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Applied and Environmental Microbiology, September 2000, p. 4105-4111, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
In Situ Reproductive Rate of Freshwater
Caulobacter spp.
Jeanne S.
Poindexter,1,*
Kanan P.
Pujara,1 and
James T.
Staley2
Barnard College, Columbia University, New
York, New York,1 and University of
Washington, Seattle, Washington2
Received 4 August 1999/Accepted 22 June 2000
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ABSTRACT |
Electron microscope grids were submerged in Lake Washington,
Seattle, Wash., in June 1996 as bait to which Caulobacter
sp. swarmers would attach and on which they would then reproduce in situ. Enumeration of bands in the stalks of attached cells implied that
the caulobacters were completing approximately three
reproductive cycles per day. A succession of morphological types of
caulobacters occurred, as well as an episode of bacteriovore
grazing that slowed the accumulation of caulobacters and prevented the
aging of the population.
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TEXT |
To elucidate roles of bacteria in
natural habitats, a wide variety of chemical and microscopical methods
are now available that allow quantitative determinations of metabolic
rates and estimations of viability. However, correlation of metabolic
activity and reproductive potential with bacterial multiplication in
situ remains elusive because the vast, diverse, and perpetual
transformations effected by bacteria in nature are typically achieved
by populations that do not fluctuate significantly in size. Some
bacteriologists attribute this seeming paradox to the presence of high
proportions of quiescent (i.e., not dividing), moribund (i.e., not
active), or dead bacteria (9, 25), while others attribute
the constancy of population size of lively, healthy, metabolically
active bacteria to bacteriovory (13).
The present study was designed to determine the in situ reproductive
rate of just one genus, Caulobacter, by exploiting the unique cell cycle marker, the stalk band, that is added to a
caulobacter stalk once during each cell cycle (19, 24).
Because of the stalk bands, a caulobacter cell that has divided several
times since it was a flagellated swarmer cell incapable of cell
division looks different from a stalked cell that has not yet divided. In artificial cultures, attachment by cells of dimorphic prosthecate bacteria is initiated predominantly in the swarmer stage, whether the
substratum is glass (8, 12), cellulose fibers
(26), the holdfasts of other cells (10, 15), or
electron microscope (EM) grids (5) (reviewed in reference
16). By allowing caulobacters in Lake Washington,
Seattle, Wash., to attach to submerged EM grids and examining the
attached populations at intervals, we expected to find that the maximum
number of bands per stalk would increase in proportion to the duration
of submersion and that that number could be used to infer the number of
reproductive cycles occurring daily.
The results revealed that caulobacters completed three reproductive
cycles per day in Lake Washington in June 1996. In addition, direct
microscopical evidence was obtained showing that attached caulobacters
are susceptible to bacteriovore grazing.
Exposure, retrieval, and examination of substrata.
Formvar-coated nickel grids on sheets of Parafilm were attached to
glass microscope slides by tape wound around the Parafilm. The slides
were placed vertically in plastic slide boxes from which most of the
bottoms had been cut out and whose corners had been fitted with plastic
fishing line tethers. Five slides were placed in each of two such
boxes; each box was enclosed within a ZipLoc plastic bag and lowered
into the water; the bag was opened underwater, and the slide box was
removed. The tethers were attached to the wooden supports of a marine
light located approximately 100 m from shore so that the boxes
hung suspended slightly more than 1 m below the lake surface, one
on the south side of the light (site 1) and one on the north side (site
2). Grids were retrieved from site 1 after 1, 2, 3, 4, and 7 days of
submersion and from site 2 after 1, 2, 3, and 4 days; one slide had
fallen out of the box at site 2 between days 1 and 2. Grids were not retrieved on days 5 and 6.
To retrieve a slide of grids, a ZipLoc bag containing a plastic slide
box with lid was lowered into the water. The bag was opened beneath the
surface, a slide was transferred from the in situ slide box to the
retrieving box, the retrieving box was closed with its lid, the bag was
snapped closed, and the bag-box-slides assembly was lifted out of the
water. The use of tightly closed plastic bags to introduce and retrieve
the grids prevented contact of the grids with caulobacters present at
and near the air-water interface of the lake, a problem not recognized
in an earlier study (7). Within 15 to 20 min, the bag was
opened and the water was allowed to drain out, the slide box lid was
removed, and the slide was dipped in a Coplin jar containing 0.2%
potassium phosphotungstate, pH 7. The surface of each grid was drained
dry with a tissue, and the slide and grids were allowed to air dry.
Grids were examined in a JEOL 1200 tranmission EM or a Philips 208S
transmission EM operated at 80 kV and 60 kV, respectively.
Each grid
was used only once; parallel rows of grid openings were
scanned in a
pattern that ensured that no opening on any grid
was viewed more than
once. Every identifiable caulobacter cell
encountered was recorded
photographically, even if the full length
of its stalk was not
observable (e.g., because it was embedded
in debris or lay over a grid
bar). All other organisms were enumerated,
as well, but only
occasionally photographed. Stalk bands were
enumerated on the negative
films and on contact prints examined
with the aid of a dissecting
microscope.
Attachment and reproduction of caulobacters.
Stalked
caulobacters (swarmers cannot be identified as caulobacters) accounted
for the majority (59%) of cells attached to the grids from two to
seven days of submersion. Examples of attached caulobacters are
illustrated in Fig. 1 through
4.
The numbers of caulobacters encountered on the grids are shown in Table
1 and Fig.
5, which combine the observations on
grids from both sites because there was no significant difference
between sites for the first 4 days. As the numbers of caulobacters
increased, it was clear that the accumulating cells comprised both new
arrivals (cells with short stalks and zero or one band per stalk) and
stably attached cells that were dividing in place. Cells with
relatively short stalks without bands were found on every grid, but
cells with long stalks with more than three bands were not found until the 4th day. These observations strongly imply that attachment to
natural substrata is initiated by swarmers, as it is in monocultures.
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FIG. 1.
Stalked vibrioid caulobacter cells attached to grids at
2 (A to C) and 4 (D and E) days of submersion in Lake Washington,
displaying one (A), two (B), three (C), four (D), and six (E) stalk
bands (marked by arrows in panels A to C). PHB, granule with the
appearance of poly- -hydroxybutyrate; Pn, granule with the appearance
of inorganic polyphosphate. Each marker is 1 µm.
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FIG. 2.
Stalked vibrioid caulobacter cells attached to grids at
4 days of submersion in Lake Washington, displaying eight (A) and nine
(B) stalk bands. Each marker is 1 µm.
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FIG. 3.
Stalked caulobacter cells attached to grids at 7 days of
submersion in Lake Washington, displaying various numbers of stalk
bands. (A) cluster of vibrioid (V) cells; (B) cluster of predominantly
fusiform (F) cells. SR, stalk remnant. Arrows indicate bands at the
ends of damaged regions of stalks. Each marker is 1 µm.
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FIG. 4.
(A) Stalked caulobacter cells and stalk remnants
attached to grids at 7 days of submersion in Lake Washington. (B to D)
The stalk remnants are shown at higher magnification. The stalk band
typically separates the intact region of the stalk remnant from the
collapsed, gnawed end that was previously connected to the cell
(arrows). The number of stalk bands remaining is indicated at the
gnawed end of each remnant; the numbers (1, 3, 5, and 7) suggest that
grazing was not selective for cells of a particular age. Each marker is
1 µm.
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FIG. 5.
Numbers of stalked caulobacter cells found attached to
grids on each day of submersion in Lake Washington. Cell numbers were
totaled for the two sites and normalized per grid for each day's
collection. Each wide bar represents the total number of caulobacter
cells, of all three types, and includes cells whose band numbers could
not be determined (see text).
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Three cell types of caulobacters appeared: vibrios first and then
fusiform cells and a bacteroid type, which was infrequently
encountered. During the first 4 days, only vibrioid caulobacters
appeared on the grids in significant numbers. Attached vibrios
with one
or two bands per stalk were detected on day 2. Two- and
three-banded
stalks were present on day 3. By day 4, accumulation
of caulobacters
and other bacteria was considerably heavier on
the grids, with one to
five cells in many of the openings. The
most common band number on day
4 was still only two, but five-
and six-banded stalks were as numerous
as were two- and three-banded
stalks on day 3. On day 4, the maximum
number of bands was nine.
On the conservative assumptions that the
earliest vibrioid caulobacter
swarmers attached during the first 1 to 2 days and had two bands
per stalk by day 2 and nine by day 4, and that
one or two bands
were installed during the first cell cycle and one
band was installed
during each subsequent cycle (
19),
the vibrioid caulobacters
completed at least eight cycles between days
1 and 4 (2.7 cycles/day)
or seven cycles between days 2 and 4 (3.5 cycles/day).
Few fusiform caulobacters with banded stalks were detectable on
day 4, but on day 7 the fusiform cells were more abundant
than vibrios
(Table
1; Fig.
5); by that time they displayed up
to nine bands
per stalk. Using the same assumptions as for the
vibrios, if most of
the fusiform cells attached after day 4 and
survived until day 7, then
they appeared to have completed nine
cycles within those 3 days (three
cycles/day).
The vibrioid and fusiform caulobacters thereby displayed similar in
situ reproductive rates. Three cycles per day (an average
of 8 h
per cycle) is well within the physiologic capabilities
of bacteria that
exhibit generation times averaging 4 to 5 h in
pure cultures.
Whether reproduction on the submerged grids occurred
throughout each
day or only during a part of the day cannot be
inferred from this
study.
Episode of predation.
On day 7, there was clear evidence of
intensive grazing at some time between 4 and 7 days, as well as a shift
within the caulobacter population from predominantly vibrioid to
predominantly fusiform cells. The most reasonable interpretation
of the significant change in caulobacter populations on the grids is
that the once-predominant vibrioid caulobacters were grazed after
grids were retrieved on day 4; continued arrival of vibrios was
accompanied by fusiform cells more frequently than before day 4. The
grazing was evident as remnants of stalks, which remained attached to
the grids like rooted stems of plants from which the fruits had been
plucked. Typically, one end of the stalk remnant had collapsed, and the remaining bleb usually terminated at a stalk band (arrows, Fig. 4).
Other damaged regions of stalks were also typically bounded by
bands (arrows, Fig. 3), implying that the band can serve to seal off
such regions, as suggested by Schmidt (21).
Cell-free stalks are not encountered in monocultures of caulobacters,
whether the cells are grown suspended (
19) or attached
to
glass beads (
24) and whether they are grown in batch or
perpetual
cultures (
19). In monocultures, stalks with as
many as 13 to
19 bands have been reported. To remove stalks from cells
requires
either shearing in a blender (
6,
18,
21,
22) or a
stalk
abscission mutation (
17,
18). A vigorous mechanical
force
must have been exerted on individual stalked cells on the grids,
a force that was not exerted on the entire population; stalk remnants
were found among cells with various numbers of stalk bands (Fig.
3 and
4). Bands still visible in the stalk remnants were enumerated
and
ranged from zero to eight bands per fragment, suggesting that
the
feeders fed at random among caulobacters of different
ages.
Other observations.
The vertical position of the slides was
effective in avoiding the accumulation of debris, dead microorganisms,
and nonadhesive microorganisms that settle by gravity. The
result was slide (and grid) surfaces free of macroscopic deposits of
material that accumulated on horizontal surfaces such as the rim of the
slide box. Microscopically, noncellular debris accumulated only slowly
and did not significantly interfere with observations, even after 7 days of submersion.
Besides caulobacters, two other morphologically distinctive
microorganisms were encountered: a sheathed rod, presumably
Sphaerotilus sp., and a diatom, presumably (from its shape
and manner of attachment)
Diatoma sp. All other attached
organisms were bacteria, predominantly
rods, but occasionally a coccus;
bacteriophage virions were detected
only once, as a cluster of
podovirus-like particles surrounding
a flagellated rod. All of the
microbial cells appeared to have
attached actively, not to have settled
passively on the grids.
The diatom and the
Sphaerotilus were
attached by puddles of adhesive
material from one point only on each
organism's surface. Like
caulobacters,
Sphaerotilus
initiates attachment in the swarmer
stage (
2); it appeared
to reproduce in situ on our grids, accumulating
up to six cells within
each sheath. Single rod-shaped bacteria
were typically flagellated,
cocci were surrounded by fimbriae
and/or fibrous extracellular slime,
and the stalked caulobacters
exhibited a progressively longer mean
stalk length as well as
increasing band numbers. Dividing cells were
encountered at frequencies
well above those usually reported for
aquatic assemblages (
3,
11), which may well include cells
not actively reproducing in
situ (
25). The metabolic
activity of the attached bacteria was
further evidenced by the presence
of reserve granules in many
cells. Electron-translucent granules
were interpreted as carbon
reserves, either polysaccharide or
poly-
-hydroxybutyrate, and
sharp-edged, electron-dense granules were
interpreted as polyphosphate.
Both types of granules were present
in many cells and in nearly
all of the stalked cells (Fig.
1D and E.).
We infer from all of these appearances that the bacteria found on the
grids were alive and were metabolically active, reproducing
members of
the microbial community of Lake
Washington.
Interpretations.
The reproductive rate of three cycles per day
inferred for caulobacters in this study is within the range of 2 to
10 h per generation calculated for aquatic bacteria that developed
as colonies on submerged glass microscope slides (1). It is
possible that these observations reflect peak activity of the
caulobacters and that a similar study conducted periodically during a
full year's turn of seasons would reveal whether the reproductive rate
varies with water temperature or other environmental variables.
The succession from vibrioid to fusiform caulobacters was probably
fortuitous. In a companion study of caulobacters in Lake
Washington
from February through August 1996 (to be reported separately),
there
was an observable succession of
Caulobacter species, with
vibrioid types predominating from March through early June, fusiforms
predominating in early summer, and then a second bloom of different
vibrioid species in late July and August. That this succession
was
reflected on the grids in mid-June is nevertheless regarded
as pure
coincidence and a result of uneven and varying distribution
of
caulobacter types within the lake as well as with time and
season.
None of the physical (temperature or turbidity), chemical
(salinity, nitrate, phosphate, or silicon) or biotic
(chlorophyll,
algae, protozoa, invertebrate animals, or total
chemoheterotrophic
aerobic bacteria) parameters monitored during the
long-term study
varied significantly within the short period of
submersion of
the
grids.
Unexpectedly, the observations reported here provided direct evidence
that periodic waves of bacteriovory can account for
lack of massive
accumulation (blooming) of bacterial populations,
even while they are
reproducing at a significant rate. Although
caulobacter swarmers are
probably susceptible to ingestion by
flagellates and by ciliates,
amebae and ciliates have been observed
to ignore prosthecate bacteria
as they meander over mixed bacterial
populations attached to glass
slides (Poindexter, unpublished
data). Amebae, in particular, appear to
react to the prostheca
as an extension of the cell, which is then
judged to be a particle
too large to engulf, as though it were an
inedible filamentous
bacterium (see references
4,
14, and
23). The water temperature
at the
submersion sites ranged from 16.4 to 18.7°C (average, 17.5°C)
at
mid-day. The temperature had risen from 7°C in early February,
and
the rise in temperature during the spring had been accompanied
by a
bloom of small animals, especially in May, most of which
fed on
suspended microbes and had nearly decimated the phytoplankton
community
by mid-June. Two types of invertebrate animals were
present that could
have gnawed caulobacter cells from their stalks:
tardigrades and
rotifers. Because the tardigrades occurred much
closer to shore than
the submersion sites, W. T. Edmondson (personal
communication)
recommended that the grazing episode be attributed
to rotifer feeding.
Some rotifers are sessile and feed on suspended
particles swept into
the gullet on feeding currents generated
by cilia, but others are
mobile and feed by means of protruding
mouth parts capable of grinding
and gnawing prey. Stalked caulobacter
cells tenaciously attached to a
large (relative to rotifers) substratum
apparently were susceptible to
such
predators.
This view of natural populations is consistent with experimental
studies with natural bacterial populations (see, e.g., reference
20) as well as with the inferred role of grazing in
the maintenance
of stable climax communities in municipal water and
sewage treatment
facilities. It is not consistent with the view that
populations
of bacteria that are stable in density in their natural
habitats
are necessarily quiescent. On the contrary, such populations
may
provide a significant, or at least a supplemental, proportion
of
the diet of animals as well as of protozoa while simultaneously
contributing to the biogeochemical transformations for which they
are
known to be
responsible.
| |
ACKNOWLEDGMENTS |
The outdoor work in this study was conducted during 1996 while
J.S.P. was on academic leave, generously hosted by J.T.S. and his
laboratory at the University of Washington in Seattle. The boat used
was made available through the generosity of the laboratory of W. T. Edmondson, Department of Zoology, University of Washington, Seattle.
The work was supported in part by a Barnard College Faculty Grant to
J.S.P.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Barnard College, Columbia University, 3009 Broadway, New York, NY 10027-6598. Phone: (212) 854-1415. Fax: (212)
854-1950. E-mail: jpoindexter{at}barnard.edu.
This report is dedicated to the memory of W. T. Edmondson,
who so faithfully defended the quality of life of the inhabitants of
Lake Washington for most of the twentieth century.
| |
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Applied and Environmental Microbiology, September 2000, p. 4105-4111, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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