids suggested that some are
most stimulating for cellular growth, a
conclusion corroborated by morphological
findings, histology, and electron-microscopy (Isenberg, et al., 1965, 1967). Thus,
casein hydrolysate-grown cells, with an
index of mineralization approximately
half that of the lactate control, hardly resembled mineralizing Hymenomonas. Electron-microscopy confirmed the inability of
casein hydrolysate-nurtured organisms to
produce but occasional coccoliths, with
most attempts failing. The cellular organelles reflected the non-mineralizing
state, although all requirements for optimal deposition of mineral were present.
Histochemical and autoradiographic investigations agreed with these findings. The
results with other organic and amino acids
corroborated their indices of mineralization.
Interference with deposition of mineral
in this protist, summarized here, probably
constituted only a fraction of the conditions which can achieve this end. Perhaps
the most striking suggestion was that deposition of mineral at the protistal level
need not be essential for the survival and
proliferation of the microorganisms so engaged; deposition of mineral by coccolithophorids in nature may well be a sometime activity in which these protists engage
only when environmental and physiological conditions are propitious and quite
narrowly defined. These studies suggested
further that the capacity to capture and
deposit mineral in a manner consistent
with the general theory of biological calcification enunciated by Moss arose in eucaryotic if not procaryotic protists. Indeed,
these investigations permit a narrower
view of calcification, if not of mineral deposition in general. For to the requirement
of a preformed, two-component, organic
matrix consisting of an organized, usually
fibrous, phase and an unorganized, sulfurcontaining, ground substance, one can now
763
add a requirement for hydroxyproline,
heretofore usually confined to various vertebrate and invertebrate collagens. Its
presence in a specific organic fraction of
coccoliths, and evidence that it is a constituent only of certain specialized subcellular organelles in actively mineralizing Hymenomonas, are very suggestive of an association between this imino acid and
Ca+2-binding. Hydroxyproline is, after all,
a characteristic constituent of collagen, the
protein most suspect of this role in deposition of hydroxyapatite in vertebrates.
However, experiments in our laboratory
with algae implicated this acid in encrustation of cell walls with aragonitic CaCO3.
Thus, hydroxyproline may well be among
the general requirements of the organic site
capable of admitting the intimate combination between the preformed matrix and
the mineral crystals involving Ca+2 regardless of the anion.
Observations that growth and mineral
deposition are distinct and separated activities of Hymenomonas and that hydroxyproline along with sulfated and nonsulfated carbohydrate moieties are essential for the latter process, led us to challenge this species with its own coccoliths as
the sole source of Ca+2. Coccolith-Ca+2
was added in the same concentration as
Ca40 in the same defined mineralizing
medium. Ca45-enriched coccoliths served
as an additional method for monitoring
these events. Growth, measured by the determination of total cellular nitrogen, attained the same levels as in controls with
ionic Ca+2. Coccolithogenesis, however,
was absent. Supernatants of coccolithCa+2- and ionic Ca+2-supplemented Hymenomonas cultures concentrated by lyophilization were examined for the presence
of agents, chelators or enzymes, which removed coccolith Ca+2, either by sequestration of the cation or decomposition of the
coccolithic organic phase by incubation
with cell-free Ca45 coccoliths. No activity
of either sort could be demonstrated. Controls which consisted of the defined medium with varying concentrations of ionic
Ca+2, a 1.8% NaCl buffer, and water, indi-
764
HENRY D. ISENBERG AND LEROY S. LEVINE
cated that release of Ca+2 from coccoliths
was a physicochemical activity which depended to an appreciable degree on the
concentration of Ca+2 in the liquid milieu
and somewhat on the ionic strength of the
solutions. The liberation of Ca4r> was
greatest in the media containing no Ca+2
ion and in distilled water. It decreased in
proportion to added ionic Ca+2. In the
NaCl buffer, release of Ca+2 was less than
in water and Ca+2-free growth medium,
and approached values attained with the
addition of 10—* M Ca+2.
CONCLUSIONS
These findings suggest that this coccolithophore does not engage directly or actively in the removal of any constituent ol
the coccoliths. Liberation of coccolithic
constituents is apparently a physical
phenomenon subject to the nature and
concentration of dissolved ions. The micro-organism may speed this process by removing Ca+2 or CO8~2 thus preventing
the establishment of an equilibrium between coccolith-bound and dissolved mineral, since we established (Isenberg, et al,
1965) that the process of complete solution
of coccoliths cannot be brought to completion quickly in a static aqueous system.
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