Effect of continuous light on diurnal rhythms in Cyanothece sp. ATCC 51142
© Elvitigala et al. 2009
Received: 23 December 2008
Accepted: 15 May 2009
Published: 15 May 2009
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© Elvitigala et al. 2009
Received: 23 December 2008
Accepted: 15 May 2009
Published: 15 May 2009
Life on earth is strongly affected by alternating day and night cycles. Accordingly, many organisms have evolved an internal timekeeping system with a period of approximately 24 hours. Cyanobacteria are the only known prokaryotes with robust rhythms under control of a central clock. Numerous studies have been conducted to elucidate components of the circadian clock and to identify circadian-controlled genes. However, the complex interactions between endogenous circadian rhythms and external cues are currently not well understood, and a direct and mathematical based comparison between light-mediated and circadian-controlled gene expression is still outstanding. Therefore, we combined and analyzed data from two independent microarray experiments, previously performed under alternating light-dark and continuous light conditions in Cyanothece sp. ATCC 51142, and sought to classify light responsive and circadian controlled genes.
Fourier Score-based methods together with random permutations and False Discovery Rates were used to identify genes with oscillatory expression patterns, and an angular distance based criterion was applied to recognize transient behaviors in gene expression under constant light conditions. Compared to previously reported mathematical approaches, the combination of these methods also facilitated the detection of modified amplitudes and phase-shifts of gene expression. Our analysis showed that the majority of diurnally regulated genes, essentially those genes that are maximally expressed during the middle of the light and dark period, are in fact light responsive. In contrast, most of the circadian controlled genes are up-regulated during the beginning of the dark or subjective dark, and are greatly enriched for genes associated with energy metabolism. Many of the circadian controlled and light responsive genes are found in gene clusters within the Cyanothece sp. ATCC 51142 genome. Interestingly, in addition to cyclic expression patterns with a period of 24 hours, we also found several genes that oscillate with an ultradian period of 12 hours, a novel finding among cyanobacteria.
We demonstrate that a combination of different analytical methods significantly improved the identification of cyclic and transient gene expression in Cyanothece sp. ATCC 51142. Our analyses provide an adaptable and novel analytical tool to study gene expression in a variety of organisms with diurnal, circadian and ultradian behavior.
Temporal periods of light have provided a significant abiotic selection pressure and greatly influenced evolution on earth. Accordingly, diurnal changes in various biological activities have been observed in different organisms, ranging from bacteria to mammals [1–4]. Central for the capacity to anticipate such environmental changes is an internal clock that controls circadian gene expression and orchestrates cell physiology in synchrony with a day and night cycle [5–8].
Cyanobacteria are the only prokaryotes with a circadian clock . These organisms thrive in environments as diverse as fresh and salt waters, glaciers, deserts, and hot springs, where they play a major role in global carbon sequestration and oxygen production. In addition, some cyanobacteria, such as Cyanothece sp. ATCC 51142 (Cyanothece 51142 hereafter) are able to fix atmospheric nitrogen and contribute to a great extent to the marine nitrogen cycle . Cyanothece 51142 is a unicellular cyanobacterium that separates oxygenic photosynthesis and nitrogen fixation temporally, thereby circumventing the irreversible inactivation of the oxygen sensitive nitrogenase enzyme . In Cyanothece 51142, photosynthesis occurs during the day and nitrogen fixation during the night of a diurnal cycle .
Previous work has focused on the identification of different clock components such as kaiA, kaiB and kaiC, and several detailed mathematical models describing the interactions between oscillator components have been published [13–15]. Most of these studies are based on modeling transcriptional and translational feedback loops as well as post-transcriptional modifications of clock genes. In other work, global DNA microarray analysis of Synechocystis sp. PCC 6803, a non-diazotrophic strain, under constant light conditions determined the extent of circadian control at the transcriptional level, leading to the prediction of 2–7% of cyclic genes . In contrast, microarray analyses of Cyanothece 51142 under alternating light and dark conditions revealed that ~30% of the genes in the genome are significantly diurnally regulated , whereas only 10% of these genes are circadian controlled . However, a detailed analytical approach to distinguish between diurnal and circadian regulated genes in Cyanothece 51142 is still outstanding.
Organisms such as Cyanothece 51142 perform different cellular processes at distinct phases of the diurnal cycle and thus depend on tightly regulated expression programs. On the other hand, the capability to adjust to periodic changes in their environment, in particular to alterations of light intensity and quantity, provides an important basis for the ecological success of these organisms. Light is an essential abiotic factor, not only because cyanobacteria obtain their entire energy through photosynthesis, but also for the activation and regulation of many central metabolic processes. In order to understand how Cyanothece 51142 balances gene expression under nitrogen-fixing conditions in consecutive light and dark cycles with altered physiological requirements under constant light conditions, we analyzed data sets from two different microarray experiments, to elucidate details beyond of what has been found in the former studies [16–18].
For this study we developed various advanced mathematical tools to identify rhythmic and transient patterns of gene expression. We demonstrate that a combination of different analytical approaches substantially improves the distinction between transient and cyclic gene expression. Our analysis revealed that a high percentage of the previously observed strongly diurnally regulated genes  are significantly affected under constant light conditions. Interestingly, the majority of circadian controlled genes in Cyanothece 51142 are maximally expressed during the subjective dark and largely comprised of genes related to energy metabolism. Furthermore, we found a significant number of genes that show ultradian rhythms in their expression and oscillate with a period of 12 hours. This study provides a novel and adaptable analytical tool for comparison of data from different microarray experiments to study gene expression in a variety of organisms with diurnal, circadian and ultradian behavior.
We have analyzed two independent Cyanothece 51142 microarray data sets that were performed over two consecutive diurnal periods with a sampling rate of every four hours and a shift in sampling time of one hour between the experiments. The studies were conducted using Agilent http://www.agilent.com custom-made two-channel microarrays. The work of Stöckel et al.  focused on global gene expression in Cyanothece ATCC 51142 under alternating 12 hour light and dark cycles, while Toepel et al.  investigated changes in gene expression under a 12 hour light and 12 hour dark period followed by a constant light period of 24 hours. In both experiments Cyanothece 51142 cells were grown under nitrogen-fixing conditions.
In Stöckel et al.  diurnal regulated genes were classified using a correlation network and a fold-change cutoff of 1.3, in which genes with a period other than 24 hours were excluded, since they did not correlate with the main oscillatory network. In Toepel et al. [17, 18] cyclic genes were identified using a 2-fold cutoff for maximal changes in gene expression. Although the main oscillatory behaviors are detectable using these methods, alterations in gene expression such as changes of amplitudes and phase-shifts were not observed, especially because the altered light conditions were applied only for a short period of time. Therefore we proposed a combination of an angular distance based criteria to characterize such alterations in gene expression.
The two main frequencies of 12 and 24 hours were used for further Fourier score calculations. For the determination of cyclic behavior, a Fourier score cutoff of 6.5 corresponding to a False Discovery Rate of 2% under alternating light and dark conditions and of 3% under continuous light was used. Based on this analysis, 659 genes that oscillate with a period of 24 hours and 9 genes that cycle with a period of 12 hours were identified as common in both microarray datasets. An additional 1551 genes that oscillate with a period of 24 hours and 83 with a period of 12 hours were detected only in the Stöckel et.al data set. All of these genes were used for further analyses (see below).
Average distance from different light and dark periods in each of the four classified gene groups.
Stöckel et al. data set 
Toepel et al. data set 
Based on the Fourier score and angular distance based methods, four major groups of genes could be identified. The genes in group 1 continued to oscillate under continuous light and were classified as circadian controlled genes, whereas genes from group 2 were light responsive and showed oscillating expression only under alternating light and dark cycles. Both groups represent genes that cycle with a 24 hour period. In contrast, genes from group 3 and 4 oscillate with an ultradian period of about 12 hours, with genes from group 3 being circadian controlled and the genes from group 4 light responsive (Table 1, [see Additional files 1, 2]).
Taken together, the combination of the angular distance and Fourier Score based methods lent an additional level of confidence to the identification of cyclic expressed genes in Cyanothece 51142. These analyses uncovered that most of the previously identified diurnal genes are indeed light responsive.
Considering the central role of photosynthesis in the cellular metabolism of Cyanothece 51142 and other cyanobacteria, a great impact of light on gene expression was anticipated. We found that the expression of the majority of regulatory genes is light responsive, presumably the result of comprehensive changes that take place in order to adjust to altered light conditions [see Additional file 1]. In addition, the expression of many genes related to transcription and translation is light regulated and likely accounts for a higher turnover of proteins under continuous light. Our analyses also revealed that several genes associated with photosynthesis, especially with photosystem II (PSII) such as psbE, psbF, and psbJ are circadian controlled [see Additional file 1]. The PsbJ protein in Synechocystis sp. PCC 6803 has been shown to control the amount of functionally assembled PSII complexes in the thylakoid membrane . Moreover, psbE and psbF which encode the α and β subunits of cytochrome b559 are required to stabilize the reaction center of PSII . Interestingly, a number of subunits of the ATP-synthase complex, including AtpF and AtpH, which are encoded by multicopy genes revealed that one gene copy is light responsive while the other gene copy was found to be circadian controlled [see Additional file 1]. The gene atpH has been identified as clock controlled in previous studies of Synechocystis sp. PCC 6803 .
A more detailed analysis of different metabolic processes revealed that nitrate and sulfate assimilation are largely under circadian control [see Additional file 1]. Furthermore, the genes hoxEFUYH encoding the bidirectional hydrogenase show a significant cyclic behavior in their expression [see Additional files 3ab. In comparison, a recent microarray analysis performed in Cyanothece 51142 over 24 hours under alternating 6 hours light/dark conditions  led the authors to the conclusion that the expression of the hox genes under their conditions are not circadian. However, an alternative interpretation of their data (Figure 3B in ) suggests that the hoxE gene does in fact show an oscillatory expression with a period of 24 hours, which would support our findings. This is also supported by previous studies in Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 which uncovered a strong circadian component in the expression of different hox-genes [5, 22]. In total, our study revealed that 9 out of 17 genes involved in hydrogen metabolism in Cyanothece 51142 were expressed at the same level during the dark and subjective dark and therefore classified as circadian controlled. In contrast, the expression of hupS and hupL encoding subunits of the uptake hydrogenase are severely affected during the subjective dark, with hupS showing an 8-time lower expression level (see Additional files 3C,D). Earlier studies of different Nostoc species uncovered that the expression of hupL is substantially stimulated by supplemented hydrogen [23, 24]. Accordingly, the reduced transcript abundance of hupS and hupL is presumably coupled to a decline in the activity of the nitrogenase enzyme, which has been observed during the subjective dark period under continuous light conditions . Furthermore, even though the genes involved in nitrogen fixation are organized in a single cluster consisting of two adjacent regulons on opposite strands in the genome , different expression profiles for many of those genes were observed under continuous light. The genes nifB, nifS, nifE, nifN, nifX and nifW, which are involved in biogenesis and assembly of the Mo-Fe cofactor, revealed a more than two fold increase in their expression levels  in addition to a shift in phase of about four hours under continuous light conditions [see Additional files 4ab vs. 4cd].
In fact, several light responsive and circadian controlled genes are found in clusters of three or more genes throughout the Cyanothece 51142 genome [see Additional files 1, 5 and 6]. Such clustering of similarly regulated genes into different regions of the genome might provide an opportunity for the cell to control the gene expression more efficiently. On the other hand, additional diurnal and circadian controlled genes can be identified which might not have been detected due to low amplitudes of oscillations. Thus, there is a high probability that an unclassified gene within a group of genes assigned to one of the two categories belongs to the same class.
In this work we compared two independent microarray experiments to study changes in gene expression under alternating and constant light conditions in Cyanothece 51142. Two mathematical approaches were applied to differentiate transient behaviors from cyclic gene expression. Fourier score based methods can successfully be used for identification of cyclic behaviors but they fail to detect some transient behaviors such as shifts of the expression peaks or smaller amplitudes under constant light conditions. Therefore, an angular distance based method was applied which improved the detection of such changes. These methods are valid for distinguishing transient from cyclic behaviors even for the limited number of time points in one of the different light treatments. This approach provides an adaptable tool for future studies of diurnal, circadian and ultradian rhythms in various organisms. Furthermore, the combined analysis of two different microarray data sets enabled the extraction of novel details about diurnal and circadian regulation in Cyanothece 51142 which would not be obtained from the individual experiments alone.
For the Stöckel et al. microarray data set , Cyanothece 51142 cultures were grown in ASP2 medium without NaNO3  at 30°C with air bubbling in alternating 12 hour light and dark cycles with 50 μmol photons/m2*s of white light. After 7 days, 150 mL samples were collected every 4 hours for 2 days, starting with 1 hour into the dark period (time point D1). In total, 12 samples were collected.
For the Toepel et al. data set , Cyanothece 51142 cultures were grown in an airlift bioreactor (6L BioFlo 3000; New Brunswick Scientific, Edison, NJ) in ASP2 medium without NO3 at 30°C in 12 hour light and 12 hour dark cycles. The culture was illuminated by two light-emitting-diode panels using orange (640 nm) and red (680 nm) light, yielding an intensity of 100 μmol photons/m2*s inside the bioreactor. The cultures were grown for 5–6 days under alternating light and dark conditions prior to collecting samples for every 4 hours over a time period of 2 days, starting at the transition from dark to light (time point L0) for 24 hours followed by 24 hours of continuous light.
In previous studies [16–18], the data were processed and analyzed differently, which made a direct comparison of the results difficult. Therefore, in this communication both data sets have been combined and analyzed uniformly. The raw data were normalized using the LOWESS normalization algorithm to avoid a systematic intensity based bias which is commonly observed with two-channel microarrays  and preliminary data processing of both data sets was performed according to .
where ω = 2πf.
Initial Fast Fourier Transform calculations indicated existence of oscillations with 24 h and 12 h periods. Therefore Fourier score calculations were performed using those frequencies.
If a given signal consists of a dominant cyclic component of the corresponding frequency, a larger Fourier score is expected. The significance of the Fourier score was quantified by comparing the Fourier score of the original signal with scores of large collection of random signals. These random signals were obtained by using different permutations of the original signal. The original gene expressions were scaled to have a unit standard deviation, which enabled direct comparisons of Fourier scores from different genes.
where x1 and x2 are the vectors from two different 12 hour periods. D1,2 can have any value between 0 and 2, with 0 representing vectors with the same direction and 2 representing vectors with the opposite direction.
We thank Wenxue Wang and all members in the Pakrasi Lab for collegial discussions. This work is part of a Membrane Biology EMSL Scientific Grand Challenge project at the W. R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research (BER) program located at Pacific Northwest National Laboratory. PNNL is operated or the Department of Energy by Battelle. The project is also partially supported by the National Science Foundation FIBR program under grant number 0425749 as well as the DOE-BES program.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.