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Applied and Environmental Microbiology, August 1999, p. 3651-3659, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Diel Rhythms in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase
and Glutamine Synthetase Gene Expression in a Natural Population of
Marine Picoplanktonic Cyanobacteria (Synechococcus
spp.)
Michael
Wyman*
Department of Biological Sciences, University
of Stirling, Stirling FK9 4LA, United Kingdom
Received 12 March 1999/Accepted 24 May 1999
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ABSTRACT |
Diel periodicity in the expression of key genes involved in carbon
and nitrogen assimilation in marine Synechococcus spp. was
investigated in a natural population growing in the surface waters of a
cyclonic eddy in the northeast Atlantic Ocean.
Synechococcus sp. cell concentrations within the upper
mixed layer showed a net increase of three- to fourfold during the
course of the experiment (13 to 22 July 1991), the population
undergoing approximately one synchronous division per day. Consistent
with the observed temporal pattern of phycoerythrin (CpeBA)
biosynthesis, comparatively little variation was found in
cpeBA mRNA abundance during either of the diel cycles
investigated. In marked contrast, the relative abundance of transcripts
originating from the genes encoding the large subunit of ribulose
bisphosphate carboxylase/oxygenase (rbcL) and glutamine
synthetase (glnA) showed considerable systematic temporal
variation and oscillated during the course of each diel cycle in a
reciprocal rhythm. Whereas activation of rbcL transcription was clearly not light dependent, expression of glnA
appeared sensitive to endogenous changes in the physiological demands
for nitrogen that arise as a natural consequence of temporal
periodicity in photosynthetic carbon assimilation. The data presented
support the hypothesis that a degree of temporal separation may exist between the most active periods of carbon and nitrogen assimilation in
natural populations of marine Synecoccoccus spp.
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INTRODUCTION |
Marine Synechococcus spp.
are among the most abundant and cosmopolitan members of the
photosynthetic picoplankton: a taxonomically mixed assemblage of small
(<2.0-µm diameter) prokaryotic and eukaryotic microorganisms that
account for the major fraction of primary production in the world's
open oceans (40). Although genetically divergent (14,
51, 54, 66, 67), oceanic strains of Synechococcus spp.
belong to a physiologically coherent group (marine cluster A) of
cyanobacteria within the unicellular order
Chroococcales (59). All known isolates are
non-nitrogen-fixing, obligate photoautotrophs (59, 62) that
produce spectroscopically distinct biliproteins (including
phycoerythrin [PE]) as accessory components of their light-harvesting
apparatuses (1, 35, 36). Many are also capable of
flagellum-free swimming motility and, in one demonstrated case at
least, display positive chemotaxis toward a variety of organic and
inorganic nitrogenous compounds (64).
Following their first description in 1979 (22, 61), a
reasonable (if incomplete) understanding has developed of the
biological and physicochemical factors that regulate the growth and
productivity of marine Synechococcus spp. in the world's
oceans (18, 19, 23, 24, 29, 46, 47, 56, 60, 65, 66). While
there are undoubted exceptions to the paradigm, these organisms are generally at their most conspicuous (but not necessarily their most
productive) in oligotrophic surface waters, where the limited supply of
inorganic nutrients appears to exclude competition in the form of
larger and potentially faster-growing eukaryotic phytoplankton (19, 23, 62).
Estimates of Synechococcus sp. growth rates in open waters
are somewhat variable, but for active populations they tend to cluster
around a mean of about one division per day (7, 18, 29, 56).
Like many groups of marine phytoplankton, there is mounting evidence
that the daily progression through the Synechococcus sp.
cell cycle may be entrained in situ by the natural diel alternation in
irradiance. While a number of previous studies have clearly hinted at
the phenomenon (7, 8, 25, 62), the recent cytological
investigation conducted by Vaulot and coworkers (56) was the
first to confirm that DNA replication and cell division can become
tightly synchronized in Synechococcus spp. growing in open
waters. If such rhythmic behavior is a common feature of natural
populations, an obvious rationale can be developed to explain several
earlier reports (20, 25, 34) of diel oscillations in cell
rRNA content and macromolecular composition in these organisms. Like
the diel phasing of DNA replication and cytokinesis (56),
temporal patterns of this type are an entirely predictable feature of
synchronously dividing populations.
Diel rhythms in the synthesis and accumulation of various mRNAs in
natural populations of Synechococcus spp. and other marine cyanobacteria have also been described (10, 26, 37, 38, 70),
but whether these might have their origin in cell cycle-related events
has not received consideration. Cell division in the marine Synechococcus sp. strain WH7803 is known to be under
circadian control (50), and there is convincing preliminary
evidence that expression of the rbcL gene (encoding the
large subunit of the Calvin cycle enzyme, ribulose bisphosphate
carboxylase/oxygenase [RubisCO]) may also be regulated in this way
(37). It is an open question, however, whether
rbcL transcription in Synechococcus spp. is
controlled directly by as-yet-unidentified clock genes or is regulated
in response to temporal oscillations in other metabolic processes that
may themselves be circadian in nature.
The RubisCO genes of marine Synechococcus spp. are
phylogenetically distinct from those of other cyanobacteria and may
have been acquired comparatively recently by lateral gene transfer (63). In the marine Synechococcus sp. strain
WH7803, both RubisCO subunits are cotranscribed with a homologue of the
Synechococcus sp. strain PCC7942 ccmK gene
(63), which encodes a structural component of the
carboxysome, the site of CO2 fixation in cyanobacteria. RubisCO expression is enhanced considerably by light and, as in other
cyanobacteria (11, 27), appears to be regulated primarily at
the level of transcription. In this regard, the temporal pattern of
RubisCO expression displayed by Synechococcus sp. strain
WH7803 in culture closely parallels that seen in natural populations of
photosynthetic picoplankton growing in subtropical oceanic surface
waters (37, 38).
By analogy to the adaptive strategies adopted by other cyanobacteria
growing at low concentrations of combined nitrogen, the principal route
of inorganic nitrogen assimilation in marine Synechococcus spp. is thought to occur via the glutamine synthetase (GS)-glutamate synthase pathway and to be dependent upon photosynthetically generated ATP and reductant (31, 53). While the existence of
alternative pathways has not been excluded experimentally, from what is
known of the kinetic properties of these systems in other cyanobacteria (32) it appears unlikely they could make other than a very
minor contribution to N assimilation in Synechococcus spp.
at the low ambient nitrogen concentrations typical of oceanic surface waters.
The pathways of carbon and nitrogen assimilation are metabolically
linked at the level of GS in cyanobacteria, and the enzyme is
extensively regulated in response to a variety of environmental signals. GS is rapidly inactivated in the dark and also by ammonium ions in the well-studied model system Synechocystis sp.
strain PCC6803 and several other cyanobacteria (45, 49). The
nature of the reversible modification system present in
Synechocystis sp. strain PCC6803 is distinct from that
described for the GS of enteric bacteria, however, in that
adenylylation is not involved. Nevertheless, some elements of the
signal transduction system that lead to the activation or inactivation
of GS do appear to be conserved between cyanobacteria and enteric
bacteria. The regulatory protein PII (encoded by glnB) is
present in both groups but in cyanobacteria is activated by
phosphorylation rather than uridinylylation (16). As with
GS, activation of PII is regulated by nitrogen availability and is
dependent upon photosynthetic electron transport (17, 45).
Transcription of glnA and glnB in
Synechococcus sp. strain PCC 7942 and
Synechocystis sp. strain PCC 6803 is regulated by the
positive transcription factor NtcA, a functional analogue of the
nitrogen regulator of enteric bacteria, NtrC (17, 57). NtcA
is widely distributed among cyanobacteria (including the marine strain
Synechococcus sp. strain WH7803 [28]) and
in the absence of ammonium binds to the promoter regions of a suite of N-regulated genes, including glnA and glnB.
Transcription of glnA increases two- to threefold in cells
of Synechococcus sp. strain PCC7942 switched from ammonium-
to nitrate-containing growth medium and by a somewhat greater margin
(5- to 10-fold) during nitrogen deprivation (13).
Enhanced glnA transcription in the absence of ammonium leads
to parallel increases in GS synthesis and activity (12, 13), and a similar correlation among nitrogen regimen, glnB mRNA
abundance, and PII protein levels has been reported recently for
Synechocystis sp. strain PCC6803 (16, 52).
Expression of glnA at the transcriptional level is not light
dependent in Synechococcus sp. strain PCC 7942 (6), but like psbA (encoding the D2 protein of
photosystem II) and the rbcL gene of marine
Synechococcus spp. (37), there is some evidence
for circadian control (30).
Establishing how temporal and spatial variability in the environment
structures the molecular biological responses of natural populations of
marine microorganisms is a central theme in contemporary biological
oceanography. The present field-based study was designed to examine the
temporal (diel) periodicity in the abundance of three different
Synechococcus sp. mRNAs that encode proteins central to
photosynthetic light harvesting (PE; cpeBA), carbon fixation (RubisCO; rbcL), and nitrogen assimilation (glutamine
synthetase; glnA). The aims of the investigation were to
assess the extent to which the three genes might be differentially
expressed during the diel cycle and to relate the findings to the
temporal pattern of Synechococcus sp. population growth and
cell division in situ. The data reported reveal a quasireciprocal
rhythm in the relative abundance of rbcL and glnA
mRNAs and point to at least some degree of temporal separation between
the most active phases of C and N assimilation in natural populations
of Synechococcus spp.
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MATERIALS AND METHODS |
Observations were made during a 10-day period in July 1991 at a
series of drifting stations located within a cyclonic eddy in the
northeast Atlantic Ocean (Fig. 1). The
study site was selected during passage of the research vessel (RRS
Charles Darwin; cruise CD61) to the sea area to the south of
Iceland following receipt of thermal infrared satellite images of the
region on 10 July 1991. During the previous month an extensive
mesoscale (~250,000-km2) bloom of coccolithophorid algae
had occurred in these subpolar waters (15, 21), but by the
time the research vessel arrived on station (12 July 1991) all surface
expression of the bloom had disappeared (21). Mapping of
surface hydrographic and biological properties within the eddy revealed
that the postbloom phytoplankton community comprised a mixed assemblage
of micro- and picophytoplankton dominated by diatoms and
microflagellates and by Synechococcus spp., respectively.
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FIG. 1.
Track of the ARGOS drifter deployed on 13 July 1991 (Julian day 194). The position of the drifter at 0000 hours on each
subsequent day is indicated by the day number. (Data courtesy of Bob
Barrett and Robin Pingree.)
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A drogued (10 m) ARGOS drifter was deployed at the center of the eddy
(61°05'N, 20°W) on 13 July 1991. Thereafter, the research vessel
was operated in Lagrangian mode by maintaining close station with the
drifter until the morning of 22 July 1991 (for further details of the
study site and its hydrographic properties, see reference
21). Plankton samples were obtained from
near-surface (~5 m) waters by using an outlet of the ship's nontoxic
seawater supply (residence time from inlet to outlet, ~2 min) or from
discrete depths by using 30-liter GO-Flo bottles (General Oceanics,
Miami, Fla.) deployed from a Kevlar line. Surface nutrient
concentrations were determined continuously while on station with a
Technicon autoanalyzer and standard analytical procedures (5,
21).
Synechococcus sp. cell counts.
The abundance of
Synechococcus sp. cells was estimated by epifluorescence
microscopy (19). Seawater samples of known volume (10 to 100 ml) were prescreened through a 250-µm-mesh plankton net, and
phytoplankton cells were collected by filtration on 25-mm-diameter, 0.2-µm-pore-size polycarbonate membranes (Nuclepore Corp.) overlaying a Whatman GF/F support filter. When necessary (i.e., during times of
excessive ship movement), filtered samples mounted on glass slides in
nonfluorescent immersion oil were stored in darkness at 4°C, but in
all cases they were counted onboard ship within 24 h of collection.
Determination of PE concentrations.
Synechococcus sp.
PE concentrations were determined as previously described with
Synechococcus sp. strain WH7803 cells as the reference
standard (69). Seawater samples (1 to 2 liters) were
fractionated through 47-mm-diameter 2.0- and 0.6-µm-pore-size polycarbonate filters (Poretics Corp.) under gentle vacuum (<10 cm of
Hg), and Synechococcus sp. cells retained on the
0.6-µm-pore-size filter were washed into 1.5 ml of Whatman
GF/F-filtered seawater (collected from a depth of 100 m). Owing to
equipment failure, it was not possible to carry out PE analyses at sea.
The concentrated samples were centrifuged at 13,000 × g for 1 min, the seawater supernatant was aspirated, and the
pelleted phytoplankton cells were taken up in 1 ml of 50% (vol/vol)
aqueous glycerol and stored in darkness at 
20°C. PE concentrations
were estimated on return to the laboratory with a Perkin-Elmer LS5
spectrofluorimeter and the instrument settings and correction
procedures previously reported (69).
RNA extraction and Northern analysis.
Seawater samples (10 to 20 liters) were fractionated through 90-mm-diameter, 2.0- and
0.6-µm-pore-size polycarbonate filters (Poretics Corp.) at negative
vacuum pressures of 10 and 60 cm of Hg for the 2.0- and
0.6-µm-pore-size filters, respectively. Routinely, this fractionation
procedure results in the retention of greater than 90% of the original
Synechococcus sp. biomass on the 0.6-µm-pore-size filter
whereas eukaryotic cells are either trapped by the 2.0-µm-pore-size
filter or disrupted by the high vacuum pressure employed during
filtration through the 0.6-µm-pore-size filter. The efficiency of the
procedure was monitored by epifluorescence microscopic examination of
the cell material retained on the 0.6-µm-pore-size filter. In all
cases, the only intact chlorophyll-containing cells observed also
exhibited PE fluorescence; i.e., only Synechococcus sp.
cells were present in the 0.6- to 2.0-µm size-fractionated samples
under the conditions applied.
Sample volume was determined by the amount of seawater that could be
filter fractioned within 15 min of sample collection,
and all nocturnal
samples were processed in a darkened laboratory.
Synechococcus sp. cells retained on the 0.6-µm-pore-size
filter
were washed with 5 ml of TEN buffer (10 mM Tris-HCl [pH 8.0],
1 mM EDTA, 250 mM NaCl), resuspended in 1 ml of ice-cold extraction
buffer (100 mM LiCl, 50 mM Tris-HCl [pH 7.5], 1 mM EGTA, 1%
[wt/vol]
sodium dodecyl sulfate [SDS]), and snap frozen prior to
storage
at
20°C until further processing
ashore.
Total RNA was isolated by a hot-phenol extraction procedure
(
70).
Synechococcus sp. cells were homogenized in
extraction
buffer at 65°C for 1 to 2 min and deproteinized with an
equal
volume of hot (65°C) phenol-chloroform-isoamyl alcohol
(25:24:1)
for 5 min. The aqueous phase was recovered by centrifugation
(13,000
×
g for 5 min) and reextracted with
chloroform-isoamyl alcohol
(24:1) at ambient temperature. Nucleic acids
were precipitated
from the aqueous phase with 2.5 volumes of 100%
ethanol at
20°C
for 24 h and recovered by centrifugation
(13,000 ×
g for 20 min).
The pelleted material was
washed in 75% (vol/vol) ethanol and
taken up in 100 µl of DNase
buffer (100 mM sodium acetate, 10
mM MgCl
2). DNA was
hydrolyzed at 37°C for 30 min in the presence
of 10 U of DNase (RNase
free; Boehringer Mannheim). Following
inactivation and removal of DNase
by phenol-chloroform extraction,
RNA was pelleted by ethanol
precipitation and taken up in 50 µl
of diethylpyrocarbonate-treated
deionized water. RNA concentrations
were determined by absorbance at
260 nm, and the integrity of
the samples was confirmed by
electrophoresis through formaldehyde
agarose gels stained with ethidium
bromide (
3).
Northern dot blots were performed as described previously
(
70) following heat denaturation of samples (1 to 2 µg of
RNA)
in 200 µl of 5× SSC (1× SSC is 0.15 M NaCl, 15 mM Na citrate,
pH 7.5)-10 mM EDTA at 65°C for 15 min. Following transfer under
gentle vacuum (<4 cm of Hg) to positively charged nylon membranes
(Boehringer Mannheim), the blots were air dried and the RNA was
immobilized by UV irradiation at 305
nm.
Dot blots were hybridized at high stringency with double-stranded DNA
probes derived from the oceanic cyanobacterium
Synechococcus sp. strain WH8103 (
68,
71). The probes employed were (i) a
699-bp
BamHI-
BglII fragment of pRBGL1.4, which
includes the 5'
region and the first 630 nucleotides of
rbcL; (ii) an
EcoRI-
SalI
fragment of
pPE1.3, which includes 198 bp of upstream sequence,
the coding region
of
cpeB, and the 5' end of
cpeA of the class
1 PE
operon; and (iii) the
EcoRI insert of pGlnA1.1, which
includes
most of the coding region of
glnA plus 350 bp of
upstream sequence.
Probe DNA was isolated in low-melting-point agarose
following
restriction digestion of plasmid DNA, and the recovered
fragments
were purified with glass milk. DNA was labelled with
digoxigenin-dUTP
(Boehringer Mannheim) by random priming according to
the manufacturer's
recommendations.
Membranes were prehybridized for 4 h at 55°C in a solution
containing 50% (vol/vol) formamide, 5× SSC, 0.1% (wt/vol) SDS,
1%
(wt/vol) blocking reagent (Boehringer Mannheim), and 5% (wt/vol)
dextran sulfate. Probe DNA (25-ng/ml final concentration) and
sheared
salmon sperm DNA (100-µg/ml final concentration) were
denatured at
100°C for 10 min and added to fresh solution prior
to overnight
hybridization at 55°C.
Northern dot blots were stringency washed twice in 2× SSC-0.1%
(wt/vol) SDS at ambient temperature for 5 min and twice again
at 65°C
in 0.2× SSC-0.1% (wt/vol) SDS for 30 min. Hybrids were
detected by
immunochemistry with alkaline phosphatase-conjugated
anti-digoxigenin
and the chemiluminescent substrate AMPPD (Tropix
Inc.) in conjunction
with Amersham Hybond enhanced chemiluminescence
film (
70).
The resulting luminographs were quantified by densitometry
with a GDS
8000 gel documentation system (UVP Ltd.) and the Gel
Works 1D Advanced
software
package.
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RESULTS |
Filter fractionation of Synechococcus spp.
An
essential requirement of several of the analyses carried out during the
present study was the development of a rapid isolation procedure to
concentrate Synechococcus sp. cells from bulk phytoplankton samples. The differential-fractionation technique employed was a minor
refinement of an earlier protocol (69) that had been field
tested during previous cruises in the northeast Atlantic Ocean (RRS
Discovery cruise D189 and RRS Charles Darwin
cruise CD47) and also by others for phytoplankton communities sampled off the North Carolina coast (25). In confirmation of the
absence of eukaryotic algae from the fractionated samples prepared
for the Northern analyses, only 16S and 23S rRNA bands were detected on
denaturing RNA gels (Fig. 2) plus an
additional band of intermediate molecular mass which is a
diagnostic degradation product of the 23S rRNA subunit found in
cyanobacterial RNA samples extracted in buffers containing low
concentrations of Mg2+ ions (25, 48).
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FIG. 2.
Ethidium bromide-stained formaldehyde agarose gel of
filter-fractionated RNA samples collected on 20 July 1991 (diel 2). The
lane on the far right contains 5 µg of RNA isolated from the marine
Synechococcus sp. strain WH8103.
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Temporal variability in Synechococcus sp. abundance and
PE content.
Within the eddy, Synechococcus sp. cell
concentrations were elevated (~2-fold) in comparison to those of
surrounding waters and showed a net increase of 3- to 4-fold between
the first and last days on station (Table
1). During this period, the sea surface temperature increased by ~1°C, leading to the development of some weak secondary structure in the upper 10 m of the water column toward the end of the study (Table 1 and Figure
3, top). Nitrate concentrations within
the upper mixed layer were within the range of 0.6 to 2.1 µM, whereas
ammonium concentrations increased with depth from the surface (<0.1
µM) to reach 0.15 to 0.2 µM near the base of the mixed layer
(5).
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TABLE 1.
Temporal variability in Synechococcus sp. cell
numbers within the surface mixed layer of a cyclonic eddy in the
northeast Atlantic during July 1991
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FIG. 3.
(Top) Vertical distribution of temperature (dotted
lines) and Synechococcus sp. cells (± standard error
[SE]) on 16 July (open circles) and 21 July (dotted circles) 1991. (Bottom) Diel variation in Synechococcus sp. cell counts
(open circles; ±SE) and PE content (solid circles) on 19 to 20 July
1991 (diel 2). The solid rectangle at the top of the figure indicates
the duration of the nocturnal period. GMT, Greenwich mean time.
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Depth profiles of
Synechococcus sp. cell numbers revealed
that the population was homogeneously distributed throughout the
surface mixed layer, with very few cells occurring at depths below
the
seasonal thermocline at approximately 25 m (Fig.
3, top).
The
increase in cell concentrations seen in surface waters, therefore,
was
indicative of similar changes in the size of the
Synechococcus sp. population that occurred throughout the
mixed layer. As suggested
by the increase in
Synechococcus
sp. cell concentrations during
the period on station, the contribution
of the picoplankton size
class (0.2 to 2.0 µm) to water column
integrated primary production
(0 to 40 m) increased from 15 to
17% at the start of the experiment
to 24 to 29% between 18 and 22 July 1991 (
5).
Synechococcus sp. cell division was phased to the brief
nighttime period that is characteristic of northern latitudes during
the summer months, the population undergoing approximately one
division, and hence one round of the cell cycle, per day (Fig.
3,
bottom). Consistent with the observed diel pattern of growth
and cell
division, the PE content of
Synechococcus sp. cells
increased
throughout the daylight hours but was reduced by about half
following
the late-evening/nocturnal separation of daughter cells (Fig.
3,
bottom).
Although the numbers of
Synechococcus sp. cells
approximately doubled between evening and nighttime samples, the net
generation
time of the population was on the order of 4 to 5 days
(Table
1). Since the study was conducted within the same water mass
and
the depth of the mixed layer did not change significantly
during the
period on station (Fig.
3, top; see also ref.
5),
this discrepancy (and the net losses in
Synechococcus sp.
cell
numbers observed during the daylight hours [Fig.
3, bottom])
suggests
that the population was being actively
grazed.
Diel variability in Synechococcus mRNA
abundance.
Changes in the relative abundance of three
Synechococcus sp. mRNAs were measured over the course of two
diel cycles (17 to 18 and 19 to 20 July 1991) toward the end of the
period on station. Comparatively little temporal variability in
cpeBA mRNA levels was observed; transcript levels oscillated
somewhat (dynamic range, ~2), but no consistent evidence of a
distinct temporal pattern was evident during either diel cycle (Fig.
4a and b). By contrast, marked temporal
variability in rbcL and glnA expression occurred and resulted in about an order of magnitude difference in mRNA levels
between the respective daily maxima and minima in the abundance of both
transcripts (Fig. 4c to f).
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FIG. 4.
Diel variation in the abundance of cpeBA mRNA
(a and b), rbcL mRNA (c and d), and glnA mRNA (e
and f) at the northeast-Atlantic Lagrangian station 17 to 18 July
(left-hand panels) and 19 to 20 July (right-hand panels) 1991. The
solid rectangles at the tops of the upper panels indicate the nocturnal
periods. GMT, Greenwich mean time.
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RubisCO mRNA increased in abundance during the short nighttime period
to reach a maximum by midmorning (~0800). Transcript
levels declined
steadily thereafter before increasing once more
from late afternoon
(~1800) to dusk. While the abundance of glutamine
synthetase
transcripts varied over approximately the same dynamic
range, the
temporal periodicity in
glnA mRNA was distinct from
that
observed for
rbcL. The peak in
rbcL mRNA
abundance was observed
about 4 h postdawn, whereas during both
diel cycles the highest
abundance of
glnA transcripts
occurred at or around local midday
(~1300 Greenwich mean time). The
abundance of
glnA transcripts
showed only small increases
during the first few hours of the
diel cycle before increasing sharply
(~2-fold) to the daily maximum.
Thereafter,
glnA mRNA
levels declined throughout the afternoon
and evening to reach a minimum
that was coincident with the initiation
of cell division around
dusk.
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DISCUSSION |
Diel patterns of Synechococcus sp. cell division and PE
synthesis.
The entrainment of Synechococcus sp. cell
division to a particular phase of the diel cycle (Fig. 3b) has been
documented previously for both coastal and open waters (7, 8, 42,
56, 62). In agreement with the observations reported here, these
earlier studies indicate that oceanic populations most frequently
undergo division during the late evening or night. It is intriguing,
therefore, that cell division in rapidly growing coastal communities is
initiated somewhat earlier in the diel cycle (8, 62). While
such contrasting behavior may point to fundamental differences in cell
cycle control between coastal and oceanic Synechococcus
spp., the few seasonal records available provide some evidence that the
peak in the frequency of dividing cells is shifted to a later phase in
the diel cycle in coastal waters during the autumn and winter months
(8, 62). Perhaps under these circumstances, in situ growth
rate (one or fewer divisions per day) and environmental constraints on
the diel phasing of the cell cycle more closely mirror those typical of
Synechococcus sp. populations growing in open waters under presumably less favorable physicochemical conditions. Such a contention is certainly supported by the observation that phosphate additions to
surface seawater samples from the Mediterranean Sea advanced subsequent
progression through the Synechococcus sp. cell cycle by
several hours (56).
Two distinct temporal patterns of DNA synthesis have been described for
Synechococcus spp. growing in laboratory culture under
alternating light-dark cycles (
2,
4), but neither pattern
is
consistent with the development of true cell cycle synchrony.
In both
coastal and oceanic isolates a significant proportion
of cells enter
the light cycle in G
2 having already completed
progression
through S during the latter half of the previous photoperiod.
In the
natural populations studied by Vaulot and colleagues, by
contrast, all
Synechococcus sp. cells entered the photoperiod
in
G
1 having progressed through G
2/M during the
previous evening
and night (
56). Similar significant
differences between the
cell cycle behavior of laboratory cultures and
that of natural
populations have also been reported for the related
photosynthetic
prokaryote
Prochlorococcus marinus.
Progression through the cell
cycle is highly synchronized to the diel
periodicity in irradiance
in natural populations of
P. marinus but, as with
Synechococcus spp., this behavior
is not reproduced under standard laboratory
conditions (
55,
56).
The apparent discrepancy between laboratory models of
Synechococcus sp. cell division and field observations
remains unexplained
but might be reconciled by examining the quantum
requirement for
passage through the light-dependent cell cycle
checkpoints in
G
1 and G
2 originally proposed by
Chisolm and coworkers (
2,
4). If the quantum requirement for
progression through G
1 into
S phase is significantly higher
than that required for progression
through G
2/M, then under
natural illumination (in which, as Vaulot
et al. [
56]
point out, incident irradiance shows a sinusoidal
temporal
distribution) cells may become stalled in G
1 during the
last few hours of fading daylight, whereas the dark block in
G
2 might still be passed. Quite clearly, such a scenario
could lead
to the rapid establishment of cell cycle synchrony under
natural
illumination because, irrespective of when division took place
during the latter half the previous diel cycle, all cells should
enter
the next diurnal period in G
1. It is not an absolute
requirement
of this model to propose the existence of two distinct
light control
circuits to regulate the G
1 and
G
2 checkpoints in order to promote
cell cycle synchrony
under natural illumination. Newly born cells
in G
1 have
approximately half the pigment complement and, presumably,
half the
light absorption capacity of G
2 cells that are just about
to undergo division (Fig.
3,
bottom).
In addition to the diel periodicity in DNA content that results from
cell cycle synchronization (
56), the entrainment of
Synechococcus sp. cell division to the natural photoperiod
predicts
a number of other cell cycle-related effects on the daily
pattern
of macromolecular synthesis. The most obvious of these is the
exponential increase in stable RNA (rRNA and tRNA) and protein
content
that must accompany progression through the cell cycle.
Bulk
measurements of biosynthetic processes performed on natural
populations
are unlikely to resolve such temporal changes in cell
composition,
however, unless they are corrected for (or are independent
of) loss
terms such as grazing and advection as well as the influence
of other
members of the planktonic community. Nevertheless, where
specific
estimates of
Synechococcus sp. cell composition have
been
attempted with natural populations the expected temporal
pattern of
macromolecular synthesis has emerged (
20,
25).
As a further case in point, the cell content of the biliprotein PE was
measured directly during the present study and found
to increase in a
quasilogarithmic fashion throughout the daylight
hours (Fig.
3,
bottom). Assuming that the
Synechococcus sp. population
within the eddy was in balanced growth (a not-unreasonable assumption
given the comparative lack of variability in the environment),
this
suggests a more or less constant rate of synthesis (relative
to the
general protein pool) during all phases of the cell
cycle.
Perhaps as a consequence of the comparatively short duration of the
nocturnal period, the decline in cell PE content after
dusk could be
largely explained by the effect of dilution through
cell division
rather than by a nighttime decrease in the net rate
of synthesis.
Periodicity in energy supply need not in fact lead
to very large
variations in the rate of protein synthesis, at
least not in
cyanobacteria grown in light-dark cycles with comparatively
long light
periods (
41). At lower latitudes, however, the nocturnal
rate of PE synthesis in marine
Synechococcus spp. does
appear
to be somewhat lower even after allowance for dilution effects
following cell division (
68). There is also some evidence
that
the rate of
Synechococcus sp. ribosome synthesis
declines during
the rather longer hours of darkness experienced at more
southerly
locations during the summer months (
25).
Temporal variation in gene expression.
Consistent with the
diel pattern of PE synthesis, the relative abundance of
Synechococcus sp. cpeBA mRNA showed comparatively little temporal variability when normalized, as in the present case, to
total RNA. Since cell ribosome content must also be a temporal variable
in synchronously dividing populations, the comparative lack of
variability in the normalized abundance of cpeBA mRNA suggests that either the PE operon was expressed constitutively or the
cpeBA message is unusually stable. In fact, the half-life of
cpeBA mRNA in the marine cyanobacterium
Synechococcus sp. strain WH7803 is of the same order (10 to
15 min) as those determined for several other genes (9, 28,
68), and there is no a priori reason to believe that the natural
population should differ markedly in this regard.
The pronounced diel variability and periodicity in
Synechococcus sp.
rbcL and
glnA mRNAs,
by contrast, is much more consistent
with the operation of specific
controls at the transcriptional
and/or posttranscriptional levels.
Although some of the temporal
variability in mRNA abundance may reflect
global changes in transcription
rates as the population progressed
through the cell cycle, such
effects are perhaps as likely to influence
the overall abundance
of transcripts from the PE operon as they are to
influence the
diel pattern of RubisCO or GS expression. It is
significant, therefore,
that the temporal periodicities in
Synechococcus sp.
rbcL and
glnA mRNAs
remain markedly conservative features of the natural
population when
each is normalized to
cpeBA mRNA (Fig.
5). Irrespective
of whether the PE operon
was truly constitutively expressed or
not, it is clearly apparent that
rbcL and
glnA were differentially
regulated
during the diel cycle, both with respect to each other
and with respect
to
cpeBA.
View larger version (25K):
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|
FIG. 5.
Histogram showing temporal variation in the abundance of
Synechococcus sp. rbcL (a) and glnA
(b) transcripts. Data from both diel studies are included and are
normalized to the diel variability in cpeBA mRNA abundance.
Both data sets are fitted with fourth-order polynomial regression
curves (r = 0.675 [a] and 0.644 [b]). GMT,
Greenwich mean time.
|
|
The diel variability in
rbcL mRNA abundance observed in this
subpolar
Synechococcus sp. population differed somewhat from
that shown previously for picoplankton-dominated surface waters
from
coastal and offshore sites in the Gulf of Mexico (
38,
39).
Whereas these earlier studies reported a midday maximum in transcript
abundance, the temporal pattern of RubisCO expression revealed
here is
more reminiscent of the diel rhythm in
rbcL mRNA found
by
these same investigators in a more detailed recent study
(
37).
However, unlike the northeast Atlantic
Synechococcus sp. population,
in which
rbcL mRNA
increased from late afternoon to reach near-maximal
levels by daybreak,
there was no evidence of a comparable nighttime
increase in transcript
abundance in the subtropical populations
studied by Pichard et al.
(
37). Nevertheless, the periodicity
(i.e., the temporal
evolution of maxima and minima) in
rbcL mRNA
observed in the
present investigation was very similar to that
found in the studies of
Paul and coworkers described above and
by Watson and Tabita
(
63) and Pichard et al. (
37) for laboratory
cultures of
Synechococcus sp. strain WH7803 and
P. marinus,
respectively.
The
rbcL gene of
Synechococcus sp. strain WH8103
is 97% identical at the nucleotide level and 99% at the amino acid
level
(GenBank accession no.
AF148536 [
68]) to the
homologue present
in strain WH7803. Neither sequence is particularly
closely related
to the
rbcL gene probe derived from the
freshwater cyanobacterium
Synechococcus sp. strain PCC6301
used by Pichard and coworkers
(
37-39) to examine gene
expression in subtropical picoplankton.
It is of interest, therefore,
that similar diel rhythms in
rbcL expression have been
observed for both sequence types even though
the target organisms
recognized by each probe are likely to be
somewhat
different.
In marked contrast to the temporal pattern of
rbcL
expression, maxima in
glnA transcript levels occurred some 5 to 6 hours
later in the diel cycle (i.e., at the midpoint of the
daylight
period) and coincided with the approach of the daily minimum
in
rbcL mRNA abundance. Whether similar periodic differences
also
occurred in the diel patterns of RubisCO and glutamine synthetase
translation and activity was not determined, but the differential
accumulation of their respective mRNAs is certainly consistent
with
such a temporal separation. For RubisCO at least, shifts
in
rbcL transcription have been shown to be closely linked to
temporal variations in the rate of C fixation (
37), but no
comparable
field investigations have been attempted to examine the diel
pattern
of nitrogen assimilation in these
organisms.
Diurnal periodicity in photosynthetic potential may be a rather general
feature of natural populations of
Synechococcus spp.,
since
very similar rhythmic behavior (characterized by a midday
peak in
CO
2 fixation capacity) to that found by Pichard et al.
(
37) has been noted by others under widely different
environmental
conditions (
43,
44). Not least because
variation in the intracellular
C/N ratio is of such key importance in
regulating the primary
N-assimilatory enzyme, glutamine synthetase
(
12,
32,
33,
53), temporal periodicity of this kind in C
fixation must also
play some role in determining the pattern of N
assimilation in
natural populations if, as would appear to be the case,
diel rhythms
in RubisCO expression are the norm rather than the
exception in
Synechococcus spp. (
37-39,
63).
In cyanobacteria, as in the well-studied prokaryotic models
Escherichia coli and
Bacillus subtilis,
expression of glutamine
synthetase and other nitrogen-assimilatory
enzymes is regulated
by signal transduction systems sensitive to the
balance of C and
N entering intermediary metabolism (
12,
13,
53,
57). Transcription
of
glnA is enhanced under
physiological conditions in which the
ratio between the relative rates
of C and N assimilation increases
to the point where intracellular
metabolites at the amino level
of reduction become depleted, i.e.,
during periods of N starvation
or, conversely, as a result of increases
in the prevailing rate
of C fixation. The combined effects of energy
limitation and the
catabolism of C reserves during the nocturnal phase
of the diel
cycle predicts that natural populations of
Synechococcus spp.
are likely to enter the diurnal period in
a C-depleted state.
Under such circumstances, it is perhaps not too
surprising that
transcription of the RubisCO operon (and hence C
fixation potential)
should be transiently up-regulated in anticipation
of, and during,
the first few hours of
daylight.
While specific feedback controls of this kind are not necessarily
excluded, they are insufficient to explain why the diel
rhythm in
rbcL mRNA abundance is also maintained in
Synechococcus spp. held in constant light (
37),
i.e., under experimental conditions
that might be expected to eliminate
the temporal oscillations
in carbon reserves and cell C/N ratio
experienced by the population
in situ. This type of rhythmic behavior
clearly suggests that
some element of circadian control may be involved
in the regulation
of the
Synechococcus sp. RubisCO operon
(
37). It does not follow,
however, that circadian control
need invoke clock-regulated
trans-acting
elements that
interact with the
rbcL promoter directly (
30).
From an organizational standpoint at least, one might argue that
coordinating RubisCO expression with the cell cycle probably offers
a
more parsimonious molecular solution to address the same problem
(i.e.,
one of intermittent energy supply) if, as in
Synechococcus sp. strain WH7803, cell division is itself controlled by a circadian
oscillator (
50).
Whether
rbcL is regulated directly or indirectly by a
circadian rhythm, the fact remains that there is a dramatic increase
in
the capacity for C fixation in natural populations of
Synechococcus spp. as the solar altitude increases toward
midday (
37,
38,
43,
44). Not least because this accelerating
supply of newly
synthesized carbon skeletons should rapidly deplete the
available
pool of intracellular nitrogen, it is the likely imbalance in
the cell C/N ratio that arises from the temporal periodicity in
Synechococcus sp. C fixation rates that also provides the
most
conservative interpretation of the diel pattern of
glnA
mRNA abundance.
The rhythm in
glnA mRNA abundance observed
was clearly distinct
from that for
rbcL and is at least
consistent with the notion
that a shift up in glutamine synthetase
synthesis and activity
may occur during the latter half of the diurnal
period in natural
populations of
Synechococcus spp. as a
direct result of the enhanced
rates of C assimilation somewhat earlier
in the diel
cycle.
An element of temporal periodicity in N-assimilation rates is a not
entirely unexpected feature of synchronously growing populations
of
microorganisms like
Synechococcus spp., since the bulk of
the
cell's macromolecules are produced during the latter half of the
cell cycle. The growth of the individual, like that of the population
in general, is an exponential function, and hence the cell's demand
for nutrients (including nitrogen) must also increase in a
quasilogarithmic
manner as the cell cycle progresses. This feature of
the daily
pattern of macromolecule biosynthesis in natural populations
of
Synechococcus spp. has been noted by others
(
34) and is further
illustrated by examination of the diel
pattern of PE production
reported here (Fig.
3, bottom). Very little
change in cell PE
content was observed during the hours up to midday
compared with
the near doubling in concentrations that occurred
thereafter,
prior to the nocturnal separation of daughter
cells.
A number of features emerge from the present study that may have wider
implications for our understanding of the factors that
regulate carbon
and nitrogen assimilation in natural populations
of marine
Synechococcus spp. Endogenous changes in physiological
demands that arise as an inevitable consequence of the daily
progression
through the cell cycle are, perhaps, as important in
determining
the periodic abundance of specific mRNAs in these organisms
as
are changes in exogenous environmental variables, such as the
diel
alternation in irradiance. The differential expression of
Synechococcus sp.
rbcL and
glnA mRNAs
reported here clearly suggests
that these key C- and N-assimilatory
enzymes may be maximally
expressed at different periods during the cell
cycle in a manner
analogous to the reciprocal control of RubisCO and
nitrogenase
gene expression found in the diazotrophic cyanobacterium
Synechococcus sp. strain RF-1 (
11). Whether, like
that of RubisCO (
37),
the activity of glutamine synthetase
in marine
Synechococcus spp.
also shows some diel
variability requires further investigation,
since the occurrence of
temporal changes in nitrogen assimilation
capacity is likely to modify
our general overview of (i) niche
specialization among marine
phytoplankton and (ii) the specific
role that these picoplanktonic
cyanobacteria may play in the nutrient
dynamics of the surface
oceans.
| |
ACKNOWLEDGMENTS |
This work was supported by research grants from the Natural
Environment Research Council (NERC), the Joint Environment Programme of
National Power and Powergen, and the University of Stirling.
I acknowledge access to ship time during the NERC Biogeochemical Ocean
Flux Study and the encouragement and support of R. P. Harris, M. Whitfield, and others at Plymouth Marine Laboratory and the Marine
Biological Association.
| |
FOOTNOTES |
*
Mailing address: Department of Biological Sciences,
University of Stirling, Stirling FK9 4LA, United Kingdom. Phone: 44 1786 467784. Fax: 44 1786 464994. E-mail:
michael.wyman{at}stir.ac.uk.
| |
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Applied and Environmental Microbiology, August 1999, p. 3651-3659, Vol. 65, No. 8
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
