Microbiology (2004), 150, 383–390
DOI 10.1099/mic.0.26846-0
Characterization of cyst cell formation in the
purple photosynthetic bacterium Rhodospirillum
centenum
James E. Berleman and Carl E. Bauer
Department of Biology, Indiana University, Jordan Hall, Bloomington, IN 47405, USA
Correspondence
Carl E. Bauer
[email protected]
Received 16 October 2003
Revised
13 November 2003
Accepted 14 November 2003
Rhodospirillum centenum is an anoxygenic photosynthetic bacterium that is capable of
differentiating into several cell types. When grown phototrophically in liquid, cells exhibit a vibrioid
shape and have a single polar flagellum. When grown on a solid surface, R. centenum will
differentiate into rod-shaped swarm cells that display numerous lateral flagella. Upon starvation
for nutrients, R. centenum also forms desiccation-resistant cysts. In this study, it was determined
that R. centenum has heat- and desiccation-resistance properties similar to other cyst-forming
species. In addition, microscopic analyses of the morphological changes that occur during cyst
cell development were performed. It was observed that R. centenum typically forms multi-celled
clusters of cysts that contain from four to more than 10 cells per cluster. It was also determined
that cell density has a minor effect on the percentage of cyst cells formed, with cell densities
of 105–107 cells per 5 ml spot yielding the highest percentage of cyst cells. The striking
similarities between the life cycle of R. centenum and the life cycle exhibited by Azospirillum
spp. are discussed.
INTRODUCTION
Bacteria utilize several strategies for surviving environmental stresses, one of which is to form a metabolically
dormant resting cell. By reducing the rate of metabolism,
cells avoid starvation. In addition, the formation of complex
cell structures, such as the cortex and coat of endospores,
can protect a dormant cell against a variety of harmful
environmental stimuli such as heat, desiccation and exposure to ultraviolet light (Nicholson et al., 2002; Takamatsu &
Watabe, 2002). The endospore formed by Gram-positive
bacteria, such as Bacillus subtilis, is the paradigm of
prokaryotic resting cells, in both its dramatic resistance
properties and in our understanding of endospore formation (Sonenshein, 2000; Stephenson & Hoch, 2002).
However, other resting cell strategies exist, such as the
cyst cells formed by Azotobacter vinelandii (Sadoff, 1973).
As with endospores, cyst cell development begins with a
nutritional shift-down. In Azotobacter vinelandii, the entire
cell rounds up to form a large, spherical cell which is
characterized by the presence of intracellular storage
granules of poly-hydroxybutyrate (PHB) and a complex
outer coat. The thick, multi-layered coat consists of an
inner ‘intine’ layer composed of lipids and carbohydrates
and an outer ‘exine’ layer composed of lipoproteins and
lipopolysaccharides (Pope & Wyss, 1970). Azotobacter
vinelandii cysts are highly resistant to desiccation but,
Abbreviation: PHB, poly-hydroxybutyrate.
0002-6846 G 2004 SGM
unlike endospores, they have minimal resistance to heat
(Socolofsky & Wyss, 1962). p Cyst formation has been
reported in a wide range of proteobacteria, including the
purple photosynthetic bacterium Rhodospirillum centenum
(Favinger et al., 1989). Other cyst-forming species include
members of the genera Azospirillum (Tarrand et al., 1978),
Methylobacter (Whittenbury et al., 1970) and Bdellovibrio
(Tudor & Conti, 1977). Azospirillum brasilense mutants
that are impaired in cyst formation affect the efficiency
of root colonization and nitrogenase activity (Katupitiya
et al., 1995; Pereg Gerk et al., 2000). Furthermore,
Azospirillum association with plant roots has been shown
to stimulate crop growth, so a study of cyst formation may
have practical agricultural applications (Okon & Itzigsohn,
1995; Steenhoudt & Vanderleyden, 2000). Even though cyst
formation is a process that is integral to the biology of
several diverse species, there is little known about the
molecular mechanisms of cyst cell development.
R. centenum exhibits a complex life cycle consisting of three
distinct cell types; swim cells, swarm cells and resting cysts
(Favinger et al., 1989; Ragatz et al., 1995). When grown
in liquid medium, the predominant cell type is the swim
cell, which is vibrioid in shape and motile via a single
polar flagellum. When grown on agar-solidified or viscous
medium, the cells differentiate into rod-shaped swarm
cells that express numerous lateral flagella. Entire colonies
of swarm cells are capable of rapid movement across a
solid surface (Jiang et al., 1998) similar to the swarming
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J. E. Berleman and C. E. Bauer
behaviour of Proteus mirabilis (Bisset, 1973). In addition to
swim and swarm cells, R. centenum also forms clusters of
cyst cells when depleted of nutrients. The cysts formed by
R. centenum are similar to those of Azotobacter (Sadoff,
1975) and Azospirillum spp. (Sadasivan & Neyra, 1985). Like
those of R. centenum, certain Azospirillum spp. also exhibit
swim and swarm cells (Moens et al., 1996), although
Azotobacter spp. do not. Indeed, recent 16S rRNA gene
phylogenetic analysis has determined that R. centenum
forms a clade with Azospirillum spp. and thus may be
considered a photosynthetic-capable Azospirillum-like
organism (Stoffels et al., 2001). These similarities led us to
examine more carefully the formation of R. centenum cyst
cells, to determine how the physiology of the R. centenum
resting cysts compares to the well-described physiology of
Azotobacter cysts.
METHODS
Bacterial strains and growth conditions. Wild-type R. centenum
strain ATCC 51521 was the sole bacterial strain used throughout
this study. Culturing of R. centenum was performed at 37 uC under
aerobic growth conditions in nutrient-rich CENS liquid medium.
For rapid induction of cyst cells, agar-solidified CENBA medium
and aerobic incubation at 42 uC were used. CENBA is a variation
of the previously described CENMED minimal growth medium
(Favinger et al., 1989) in which 20 mM butyrate substitutes for
20 mM pyruvate as the sole carbon source.
Colony morphology. For analysis of colony morphology, an over-
night R. centenum culture was harvested and washed three times
in phosphate buffer (40 mM KH2PO4/K2HPO4; pH 7?0). Serial dilutions appropriate to yielding isolated colonies were pipetted onto
agar-solidified CENS medium and incubated at 42 uC. After 10 days
growth of the original colonies, dilutions of fresh cultures were
pipetted onto the same plates to allow direct comparison of mature
and young colonies. Colonies were photographed using a Sony
DSC-F707 digital camera.
Phase-contrast microscopy. For characterization of individual
cells during the encystment process, R. centenum cultures were
harvested and washed three times in phosphate buffer. Aliquots
(5 ml) of cell suspensions were pipetted onto agar-solidified CENBA
medium. For characterization of the cyst developmental cycle, cells
were scraped from CENBA plates with wet mounts prepared at 12 h
intervals over a 96 h period. Individual cells were viewed with a
Nikon E800 light microscope equipped with a 1006 Plan Apo oil
objective. Image capture was carried out with a Princeton Instruments
cooled charge-coupled device (CCD) camera and METAMORPH
imaging software, v.4.5.
Electron microscopy. R. centenum cultures were harvested and
washed three times in phosphate buffer and then pipetted onto
CENBA plates in 5 ml aliquots. After 1, 2 and 3 days incubation, the
cell spots were harvested, fixed in 5 % glutaraldehyde/100 mM
HEPES/2 mM MgCl2 and analysed by transmission electron microscopy as described previously (Favinger et al., 1989). Mature colonies of R. centenum were analysed by scanning electron microscopy,
performed as described previously (Nickens et al., 1996).
Analysis of cyst development, desiccation resistance and
density dependence. For analysis of the maturation of R. cente-
num cyst cells, wild-type R. centenum cultures were harvested and
washed three times in phosphate buffer and 5 ml spots were pipetted
onto CENBA plates. Spots were subsequently harvested daily for
384
6 days and resuspended in 1 ml phosphate buffer. Resuspended cells
were sonicated for 5 s at low power (30 % output, using a Microson
ultrasonic cell disrupter) to disperse clumps [previous experiments
by our group had shown that low-power sonication did not reduce
the number of colony-forming units (c.f.u.) for vegetative cells but
did effectively disperse clumps of cysts as evidenced by 10- to 100fold increases in c.f.u. after sonication of cyst-induced cultures]. To
quantitate the total number of viable cells (vegetative cells plus cyst
cells), the resuspended cells were serially diluted onto CENS plates
and incubated at 42 uC for 3 days. To quantitate the number of cyst
cells, replicates of the total viable cell diluents were pipetted onto
0?45 mm filters, dried for 20 min at 22 uC, then desiccated at 42 uC
for 3 days. Desiccated filters were then placed onto CENS plates for
2 days at 42 uC to allow outgrowth of surviving cells. Total colonies
before and after desiccation were counted, with analysis repeated in
triplicate.
For comparison of vegetative and cyst cell resistance to prolonged
periods of desiccation, we analysed the desiccation resistance of both
exponentially growing R. centenum cultures and cultures induced to
form cysts. Cells were induced to form cysts by spotting washed cells
of R. centenum onto CENBA medium as described above. Cystinduced spots were harvested after 5 days incubation on CENBA
and resuspended in 1 ml phosphate buffer. Resuspended cells were
sonicated to disperse clumps, serially diluted in phosphate buffer
and plated onto CENS plates to obtain a total viable cell count.
Mid-exponential-phase cultures of exponentially growing R. centenum
cells were obtained by growth in liquid CENS medium. Cells were
harvested by centrifugation, washed three times in phosphate buffer
and subjected to the same treatment as the cyst culture preparations.
To obtain the number of desiccation-resistant cells, serial dilutions
were also pipetted onto 0?45 mm filters as described above and
desiccated for various lengths of time (1–14 days). Desiccated filters
were placed onto CENS agar medium to allow outgrowth of surviving
cells. After 2 days incubation at 42 uC, colony counts were obtained
from dilutions which yielded defined colony growth. Analysis was
performed in triplicate for each culture. Desiccation resistance was
calculated by dividing the number of c.f.u. after desiccation by the
number of c.f.u. prior to desiccation. Cell counts were then normalized
to a total cell count of 56108 cells (actual total cell counts ranged from
1?66108 to 6?66108).
For analysis of density dependence on the formation of cysts, wild-type
R. centenum vegetative cells were grown overnight in CENS medium,
washed three times in phosphate buffer and concentrated to a final cell
density of 261010 cells ml21. Serial dilutions were pipetted as 5 ml
spots onto CENBA medium to yield inoculums of 16101–16108 cells
per spot. After 5 days incubation at 42 uC, cell spots were harvested
and resuspended in 1 ml phosphate buffer. Resuspensions were then
sonicated to disperse clumps and serially diluted onto CENS plates and
onto 0?45 mm filters to assay desiccation-resistance levels. Filters were
desiccated then placed onto CENS plates as described above. The
number of c.f.u. before and after desiccation was determined from
three replicates of each dilution.
Heat resistance. Vegetative cells were harvested from a wild-type
R. centenum culture grown at 37 uC in liquid CENS medium to an
optical density at 650 nm of 0?2. Cells were washed three times and
then resuspended in 1 ml phosphate buffer. Cyst-induced cultures
were prepared by pipetting 5 ml spots of cells onto CENBA plates.
The cells were then harvested after 5 days incubation at 42 uC and
resuspending in 1 ml phosphate buffer. Cyst-induced and vegetative
cell cultures were dispersed by brief sonication at low output power
for 5 s with 100 ml aliquots incubated at 52 or 57 uC for 0, 15, 30
and 60 min. At each time point, surviving heat-treated vegetative
and cysts cells were serially diluted in phosphate buffer and plated
onto CENS medium to determine the number of surviving c.f.u.
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Microbiology 150
The life cycle of Rhodospirillum centenum
RESULTS
Colony alteration during cyst development
Growth of R. centenum on agar-solidified CENS medium
for a 3 day period leads to the production of red convex
colonies (Fig. 1a, left). However, when colonies are incubated for longer durations (1–2 weeks), they develop a
complex morphology composed of multiple tiers and
striated ridges that protrude up and away from the agar
surface (Fig. 1a, right). Significant changes in colony
morphology have been observed for several developing
bacteria, such as spore-forming Bacillus spp. (Branda et al.,
2001) and cyst-forming Azospirillum spp. (Sadasivan &
Neyra, 1987). None of these colony alterations are as
dramatic as the elaborate fruiting bodies formed by some
Myxobacteria spp. (Dworkin, 2000), which develop myxospores at aerial extensions of the fruiting-body structures.
Alteration in colony morphology corresponds well with
the appearance of cyst cells in R. centenum, but cysts are
present throughout the entire colony as assayed by phasecontrast microscopy of dissected colony sections (data
not shown). Scanning electron microscopy analysis of
R. centenum cells from aged (1–2 week) colonies shows that
vegetative and cyst cell types exist in a heterogeneous
mixture throughout the colony (Fig. 1b), indicating that
the process of cyst formation does not require elaborate
cellular aggregation as does fruiting-body formation in
Myxobacteria.
Wet mount microscopy, scanning electron microscopy
(Fig. 1b) and thin-section transmission electron microscopy
(Fig. 1c, d) all indicate that there are variable numbers of
individual cellular units present in a single cyst ‘cluster’ as
constituted by a well-defined intine–exine border. The
number of cells per cyst cluster varies from approximately
four cells that are observed soon after the colony enters cyst
formation (Fig. 1c) to more than 10 cells per cyst cluster as
the colony continues to age (Fig. 1d). Cysts comprising
large clusters of cells, and cysts with multiple outer coats,
have been observed in both Azotobacter vinelandii (Cagle
& Vela, 1972) and Azospirillum brasilense (Sadasivan &
Neyra, 1987). However, in these species, large cyst clusters
are a rare occurrence with a ‘typical’ cyst containing only
one cell. Another interesting aspect of the R. centenum cyst
ultrastructure is the relatively thin outer coat. In Azotobacter
Fig. 1. Macroscopic and microscopic morphology of R. centenum cyst formation. (a) Typical colony morphology of R.
centenum grown on nutrient-rich CENS medium. The shiny, convex colonies on the left are typical 3-day-old R. centenum
colonies. The large, striated colony on the right is typical of a mature 2-week-old colony that is undergoing differentiation into
cyst cells. (b) Scanning electron micrograph of R. centenum mature colony, showing a heterogeneous array of vibrioid-shaped
vegetative cells and clusters of spherical cyst cells. (c) Thin-slice transmission electron micrograph depicting the ultrastructure
of recently differentiated cyst cells, with three cells all surrounded by a continuous outer coat. The large electron-transparent
granules in the cytosol of each cyst cell presumably consist of the polymer PHB. (d) Transmission electron micrograph
depicting an older ‘giant’ cyst containing more than 10 cells surrounded by a common outer coat.
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J. E. Berleman and C. E. Bauer
vinelandii, the intine and exine layers can reach over 0?5
microns in thickness (Pope & Wyss, 1970), whereas in
R. centenum cysts the exine is less than 0?1 microns in
thickness, and the intine layer may only exist in the
intercellular gaps present in each cyst cluster. There is also
a reduction in the size of the intracellular PHB storage
granules as the cysts progress from early to late cyst forms
(Fig. 1c, d).
Cellular maturation of cysts
We also analysed the maturation of cysts by visually
monitoring changes in cell morphology that occur after
induction of cyst formation on butyrate-containing
medium. To induce cyst development, we transferred cells
to CENBA minimal medium, which contains 20 mM
butyrate as the sole carbon source. Growth with butyrate
derivatives as a sole carbon source has been shown to
rapidly induce cyst formation in Azotobacter vinelandii
(Stevenson & Socolofsky, 1966) and Azospirillum brasilense
(Sadasivan & Neyra, 1985) in 2–3 days, as well as in
R. centenum (Favinger et al., 1989). Wet mounts were then
prepared at 12 h intervals over a 3 day period to observe
Fig. 2. Stages of cyst cell development. (a) Phase-contrast
microscopy depicting representative stages in R. centenum cyst
formation. The panels labelled with white numbers show
demonstrative cell types observed at 12 h time points after
induction of cyst formation by a shift to butyrate-based CENBA
medium. The panel with the black number depicts germinating
cells 12 h after plating cyst cells onto rich CENS medium.
(b) Thin-slice transmission electron micrographs portraying the
ultrastructure of representative stages of cyst cell development
in R. centenum. i And ii are from 1 day after cyst induction; iii
and iv are from 2 days after cyst induction; v and vi are from
3 days after cyst induction.
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morphological changes (Fig. 2a). At time zero, the culture
consists of 100 % vibrioid cells typical of vegetative swim
cells. The first step in cyst formation becomes visible after
12 h, which is the appearance of intracellular granules. The
light-refractile granules can be as large as the width of the
cell and presumably consist of the polymer PHB, which has
been shown to accumulate to up to 20 % of the dry weight
of cysts in R. centenum (Stadtwald-Demchick et al., 1990) as
well as in cysts from other species (Stevenson & Socolofsky,
1973). After 24 h, the intracellular granules appear larger
as cells take on an oblong shape. At this stage, motility
becomes lethargic and by 36 h post-induction encysting
cells lose motility concurrent with the accumulation of
ejected flagella in the culture medium as observed by
flagella staining (data not shown). From 36 to 48 h postinduction, cell division can be seen, but daughter cells no
longer separate as they would in normal vegetative growth.
Instead, cells divide but remain attached. At this time
there is also an increase in the refractile aspect of the outer
wall presumably due to the synthesis of the intine–exine
outer coat. At 60 and 72 h, the outer coat becomes
increasingly refractile, with subsequent cell divisions occurring that result in multi-celled cysts that are typical of
R. centenum. Thin-slice transmission electron microscopy
analysis (Fig. 2b, i–vi) also shows the ultrastructure of cells
through these stages of cyst formation. Notable changes are
the formation of large intracellular granules, alteration in
cell shape and the formation of a thick outer coat during the
development of cysts.
To better understand the process of cyst maturation, we
assayed for the appearance of desiccation resistance after
induction of cyst formation. For this analysis, we induced
cyst formation in wild-type R. centenum by shifting to
growth on butyrate-containing plates and then harvested
cells every 24 h for 6 days of incubation on CENBA
medium. After dispersal, the cells were serially diluted and
then plated either onto CENS plates, to determine total
viable cell count (vegetative plus cyst cells), or onto 0?45 mm
filters. The filters were desiccated for 4 days at 42 uC prior
to moving the filters onto CENS plates to allow outgrowth
of surviving cells and determine the number of desiccationresistant cells. Thus, a ratio of desiccation-resistant cells
versus total viable cell counts gives an indication as to the
rate of mature cyst development. The graph in Fig. 3
indicates that there are a negligible number of desiccationresistant cells prior to the shift to butyrate-containing
medium. However, after incubation on butyrate, there was
a steady increase in the number of desiccation-resistant cells
which plateaus after 5 days. Thus, although mature cysts
are microscopically observed within 72 h of induction, the
maximum level of desiccation resistance is not achieved
until 5 days after induction. This is probably due to a lack
of synchronicity in the process of cyst formation, as the
total number of mature cysts visible through microscopic
analysis continues to increase over 3–5 days after induction. Indeed, Fig. 2(a) shows a field of aggregated multicelled cysts commonly seen 120 h after induction, which
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The life cycle of Rhodospirillum centenum
Fig. 3. Development of desiccation resistance in R. centenum.
Cyst cell formation was induced by pipetting 5 ml spots
containing ~56107 cells of R. centenum onto agar-solidified
CENBA minimal medium with butyrate as the sole carbon
source. Cell spots were harvested at 1 day intervals for 6 days.
n, Total number of harvested cells at each time point. #,
Number of cells resistant to 4 days of desiccation. At time
zero, the cultures consist of 100 % vegetative cells, which are
destroyed rapidly by desiccation. Incubation on CENBA
medium results in a rapid increase in desiccation resistance,
with the maximum level of desiccation resistance reached after
5 days induction.
corresponds with the maximum desiccation resistance
observed in this organism.
Duration of desiccation resistance
Azotobacter vinelandii forms cysts that are resistant to
desiccation for up to several months (Sadoff, 1975). To
determine the extent of desiccation resistance in R.
centenum, we subjected both vegetative and cyst cell cultures to periods of prolonged desiccation. Cultures were
harvested and washed in phosphate buffer as before. Serial
dilutions were pipetted directly onto 0?45 mm filters and
placed in sterile Petri dishes where they were desiccated
from 0 to 14 days prior to moving the filter onto CENS
agar medium to allow outgrowth of surviving cells and
assess the number of c.f.u. As shown in Fig. 4 (n), there is
complete loss of viability of vegetative cells after just 1 day
of desiccation. In the case of R. centenum cyst cultures
(Fig. 4, #), there was an initial decrease in viable cell
counts to 33?8 % of the total after 1 day of desiccation that
we attribute to destruction of the vegetative cells that
remained in the cyst-induced culture. This initial decline
was followed by a more gradual decline over the next 14
days, with 3?2 % of the cells surviving 2 weeks of desiccation. Extrapolation of the slope of the curve indicates that
R. centenum cyst cells should survive desiccation for over
3 months.
Heat resistance
The endospores formed by Gram-positive bacteria typically
exhibit resistance to high temperatures; however, cysts that
are formed by Gram-negative species such as Azotobacter
or Azospirillum typically have only moderate resistance to
heat (Socolofsky & Wyss, 1962; Sadasivan & Neyra, 1987).
To test the survivability of R. centenum cells to heat, we
incubated vegetative and cyst cell cultures at 52 and 57 uC
for varying lengths of time. These assay temperatures are
5 and 10 uC above the maximum vegetative growth temperature in this species, of 47 uC (Stadtwald-Demchick
et al., 1990). The survivability results shown in Fig. 5
indicate that there is only a slight loss in the viability of cystinduced cultures when incubated at 52 uC for as long as
30 min (81?8 % survival) and that after 1 h incubation
at this temperature there is still 6?5 % survival. In contrast,
incubation of vegetative cell cultures at 52 uC leads to
Fig. 4. Duration of R. centenum desiccation
resistance. n, Vegetative cell cultures; #,
cyst-induced cultures. Cells were subjected
to prolonged periods of desiccation, with
survivability assayed after 0, 1, 2, 3, 4, 5, 7
and 14 days. Vegetative cell cultures rapidly
lose viability after 24 h desiccation. Cystinduced cultures gradually lose viability, but
are desiccation-resistant for at least 14
days. The experiment was repeated in
triplicate, with the c.f.u. from each replicate
normalized to a total cell count of 56108
at time zero.
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J. E. Berleman and C. E. Bauer
Fig. 5. Heat resistance of R. centenum. # And n, cystinduced and vegetative cell cultures, respectively, of R. centenum incubated at 52 6C for the times indicated. $ And m,
cyst-induced and vegetative cell cultures, respectively, of
R. centenum incubated at 57 6C for the times indicated. The
experiment was repeated in triplicate, with the c.f.u. from each
replicate normalized to a total cell count of 56108 at time
zero.
significant loss of viability, with only 0?0024 % of cells
surviving the first 30 min of incubation at 52 uC and only
0?000021 % of cells surviving 1 h of incubation at this
temperature. At 57 uC, both vegetative and cyst cells exhibit rapid loss of viability over a 30 min period. Thus,
R. centenum cyst cells, like the cysts from other species, have
only a modest resistance to elevated temperatures.
Involvement of cell density on cyst formation
Induction of endospores and myxospores has been shown
to require a high cell density for development to proceed
when a nutritional step-down occurs (Grossman & Losick,
1988; Kaplan & Plamann, 1996). We addressed whether cell
density affects induction of cyst formation in R. centenum.
For this assay, R. centenum cultures were harvested by
centrifugation, washed and serially diluted. Serial dilutions
of the R. centenum cell resuspensions were then pipetted
onto butyrate-containing plates as 5 ml spots with initial
inoculums ranging from 16101 to 16108 cells per spot.
Cells were incubated for 5 days to promote cyst formation.
We then assayed for the percentage of cyst formation by
resuspending the cell spots in 1 ml phosphate buffer and
assaying for desiccation resistance (cyst cells) versus total
viable cells (cyst cells plus vegetative cells). The result of
this analysis (Fig. 6) shows that the percentage of cyst cell
formation ranges from 18?7 to 51?9 % of the total viable
cell count and occurs maximally at initial cell densities of
105–107. Interestingly, there was no detectable minimum
cell density requirement for cyst formation under these
conditions, indicating that a cell density signal may not be
essential for encystment to proceed on CENBA medium.
The decrease in the percentage of cysts formed at high cell
density (108 cells per spot) is probably due to a lack of
sufficient nutrient availability to enable efficient encystment.
Germination of cysts
Resting-cell formation is ultimately futile without subsequent regeneration of the vegetative cell. To analyse the
process of germination in R. centenum, we desiccated cystinduced cultures on 0?45 mm filters for 4 days, then shifted
desiccated filters to a rich growth medium. In R. centenum,
germination of cyst cells occurs much more rapidly than
the formation of cyst cells. Within 6 h of plating onto rich
growth medium, cyst cells can be observed through phasecontrast microscopy which show a decrease in the width
and brightness of the highly refractile outer coat, presumably as a consequence of the degradation (or salvaging) of
the outer coat. Individual cells within a cyst cluster appear
smaller than the coat that surrounds them but there is
neither movement nor cell division observed at this stage.
After 12 h, there is an appearance of empty coats (husks)
and ‘free’ cells which are predominantly oblong in shape
Fig. 6. Effect of cell density on cyst formation. Serial dilutions of R. centenum cultures
were pipetted onto CENBA plates in 5 ml
spots to induce cyst formation. Initial inoculums range from 101 to 108 cells per spot.
Cells spots were harvested after 5 days
incubation on CENBA medium. For each
harvested cell spot, the total number of c.f.u.
was determined and compared to the
number of c.f.u. after 4 days desiccation to
give the percentage of desiccation-resistant
cells at each cell density.
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Microbiology 150
The life cycle of Rhodospirillum centenum
(Fig. 2a). Emerging cells are mostly pre-divisional cells and
still contain large granules of PHB. After 18–24 h, return of
the vegetative cell is observed with a recurrence of vibrioid
and spiral shapes, an absence of intracellular granules and
an active cell motility.
To better define the rate of germination, we resuspended
desiccated cysts into liquid CENS medium and then
followed germination by assaying for desiccation resistance
from cells that were harvested at 12 h time points after
induction of germination. By this assay, we observed that
only 0?0001 % of cells are desiccation-resistant 12 h after
inducing germination. Twenty-four hours after induction,
desiccation resistance has further decreased to the point
where it is indistinguishable from vegetative cell cultures
prior to cyst induction.
DISCUSSION
For a photosynthetic bacterium, R. centenum has an unusual
life cycle (Fig. 7). It is the only photosynthetic organism for
which swim cell and swarm cell differentiation has been
observed (Nickens et al., 1996). Swarm cell differentiation
allows rapid movement of R. centenum colonies across a
solid surface, which could provide an advantage by allowing cells to migrate to optimal light conditions in a microbial mat (Ragatz et al., 1995). The formation of cysts is a
developmental trait that has not been described for any
other anoxygenic photosynthetic species. The ability to
Fig. 7. Diagram of the R. centenum life cycle, depicting swim
cells, swarm cells and several stages of cyst formation. Swim
cells are present in liquid-grown cultures of R. centenum,
swarm cells are formed when R. centenum is grown on solid
or viscous medium. A nutritional shift-down induces cyst cell
formation. Development of cyst cells begins with the accumulation of intracellular granules of PHB, followed by a change in
cell shape, outer-coat formation and further divisions resulting
in multi-celled cysts typical of this species. On return to
nutrient-rich medium, cells emerge from the outer coat and the
vegetative cell is regenerated.
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form desiccation- and heat-resistant cyst cells is presumably important for the survivability of R. centenum in the
ecological niches that this organism is known to inhabit.
Indeed, strains of R. centenum are typical isolates of hot
springs and factory run-offs (Nickens et al., 1996). Successful enrichments of R. centenum have been reported using
hot spring inoculums that were obtained from pools at
temperatures as high as 58 uC. This is despite the fact
that R. centenum isolates obtained from such enrichments
have a maximum vegetative growth temperature of 47 uC.
Presumably, the original enrichment inoculums contained
cysts that allowed survivability at these elevated temperatures.
Phylogenetic trees based on 16S rRNA gene sequences have
shown that R. centenum is closely related to species of the
genus Azospirillum (Stoffels et al., 2001). This casts a new
light on the developmental cycle observed with R. centenum
since the physiological traits of R. centenum share a number
of similarities with Azospirillum spp. R. centenum and
Azospirillum brasilense each exhibit similar swim cells with
distinct polar flagella, as well as swarm cells that exhibit
surface motility through the production of distinctly
different lateral flagella (Jiang et al., 1998; McClain et al.,
2002; Moens et al., 1996; Scheludko et al., 1998). Likewise,
Azospirillum brasilense also undergoes cyst formation as
has been characterized in R. centenum (Sadasivan & Neyra,
1985; Sadasivan & Neyra, 1987; Stadtwald-Demchick et al.,
1990).
Although there are several similarities between R. centenum
and Azospirillum cysts, there is an important difference
between these genera. For example, Azospirillum spp. typically contain only one cell per cyst coat. This is in contrast
to the large cyst cell clusters that are typically formed by
R. centenum. Indeed the number of cells per coat in
R. centenum appears to increase as the cysts mature from
an initial grouping of four cells per cyst to more than 10
cells per cyst coat as the cysts mature. One possibility is
that the increasing number of cells is due to a delay in
cyst-induced inhibition of chromosome replication in
R. centenum leading to a large grouping of cells as the
chromosomes subsequently segregate into daughter cyst
cells. The appearance of multi-bodied cysts has been
observed in both Azotobacter vinelandii and Azospirillum
brasilense, but in these species it is a rare occurrence. Similar
to myxobacteria, the formation of cyst clusters may be
rationalized as a mechanism for ensuring a high cell density
upon germination. The benefit for R. centenum could be
the ability to quickly develop a cell density high enough to
enable the colony-wide motility of swarm cells.
In addition to defining the physiology of R. centenum cyst
cell development, we have begun performing a detailed
genetic analysis of cyst development in this species.
Mutations can be readily obtained that either significantly
overproduce or fail to produce cysts. Characterization
of these mutational events should shed light on the cyst
developmental cycle in this, and in other, cyst-producing
species.
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389
J. E. Berleman and C. E. Bauer
ACKNOWLEDGEMENTS
Pope, L. M. & Wyss, O. (1970). Outer layers of the Azotobacter
vinelandii cyst. J Bacteriol 102, 234–239.
We thank F. Rudolf Turner for his aid in preparation and examination of electron micrographs. We would also like to thank Diane
Bodenmiller for her editorial comments. J. E. B. was supported by a
National Institutes of Health Training grant, GM007757.
Ragatz, L., Jiang, Z. Y., Bauer, C. E. & Gest, H. (1995). Macroscopic
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