Download Multicellular life cycle of magnetotactic prokaryotes

FEMS Microbiology Letters 240 (2004) 203–208
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Multicellular life cycle of magnetotactic prokaryotes
Carolina N. Keim a,b, Juliana L. Martins b, Fernanda Abreu b,
Alexandre Soares Rosado b, Henrique Lins de Barros c, Radovan Borojevic a,
Ulysses Lins b,*, Marcos Farina a
b
a
Instituto de Ciências Biomédicas, Bloco F, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
c
Centro Brasileiro de Pesquisas Fı́sicas/CNPq, Rua Xavier Sigaud, 150, Urca, Rio de Janeiro, RJ, Brazil
Received 16 April 2004; received in revised form 20 September 2004; accepted 22 September 2004
First published online 8 October 2004
Edited by S. Silver
Abstract
Most multicellular organisms, prokaryotes as well as animals, plants and algae have a unicellular stage in their life cycle. Here, we
describe an uncultured prokaryotic magnetotactic multicellular organism that reproduces by binary fission. It is multicellular in all
the stages of its life cycle, and during most of the life cycle the cells organize into a hollow sphere formed by a functionally coordinated and polarized single-cell layer that grows by increasing the cell size. Subsequently, all the cells divide synchronously; the
organism becomes elliptical, and separates into two equal spheres with a torsional movement in the equatorial plane. Unicellular
bacteria similar to the cells that compose these organisms have not been found. Molecular biology analysis showed that all the
organisms studied belong to a single genetic population phylogenetically related to many-celled magnetotactic prokaryotes in the
delta sub-group of the proteobacteria. This appears to be the first report of a multicellular prokaryotic organism that proliferates
by dividing into two equal multicellular organisms each similar to the parent one.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Cell division; Life cycle; Magnetotactic bacteria; Magnetotaxis; Many-celled magnetotactic prokaryote; Magnetotactic multicellular
aggregate; Multicellularity; Microscopy; DGGE
1. Introduction
Magnetotactic bacteria are gram-negative microorganisms that orient passively along magnetic fields while
swimming propelled by flagella. The magnetic orientation is due to the presence of membrane-bounded magnetic crystals, called magnetosomes, in the cytoplasm,
composed of either magnetite (Fe3O4) or greigite
(Fe3S4) [1]. Most magnetotactic bacteria are unicellular,
but spherical organisms composed of several prokary*
Corresponding author. Fax: +55 21 2560 8344.
E-mail address: [email protected] (U. Lins).
otic cells have also been described [2–5]. These magnetotactic multicellular organisms are highly motile, showing
a complex swimming behavior consisting of a forward
movement in the direction of the magnetic field and a
backward movement in the opposite direction, indicating that the flagellar movement in the whole organism
is coordinated [2–4]. Their cells are Gram-negative
[2,3,5] and contain electron-dense particles corresponding to iron sulfide magnetosomes [1]. Adding distilled
water to the samples causes disaggregation of the organism and loss of motility [2,4,5].
High numbers of these organisms were found in
Araruama lagoon, a hypersaline lagoon near Rio de
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.09.035
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C.N. Keim et al. / FEMS Microbiology Letters 240 (2004) 203–208
Janeiro, Brazil. Transmission electron microscopy of
ultra-thin sections and freeze-fracture replicas showed
that the cells are arranged side by side around an internal compartment, which is acellular, such that all cells
maintain contact with both the external environment
and the internal compartment. The cells are tightly
bound to each other and have a pyramidal shape that allows them to fit in the spherical organism. The high level
of structural organization, the interdependence of the
cells suggested by loss of motility when the organism
disaggregate, the cell coordination required for the complex swimming behavior and the orientation of magnetic
crystals to give a net magnetic moment, shows that this
organism is in fact a highly organized prokaryotic multicellular organism [3].
Most prokaryotes are unicellular throughout their
cell cycle. On the other hand, some bacteria show a
complex life cycle that involves cell communication
and coordination. The best-known examples are the
myxobacteria that are able to form fruiting bodies containing thousands of cells [6]. Although magnetotactic
multicelullar organisms were first reported more than
20 years ago [5], their mode of proliferation remained
unknown. Here, we describe the life cycle of magnetotactic multicellular organisms from Araruama lagoon,
which is different from previously known ones in that
there is no unicellular stage and the development is
restricted to coordinated cell division and cell
movements.
2. Materials and methods
Samples of water and sediment were collected in
Araruama Lagoon (22° 50 0 2100 S 42° 13 0 4400 W) and maintained in bottles in the laboratory for a few days. The
magnetotactic multicellular organisms were magnetically isolated [7] and processed for both scanning and
transmission electron microscopy as described [3].
Organism diameters were measured on magnification
calibrated images from unfixed organisms produced in a
Zeiss Axioplan II optical microscope using the Nomarski interferential contrast mode. The cell number of each
organism was evaluated using the bright field mode after
the organisms have disaggregated. Video records were
obtained using a JVC camera in the same microscope
and the analogical signal was digitized. For confocal
laser scanning microscopy, living organisms were
stained with the dye FM 1-43 (Molecular Probes Inc.,
USA), which binds to membrane lipids. They were observed in a Zeiss LSM 510 META confocal laser scanning microscope (Oberkochen, Germany) using a 488
nm excitation wavelength.
For DNA analysis, the magnetically separated organisms were further purified using a rare-earth strong magnet glued in the lateral wall of the tube in the proper
orientation. After some minutes, the water was removed
leaving a small pellet next to the magnet. Cells were frozen and thawed and directly used for polymerase chain
reaction (PCR) amplification. The universal 16S rDNA
primers used were 968F and 1401R (E. coli numbering
[8]). A GC clamp (a 40-nucleotide GC-rich sequence
attached to the 5 0 end of primer) was added to the forward primer to improve the resolution of bands in the
DGGE (denaturating-gel gradient electrophoresis) gel.
PCR amplifications were performed using a thermal
cycler, and DGGE was carried out with the PCR products as described [9]. The stained gels were scanned on a
Storm Gel and Blot Imaging System (Amersham Pharmacia Biotech, Freiburg, Germany). The PCR product
was purified by the Wizard PCR Preps DNA Purification System (Promega, Madison, USA) and then
sequenced using an ABI PRISM model 373 automatic
sequencer with a BigDye Terminator Cycle sequencing
kit (PE Biosystems, CA, USA). Sequence identification
was done using the BLAST-N of the National Center
for Biotechnology Information (NCBI). The sequence
has been deposited in the GenBank Database under
Accession No. AY576052.
3. Results
The size distribution of magnetotactic multicellular
organisms from Araruama lagoon showed a single peak
in the frequency histogram plot, indicative of the presence of a single population of organisms (Fig. 1). In
some freshly collected samples, as in the case described
in Fig. 1(b), the histogram of the cell number of disrupted organisms had two peaks. This distribution
could be caused either by the presence of two distinct
populations of magnetotactic multicellular organisms
or two stages of the life cycle of a single population.
Denaturing-gel gradient electrophoresis (DGGE) profiles showed only one band with the same melting behavior in all samples analyzed (data not shown), suggesting
that all the magnetically concentrated organisms and
their cells were from the same genetic group. Preliminary 16S rRNA sequencing data indicates that the magnetotactic multicellular organisms from Araruama
lagoon belong to the delta subgroup of the proteobacteria (Accession No. AY576052). The phylogenetic analysis using BLAST-N showed that the fragment amplified
with R1401 and F968GC was most closely related to
16S rDNA sequences of uncultured bacteria found in
marine sediments (87.82–87.76% similarity) and 87%
similar to the many-celled magnetotactic prokaryotes
clone MMP 1991 [10].
Cells in living spherical organisms were arranged
radially around a small central space (Fig. 2), as described for fixed organisms prepared for transmission
electron microscopy [3]. Freshly collected samples
C.N. Keim et al. / FEMS Microbiology Letters 240 (2004) 203–208
205
Fig. 1. Populations of magnetotactic multicellular organisms from Araruama lagoon. (a) Distribution of magnetotactic multicellular organisms
diameters in lm; (b) Number of cells within magnetotactic multicellular organisms. Observe two peaks, which would indicate the presence of two
populations in this sample.
Fig. 2. Confocal laser scanning image of a living magnetotactic
multicellular organism stained with FM 1-43, a lipophilic fluorescent
dye. Observe the radial arrangement of the cells and an internal
compartment at the center of the organism.
contained also organisms with diverse morphologies besides the common spherical shape, suggesting a sequence
of multicellular life cycle stages, as illustrated in Fig. 3.
For most of the lifetime, the magnetotactic multicellular
organisms were spherical (Fig. 3(a)), similar to the previously described ones [2–5]. They appear to grow by
enlarging the cell size, but not their number (Figs. 3(a)
and (b)). This is consistent with the previous observation
that the volume of the whole organism is proportional
to the volume of cells [4]. After that, cells would divide
synchronously but would remain together, maintaining
their general arrangement. In this part of the cycle, the
organism would present a double number of smaller
cells (Fig. 3(c)). Subsequently, the magnetotactic multicellular organisms became elliptical (Fig. 3(d)). In the
next step, the equatorial region progressively narrowed,
the organisms became eight-shaped, as two attached
organisms (Fig. 3(e)). In this stage, the two halves were
about the same size and shape. The constriction between
them was followed by a slight torsion of one half in relation to the other. This torsion would be the best way to
separate the two daughter-organisms maintaining the
cells close to each other. Finally, at the end of the cycle,
the eight-shaped organism would split into two equal
smaller spherical organisms (Fig. 3(f)). We observed under the light microscope some organisms splitting into
two new organisms after staying eight-shaped for up
to two hours. We do not know whether the division in
the natural environment lasts all this time, since they
seem to be anaerobic and observation in the laboratory
was done without providing anoxia. Fig. 4 shows a series of micrographs of the division of a single magnetotactic multicellular organism obtained from a video
record.
Transmission electron microscopy showed that some
magnetotactic multicellular organisms had cells containing invaginations of the cell membranes indicative
of concomitant cell division in two or more cells of
the same organism (Fig. 5). Because ultra-thin sections
(ca. 60 nm thick) were observed, it is possible that all
cells in these organisms are dividing, but membrane
invaginations were not seen in all of the cells because
of different cutting planes. Moreover, the two peaks
observed in some of the cell counts (Fig. 1(b)) and
the clearly different number of cells in organisms observed by scanning electron microscopy (Figs. 3(b)
and (c)) corroborates the hypothesis that cell divisions
are synchronous in magnetotactic multicellular organisms. The invaginations were oriented radially and always began at the part of the cell that has direct
contact with the external environment (Fig. 5). This
mechanism of cell division preserves the general organization of the organism because it maintains all cells
arranged radially.
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Fig. 3. Scanning electron micrographs of selected individuals arranged to illustrate the presumed sequence of the life cycle of the magnetotactic
multicellular organisms. Initially (a), the organism is small and spherical; as it grows (b) their cell size enlarges, but not the cell number. Later (c), cells
synchronously divide without separating and the organism contains a larger number of smaller cells. In the next step (d), the magnetotactic
multicellular organisms become elliptical and then (e) eight-shaped, as two attached organisms. Finally (f), the eight-shaped organism splits into two
equal organisms. Scale bar: 4 lm.
Fig. 4. Light microscopy sequence of a single dividing magnetotactic multicellular organism showing the final steps in the organism division.
Initially, the organism is at the eight-shaped stage, and the constriction between the two halves seems to increase with time. Finally, the organism split
into two organisms that swim independently. Bar = 10 lm.
4. Discussion
Fig. 5. Ultra-thin section of a magnetotactic multicellular organism
observed by transmission electron microscopy showing invaginations
(arrows) indicative of cell division in four of the seven cells seen in
longitudinal view. The invaginations arise in the part of the membrane
that has direct contact with the environment. The magnetosomes are
observed in both sides of the invaginations, showing that both
daughter-cells receive the magnetosomes from the mother-cell.
Bar = 1.0 lm.
In unicellular magnetotactic bacteria, cell division results in partitioning of the magnetosomes, of the other
intracellular inclusions such as polyhydroxyalkanoates,
and possibly also flagella between the two daughter-cells
[11]. Similarly, in the magnetotactic multicellular organisms the magnetosomes were disposed in both sides of
the dividing cells, showing that they were distributed
to the two daughter-cells during cell division and that
their magnetic polarity in relation to the flagella is maintained (Fig. 5). The magnetic polarity of magnetic crystals in magnetotactic bacteria seems to be an epigenetic
heritable trace [12].
Considering that the magnetic field polarity of each
magnetosome is kept during the cell division, the
maintenance of the magnetic polarity in the whole
daughter-organisms is complicated by the fact that some
cells (those next to the splitting site) must change significantly their position in the whole organism (up to 90°
from the original position). Thus, these organisms
should coordinate cell division and relative position of
C.N. Keim et al. / FEMS Microbiology Letters 240 (2004) 203–208
the daughter-cells to keep the magnetic polarity of the
whole organism as well as generating two magnetotactic
organisms with the same magnetotactic behavior as the
mother-organism.
Electron micrographs (Fig. 3) show that cells are arranged in a roughly helical distribution. Based on the
helical organization of cells we hypothesize for the cell
rearrangements during division to explain the maintenance of the magnetic moment after several generations.
The axis of the helix would define a polar axis parallel to
the direction of movement. Cell division planes would
be aligned perpendicularly to the direction of the helix
trace, which would maintain the general cell arrangement in the organism. During the organism division,
the cells from different turns of the helix would slide in
relation to each other, causing the organism to become
elliptical, then eight-shaped as seen in Figs. 3(d) and
(e). All cells have their magnetic moments pointing in
a specific direction along the helix trace. The projection
of the magnetic moment of each cell in the plane perpendicular to the axis would cancel out with the projected
magnetic moment of another cell in the opposite side
of the organism. In contrast, the components of the
magnetic moments parallel to the polar axis point in
the same direction. Thus, the net magnetic moment of
the whole organism would be generated by the sum of
the individual cell magnetic moment projection component in the direction parallel to the polar axis. Consequently, after the separation, each new organism
would present the same radial–helical distribution of
cells as the mother organism, with the net magnetic moment parallel to the polar axis.
Helical organization is found in early developmental
stages during the cleavage in several major invertebrate
animal groups, assembled under the name Spiralia. In
this case, it is determined by the division plane of blastomeres, and depends upon the positioning of centrosomes [13]. Since in magnetotactic organisms the
helical organization is present probably before the cell
division, and their cells have no centrosomes, this order
may correspond to the best spatial accommodation of
cells with finely tuned forms, or may be caused by special adhesive properties of the cells that enable each cell
to find and maintain its position in the whole multicellular body.
The similarity of this organism with the previously
studied magnetotactic multicelular organisms [2–5] is
great: they are all spherical organisms composed of multiple Gram-negative prokaryotic cells containing iron
sulfide magnetosomes. Interestingly, Rodgers et al. [2]
reported the existence of elliptical organisms, and Lins
and Farina [4] observed eight-shaped organisms in their
samples. These observations are in accordance to Figs.
3(d) and (e), respectively, suggesting that the life cycle
described here is also the life cycle of these previously
described organisms.
207
We hypothesize that the absence of a one-cell stage in
the life cycle of magnetotactic multicellular organisms is
caused, at least in part, by the need to maintain the content of the internal compartment isolated from the environment. Another possibility is to maintain the organism
always too large to be preyed by most bacteria-grazing
protist populations [14].
The life cycle of this prokaryotic organism is completely multicellular, generating directly two fully
organized new bodies through a rather unusual morphogenetic process. As far as we know, the organism
described here is the first multicellular prokaryote that
divides in two identical ‘‘adult’’ daughter-organisms.
Besides, this is different from most other multicellular
prokaryotic or eukaryotic organisms, which present
at least one part of their life cycle in a unicellular
form, from which the step-by-step ontogenic processes
generate the specific body-plan of the new adult
organisms.
Acknowledgements
We thank R.C.C. Manso for finding this new collecting site and Laboratório de Ultraestrutura Celular Hertha Meyer (UFRJ) for microscopy facilities. FAPERJ
(PRONEX), FUJB, CAPES-PROCAD and CNPq Brazilian financial programs supported this work.
Appendix A. Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/
j.femsle.2004.09.035.
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