- Email: [email protected]
Organization of cells in magnetotactic multicellular aggregates
Microbiol. Res. (1999) 154,9-13 http://www.urbanfischer.de/journals/microbiolres ©
Urban & Fischer Verlag
Organization of cells in magnetotactic multicellular aggregates Ulysses Linsl, Marcos Farina2 I
2
Setor de Microscopia Eletr6nica e Departamento de Microbiologia General, Instituto de Microbiologia Professor Paulo de Goes - UFRJ - 21941-590, Rio de Janeiro - RJ, Brasil Departamento de Anatomia - Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro - CCS - Bl. F - 21941590, Rio de Janeiro - RJ, Brasil.
Accepted: February 14, 1999
Abstract Magnetotactic multicellular aggregates (MMAs) live in sulfurrich marshes and consist of aggregates of motile bacterial cells. We studied the cellular organization of magnetotactic multicellular aggregates with light and scanning electron microscopy. All MMAs, collected at the same site, were of one morphological type, and presented diameter from 2.2 11m to 4.6 11m, with a mean of 3.1 11m. Larger MMAs presented larger cells than smaller MMAs, not higher number of them. Cells appeared arranged in helical-like patterns within most of the aggregates. We estimated volumes from diameter measurement, and proposed a linear correlation between MMA volume and mean cell volume. MMA volumes ranged from 5.3 to 49.9 11m3 with a mean of 16.9 11m3 and a standard deviation of 8.0 11m3, whereas cell volumes ranged from 0.16 to 1.15 11m3 with a mean of 0.33 11m3 and a standard derivation of 0.2 11m3. Besides, scanning electron microscopy images of slightly disorganized MMAs revealed specialized regions between cells, where bacterial surfaces are in close contact to each other. This information suggests that MMAs are highly organized aggregates of bacterial cells capable of coordinated response to magnetic fields. Keywords: magnetotactic bacteria - scanning electron microscopy - magnetotaxis - cell division
MMAs present hundreds of magneto somes that are organelles composed of a magnetic crystal enveloped by a membrane (Gorby et al. 1988; Stolz 1993). Magnetosomes contain iron sulfides as mineral phases (Farina et al. 1990; Mann et al. 1990; Posfai et al. 1998), but can also incorporate other metals like copper (Bazylinski et al. 1993). They impart aggregates a magnetic moment that is responsible for orientation to magnetic field lines, and hence, for magnetotactic behavior of MMAs. Few ultrastructural data are available on MMAs (Farina et at. 1983). Rodgers et al. (1990) described intercellular structure of MMAs, and proposed the existence of intercellular junctions for connection between adjacent cells. In this work, we observed contacts between adjacent cells in MMAs briefly exposed to distilled water before chemical fixation. To our knowledge, no systematic size measurements were done in MMAs. So, we studied MMAs with light microscopy and scanning electron microscopy, and observed a direct relationship between MMA volume and mean cell volume. This indicates that higher volume MMAs have cells with higher volume, and not a higher number of cells. These results suggest that smaller MMAs are younger than larger MMAs.
Introduction Magnetotactic multicellular aggregates (MMAs) are motile microorganisms that live in sulfur-rich marshes (Lins de Barros et al. 1990a), and consist of aggregates of gram negative bacterial cells (Farina et al. 1983). Corresponding author: M. Farina (e-mail: [email protected]) 0944-5013/99/154/01-009
$12.00/0
Materials and methods Sediments were always collected at the same site from a brackish water lagoon (Lagoa Rodrigo de Freitas) in Rio de Janeiro city and stored in bottles, under dim light and room temperature, for several weeks. For magnetic enrichment of MMAs, a specially designed glass chamber Microbiol. Res. 154 (1999) 1
9
-+ B
2
---+
Fig. 1. Diagram that summarizes enrichment procedure used for studying MMAs. Briefly, bottles (1), filled with lagoon water (2) and sediment (3), were stored in the laboratory for several weeks. For enrichment, special glass devices (4) were filled and exposed to a properly aligned magnetic field (B) for 20 minutes. Enriched MMAs were withdrawn with a capillary tube (5) and processed for light or scanning electron microscopy (see Materials and methods for details).
Fig. 3. Scanning electron microscopy image of two MMAs. Note that larger cells are present in the larger MMA, and smaller cells are present in the smaller one. MMA volume is proportional to cell volume. Bar = 2 J.lm.
. .
, • .' • , ,. •• •
-
•
..
.'
....... -... ..
........ ..
..'.
~
•
•
~.
.. .-..
tI• • ~
'. #I
•• •
,,'t
~,
...
.,""....
••
with capillary end (Fig. 1) was filled with sediment and lagoon water, and then exposed to a properly aligned magnetic field for 20 minutes. Enriched MMAs were collected with a capillary tube and used in light and scanning electron microscopy studies. For light microscopy, MMAs were placed on a slide and observed on an Axioplan light microscope equipped with Nomarski interference contrast optics. For scanning electron microscopy, MMAs were concentrated at the edge of a drop on a coverslip previously treated with poly-L-lysine to improve attachment. MMAs were fixed in 2.5% glutaraldehyde in 100 mM cacodylate buffer, pH 7.2, post-fixed in buffered 1% OS04 solution, dehydrated in ethanol, critical point dried with CO 2 and gold sputtered in a Balzers apparatus. Observation was done at 40 kV with a JEOL 100 ex transmission electron microscope equipped with an ASID-4D scanning accessory. Observation of whole cells was done with a Zeiss 912 transmission electron microscope equipped with an Omega filter (Reimer 1991). Inelastically scattered electrons were used to get information of the distribution of the magnetosomes inside cells of MMAs. Measurements of MMAs and their cells were done with a Nikon profile projector on scanning electron microscope negatives. Only cells on the central
Fig. 2. Electron spectroscopic image (LiE = 56 eV) of cells of a MMA. Note the presence of numerous magnetosomes both in chains and in clusters. Bar = 0.5 J.lm.
10
Microbiol. Res. 154 (1999) 1
... . 20
. >. CJ
e
G:I ::l
~
10
""
.
'
.s
0
2.0
2.8
24
3.2
.
36
4.0
4.4
n
Diameter (Jun)
4.8
Fig. 4. Histogram of diameters of MMAs measured on magnification caIibrated scanning electron micrographs. Note that diameters form one major group, with most of the values around 3.0/lm. Dashed line represents the gaussian fit for plotted values.
60
0
~o
-3. -
....
G)
E ::l
40
0 0
30
g
« ~
0 0
20
0
::E
o
0 0
0
0
0°
10
4»
0
o
(>
Fig. 5. Scatter plot of the mean cell volume of each MMA and the total aggregate vol1.2 ume. A linear correlation (r = 0.75) between MMA volume and mean cell volume is proposed.
0
0 0.0
O.
0.6
0.9
Mean cell volume (j..Lm.3) region of the scanning electron microscope images of MMAs were measured. In this situation, the longer cell axis is almost parallel to the plane of the micrograph. Latex beads (diameter 1.13 ~m, sd 0.09 ~m; Poly sciences) were used for magnification calibration. Volumes of MMAs were estimated by a spherical geometry. Volumes of cells were estimated by the arithmetic mean of volumes for cylinder and ellipsoid geometry as this shape fitted properly to profiles of cells. Over 100 MMAs and their respective cells were measured.
Results MMAs obtained by magnetic concentration presented an active movement when an external field was applied. They swam in a helical trajectory towards the drop edge and then moved back and forth in a "ping-pong" movement. At the edge of the drop, several MMAs exhibited a spinning movement. When distilled water was added, larger MMAs disrupted first, while smaller MMAs resisted longer to disorganization. After disruption, cells did not present motility in all cases observed. Disrupted Microbiol. Res. 154 (1999) 1
11
We observed a spatial disposition of cells in a helicallike pattern within most MMAs (Fig. 6). Observations of larger MMAs indicated that adjacent cells appeared associated with each other by adhesion regions (Fig. 7). These regions were probably uncovered because a small, osmotically induced, disorganization of the whole structure of the aggregates was done.
Discussion
Fig.6. Scanning electron microscopy image of a MMA. Note the disposition of cells in a helical-like pattern. Bar = 1 /lm. Fig. 7. Scanning electron microscopy image that shows a relatively large MMA with adhesion regions (arrowhead) between adjacent cells. Bar = 0.5 /lm.
cells imaged by electron spectroscopic imaging technique contained numerous magneto somes (Fig. 2). Some cells contained magneto somes disposed in linear chains. Smaller MMAs presented smaller cells whereas larger MMAs presented larger ones as observed by light (not shown) and scanning electron (Fig. 3) microscopy. Diameters were distributed between 2.2 !lm and 4.6 !lm with a mean of 3.1!lm and a standard deviation of 0.44!lm (Fig. 4). The histogram distribution provided a good gaussian fit. There was a direct relation between volumes of MMAs and volumes of their cells. MMAs volumes and cell volumes presented a good linear fit (Fig. 5). MMA volumes ranged from 5.3 to 49.9 !lm3 with a mean of 16.9 !lm3 and a standard deviation of 8.0 !lm3, whereas cell volumes ranged from 0.16 to 1.15 !lm3 with a mean of 0.33 !lm3 and a standard deviation of 0.2 !lm3. We could not directly count the number of cells in enough MMAs. 12
Microbiol. Res. 154 (1999) 1
Magnetotactic bacteria vary considerably in cell size and number of magnetosomes. Bacteria, up to 15!lm long, and with more than 5000 magnetosomes, were reported (Vali and Kirschvink 1990). In MMAs, there are differences between our diameter measurements and literature data. We measured maximum values of 4.6 !lm, and literature data report values up to 12!lm (Rodgers et at. 1990). These size differences may be because of different MMA populations in different environmental and growth conditions. In our experimental conditions, however, it is possible that some shrinkage of MMAs happened during dehydration and critical point drying steps of SEM preparation (Boyde and Willams 1971; Dykstra 1992). On the other hand, literature data (Rodgers et at. 1990) may be overestimated because they were obtained with phase-contrast microscopy, which is a limited technique for measuring small objects (Fran90n 1962). Volumes of MMAs were proportional to the volume of their cells. It is unlikely that more than one population of MMAs was present in samples because we were careful in measuring only similar morphological types of aggregates, since in a previous paper (Lins de Barros et at. 1990b) we described two different morphological types of MMAs present in samples collected at the site. Besides, in the histogram of diameters only one major group was observed as shown in the gaussian fit of Fig. 4. This also suggests that there is only one population. Detailed ultrastructural observations associated with molecular systematic studies based on amplification and sequencing of 16S rRNA genes (DeLong et at. 1993; Spring et at. 1992, 1993, 1998) or cultivation could give new insights in this question. Smaller MMAs could represent younger aggregates and larger MMAs older ones for the range of measured diameters. Larger MMAs disrupt before smaller ones in both hypotonic and hypertonic solutions, which suggests a disorganization related to intercellular connections in the aggregate, probably because it becomes difficult to keep cell surface contacts in larger aggregates. Magnetotaxis is a way of guiding cells to regions with available iron, oxygen and sulfide in best concentrations (Frankel et al. 1997). With our isolation method (Fig. 1),
we never found either free swimming cells of MMAs or even very small MMAs (diameter < 2.2!lm ). It is possible that cells do not separate from MMAs, even in cell division, and therefore we could not magnetically isolate independent cells. Each cell of a MMA grows continuously, increasing in volume, and thus contributing to the enlargement of the MMA to an upper size limit. After MMA reaches this limit, cells could coordinately and simultaneously divide. Observation of MMAs with oval shape supports the hypothesis of division of MMAs (Rodgers et al. 1990). Rarely, we observed what could be a division of a MMA. The "dividing" MMA appeared as two aggregates symmetrically attached to each other in the middle plane. Alternatively, availability of contact areas between adjacent cells would limit MMA size in larger aggregates. In these MMAs, cells would keep cellular connections until the whole aggregate reaches an unstable or transitory state when it disorganizes into cells that can divide individually. Both hypotheses do not conflict with results in Fig. 5, as we did not observe MMAs with diameters outside of the range expressed in the figure, and we never observed a complete division of a whole MMA. On the other hand, the linear correlation proposed between MMA volume and cell volume has to be interpreted as an approximation, valid only for cells and aggregates observed after magnetic concentration. It is possible that MMAs outside the range expressed in Fig. 5 are either non-magnetic or represent transitory situations which are difficult to observe. As a consequence, extrapolation of the linear correlation outside the data presented in the figure has no practical meaning. The life cycle of MMAs is yet to be determined.
Acknowledgements This work was supported by: CAPES, PRONEX, and TWAS.
References Bazylinski, D. A, Garratt-Reed, A J., Abedi, A, Frankel, R. B. (1993): Copper association with iron sulfide magnetosomes in a magnetotactic bacterium. Arch. Microbiol. 160, 35-42. Boyde, A., Williams, R. A. D. (1972): Estimation of the volume of bacterial cells by scanning electron microscopy. Arch. Oral BioI. 16,259-267. DeLong, E. F., Frankel, R. B., Bazylinski, D. A (1993): Multiple evolutionary origins of magnetotaxis in bacteria. Science 259,803-806. Dykstra, M. J. (1992): Scanning electron microscopy. In: Bio-
logical electron microscopy. Theory, techniques and troubleshooting. Plenum Press, New York, 223-246. Farina, M., Lins de Barros, H. G. P., Esquivel, D. M. S., Danon, J. (1983): Ultrastructure of a magnetotactic microorganism. BioI. Cell. 48, 85-88. Farina, M., Esquivel, D. M. S., Lins de Barros, H. G. P. (1990): Magnetic iron-sulphur crystals from a magnetotactic microorganism. Nature 343, 256-258. Franc;:on, M. (1962): Progress in microscopy. Row Peterson, Evanston, Illinois. Frankel, R. B., Bazylinski, D. A., Johnson, M. S., Taylor, B. L. (1997): Magneto-aerotaxis in marine coccoid bacteria. Biophys. J. 73, 994-1000. Gorby, Y. A., Beveridge, T. J., Blakemore, R. P. (1988): Characterization of the bacterial magnetosome membrane. J. Bacteriol. 170, 834- 841. Lins de Barros, H. G. P., Esquivel, D. M. S., Farina, M. (l990a): Magnetotaxis. Science Progress (Oxford) 74, 347-359. Lins de Barros, H. G. P., Esquivel, D. M. S., Farina, M. (1990 b): Biomineralization of a new material by a magnetotactic microorganism. In: Iron Biominerals (Eds: Frankel, R. B., Blakemore, R. P.). Plenum Press, New York, 257-268. Mann, S., Sparks, N. H. c., Frankel, R. B., Bazylinski, D. A., Jannasch, H. W. (1990): Biomineralization of ferrimagnetic greigite (Fe 3S4) and iron pyrite (FeS 2) in a magnetotactic bacterium. Nature 343, 258-261. Posfai, M., Buseck, P. R., Bazylinski, D. A., Frankel, R. B. (1998): Reaction sequence of iron sulfide minerals in bacteria and their use as biomarkers. Science 280, 880-883. Reimer, L. (1991): Energy-filtering transmission electron microscope. Adv. Electron Phys. 81,43-126. Rodgers, F. G., Blakemore, R. P., Blakemore, N. A., Frankel, R. B., Bazylinski, D. A., Maratea, D., Rodgers, C. (1990): Intercellular structure in a many-celled magnetotactic prokaryote. Arch. Microbiol. 154, 18-22. Spring, S., Amann, R., Ludwig, w., Schleifer, K., Petersen, N. (1992): Phylogenetic diversity and identification of nonculturable magnetotactic bacteria. System. Appl. Microbiol. 15,116-122. Spring, S., Amann, R., Ludwig, w., Schleifer, K., Gemerden, H., Petersen, N. (1993): Dominating role of an unusual magnetotactic bacterium in the microaerobic zone of a freshwater sediment. Appl. Environ. Microbiol. 59, 2397-2403. Spring, S., Lins, U., Amann, R., Schleifer, K., Ferreira, L., Esquivel, D., Farina, M. (1998): Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes. Arch. Microbiol. 169, 136-147. Stolz, J. F. (1993): Magnetosomes. J. Gen. Microbiol. 139, 1663-1670. Yali, H., Kirschvink, J. L. (1990): Observations of magnetosome organization, surface structure, and iron biomineralization of undescribed magnetic bacteria: evolutionary speculations. In: Iron Biominerals (Eds: Frankel, R. B., Blakemore, R. P.). Plenum Press, New York, 97-115.
Microbia!. Res. 154 (\ 999) 1
13
Urban & Fischer Verlag
Organization of cells in magnetotactic multicellular aggregates Ulysses Linsl, Marcos Farina2 I
2
Setor de Microscopia Eletr6nica e Departamento de Microbiologia General, Instituto de Microbiologia Professor Paulo de Goes - UFRJ - 21941-590, Rio de Janeiro - RJ, Brasil Departamento de Anatomia - Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro - CCS - Bl. F - 21941590, Rio de Janeiro - RJ, Brasil.
Accepted: February 14, 1999
Abstract Magnetotactic multicellular aggregates (MMAs) live in sulfurrich marshes and consist of aggregates of motile bacterial cells. We studied the cellular organization of magnetotactic multicellular aggregates with light and scanning electron microscopy. All MMAs, collected at the same site, were of one morphological type, and presented diameter from 2.2 11m to 4.6 11m, with a mean of 3.1 11m. Larger MMAs presented larger cells than smaller MMAs, not higher number of them. Cells appeared arranged in helical-like patterns within most of the aggregates. We estimated volumes from diameter measurement, and proposed a linear correlation between MMA volume and mean cell volume. MMA volumes ranged from 5.3 to 49.9 11m3 with a mean of 16.9 11m3 and a standard deviation of 8.0 11m3, whereas cell volumes ranged from 0.16 to 1.15 11m3 with a mean of 0.33 11m3 and a standard derivation of 0.2 11m3. Besides, scanning electron microscopy images of slightly disorganized MMAs revealed specialized regions between cells, where bacterial surfaces are in close contact to each other. This information suggests that MMAs are highly organized aggregates of bacterial cells capable of coordinated response to magnetic fields. Keywords: magnetotactic bacteria - scanning electron microscopy - magnetotaxis - cell division
MMAs present hundreds of magneto somes that are organelles composed of a magnetic crystal enveloped by a membrane (Gorby et al. 1988; Stolz 1993). Magnetosomes contain iron sulfides as mineral phases (Farina et al. 1990; Mann et al. 1990; Posfai et al. 1998), but can also incorporate other metals like copper (Bazylinski et al. 1993). They impart aggregates a magnetic moment that is responsible for orientation to magnetic field lines, and hence, for magnetotactic behavior of MMAs. Few ultrastructural data are available on MMAs (Farina et at. 1983). Rodgers et al. (1990) described intercellular structure of MMAs, and proposed the existence of intercellular junctions for connection between adjacent cells. In this work, we observed contacts between adjacent cells in MMAs briefly exposed to distilled water before chemical fixation. To our knowledge, no systematic size measurements were done in MMAs. So, we studied MMAs with light microscopy and scanning electron microscopy, and observed a direct relationship between MMA volume and mean cell volume. This indicates that higher volume MMAs have cells with higher volume, and not a higher number of cells. These results suggest that smaller MMAs are younger than larger MMAs.
Introduction Magnetotactic multicellular aggregates (MMAs) are motile microorganisms that live in sulfur-rich marshes (Lins de Barros et al. 1990a), and consist of aggregates of gram negative bacterial cells (Farina et al. 1983). Corresponding author: M. Farina (e-mail: [email protected]) 0944-5013/99/154/01-009
$12.00/0
Materials and methods Sediments were always collected at the same site from a brackish water lagoon (Lagoa Rodrigo de Freitas) in Rio de Janeiro city and stored in bottles, under dim light and room temperature, for several weeks. For magnetic enrichment of MMAs, a specially designed glass chamber Microbiol. Res. 154 (1999) 1
9
-+ B
2
---+
Fig. 1. Diagram that summarizes enrichment procedure used for studying MMAs. Briefly, bottles (1), filled with lagoon water (2) and sediment (3), were stored in the laboratory for several weeks. For enrichment, special glass devices (4) were filled and exposed to a properly aligned magnetic field (B) for 20 minutes. Enriched MMAs were withdrawn with a capillary tube (5) and processed for light or scanning electron microscopy (see Materials and methods for details).
Fig. 3. Scanning electron microscopy image of two MMAs. Note that larger cells are present in the larger MMA, and smaller cells are present in the smaller one. MMA volume is proportional to cell volume. Bar = 2 J.lm.
. .
, • .' • , ,. •• •
-
•
..
.'
....... -... ..
........ ..
..'.
~
•
•
~.
.. .-..
tI• • ~
'. #I
•• •
,,'t
~,
...
.,""....
••
with capillary end (Fig. 1) was filled with sediment and lagoon water, and then exposed to a properly aligned magnetic field for 20 minutes. Enriched MMAs were collected with a capillary tube and used in light and scanning electron microscopy studies. For light microscopy, MMAs were placed on a slide and observed on an Axioplan light microscope equipped with Nomarski interference contrast optics. For scanning electron microscopy, MMAs were concentrated at the edge of a drop on a coverslip previously treated with poly-L-lysine to improve attachment. MMAs were fixed in 2.5% glutaraldehyde in 100 mM cacodylate buffer, pH 7.2, post-fixed in buffered 1% OS04 solution, dehydrated in ethanol, critical point dried with CO 2 and gold sputtered in a Balzers apparatus. Observation was done at 40 kV with a JEOL 100 ex transmission electron microscope equipped with an ASID-4D scanning accessory. Observation of whole cells was done with a Zeiss 912 transmission electron microscope equipped with an Omega filter (Reimer 1991). Inelastically scattered electrons were used to get information of the distribution of the magnetosomes inside cells of MMAs. Measurements of MMAs and their cells were done with a Nikon profile projector on scanning electron microscope negatives. Only cells on the central
Fig. 2. Electron spectroscopic image (LiE = 56 eV) of cells of a MMA. Note the presence of numerous magnetosomes both in chains and in clusters. Bar = 0.5 J.lm.
10
Microbiol. Res. 154 (1999) 1
... . 20
. >. CJ
e
G:I ::l
~
10
""
.
'
.s
0
2.0
2.8
24
3.2
.
36
4.0
4.4
n
Diameter (Jun)
4.8
Fig. 4. Histogram of diameters of MMAs measured on magnification caIibrated scanning electron micrographs. Note that diameters form one major group, with most of the values around 3.0/lm. Dashed line represents the gaussian fit for plotted values.
60
0
~o
-3. -
....
G)
E ::l
40
0 0
30
g
« ~
0 0
20
0
::E
o
0 0
0
0
0°
10
4»
0
o
(>
Fig. 5. Scatter plot of the mean cell volume of each MMA and the total aggregate vol1.2 ume. A linear correlation (r = 0.75) between MMA volume and mean cell volume is proposed.
0
0 0.0
O.
0.6
0.9
Mean cell volume (j..Lm.3) region of the scanning electron microscope images of MMAs were measured. In this situation, the longer cell axis is almost parallel to the plane of the micrograph. Latex beads (diameter 1.13 ~m, sd 0.09 ~m; Poly sciences) were used for magnification calibration. Volumes of MMAs were estimated by a spherical geometry. Volumes of cells were estimated by the arithmetic mean of volumes for cylinder and ellipsoid geometry as this shape fitted properly to profiles of cells. Over 100 MMAs and their respective cells were measured.
Results MMAs obtained by magnetic concentration presented an active movement when an external field was applied. They swam in a helical trajectory towards the drop edge and then moved back and forth in a "ping-pong" movement. At the edge of the drop, several MMAs exhibited a spinning movement. When distilled water was added, larger MMAs disrupted first, while smaller MMAs resisted longer to disorganization. After disruption, cells did not present motility in all cases observed. Disrupted Microbiol. Res. 154 (1999) 1
11
We observed a spatial disposition of cells in a helicallike pattern within most MMAs (Fig. 6). Observations of larger MMAs indicated that adjacent cells appeared associated with each other by adhesion regions (Fig. 7). These regions were probably uncovered because a small, osmotically induced, disorganization of the whole structure of the aggregates was done.
Discussion
Fig.6. Scanning electron microscopy image of a MMA. Note the disposition of cells in a helical-like pattern. Bar = 1 /lm. Fig. 7. Scanning electron microscopy image that shows a relatively large MMA with adhesion regions (arrowhead) between adjacent cells. Bar = 0.5 /lm.
cells imaged by electron spectroscopic imaging technique contained numerous magneto somes (Fig. 2). Some cells contained magneto somes disposed in linear chains. Smaller MMAs presented smaller cells whereas larger MMAs presented larger ones as observed by light (not shown) and scanning electron (Fig. 3) microscopy. Diameters were distributed between 2.2 !lm and 4.6 !lm with a mean of 3.1!lm and a standard deviation of 0.44!lm (Fig. 4). The histogram distribution provided a good gaussian fit. There was a direct relation between volumes of MMAs and volumes of their cells. MMAs volumes and cell volumes presented a good linear fit (Fig. 5). MMA volumes ranged from 5.3 to 49.9 !lm3 with a mean of 16.9 !lm3 and a standard deviation of 8.0 !lm3, whereas cell volumes ranged from 0.16 to 1.15 !lm3 with a mean of 0.33 !lm3 and a standard deviation of 0.2 !lm3. We could not directly count the number of cells in enough MMAs. 12
Microbiol. Res. 154 (1999) 1
Magnetotactic bacteria vary considerably in cell size and number of magnetosomes. Bacteria, up to 15!lm long, and with more than 5000 magnetosomes, were reported (Vali and Kirschvink 1990). In MMAs, there are differences between our diameter measurements and literature data. We measured maximum values of 4.6 !lm, and literature data report values up to 12!lm (Rodgers et at. 1990). These size differences may be because of different MMA populations in different environmental and growth conditions. In our experimental conditions, however, it is possible that some shrinkage of MMAs happened during dehydration and critical point drying steps of SEM preparation (Boyde and Willams 1971; Dykstra 1992). On the other hand, literature data (Rodgers et at. 1990) may be overestimated because they were obtained with phase-contrast microscopy, which is a limited technique for measuring small objects (Fran90n 1962). Volumes of MMAs were proportional to the volume of their cells. It is unlikely that more than one population of MMAs was present in samples because we were careful in measuring only similar morphological types of aggregates, since in a previous paper (Lins de Barros et at. 1990b) we described two different morphological types of MMAs present in samples collected at the site. Besides, in the histogram of diameters only one major group was observed as shown in the gaussian fit of Fig. 4. This also suggests that there is only one population. Detailed ultrastructural observations associated with molecular systematic studies based on amplification and sequencing of 16S rRNA genes (DeLong et at. 1993; Spring et at. 1992, 1993, 1998) or cultivation could give new insights in this question. Smaller MMAs could represent younger aggregates and larger MMAs older ones for the range of measured diameters. Larger MMAs disrupt before smaller ones in both hypotonic and hypertonic solutions, which suggests a disorganization related to intercellular connections in the aggregate, probably because it becomes difficult to keep cell surface contacts in larger aggregates. Magnetotaxis is a way of guiding cells to regions with available iron, oxygen and sulfide in best concentrations (Frankel et al. 1997). With our isolation method (Fig. 1),
we never found either free swimming cells of MMAs or even very small MMAs (diameter < 2.2!lm ). It is possible that cells do not separate from MMAs, even in cell division, and therefore we could not magnetically isolate independent cells. Each cell of a MMA grows continuously, increasing in volume, and thus contributing to the enlargement of the MMA to an upper size limit. After MMA reaches this limit, cells could coordinately and simultaneously divide. Observation of MMAs with oval shape supports the hypothesis of division of MMAs (Rodgers et al. 1990). Rarely, we observed what could be a division of a MMA. The "dividing" MMA appeared as two aggregates symmetrically attached to each other in the middle plane. Alternatively, availability of contact areas between adjacent cells would limit MMA size in larger aggregates. In these MMAs, cells would keep cellular connections until the whole aggregate reaches an unstable or transitory state when it disorganizes into cells that can divide individually. Both hypotheses do not conflict with results in Fig. 5, as we did not observe MMAs with diameters outside of the range expressed in the figure, and we never observed a complete division of a whole MMA. On the other hand, the linear correlation proposed between MMA volume and cell volume has to be interpreted as an approximation, valid only for cells and aggregates observed after magnetic concentration. It is possible that MMAs outside the range expressed in Fig. 5 are either non-magnetic or represent transitory situations which are difficult to observe. As a consequence, extrapolation of the linear correlation outside the data presented in the figure has no practical meaning. The life cycle of MMAs is yet to be determined.
Acknowledgements This work was supported by: CAPES, PRONEX, and TWAS.
References Bazylinski, D. A, Garratt-Reed, A J., Abedi, A, Frankel, R. B. (1993): Copper association with iron sulfide magnetosomes in a magnetotactic bacterium. Arch. Microbiol. 160, 35-42. Boyde, A., Williams, R. A. D. (1972): Estimation of the volume of bacterial cells by scanning electron microscopy. Arch. Oral BioI. 16,259-267. DeLong, E. F., Frankel, R. B., Bazylinski, D. A (1993): Multiple evolutionary origins of magnetotaxis in bacteria. Science 259,803-806. Dykstra, M. J. (1992): Scanning electron microscopy. In: Bio-
logical electron microscopy. Theory, techniques and troubleshooting. Plenum Press, New York, 223-246. Farina, M., Lins de Barros, H. G. P., Esquivel, D. M. S., Danon, J. (1983): Ultrastructure of a magnetotactic microorganism. BioI. Cell. 48, 85-88. Farina, M., Esquivel, D. M. S., Lins de Barros, H. G. P. (1990): Magnetic iron-sulphur crystals from a magnetotactic microorganism. Nature 343, 256-258. Franc;:on, M. (1962): Progress in microscopy. Row Peterson, Evanston, Illinois. Frankel, R. B., Bazylinski, D. A., Johnson, M. S., Taylor, B. L. (1997): Magneto-aerotaxis in marine coccoid bacteria. Biophys. J. 73, 994-1000. Gorby, Y. A., Beveridge, T. J., Blakemore, R. P. (1988): Characterization of the bacterial magnetosome membrane. J. Bacteriol. 170, 834- 841. Lins de Barros, H. G. P., Esquivel, D. M. S., Farina, M. (l990a): Magnetotaxis. Science Progress (Oxford) 74, 347-359. Lins de Barros, H. G. P., Esquivel, D. M. S., Farina, M. (1990 b): Biomineralization of a new material by a magnetotactic microorganism. In: Iron Biominerals (Eds: Frankel, R. B., Blakemore, R. P.). Plenum Press, New York, 257-268. Mann, S., Sparks, N. H. c., Frankel, R. B., Bazylinski, D. A., Jannasch, H. W. (1990): Biomineralization of ferrimagnetic greigite (Fe 3S4) and iron pyrite (FeS 2) in a magnetotactic bacterium. Nature 343, 258-261. Posfai, M., Buseck, P. R., Bazylinski, D. A., Frankel, R. B. (1998): Reaction sequence of iron sulfide minerals in bacteria and their use as biomarkers. Science 280, 880-883. Reimer, L. (1991): Energy-filtering transmission electron microscope. Adv. Electron Phys. 81,43-126. Rodgers, F. G., Blakemore, R. P., Blakemore, N. A., Frankel, R. B., Bazylinski, D. A., Maratea, D., Rodgers, C. (1990): Intercellular structure in a many-celled magnetotactic prokaryote. Arch. Microbiol. 154, 18-22. Spring, S., Amann, R., Ludwig, w., Schleifer, K., Petersen, N. (1992): Phylogenetic diversity and identification of nonculturable magnetotactic bacteria. System. Appl. Microbiol. 15,116-122. Spring, S., Amann, R., Ludwig, w., Schleifer, K., Gemerden, H., Petersen, N. (1993): Dominating role of an unusual magnetotactic bacterium in the microaerobic zone of a freshwater sediment. Appl. Environ. Microbiol. 59, 2397-2403. Spring, S., Lins, U., Amann, R., Schleifer, K., Ferreira, L., Esquivel, D., Farina, M. (1998): Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes. Arch. Microbiol. 169, 136-147. Stolz, J. F. (1993): Magnetosomes. J. Gen. Microbiol. 139, 1663-1670. Yali, H., Kirschvink, J. L. (1990): Observations of magnetosome organization, surface structure, and iron biomineralization of undescribed magnetic bacteria: evolutionary speculations. In: Iron Biominerals (Eds: Frankel, R. B., Blakemore, R. P.). Plenum Press, New York, 97-115.
Microbia!. Res. 154 (\ 999) 1
13