1. Euk Microbiol.. 44(4). 1997 pp. 3 6 6 3 7 3 Q 1997 by the Society of Protozoologists
The Occurrence of Biogenic Calcian Struvite, (Mg, Ca)NH4PO4=6H20, as Intracellular Crystals in Paramecium JOHN E. GROVER,* ALAN F. ROPE,** and EDNA S. KANESHIRO**,’ *Department of Geology, and **Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221, USA ABSTRACT. Intracellular crystals are conspicuous refractile “inclusion bodies” commonly found in many protozoans, but very few have been identified mineralogically. We have isolated crystals from axenically grown mass cultures of Paramecium tetraurelia, and purified them using differential centrifugation. The crystals’ structure and chemistry were analyzed using x-ray powder diffraction and energy-dispersive electron microprobe techniques. The morphology was studied by means of scanning electron microscopy. The crystals were identified as the orthorhombic mineral, calcian struvite, (Mg, Ca)NH4P0,.6H,O. Struvite from P. tetraurelia exhibited a variety of crystal habits, including hemimorphic forms, epitactic overgrowths and several types of twins. A linear correlation between computed hydration number and Mg content suggests that the crystal composition may reflect the range of conditions under which struvite nucleation and growth occur. The mineral struvite also occurs in association with guano and other rich organic products, and can be biologically induced to precipitate extracellularly. Extracellular struvite has been well characterized in pathogenic calculi (kidney stones) of humans and cats, where precipitation is enhanced by bacterial urease activity that produces ammonia in the urinary tract. This is the first study demonstrating that struvite is also biologically controlled to form as an intracellular mineral. These crystals may have formed within lipid-rich, membrane-bound vesicles in Paramecium. Supplementary key words. Ammonia, electron microprobe, kidney stones, lipids, orthorhombic, scanning electron microscopy, x-ray diffraction.
M
than 60 different biogenic minerals have been described from organisms representing broad phylogenetic backgrounds [14, 28, 30, 491. These minerals are often readily distinguishable from their counterparts formed under abiotic conditions [191. The formation of ‘biologically induced’ minerals is initiated by structural or chemical perturbations caused by the surfaces of organisms or their chemical by-products, whereas ‘biologically controlled’ minerals form in membranebound compartments. Despite the widespread occurrence of biogenic minerals, relatively little is known about the morphologic and crystallographic properties of nonskeletal intracellular crystals. The mineralogy of some protozoan structures, especially skeletal structures, is well understood because protists comprise the main sediment-forming orgapisms of the ocean. Silica and calcite tests, scales, spines, and coccoliths of diatoms, foraminiferans, radiolarians and other single-celled eukaryotes are abundant, widely distributed, and have played an important role in the geologic record of the earth. Intracellular crystalline inclusions and other refractile bodies are conspicuous because of the contrast between their refractive indices and that of the surrounding cytoplasm, and were first observed in protozoan cells soon after the invention of the compound microscope. However, the chemical and physical nature of most of these cytoplasmic components have yet to be elucidated. At the turn of the century, investigators characterized intracellular crystals of paramecia cells by their solubility, morphology, and optical and chemical properties using methods that are now dated. In 1893, Schewiakoff described twinning and other morphologic habits of crystals in the cytoplasm of Paramecium [43]. He also performed some chemical analyses and concluded that these crystals were composed of CaH,(PO),. In 1938, Bernheimer, using Nicol prisms attached to an ordinary microscope, concluded that the crystals in Paramecium were monoclinic in nature [2]. However, he stated that the precise chemical composition and physical constants of the crystals were needed before their identity could be established with certainty. More recently, analyses of the elemental composition of several protozoan crystals, including those of Paramecium, have been performed using in situ electron probe x-ray microORE
‘To whom correspondence should be addressed. Telephone: 5 13-5569712; Fax: 513-556-5280; Email:
[email protected]
analysis, High levels of Ca, Mg, and P were consistently detected [4, 6, 7, 18, 21, 24, 26, 33, 35, 39, 41,421. In the present study, crystals were isolated and purified from mass cultures of axenically grown P. tetraurelia cells. This enabled us to perform a variety of chemical and physical analyses that were previously not possible with intracellular crystals of Paramecium. The isolated crystals were identified mineralogically as struvite. MATERIALS AND METHODS
Organisms and crystal preparations. Mass axenic cultures of Paramecium tetraurelia, strain 51s, were grown at room temperature in an enriched crude medium containing phosphatidylethanolamine, stigmasterol and fatty acids [23, 561. Routinely, 40 Fernbach flasks containing 500 ml of medium were used for a single preparation. Cultures at late-log phase were harvested by continuous-flow centrifugation [23], washed, resuspended in 70% ethanol, and lysed with a Dounce homogenizer placed on ice. The homogenate was layered onto a 2.5 M sucrose solution in 5 0 - 4 tubes, then centrifuged in a swinging bucket rotor (HB-4 rotor, Sorvall RC-5 or RC-5B refrigerated centrifuge, Du Pont Instruments, Newtown, CT) at 4,000 X g for 10 min at 4” C. The pellet was recovered, and recentrifuged under the same conditions. The second pellet was rinsed three times with 70% ethanol to remove sucrose; and the supernates were decanted. The final purified crystal preparation was airdried. Light microscopy. Organisms at different stages of the culture cycle were air-dried on albumin-coated glass slides and permanently mounted (Permount, Fisher Scientific Co., Fair Lawn, NJ). Crystals in individual cells were counted using a Nikon Diaphot microscope (Nikon Inc., Melville, NY) equipped with Nomarski differential interference optics. Each analysis (trial) consisted of 100 cells; six independent analyses were performed, and the values were expressed as means ? standard deviations. X-ray powder diffraction and crystallographic cell parameters. Initial analyses were done using long-duration exposures on strip film, and were later confirmed with a Siemens D-500 powder diffraction unit. In both cases, crystals were airdried overnight, gently crushed to a powder, and mixed with 10-20% (v/v) of cleaned, ground acid-washed a-quartz, which served as an internal standard. Exposures on the camera film ranged from 25-66 h at 20” C; count times were 10 sec/O.Ol degree. After the material was identified as struvite, the dif-
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IN PARAMECIUM
0
2
4
6
a
AGE (days)
Fig. 2. Change in number of crystals in Paramecium cells with culture age. The number of crystals in stationary phase cells dramatically increases within 24 h following inoculation into fresh medium. Values are means; bars represent SD (n = 6 ; 100 cells per determination). The change in the number of crystals per cell is compared with concomitant changes in cell density (triangles) and the total lipidcell (squares) (from ref. 23).
Fig. 1. Photomicrograph of a flattened Paramecium cell taken under Nomarski optics showing highly refractile intracellular crystals. Bar = 10 p,m.
fraction pattern was used to determine crystallographic cell parameters. Known reflection indices were verified, and complete assignment of indices to reflections at diffraction angles (28) beyond published values was also accomplished. Scanning electron microscopy and elemental composition. Samples of isolated crystals were embedded in epoxy within small brass cylinders, and mounted on aluminum studs. The specimens were ground smooth by hand through a succession of A1,0, polishing powders down to 600 grit. The polished samples were cleaned in ethanol, dried, and coated with graphite. Alternatively, isolated crystals suspended in ethanol were placed on glass microscope slides without coverslips, air-dried, and then coated with gold. X-ray microprobe analyses of individual crystals were performed with a Stereoscan 90 scanning electron microscope (SEM) equipped with an energy-dispersive system (EDAX/EDS, Cambridge Lab., Cambridge). RESULTS Effect of culture age on crystal numbers. The highly refractile crystals within individual Paramecium cells, which numbered from zero to over 100, were readily observed by Nomarski optics (Fig. 1). The numbers of crystals per cell for these axenically grown ciliates changed with culture age (Fig. 2). When stationary-phase cells were inoculated into fresh medium, the number of crystals increased dramatically after one day, and then decreased with culture age. There was a sharp drop at day 5-6, when cultures were in late-log to early-stationary phase. Thus, crystal numbers reflected dynamic changes occurring within cells as they progressed through the culture cycle. The crystals isolated from late-log to early-stationary phase
cultures were morphologically similar in size and shape to those seen in intact Paramecium cells. Isolated crystals dissolved in HC1, HNO,, formaldehyde or saturated aqueous HgCl,, but remained intact when treated with ethanol or acetone. Thus, it is likely that crystals in cells that were subjected over long periods to high concentrations of compounds in the former group during some standard cytochemical fixation procedures would dissolve prior to microscopic examination. X-ray powder diffraction. Identification of struvite as the crystalline material isolated from P . tetraurelia was made using standard X-ray diffraction techniques (Table 1). Once the material had been identified with certainty as struvite, the diffraction pattern was used to determine crystallographic cell parameters. Known reflection indices were verified, and complete assignment of indices to reflections at higher diffraction angles (28) was also accomplished (Table 2). Energy-dispersive electron microprobe analysis. Elemental analyses of isolated crystals made on the SEM using energydispersive electron spectra showed that the major elements present were P, Mg, Ca and K (Fig. 3). The elements detected by electron microprobe analysis are listed in Table 3 according to their presumed positions in the Ca-struvite structure. Although elements in the “ideal” chemical formula for struvite (MgNH4P0,.6H,0) predominated, the other species present must be assigned to appropriate positions in the crystal structure. In struvite, Mg is in octahedral coordination with 6 H,O molecules; NH, is 5-coordinated with four oxygens of nearby water molecules, and one atomic oxygen; and P is tetrahedrallycoordinated to form PO, anion units [ l , 591. The cations (including NH,) occupy Wyckoff (2a) positions (0, y, z; %, 7 , % + z) in a structure in space group Pmn2, (C2,’; #31) having Z = 2 formula unitsfunit cell, where hydrogen bonding binds and supports the individual molecular units Mg[H20]62+, PO,3-, and NH,’ [ l , 15, 57, 581. The assignment of cations to IV-, V- or VI-coordinated sites was made on the basis of standard ion size and charge considerations [47, 48, 591 so that a structural formula could be devised and an estimate of the average hydration number made. Specifically, P and S were taken to occupy JY-coordinated sites, NH, and K occupy V-coordinated sites, and Mg, Ca, Sr, Sc, Fe and Mn occupy VI-coordinated sites.
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Table 1. X-ray powder diffraction patterns for struvite isolated from P . tetraurelia. Paramecium struvite"
Line
Authentic standardh
I'
d,,,
h
k
l
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
f-w m in-s, d w-m vvf s--, d m vvf w-f w-m, d
6.117 5.886 5.588 5.369 4.579 4.246 4.134 3.576 3.467 3.287
0
1 0 0
0 1 2 1
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
-
-
d d w-m, d m-s w-f, d m-w, d m, d vf vf, d vvf, d w, d vvf vvvf, d vvf, d vvf, d f vvf vvf
3.062 3.019 2.952 2.913 2.797 2.717 2.687 2.656 2.547 2.505 2.393 2.347 2.297 2.272 2.253 2.179 2.157 2.134 2.124
vvf vvf f, d
2.092 2.068 2.053
vf f, f,
-
d vvvf f. d f,
-
vf, vd vf, d vf, d vf, d vvf vvf f, d vf, d f, d f-w, d f-, d vf vvf vvf
-
2.014 1.978 1.958
1.874 1.852 1.849 1.846 1.823 1.809 1.800 1.791 1.761 1.736 1.713 1.680 1.659 1.651
54 55
vvf, d vf-
1.595 1.345
56
vvf
1.270
a
1
0 0 1 1 0 1 2 1 0 0 2 0 2 0 1 0 2 0 1 1 2 2 3 2 2 3 1 2 3 0 0 2 0 1 1 2 1 1 0 3 0 1 3 3 0 2 0 2 4 1 3 2 4 0 4 0 5 3 4 3 5 3 0
1
1 I 1 1 0 0 1 2 1 2 1 0 2 2 1 1 2 1 1 2 0 2 0 1 0 2 1 1 2 1 3 1 2 2 3 3 3 1 0 3 2 2 2 1 3 3 0 3 1 3 0 3 1 2 1 3 0 4 2 3 4
0 1 2 2 0 3 3 0 0 1 1 4 1 2 2 4 2 4 3 0 1 1 4 0 5 2, 1 5 4 4 0 5 4
dsm, ~~.
6.140 5.905 5.601 5.378 4.600 4.257 4.139 3.557 3.475 3.289 3.192 3.067 3.022 2.958 2.919 2.802 2.722 2.692 2.660 2.548 2.511 2.394 2.352 2.300
-
n6 29 46 20 6 100 41 5 15 32 3 6 20 36 80 53 25 79 72 7 14 11 22 4 -
2.253 2.180 2.167 2.133 2.127
8 8 8 12 16
-
-
2.069 2.054 2.046 2.014 1.983
12 25 25 21 13
1.960 1.932 1.921 1.873
30 4 9 14
3,
0 1 2 3 6 2 0 1 5 5 3 0 0, 3 4 2 2 4, 1 6 1, 4 6, 1, 0, 5, 5
-
-
1.851
9
Measured for 66 h; corrected using powdered SiO, as internal standard Powder pattern, JCPDS File 15-762 in database PDF-2 (superior). s, strong; m , moderate; w, weak; f, faint; d, diffuse.
Table 2. Crystallographic cell parameters for Paramecium Ca-stmvite. Cell parameter
a b
(4)
(4) c (A)
V (A)S a
Isolated Paramecium crystals
6.944 2 6.126 2 11.204 ? 476.606 2
0.001 0.001 0.002 0.10
Synthetic struvite standarda
6.948 2 6.135 F 11.207 ? 477.68 2
0.001 0.001 0.002 0.11
JCPDS file # 15-762 1201.
Although Sc3+may create a minor charge imbalance in VI-fold sites, accepted values of radii for this ion in different coordinations indicate that in struvite, Sc should be in octahedral coordination [47, 481. With this assignment of elements to structural sites, a structural formula could be computed for Ca-struvite. Normalization of the overall phase composition was based on total molar (P + S) = l.OOO/formula unit. The mole numbers for cations in VI- and V-fold sites were then readjusted to produce a corrected weight sum for all species analyzed by microprobe. In particK), and those of all divalent cations ular, the moles of (NH, plus Sc3+,were normalized to one formula unit in each group, as required by charge-balance considerations. In struvite, the "structural" water is integral to the overall stability of the unit cell, meaning that the structure would lose its crystallographic identity if the water were removed. Thus, we used the numbers of oxygen atoms implied by balancing the other species to compute the hydration number for each analysis. The mean of computed hydration numbers was 6.704 (range, 4.15-9.31; n = 55 analyses on 19 different crystals). Thus, the over-all mean composition of struvite isolated from axenically grown P. tetraurelia in late-log to early-stationary growth was
+
An independent analysis using inductively coupled plasma (ICP) and mass spectrometric techniques was performed by direct analysis of a sample of isolated crystals dissolved in 1 M HNO,. A Ca/Mg value of 0.084 was obtained, consistent with P
>r .-c (I)
c
a,
c " L
a,
> ._ Y
(d -
a,
LT
I
0
5 eV
10
Fig. 3. Energy-dispersive eiectron microprobe spectrum obtained using a scanning electron microscope. Elemental analysis (K, emission) identified P, Mg, and C a as major components of the isolated crystals from Paramecium.
GROVER ET AL.-STRUVITE Table 3. Elemental composition of isolated Paramecium intracellu-
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...
10
lar crystaka
Observationsb
Concentration'
m -
9-
55 55 16 2 1
8.850 0.833 0.059 0.004 0.002
LL
8
8-
.G
7-
55
0.196
Octahedral (VI) sitesd: Mg Ca Sr
sc Fe
Pentahedral (V) sites: K
2
? 2
0)
-0 K 3
65-
...
0
m
Tetrahedral (IV) sites: P S
55 4
11.931 0.007
4-
3
I
7
a Determined by energy-dispersive electron microprobe analysis. Al, attributed to residual polishing grit left on crystal surfaces, was detected in 13 of the 55 analyses. Elements detected in 55 spot analyses on 19 crystals. Weight percent (mean of 55 spot analyses on 19 crystals). Mn was sought but was not detected.
the average elemental (weight %) values obtained from the microprobe data (C&g = 0.8328/8.8502 == 0.094). The observed average hydration number, different from 6.00, is consistent with the range observed in the calculated (model) structural formula. This observation on the Paramecium crystals suggests variations in the amount of water in the lattice, beyond that required merely to support the structure. Attempts to find simple linear correlations among the original compositional parameters (elemental weight percentages) and hydration numbers showed widely varying values (Table 4). The regression for hydration number vs. Mg2+content (elemental weight percent) is notable in that there is a strong linear correlation between these two variables (r2 = 0.946) (Fig. 4), absent in all other cases. The closest competitor among single elements is hydration number vs. P5+, with r2 = 0.710. The r2 value for hydration number vs. Ca2+was only 0.041. This linear relation between hydration and Mg content thus appears to represent a natural distribution of
I
a
I
4
9
I
I
I
10
11
Fig. 4. Correlation of the degree of hydration and Mg content of individual P. tetruureliu struvite crystals. The hydration number was computed from microprobe analysis of individual samples, assuming electroneutrality and with cations assigned to crystallographic sites as described in the text. Linear regression on those data (solid line); theoretical relation between Mg2+content and hydration number for stmvite having a fixed composition for all elements except Mg to match the average values reported here (broken line).
aqueous ionic hydration numbers for Mg, [Mg2+].nH20,within the fluid of the intracellular compartment when Ca-struvite crystals were precipitated in vivo within Paramecium cells. This is consistent with published speciation models [ l , 571 and with the assembly of molecular-ionic components, Mg(H,O),Z+, PO,3-, and NH,+, into a structure known to be connected and supported by hydrogen bonds. Scanning electron microscopy. Crystals of Ca-struvite isolated from Paramecium exhibited a variety of crystal morphologies, all consistent with those described for the naturally occurring mineral (Fig. 5). Struvite belongs to the noncentrosymmetric hemimorphic class mm2 of the orthorhombjc system. The similarity of the a and b cell dimensions (6.94 A and 6.13 A, respectively) may lead to pseudohexagonal symmetry, particularly if [ h k O } forms are developed (Table 5 ) , owing to the
Table 4. Regression analysis of element concentration and the computed hydration number for Ca-struvite crystals isolated from Parurneciurn.a Dependent variable H#d
H# Mgh
H#
Mgb Cab H# H#
H# H# H# Cab
Independent variableb Mg Element sume Element sume
P P P Element sumf K Ca Sr NH4 Mg
r2
Regression equationc
0.9455 0.8758 0.7619 0.7100 0.5930 0.1800 0.1668 0.1489 0.0406 0.0067 0.0003 0.0000
H# = (20.216 ? 0.324) - (1.512 t- 0.050) X [Mg] H# = (21.206 % 0.488) - (0.512 t- 0.034) X [element sum] [Mg] = (0.2295 t 0.435) + (0.3939 % 0.030) X [element sum] H# = (20.056 t- 0.746) - (1.108 ? 0.097) X [PI
Element concentration, weight percent. Calculated as weight percent. Values in square brackets are in wt % of the element; H is the computed hydration number; error are SEM of the dependent variable and on the coefficient of the independent variable. H#, hydration number. Excluding Al. Calculated as moles. a
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Fig. 5. Crystal morphologies of Ca-struvite isolated from Paramecium cells demonstrated by scanning electron microscopy. A. Secondary struvite twin crystals growing epitaxially on { O O i ) , the negative pedion of the host “propeller” form. The principal crystallographic axes a and b are thought to be coincident (syntactic) for both host and twin forms, while the polar axis is inverted from host to twin. More complete development of this habit might be responsible for the massive twinned crystal shown in E B. Hemimorphic crystal, class mm2 (orthorhomic), showing a large {Oll)-form face truncated at either end by smaller { 102) faces and by a poorly developed positiye pedion, (001). at the top. C. Several irregular and “propeller-form” crystals exhibiting both positive, (001 ) (solid arrow) and negative {OOl } (open arrow) pedions. D. Unusual growth habits of crystals (center and bottom right) with one hemimorphic “blade” (upper right) shown in this field. E. “Propeller.”
GROVER ET AL.-STRUVITE
combination of an n-glide and a c : a ratio of 1.613, which is close to the hexagonal-closest-packed (HCP) ideal value of ( 2 G ) / 3 . Natural struvite is soft (2 on Mohs’ hardness scale) and brittle, fracturing conchoidally. It is both pyro- and piezoelectric because of the polar c axis. Struvite varies in habit [9, 361; crystals are frequently hemimorphic, terminating in domes or companion (but inequivalent) forms where 1 is replaced by - 1 . Twinning is common, with pedions {OOl} or {OOi} as the composition planes. The combination of struvite twins with hemimorphs may give rise to flattened hourglass shapes [9]. The only recorded twin law for natural struvite has one of two basal pedions, (001) or {OOl} as the composition plane, and reflection across { O O l ) as the element of twin symmetry supplementary to class mm2. Combination of struvite twins with hemimorphs was commonly observed in the present study. Paramecium struvite crystals commonly exhibited sets of “propellers” intersecting at approximately right angles to form a fourpronged cross. The extensive twinning that occurs in Paramecium crystals may explain why Bemheimer [2] was led to believe they were monoclinic. Rapid growth of struvite in Paramecium cells is suggested by morphologies such as X-shaped twinning (Fig. 5F) [5, 321. Pock-marked surface texture (Fig. 5E), often observed in isolated Paramecium struvite crystals, probably represents partial dissolution. Dissolution could have occurred in vivo or during the isolation and purification process. The small pits appear to conform approximately to pyramidal symmetry, a characteristic of etch pits described previously for natural struvite [12, 36, 521. DISCUSSION Studies on intracellular crystalline material isolated from protozoans. Crystalline refractile bodies have been successfully isolated and purified from only two marine protozoans. Biochemical analyses demonstrated that the crystalline refractile bodies contain high concentrations of purines. Specifically, guanine has been identified in the intracellular crystals from the bioluminescent dinoflagellate Gonyaulax [ l l , 16, 341, and both guanine and hypoxanthine are present in the refractile bodies of the marine ciliate Parauronema [51]. The Gonyaulax guanine crystals have been described as birhombohedral or chevrons, whereas the Parauronema refractile bodies are believed to be ultrastructurally similar to concentrically layered lamellar vesicles described in Uronema [22]. These intracellular crystals of marine protozoa are either readily dissolved during standard fixation, staining and embedding procedures, or are volatilized in the electron microscope. Crystals from the giant fresh water ciliated protozoa Spirostomum were isolated by Pautard [37, 381 in sufficient numbers to permit x-ray diffraction analysis. Pautard concluded that the Spirostomum crystals were calcite and hydroxyapatite. Subsequently these data were apparently reevaluated and the crystals are now described as dahllite [28], a colloidal form of carbon-
37 1
IN PARAMECIUM
Table 5. Computed interfacial angles near 60” for crystal faces h,k,Z, and h2k212,based on the cell parameters of Ca-struvite determined in this study. k,
h,
1 1
0 2 2 0 1 1 1
0 0 0 1 1 O 2 2 0 0 1 0 1
0 1 1 a
1,
”
2 0 5 1 0 0 3
1 1 I 1 0 2 2 0 1 0 2
h,
0 0 0 2 1 0 1 0 0 1 1
2 0 5 1 0 0 0 2 0 0 5 0 0 2 1 1 1 5
k,
I,
1 0 2 5 0 7 0 1 1 3 1 2 1 6 1 1 0 1 6 1 4 1 0 7 1 1 1 3 1 0 0 3 1 3 2 1 0 7
Interfacial angle [“I 56.890 57.302 58.066 58.210 58.632 59.598 60.048 60.457 60.476 60.756 6 1.077 61.332 61.558 61.600 61.727 61.768 62.573 62.95 1
Angle
-
60
-3.1104 -2.6984 - 1.9338 - 1.7898 - 1.3682 -0.4017 0.0485 0.4570 0.4759 0.7557 1.0766 1.3315 1.5585 1.6000 1.7275 1.7680 2.570 2.95 13
The only pair common to .
ate-apatite. Although pyrophosphate granules [6, 7, 33, 35, 421, polyphosphate heavy spherical bodies [4, 261, and other intracellular crystalline material have been examined in other protozoa, the studies on crystals from Gonyaulax, Parauronema and Spirostomum represent the major, if not the only, mineralogical studies performed o n intracellular crystals isolated from protozoan cells, and then analyzed by techniques such as x-ray diffraction. ‘Biologically induced’ struvite. Extracellular struvite has previously been identified in bat and penguin guano [9, 36, 551, and in other materials with high organic content [31]. Formation of struvite has important medical and veterinary implications because it is found in pathogenic calculi such as kidney stones that form in both human [40], and feline [3] urinary tracts. Precipitation of extracellular struvite in urea-rich environments is correlated with the presence of bacteria that secrete urease [lo, 13, 271, which converts urea to ammonia. Struvite precipitation, enhanced by urinary bacterial infections, can lead to clogging of catheters [45,541. Intracellular struvite function. The Paramecium crystals characterized in the present study were found not to be polyphosphate bodies, hydroxyapatite, guanine, or other minerals described in protozoan cells. The occurrence of struvite in vertebrate excretory products suggests that struvite serves a role in nitrogen metabolism in Paramecium. The high purine content observed in Gonyaulax and Parauronema refractile bodies suggested that these may be related structures that play a role in
t
crystal morphology showing the pitted surface texture that probably results from partial dissolution (etching) of the crystal. F. A twinned hemimorphic crystal with the crystallographic c axis presumed to be approximately parallel to the plane of the micrograph. This crystal is interpreted to be a type of cyclic twinning, with well developed ( 110) planes intersecting along a mirror plane to form the ridges, and irregular in-filling of the reentrant angles between pairs of ridges by means of face-parallel growth on other crystallographic planes, such as { 101 }. In this model, a pair of (001) surfaces would form composition planes perpendicular to the trace of the prominent ridges. Alternatively, the specimen could represent skeletal growth with mirror twinning on { O O l ) faces in the plane or the photograph (belying the massive character of this grain; see also Fig. A, C & G), or a new twin law for struvite. G . The “underside” {OOl 1, of an incomplete struvite “propeller” showing presumed growth lines radiating outward from linear skeletal cores. Crystals with this growth habit indicate rapid, and possibly nonequilibrium growth. H. Composite mass of struvite grains, with the prominent crystal exhibiting striking pseudohexagonal symmetry. The principal crystallograpic c axis for this habit is thought to be perpendicular to the “hexagonal” face; thus, the “prismatic” faces in the [OOi] zone are probably { O l O ) and (2101, developed to equivalent degrees. This combination of {Ool), (010) and (hkO) forms a credible pseudohexagonal prism. Bar = 10 pm.
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nitrogen excretion [SI] or storage [44] in these marine protozoa. Paramecium is an ammonotelic organism; it does not synthesize urea [50] and, like mammals, it lacks urease. It is generally believed that aquatic organisms smaller than a micrometer in diameter rid themselves of the toxic excretory product ammonia by simple diffusion of the compound into the aqueous milieu. Intracellular crystals have been implicated not only in excretion and nitrogen metabolism, but also in ion homeostasis, taxis (e.g. magnetite), detoxification, and nutrient storage. Growth and resorption of protozoan intracellular crystals have been reported [35, 37, 381, again suggesting homeostatic or storage functions. The possibility that the crystals serve multiple functions, such as ion homeostasis, nitrogen waste and nutrient storage, cannot be ruled out. If struvite provides a mechanism for the storage of P, Mg, and Ca in a membrane-bound vesicle for subsequent metabolic needs of the cell, the highly diffusible ammonia produced by the cell may become sequestered within these vesicles. The existence and isolation of Paramecium “clear” mutants that lack visible crystals at the light microscope level has been reported [53]. This suggests that these intracellular structures are not vital for the organism. However, these mutants have not been examined ultrastructurally and structures not visible by light microscopy may be present. Also, it is not known whether the ability of normal cells to form visible crystals gives the organism an advantage over “clear” mutants for survival under stress or nutrient-poor conditions. ‘Biological controlled’ formation of Paramecium struvite. The observations made in this study demonstrate that the number of crystals in the cells correlates with changes in the lipid content of this ciliate. It should be noted however, that there are differences in the sizes of individual crystals (Fig. l), thus total number of crystals (or crystal aggregates) may not precisely correlate with the total mass of crystals per cell. Future studies should include estimation of total crystal mass by techniques such as image processing and analysis. Given currently available data, we propose that the vesicles in which struvite forms in Paramecium also contain lipids, and that the crystals therefore reside in a cellular compartment serving a nutrient storage function. When stationary-phase Paramecium (which is a lipid auxotroph) was inoculated into fresh medium containing lipids, the cellular lipid content rapidly increased. Thus, the lipid content per cell was highest in lagphase cells, and then decreased throughout culture growth (Fig. 2) [23]. It appears that the number of pyrophosphate-containing crystals per cell is also correlated with the lipid content of another freshwater ciliate, Tetrahymena. Unlike P. retraurelia, the Tetrahymena species on which pyrophosphate granules have been studied has no dietary lipid requirements, and therefore it can grow in media lacking lipids. The amount of lipids per Tetrahymena cell increased, rather than decreased, during axenic culture growth. Thus, in Tetrahymena cells, cellular lipid content was highest in stationary phase. Likewise, it has been previously reported that the number of crystals in Tetrahymena cells increased with culture age and was highest in stationary phase cells [6,7, 351. The observations on these two ciliates, and the detection of relatively high concentrations of lipids in urinary struvite stones [25), is consistent with the suggestion that intracellular crystal formation is facilitated by lipids. Lipids can extract metals from solution, and induce crystal nucleation, especially at oiYaqueous interfaces [&8, 291. Putative inwarddirected ion pumps in the membrane of Paramecium lipid storage vesicles could then increase the P, Mg, and Ca concentrations within that compartment, and the hydrophobic environment within the vesicles would provide physical and chemical conditions appropriate for the nucleation and growth of struvite
crystals. The correlation between the degree of hydration and Mg2+ concentration observed in Paramecium struvite might, therefore, reflect intravesicular changes in the lipidwater content. Thus, this study provides evidence that crystals form over a range of physical and chemical conditions within intracellular vesicles. Release of crystals from protozoan cells has been reported. Whether this process is physiological or occurs in moribund cells under a microscope coverslip remains an open question. The intracellular digestive cycle of ciliates has been studied in detail, especially in Paramecium [I71 but there is no evidence for the fusion of crystal-containing vesicles with the phagolysosomes. Therefore, the release of intracellular crystals by ciliates probably does not involve excretion via the cytoproct (cell anus). The ion and osmoregulatory system (e.g. contractile vacuole) have not been observed to fuse with vesicles containing crystals, but exocytosis via the contractile vacuole pore cannot be ruled out. It is possible that exocytosis of crystals occurs by mechanisms similar to those by which coccoliths, trichocysts, and other large structures are discharged by protozoan cells. Struvite as a biomarker. Biologically induced extracellular precipitation of struvite is well documented. This study provides unambiguous evidence that struvite can also be formed intracellularly in a eukaryotic cell, i.e. struvite formation is also ‘biologically controlled.’ This suggests that struvite is exclusively formed by biological processes or in organic products, which makes it an attractive biomarker. We propose that struvite be considered among other biomarkers [46] in the search for evidence of life on Mars. ACKNOWLEDGMENTS We thank the following for assistance andor for making instrumentation available for this study: the late Ernest Clark, Andrew Buckley, Anne Graeme-Barber, Ben Harris, and Morris Haslop (scanning electron microscopy-elemental analysis and x-ray powder diffraction), and Joseph Caruso (inductively coupled plasma-mass spectrometry). Crystallographic analysis was camed out by JEG using the facilities in the Department of Earth Sciences, Cambridge University. Supported in part by funds from the University of Cincinnati Research Council. LITERATURE CITED 1. Abbona, E & Boistelle, R. 1979. Growth, morphology and crystal habit of struvite crystals (MgNH4PO,.6H,O). J. Crysral Growth, 46: 339-354. 2. Bernheimer, A. W. 1938. A comparative study of the crystalline inclusions of protozoa. Trans. Am. Microsc. Soc., 57:336-343. 3. Buffington, C. A , , Rogers, Q. R. & Morris, J. G. 1990. Effect of diet on struvite activity product in feline urine. Am. J. Vet. Res., 51: 2025-2030. 4. Chapman-Andressen, C. 1976. Studies on the heavy spherical (refractile) bodies of freshwater amoebae. I. Morphology and regeneration of HSBs in Chaos carolinense. Carlsberg Res. Commun.,41:191210. 5. Clapham, L., McLean, R. J. C., Nickel, J. C., Downey, J. & Costerton, J. W. 1990. The influence of bacteria on struvite crystal habit and its importance in urinary stone formation. J. Crystal Growth, 104: 475-484. 6. Coleman, J. R., Nilsson, J. R., Warner, R. R. & Batt, P. 1972. Qualitative and quantitative electron probe analysis of cytoplasmic granules in Tetrahymena pyriformis. Exp. Cell Res., 74:207-219. 7. Coleman, I. R., Nilsson, J. R., Warner, R. R. & Batt, I? 1973. Effects of calcium and strontium o n divalent ion content of refractile granules in Tetrahymena pyriformis. Exp. Cell Rex, 80: 1-9. 8. Cotmore, J. M., Nichols, G . & Wuthier, R. E. 1982. Phospholipidcalcium phosphate complex: enhanced calcium migration in the presence of phosphate. Science, 172:1339-1341. 9. Dana, E. S. 1893. The System of Mineralogy of James Dwight
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