SABBATICAL LEAVE APPLICATION- BIOLOGY
University of St. Thomas
Title of project: Role of chromosome compaction in the cyanobacterial response to
photoperiod.
Abstract of project (150 word max.; 12 point Times New Roman font, single-spaced text)
The cyanobacterium Synechococcus elongatus has circadian (daily) rhythms in behavior that
are susceptible to light and temperature cues; allowing this organism to shift behavioral
activities based upon environmental signals. The mechanism of the circadian clock that
controls these behaviors in S. elongatus has been studied intensively. However, the portion
of the clock that has received the least attention thus far, in any circadian model system, has
been the mechanism by which the circadian clock communicates with the environment to
interpret changes in day length (photoperiod), such that behavior can be appropriately
adjusted. The work proposed here will investigate whether chromosome compaction plays a
role in the ability of these organisms to adjust their behavior based on day length. This
sabbatical will 1) forward my research agenda, 2) increase our collective understanding of
the circadian mechanism, and 3) bring new research opportunities to UST students.
NARRATIVE:
a) Significance of this work: Cyanobacteria are found in nearly every habitat including most dry
land ecosystems, geothermal hot springs, caves, and polar and hot deserts (Cox et al., 1981; Priscu
et al., 1998; Brasier et al. 2002). The cyanobacterium Synechococcus elongatus is
photoautotrophic, meaning it produces its own energy directly from sunlight and obtains all its
carbon mass by fixing carbon dioxide, using photosystems similar to photosynthetic plants.
Although S. elongatus is a unicellular organism, it performs a number of complex metabolic
processes that take place at certain times of day. To efficiently regulate their cellular activities in
a temporal fashion, cyanobacteria make use of a circadian clock (Ditty et al., 2009).
The primary function of a circadian clock is to keep time on a 24-hour scale, which
provides an internal estimate of time of day based on cues from the external environment
(Pittendrigh, 1981; Johnson et al., 2003).
The circadian clock allows an organism to be
synchronized to the external environment such that it allows the organism to best fit their cellular
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functions to the environment (e.g. photosynthesis during the day (Class 1) when light is available
and purine biosynthesis at night (Class 2) (Ditty et al., 2009)).
input
oscillator
output
°C
or
LdpA
CikA
Pex
KaiA
KaiB
KaiC
SasA
RpaA
LabA
chromosome
compaction
Figure 1. Schematic of the circadian clock in cyanobacteria (from
Ditty et al., 2009)
Synechococcus elongatus has circadian rhythms that are susceptible to light and
temperature cues and can therefore shift behaviors by phase resetting, or changing when activity
is at its peak based upon environmental signals. This process is exactly the same as to how humans
shift behavior in response to travel across time zones. The genes responsible for the circadian
clock, which control the circadian rhythms in S. elongatus, have been separated into three
categories: oscillator genes, input genes, and output genes (Figure 1). The oscillator genes are
kaiA, kaiB, and kaiC, named after the Japanese word kaiten meaning cycle or “turning of the
heavens” (Ditty et al., 2003). The mechanism of the circadian clock in S. elongatus has been
studied intensively. However, the portion of the clock that has received the least attention in this
field thus far has been the mechanism by which the circadian clock communicates with the
environment through input pathways. In particular, very little is known about how the circadian
clock in cyanobacteria responds to photoperiod, or the duration of light versus the duration of
darkness within a 24-hour day. One of the current problems in the cyanobacterial circadian rhythm
field is that the vast majority of what we know about the circadian mechanism in S. elongatus has
been based solely on photoperiods that consist of 12-hours of light and 12-hours of dark
(12L:12D). Although different photoperiods have considerable impacts on the circadian activity
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of organisms in other model systems (Dunlap et al., 2004; Johnson et al., 2003; Pittendrigh, 1981),
we know nothing of how any organism responds to photoperiod at the molecular level. Therefore,
the work proposed here is of major importance to the field of circadian biology in that it will be
the first significant work to address this question. In addition, this work will expand my own
research program and foster collaboration with the lab of Dr. Stanly Williams that has the expertise
to unveil the molecular basis of photoperiod.
b) Basic description of the proposed work: Recent work from the Williams lab has begun to
shed light on the molecular mechanism of how the circadian clock conveys environmental
information to gene expression. Their work has demonstrated that changes in chromosome
compaction (defined as the physical accessibility of the chromosome for gene expression) are
observed as a function of circadian time. It was found that the chromosome is relatively diffuse
during the day and compacted at night. Therefore, it is thought that to generate circadian gene
expression, the Kai proteins may communicate temporal information through chromosome
compaction, which then controls access to gene expression based upon that compaction at certain
times of day (Smith and Williams, 2006). The research proposed here will begin to investigate if
chromosome compaction is a probable mechanism for mediating the effects of photoperiod on
gene expression.
Figure 2. Chromosome compaction of wild-type S.
elongatus under 12L:12D photoperiod. Green;
DAPI stained chromosomal DNA, Red;
autofluoresence of cell membranes (from Smith and
Williams, 2006).
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Prior research has shown that chromosome compaction is a rhythmic process during a
twenty-four hour period. It was found that in a 12L:12D photoperiod cycle, the chromosome goes
through compaction for the first 12 hours of light and then decompaction of the chromosome for
the second 12 hours of dark (Figure 2). Again, all of this research was preformed using a standard
12L:12D photoperiod, and nothing is known about the relationship between chromosome
compaction and the cellular response to other photoperiods. Therefore, this project will investigate
how chromosome compaction and phase response of the wild-type S. elongatus strain is affected
in varying photoperiods.
The methodology for this investigation is rather simple and will follow protocols published
by the Williams lab (Smith and Williams, 2006). Briefly, wild-type S. elongatus cultures will be
grown under exposure to different photoperiods; 12L:12D as a control, and 6L:18D, 8L:16D,
16L:8D, and 18L:6D for two days and then set into constant light. Cell samples will be taken every
four hours starting at the time point that the cultures go into constant light. The DNA stain DAPI
will be added to the cell suspension to track chromosome structure by visualization using
fluorescence microscopy (Smith and Williams, 2006). To begin this work, I will need to learn the
staining, microscopy, and data analysis techniques in the Williams laboratory. After developing
these skills, I plan to bring the expertise back to the Biology Department at the Unviersity of St.
Thomas as we have a fluroescence microscope such that raw chromosome compaction images will
be taken at UST. UST does not have the capability at this time to analyze the raw images, therefore
they will be sent to the Williams lab at the University of Utah for image deconvolution (where
background fluorescence is removed) and compaction indeces will be calculated. Analysis
comparison will be conducted between the different photoperiods with the 12L:12D photoperiod
serving as the control.
The data collected will be able to show whether the varying photoperiods cause the
chromosome of the S. elongatus cells to compact at different time points from the standard
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12L:12D photoperiod, and will demonstrate the mechanism of how behavior changes based upon
photoperiod. If this occurs, it will support the data that my lab has already collected indicating that
photoperiods do cause a phase shift in the cellular activity (see previous work). These results will
help to determine if chromosome compaction can be changed by the amount and duration of light
that the S. elongatus cells are receiving.
Figure 3. Change in clock activity in response to photoperiod
change in S. elongatus cells. Peak of activity occurs
approximately 4 hours early in a 6L:18D (light blue) and
approximately 4 hours late in a 18L:6D (dark blue) photoperiod
relative to the 12L:12D control (green) (Dubis and Ditty,
unpublished data).
(c) Previous work: Preliminary work by undergraduates in my lab has begun to show the effects
that various extreme photoperiods have on the circadian oscillator gene activity in cyanobacteria
(Figure 3). Wild-type S. elongatus cultures were grown in 12L:12D, 6L:18D, and 18L:6D
photoperiods for seven days. Results showed that increasing or decreasing the duration of light
(18L:6D or 6L:18D) varied when the clock was most active (i.e., relative timing) by shifting the
peak of activity by approximately 4 hours in delay and advance, respectively (Dubis and Ditty,
2007). In addition, Class 1 (day genes) and Class 2 (night genes) output genes that are controlled
by the circadian clock have also been investigated. Interestingly, preliminary results have shown
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that a Class 1 gene was affected by photoperiod, but the Class 2 gene was relatively insulated from
photoperiod (Rhein et al., 2008).
My students and I have studied the effects of photoperiod on the behavior of cyanobacteria.
This previous work has now led our project into the realm of how the cells drive this change in
behavior. Therefore, this proposed sabbatical project is a natural extension of my current research
program.
d) Specific professional goals: I have three specific goals for my sabbatical. First, I will learn the
basic fluorescence microscopy and imaging techniques that have been used in the Williams lab for
chromosome compaction studies. Second, I will apply this technique at UST to investigate the role
of chromosome compaction in the cyanobacterial response to photoperiod. Third, I plan to
disseminate these findings in the short-term at national meetings with the ultimate goal of
publication of the scientific findings in peer-reviewed journals. All these goals will facilitate the
expansion of my research program into new areas and techniques, which will allow me to provide
novel research experiences for UST undergraduates.
(e) Off-campus locations: I plan to spend approximately 2 weeks of the sabbatical in the
laboratory of Dr. Stanly Williams at the University of Utah to learn the aforementioned microscopy
techniques. Funding for the time in the Williams lab will be solicited through a UST Faculty
Development Research Assistance Grant (RAG) or from grants from the Williams lab. The
remainder of the sabbatical will be completed in my lab at UST. Raw images generated here at
UST will be sent to the Williams lab for manipulation. A letter of support for this collaboration,
along with the invitation to work in the Williams lab, is attached as Appendix 4.
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APPENDIX 1. TIMETABLE FOR THE PROJECT
2011
January: Travel to the Williams laboratory at the University of Utah to learn the proper
microscopy techniques to measure chromosome compaction.
January through May: The end of January will be designated to microscope set up and
troubleshooting here at UST. February through May will be used for the implementation of
photoperiod experiments. This time period will also be used to send raw data images back to the
Williams lab for data analysis.
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APPENDIX 2. BIBLIOGRAPHY
Works cited in narrative:
Brasier MD, Green OR, Jephcoat AP, Kleppe AK, Van Kranendonk MJ, et al. 2002. Questioning
the evidence for Earth’s oldest fossils. Nature 416:76-81.
Cox G, Benson D, Dwarte DM. 1981. Ultra structure of a cave wall cyanophyte, Gloeocapsa NS
4. Archives of Microbiology 130:165-174.
Ditty JL, Mackey SR, Johnson CH. Eds. 2009. Bacterial circadian programs. Berlin: SpringerVerlag.
Ditty JL, Williams SB, Golden SS. 2003. A cyanobacterial circadian timing mechanism. Annual
Reviews Genetics 37:513-543.
Dubis JW, Ditty JL. 2007. Effects of photoperiod on the circadian mechanism in cyanobacteria.
MN Academy of Sciences Annual Meeting. Hamline University, St. Paul, MN.
Dunlap JC, Loros JJ, DeCoursey PJ. 2004. Chronobiology: Biological Timekeeping. Sunderland,
MA: Sinauer.
Johnson CH, Elliott JA, Foster R. 2003. Entrainment of Circadian Programs. Chronobiology
International 20:741-774.
Pittendrigh CS. 1981. Circadian systems: general perspective and entrainment. In Handbook of
Behavioral Neurobiology: Biological Rhythms, ed. J Aschoff, pp. 57-80 and 95-124. New
York: Plenum.
Priscu JC, Fritsen CH, Adams EE, Giovannoni SJ, Paerl HW, et al. 1998. Perennial Antarctic
lake ice: an oasis for life in a polar desert. Science 280:2095-2098.
Rhein BA, Delaney KL, Dubis JW, Ditty JL. 2008. The effect of differing photoperiods on the
output genes, psbA1 and purF of the cyanobacterium Synechococcus elongatus. North
Central Branch American Society for Microbiology Meeting. St. Cloud State University,
St. Cloud, MN.
Smith RM, Williams SB. 2006. Circadian rhythms in gene transcription imparted by
chromosome compaction in the cyanobacterium Synechococcus elongatus. Proceedings
of the National Academy of Science USA 103:8564-8569.
Pertinent references to the field of study
Andersson CR, Tsinoremas NF, Shelton J, Lebedeva NV, Yarrow J, et al. 2000. Application of
bioluminescence to the study of circadian rhythms in cyanobacteria. Methods in
Enzymology 305: 527-542.
Dunlap JC. 1999. Molecular bases for circadian clocks. Cell 96: 271-290.
Golden SS, Ishiura M, Johnson CH, Kondo T. 1997. Cyanobacterial circadian rhythms. Annual
Review of Plant Physiology Plant Molecular Biology 48:327–354.
Ishiura M, Kutsuna S, Aoki S, Iwasaki H, Andersson CR, et al. 1998. Expression of a gene
cluster kaiABC as a circadian feedback process in cyanobacteria. Science 281: 15191523.
Iwasaki H, Williams SB, Kitayama Y, Ishiura M, Golden SS, Kondo T. 2000. A KaiCinteracting sensory histidine kinase, SasA, necessary to sustain robust circadian
oscillation in cyanobacteria. Cell 101: 223-233.
Katayama M, Kondo T, Xiong J, Golden SS. 2003. ldpA encodes an iron-sulfur protein involved
in light-dependent modulation of the circadian period in the cyanobacterium
Synechococcus elongatus PCC 7942. Journal of Bacteriology 185: 1415-1422.
Kondo T, Mori T, Lebedeva NV, Aoki S, Ishiura M, Golden SS. 1997. Circadian rhythms in
rapidly dividing cyanobacteria. Science 275: 224-227.
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Kondo T, Strayer CA, Kulkarni RD, Taylor W, Ishiura M, et al. 1993. Circadian rhythms in
prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria.
Proceedings of the National Academy of Science USA 90: 5672-5676.
Schmitz O, Katayama M, Williams SB, Kondo T, Golden SS. 2000. CikA, a
bacteriophytochrome that resets the cyanobacterial circadian clock. Science 289:765-768.
Vakonakis I, Klewer DA, Williams SB, Golden SS, LiWang AC. 2004. Structure of the Nterminal domain of the clock-associated histidine kinase SasA. Journal of Molecular
Biology 342: 9–17.
Whitton BA, Potts M. 2000. Introduction to Cyanobacteria. In The Ecology of Cyanobacteria,
ed. BA Whitton, M Potts, pp. 1-11. Amsterdam: Klewer Academic Publishers.
Xu Y, Mori T, Johnson CH. 2000. Circadian clock-protein expression in cyanobacteria: rhythms
and phase setting. EMBO Journal 19: 3349-3357.
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