Microbiology (2013), 159, 253–258
DOI 10.1099/mic.0.064220-0
Fluorescence spectroscopy study of heterocyst
differentiation in Anabaena PCC 7120 filaments
Shan Ke and Robert Haselkorn
Correspondence
Department of Molecular Genetics and Cell Biology, the University of Chicago, 920 E 58th Street,
Chicago, IL 60637, USA
Robert Haselkorn
[email protected]
Received 12 October 2012
Revised
29 November 2012
Accepted 30 November 2012
Filamentous Anabaena PCC 7120 differentiates nitrogen-fixing specialized cells called
heterocysts at regular intervals following removal of combined nitrogen from the medium.
Phycobiliproteins are degraded during differentiation. Heterocyst differentiation was followed at
the single cell level by using confocal fluorescence microscopy. The presence of an enhanced
fluorescence emission peak from allophycocyanin (APC) indicates that the degradation of the
phycobilisomes during nitrogen deprivation possibly initiates at the linker between APC and
photosystem II in a bottom-to-top disassembly model. Furthermore, the fluorescence emission
peak around 650 nm provides an advantageous marker to identify early candidates for
differentiation.
INTRODUCTION
Filamentous nitrogen-fixing cyanobacteria, of which
Anabaena PCC 7120 has become the archetypical example,
differentiate nitrogen-fixing specialized cells called heterocysts at regular intervals along each filament in suitable
medium. The length of the intervals is determined by the
interplay of regulatory proteins: a transcription factor
called HetR, two proteins that contain the amino acid
sequence RGSGR (PatS and HetN), and other transcription
factors (Flores & Herrero, 2010; Haselkorn, 1978, 2008).
The PatS and HetN peptides prevent HetR from binding to
DNA and destabilize the HetR protein (Huang et al., 2004;
Risser & Callahan, 2009; Yoon & Golden, 1998). Under our
laboratory conditions the vegetative cells between each
heterocyst divide approximately every 16 h. During that
time, a vegetative cell approximately halfway between two
heterocysts differentiates into a new heterocyst. Anabaena
PCC 7120 usually has about 5–10 % heterocysts, but that
fraction can be increased by overexpression of the hetR
gene (Buikema & Haselkorn, 2001) or reduced by
overexpression of the patS or hetN genes (Callahan &
Buikema, 2001; Yoon & Golden, 2001).
Differentiation requires the destruction of many proteins,
including the phycobiliproteins, parts of the photosystem
(PS)II reaction centre, and ribulose-1,5-bisphosphate
carboxylase oxygenase (RuBisCO), ensuring that the
heterocyst cannot generate oxygen and cannot fix carbon.
Newly expressed proteins include enzymes for the synthesis
of glycolipids and polysaccharides that form a doublelayered envelope around the heterocyst, enzymes for
Abbreviations: APC, allophycocyanin; au, arbitrary fluorescence units;
Chl, chlorophyll; C-PC, C-phycocyanin; PBS, phycobilisome; PC,
phycocyanin; PSII, photosystem II.
064220 G 2013 SGM
reorganization of metabolism to produce reductants for
nitrogen fixation, and the suite of enzymes and redox
proteins for nitrogen fixation and assimilation (Fay, 1992;
Golden & Yoon, 2003; Kumar et al., 2010). In all, HetR is
responsible for the expression of some 1500 of the 7000
genes in the Anabaena PCC 7120 genome (Buikema &
Haselkorn, 2001, 1991; Liang et al., 1992; Wolk, 1996).
The process of differentiation can be followed at the single
cell level by using confocal fluorescence microscopy.
Cyanobacteria harvest light energy across much of the
visible spectrum using phycobiliproteins organized in
cylindrical phycobilisomes (PBSs), which transfer energy
principally to PSII reaction centres. The energy transfer can
be monitored by measuring fluorescence emission from
phycobiliproteins or chlorophyll (Chl) following excitation
at a wavelength absorbed by phycobiliproteins (Glazer,
1985; Yoon & Golden, 1998). Any damage to the protein
involved in energy transfer will result in alteration in the
fluorescence emission spectra. By monitoring fluorescence
emission, we can track single cell development.
METHODS
Cell growth and induction. Anabaena PCC 7120 cultures were
grown in BG11 medium under room light (~9 mE m22 s21) and
temperature (~24 uC). They were switched from BG11 to a BG112
0.75 % agarose slab gel surface by being centrifuged and washed three
times with BG112 medium.
Microscopy and fluorescence spectroscopy. The growth and
fluorescence spectroscopy of filaments on the gel surface were
monitored with a Leica SP5 Tandem Scanner Spectral 2-Photon
confocal microscope with a Leica 663 numerical aperture 0.9 water
immersion lens. The fluorescence spectra were measured at a pinhole
value of AU 1.3, meaning that the focus thickness along the z axis was
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253
S. Ke and R. Haselkorn
about 4.5 mm. Samples were excited by the orange HeNe laser at
561 nm and the fluorescence emission spectrum was collected from
570 to 740 nm with 5.0 nm per step. Each sample was scanned 32
times and the mean of the fluorescence spectra is presented. The
fluorescence intensity was recorded using arbitrary fluorescence units
(au).
RESULTS
The absence of phycobiliprotein absorption in the heterocysts predicts that excitation at a wavelength absorbed by a
phycobiliprotein will not yield emission from Chl in the
heterocysts. This result is well known, and is shown for
single cells in a differentiated Anabaena PCC 7120 filament
in Fig. 1. The new and unexpected result is that after the
differentiation begins, emission from the developing cell
increases in intensity with a shift to shorter wavelength.
The Chl (Chl a) emission intensity eventually drops until it
is no longer visible. We interpret the short-lived increase in
emission intensity to be due to release of the phycobilisomes from the PSII centres and the loosening of their
structures, allowing some of the energy in phycobilisomes
to contribute directly to fluorescence rather than be
transferred to Chl (Gantt, 1981; Glazer, 1985). These
differences allow us to use confocal microscopy of single
cells to follow the evolution of a heterocyst by recording
the fluorescence emission spectrum as a function of time
after transfer of the cells from complete medium to one
lacking combined nitrogen. Eventually the degradation of
the pigments interrupts the energy transfer completely. As
shown in Fig. 1, at stage II of heterocyst differentiation, the
fluorescence emission has shifted to a shorter wavelength
with increasing intensity. Watching the bright spots along
the filament can directly identify early candidates for
differentiation, as reported elsewhere (Toyoshima et al.,
2010).
In Fig. 2, cell 1 is a mature heterocyst with low Chl
fluorescence. Cells 2, 3, 6, 7, 8, 10, 11 and 12 are considered
to be vegetative cells with similar fluorescence spectra. At
around 645 nm, only one peak from phycocyanin (PC) is
shown. Fluorescence emission from allophycocyanin (APC)
is on the right shoulder of the PC peak. Compared with the
fluorescence of the vegetative cells, cells 4, 5, 9 and 13 have a
more resolved fluorescence emission peak at 650 nm, which
we attribute to APC, due to decreased energy transfer from
APC to Chl a (Glazer, 1985). Another possible contributor
to this peak is C-phycocyanin (C-PC) (Gantt, 1981). These
cells with the fluorescence peak at 650 nm were considered
to be early candidates for heterocyst differentiation at stage I.
The APC peak occurs almost at the initiation of heterocyst
differentiation, much earlier than the candidates for
differentiation become bright. About four vegetative cells
separated these early candidates.
Cell 4 and cell 5 were descended from the same mother cell,
and both show the appearance of the APC peak. Later, cell
5 has differentiated into a heterocyst, while cell 4 has
reverted to a vegetative cell. We interpret these fates as
resulting from early competition, mentioned earlier by
others (Allard et al., 2007; Toyoshima, et al., 2010). Both
cell 4 and cell 5 are induced to produce the early regulator
PatS, which binds to HetR to prevent DNA binding and
consequent differentiation (Allard et al., 2007; Yoon &
Golden 2001). Here, we believe that cell 5 produces the
PatS peptide, which is exported to cell 4, causing cell 4 to
revert due to degradation of HetR, while cell 5 continues to
differentiate due to the activity of HetR. Thus, the cell fates
could be determined by a slight difference in the ability to
Fluorescence intensity (au)
300
Stage II, increase in short-wavelength
fluorescence
200
Stages III and IV, emission
from chlorophyll is lost. The
mature heterocyst has
degraded phycobilisomes,
and cannot transfer energy
to chlorophyll
Stage I, initiation of
heterocyst differentiation
100
Vegetative cell
0
570
620
670
720
Wavelength (nm)
Fig. 1. Fluorescence emission spectra of single Anabaena PCC 7120 cells in a filament. The fluorescence intensity is recorded
as au. Four stages during heterocyst differentiation can be identified. Fluorescence excitation was at 561 nm.
254
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Microbiology 159
Heterocyst differentiation
Fluorescence intensity (au)
(a)
(b) Cell 5 (early candidate for differentiation)
150
100
50
650 nm
0
570
620
670
720
Fluorescence intensity (au)
Wavelength (nm)
(c) Cell 2 (vegetative cell)
150
100
50
0
570
620
670
720
Fluorescence intensity (au)
Wavelength (nm)
(d) Cell 1 (heterocyst with low fluorescent intensity)
50
0
570
620
670
720
Wavelength (nm)
Fig. 2. Fluorescence emission of Anabaena cells in the same filament 1 day after being transferred from BG11+ liquid medium
to a BG11” gel surface. (a) Single cell images and the sampling area for the fluorescence spectroscopy. We covered the entire
individual cell areas and used a mean of 32 scans for the fluorescence spectra. The star indicates a heterocyst and the arrows
indicate early candidates for differentiation. (b–d) Fluorescence spectra of an early candidate, a vegetative cell and a heterocyst.
Fluorescence excitation was at 561 nm.
export PatS. Alternatively, there might be stochastic
differences in the amount of PatS made in the two cells.
With the same single cell tracking, we have been able to
track the growth and differentiation of all individual
Anabaena cells along the filament shown in Fig. 3 for the
next 4 days. For example, note the development of cell 9 in
Fig. 2. Its differentiation with the corresponding fluorescence during development is shown in Fig. 3. One day
after the beginning of nitrogen deprivation, cell 9 began to
show fluorescence emission at 650 nm. After 4 days, it had
differentiated into a heterocyst. Using the appearance of the
fluorescence peak at 650 nm as a measure of the candidates
for differentiation, we found that except for a few cases, all
early candidates with the fluorescence peak at 650 nm finally
differentiated into heterocysts. Furthermore, no peak was
seen at 650 nm in the vegetative cells. For example, five early
candidates for heterocysts were identified by 650 nm
fluorescence emission in the 52-cell filament shown as day
3 in Fig. 3. Their fluorescence spectra, together with those of
five random vegetative cells in the same filament, are shown
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in Fig. 4. The fluorescence difference between the early
heterocyst candidate and the vegetative cell is clearly
demonstrated.
DISCUSSION
The key findings of this report can be summarized as
follows: tracking Anabaena PCC 7120 cell evolution via
single cell fluorescence reveals a peak at 650 nm from APC
or from a possible combination of C-PC and APC. The peak
at 650 nm appears very early during differentiation and can
be used to identify early candidates for differentiation. The
presence of this emission rise at 650 nm can be attributed to
loss of energy transfer between APC and PSII. In Fig. 2, cell 4
and cell 5 were descended from the same parent cell, and
both show the appearance of the APC peak. Cell 5
differentiated into a heterocyst, while cell 4 reverted to a
vegetative cell. Fig. 2 also demonstrates that fluorescence
emission from PC is constant and that from APC increases
during the early stage of the differentiation. This means that
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255
S. Ke and R. Haselkorn
(a)
(b)
150
100
50
0
570
620
670
720
3 h after switch to BG11- gel, still a vegetative cell
150
100
50
650 nm
570
620
After 1 day, early candidacy for a heterocyst
670
720
670
720
300
200
100
0
570
620
Fluorescence intensity (au)
0
After 2 days, early stage of a heterocyst
100
50
0
570
620
720
670
After 3 days, at the stage of a heterocyst
100
50
0
570
620
670
720
Wavelength (nm)
After 4 days, the final stage of a heterocyst
Fig. 3. (a) Images of the Anabaena PCC 7120 filament and differentiation of the individual cell (arrow); (b) fluorescence
emission spectra of the individual cell (arrow) during differentiation. Fluorescence excitation was at 561 nm.
PC remains on the rod and connected with APC, with
energy transfer from PC to APC still efficient.
With these findings, it is possible to propose that the
degradation of the phycobilisomes during nitrogen deprivation initiates at the linker between the APC and PSII. The
256
linkers in the PBS of cyanobacteria and red algae play
important roles in PBS assembly and energy transfer: one
linker connects the rod with the PBS core, another linker
holds the rod together, and another linker connects APC to
PSII. The linker connecting the core of PBS with the
thylakoid membrane and PSII is considered to be the major
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Microbiology 159
Heterocyst differentiation
Fluorescence intensity (au)
(a)
140
120
100
80
60
40
20
0
570
Fluorescence intensity (au)
(b)
590
610
630
650
670
Wavelength (nm)
690
710
730
140
120
100
80
60
40
20
0
570
590
610
630
650
670
Wavelength (nm)
690
710
terminal energy emitter to PSII. During the initiation of PBS
degradation, phosphatases first dephosphorylate the linkers.
Linker dephosphorylation may act as a signal for protein
degradation (Piven et al., 2005; Dines et al., 2008).
The appearance of the fluorescence emission peak at
650 nm possibly shows that the linker between the APC
and PSII is cleaved at the start of cell differentiation.
Consequently, energy transfer is blocked between APC and
PSII and energy is released as fluorescence emission from
APC. On the other hand, the linker connecting the rod and
core is still intact and the energy from the PC absorption is
still transferred efficiently to APC. In our fluorescence
spectroscopy set-up, the sample excitation was at 561 nm,
where PC has much stronger absorption than APC. If the
linker between PC and APC was impaired, the fluorescence
emission at 650 nm should decrease. These two factors
contribute to the appearance of the APC fluorescence
emission peak at 650 nm.
Earlier papers show a model in which PBS disassembly
occurs with the loss of rod hexamers before degradation of
the core, and from rod tips down toward the core (Dines
et al., 2008 and references therein). But if degradation of
PBS involves dephosphorylation and subsequent cleavage
of the linkers from the bottom up, that would explain
the appearance of the APC fluorescence emission peak at
650 nm at the beginning of cell differentiation.
Based on the observations and discussions above, the
appearance of the APC fluorescence emission peak can be
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730
Fig. 4. Fluorescence spectra of (a) vegetative
Anabaena cells and (b) early candidates for
heterocysts in the same filament 3 days after
being transferred from BG11+ liquid medium
to a BG11” gel surface. The light lines are
from five individual cells, while the heavy line is
the mean of these five cells; error bars, SD.
used as a sign of early candidacy for heterocyst differentiation along the Anabaena PCC 7120 filament. The early
candidate is about four cells away from a mature heterocyst
under our conditions. It is interesting to find that the
fluorescence emission from PC is constant and that the
fluorescence emission from APC even increases during this
early stage. This demonstrates that PC remains on the rod
and that the energy transfer from PC to APC is still efficient.
ACKNOWLEDGEMENTS
This research was supported in part by the University of Chicago and
the Ellison Medical Foundation. We thank Dr V. Bindokas for
assistance with the fluorescence measurements.
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Edited by: C.-C. Zhang
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