Diel Regulation of Metabolic Functions of a Western Lake Erie Microcystis Bloom Informed by Metatranscriptomic Analysis

Home , Exodeoxyribonuclease I, FMN adenylyltransferase, Histidinol dehydrogenase, NdhF, Polyketide synthase, Ribonuclease Z

DIEL REGULATION OF METABOLIC FUNCTIONS OF A WESTERN LAKE ERIE MICROCYSTIS BLOOM INFORMED BY METATRANSCRIPTOMIC ANALYSIS

Emily J. Davenport

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

December 2016

Committee:

Robert Michael McKay, Advisor

George Bullerjahn

Paul Morris

Emily Davenport

Robert Michael L. McKay, Advisor

Despite the global prevalence and apparent increased frequency of freshwater cyanobacterial harmful algal blooms (cHABs), metabolic functions that promote cHAB formation and maintenance remain poorly understood. An opportunity to elucidate cellular responses over diel cycles was presented following the 2014 Toledo (OH, USA) water crisis as part of a 48-h Lagrangian survey following toxin-producing bloom biomass in western Lake

Erie. Taxonomic analysis of samples collected from 1 m depth showed the bloom to be dominated by three major phyla: Cyanobacteria (~30%), Proteobacteria (~30%), and

Bacteroidetes (~20%). Differential expression analysis of Microcystis reads revealed strong diel patterns of expression between day (10:00 h, 16:00 h) and night (22:00 h, 04:00 h) samples. Day transcripts were abundant in functions related to photosynthesis (e.g. psbA, cpcB), nutrient and energy metabolism (glnA, nirA), cell division (ftsH), heat shock response (dnaK, clpB), and nutrient and ion transporters (bicA, acrB). Night transcripts included microcystin synthesis, amino acid synthesis (carB), and pre-dawn expression of photosynthetic antennae (chlI, cbiE).

Hierarchical clustering and PCA showed a uniform grouping of expressed genes at night, whereas day transcripts were separated over the 48-h duration. Lack of uniform clustering of day transcripts suggests that the functional partitioning of gene expression during a

Microcystis bloom is not limited to circadian regulation, but also to physico-chemical changes within the environment.

I would like to dedicate this thesis to girls everywhere, you can do anything. v ACKNOWLEDGMENTS

I would like to thank Dr. Mike McKay and Dr. George Bullerjahn for inviting me into their labs, for their countless guidance, and for calmly meeting all of my crises and slowly talking me off of the ledge. Dr. Paul Morris, thank you for your input on my committee. Thanks to Dr. Tim Davis and NOAA, Taylor Tuttle, and Joshua Stough for all of their help with my methods, and friendly words of advice. To Dr. Verner Bingman, your loud greetings always found me on days I needed it most. To Dr. Hans Wildschutte and Dr. Julia Halo Wildschutte, thank you for believing in me when I doubted myself, and all of the happy hours.

My family has been very gracious to me the last two years. Thank you to my parents,

Esperanza and Jordan Davenport, for everything. Adam and Alex, thank you letting me decompress in the mountains, to Janie, for constantly bankrolling me, and Jim, who loves from a distance. To my Grandpa Juan, thank you for keeping me in your prayers. Grandma Jane, thank you for Bloody Mary Sundays.

I am beyond thankful for my friends. My Boys have provided so much comic relief, and in the direst of times, kindness, in the simple form of a group chat. To my wonderful friend

Allyse, thank you for your constant emotional availability. You have been at every one of my ups and downs and helped me get through it all with a grace I could never have found myself.

You are such a Good light. To Mandy, 3,800 miles away, yet never more than a few hours away from a text or call, thank you for truly being my soul mate. To Betty and K Dawg, thank you for being my best friends. All of the late nights together, Roomie Weekends, everything, made life perfect. I could not have asked for two better friends to be by my side. To Jet and Mikayla, sorry for all of the early mornings, and Baby Bond, thank you for letting me borrow your room. And to

Yvonne and Tony for opening their home to me.

Lastly, to Andrew Jilwan, thank you for loving me, thank you for being there. vi

TABLE OF CONTENTS

Page

CHAPTER I. INTRODUCTION………………………………………………………...... 1

1.1 Rationale of study ...... 1

1.2 Cyanobacteria ...... 1

1.3 Microcystis Aeruginosa ...... 2

1.4 Cyanotoxins: Microcystin ...... 3

1.5 Lake Erie and eutrophication ...... 6

1.6 Rationale for circadian regulation ...... 8

CHAPTER II. MATERIALS AND METHODS……………………… ...... 11

2.1 Sample collection ...... 11

2.2 Physico-chemical measurements ...... 12

2.3 Microcystin measurement ...... 13

2.4 Nucleic acid extraction and sequencing ...... 14

2.5 Bioinformatics and statistical analysis ...... 15

CHAPTER III. RESULTS………………………...... 17

3.1 Lake physico-chemical properties ...... 17

3.2 Community composition of bloom ...... 19

3.3 Metatranscriptome analysis ...... 20

3.4 Differential expression in Microcystis spp. transcripts ...... 21

3.4.1 Day group vs. night group ...... 21

3.4.2 Morning vs. afternoon ...... 24

3.4.3 Night vs. pre-dawn ...... 28 vii

CHAPTER IV. DISCUSSION ...... 31

4.1 Photosynthesis ...... 32

4.2 Biosynthesis of amino acids and proteins ...... 33

4.3 Nutrient metabolism ...... 33

4.4 Heat shock, stress response, gas vesicle, replication & division ...... 35

4.5 Toxin synthesis ...... 36

CHAPTER V. CONCLUSION……………………………...... 38

REFERENCES……………………………………………………………………………… 39

APPENDIX A ……………………………………… ...... 55

APPENDIX B …………………………………………… ...... 61

APPENDIX C ……………………………………………… ...... 67

APPENDIX D ……………………………………………… ...... ……… 86

APPENDIX E ……………………………………………… ...... ……… 91

viii

LIST OF FIGURES

Figure Page

1 General structure of microcystin ...... 5

2 Mcy operon in Microcystis spp...... 5

3 Satellite image of the western basin Lake Erie cyanobacterial harmful algal bloom

on August 3rd, 2014 ...... 12

4 Physico-chemical water data ...... 18

5 Map of Maumee Bay, western basin of Lake Erie indicating movements of bloom

during 48-hour survey ...... 19

6 Taxonomic analysis of bloom community ...... 20

7 Principal Component Analysis of Microcystis reads ...... 22

8 Heat map visualization of differential expression between Night group and

Day group ...... 24

9 Heat map visualization of differential expression between Morning samples

and Afternoon samples ...... 25

10 Heat map visualization of differential expression between Night samples

and pre-dawn sample ...... 29

CHAPTER I. INTRODUCTION

1.1 Rationale of study

The Laurentian Great Lakes are one of the largest sources of freshwater in the world, and hold the vast majority of surface freshwater in North America. Lake Erie is not only a source of potable water for millions of residents, but is also of high economic importance due to fishing and recreation. Within Ohio, cyanobacterial harmful algal blooms (cHABs) are responsible for millions in annual losses (tourism, property value, water treatment; Bingham et al., 2015).

Toxin-producing cHABs have been recurring annually in Lake Erie since the mid-1990’s, with increasing severity and duration (Michalak et al., 2013; Bullerjahn et al. 2016). The cyanobacterium Microcystis aeruginosa is capable of forming massive blooms and producing microcystin, a known hepatotoxin and potential carcinogen (Falconer, 1994; Fan et al., 2014).

The mechanisms underlying its production largely remains a mystery, but given the human health considerations, properly characterizing bloom events is warranted. This study facilitated observation of key metabolic functions to better understand the ecophysiology of an M. aeruginosa bloom over the course of a diel cycle. Specifically, a metatranscriptomic analysis was undertaken to study temporal changes in the metabolic functions of an M. aeruginosa bloom. Metatranscriptome analysis paired with environmental metadata should provide insights into factors related to bloom success as well as it’s toxicity. A better understanding of bloom metabolism will guide new strategies toward bloom mitigation and ultimately prevention.

1.2 Cyanobacteria

Cyanobacteria (blue-green algae) are some of the oldest organisms on Earth with the earliest occurrence of cells found in the fossil record in stromatolites dating 3.5 billion years old (Schopf, 2

1993). Given the long evolutionary history, the phylum contains physiologically diverse species that have successfully adapted to virtually all marine, freshwater, and terrestrial environments, even in extreme environments such as hot springs, hypersaline lakes, and deserts (Bullerjahn and

Post, 2014). While some species are capable of non-photosynthetic growth in prolonged-dark conditions via heterotrophy or chemolithotrophy (Fay, 1965; Rippka, 1972), the majority of cyanobacteria are reliant on oxygenic photosynthesis, and through this process yielded the formation of our current oxygen-rich atmosphere. Hence, evolution of aerobic life forms can be attributed to the cyanobacteria. The cyanobacteria have developed highly efficient strategies for nutrient (C, N, P) acquisition and fixation (Price et al., 1998; McKay et al., 1993; Fay, 1992,;

Flores and Herrero, 2005; Moutin et al., 2002). These adaptations allow for cyanobacteria to succeed in a wide range of environments.

1.3 Microcystis aeruginosa

Microcystis aeruginosa is a ubiquitous freshwater colony-forming species, notable for bloom formation during the summer months in temperate regions. M. aeruginosa can dominate the phytoplankton community due to a variety of adaptive strategies. In temperate freshwater systems, M. aeruginosa may over-winter in the sediments from where they can be recruited to surface waters during the spring and summer as light availability and temperatures increase

(Reynolds, 1973; Reynolds and Rogers, 1976; Reynolds et al., 1981; Brunberg and Blomqvist,

2003; Verspagen et al., 2005). Vertical recruitment into the water column is due to the presence of gas vesicles. Buoyancy from the vesicles allows cells to control light (Reynolds et al., 1987) and nutrient (Brookes and Ganf, 2001) exposure, and form surface scums as the population growth increases. The genome of M. aeruginosa is plastic due to horizontal gene transfer, transposase, and restriction modification enzymes within their genomes (Franguel et al., 2008; 3

Steffen et al., 2014). These generate diversity within the genomes of the cyanobacterial community via deletion, duplication events and incorporation of genes into the genome as an adaptive strategy proposed to maintain competitive dominance (Humbert et al., 2013). M. aeruginosa harbor a variety of nutrient uptake genes and pathways to compete for light and nutrients within the microbial community, such as maintaining uptake systems for various nitrogen (urea, ammonium) and carbon (carbon concentrating mechanisms, bicarbonate) sources

(Valladares et al., 2002; Badger and Price, 2003). M. aeruginosa are able to out-compete the microbial community for required nutrients by assimilating a broad array of N sources including ammonium and urea (Donald et al., 2011). The carbon uptake pathways and concentrating mechanisms of M. aeruginosa also promote dominance in freshwater systems, which is expected to increase occurrences of cHAB events as atmospheric levels of CO2 increase due to the increased availability of carbon species and the ability of toxin-producing strains to outcompete non-toxic strains for CO2 (Verspagen et al., 2014; Van de Waal at al., 2011). Increasing temperatures are also favorable for growth of M. aeruginosa, whose optimum growth temperature (>25°C) is higher than that of other phytoplankton species (Reynolds, 2006; Jöhnk, et al., 2008). The increasing temperatures will likely strengthen vertical stratification thereby reducing mixing and allowing phytoplankton growth at the surface to remain undisturbed and form surface blooms (Huisman et al., 2005; Paerl et al. 2006).

1.4 Cyanotoxins: Microcystin

Many cyanobacteria species are capable of producing various non-ribosomally synthesized secondary metabolites classified as toxins due to the potential harmful effects on higher organisms. Microcystis is known for producing the hepatotoxin microcystin (Figure 1;

Carmichael, 1992; Sivonen and Jones, 1999). The structure of this heptapeptide contains D- 4

amino acids at positions one and six, and a D-erythro-β-methylaspartic acid at position three, variable amino acids at positions two and four, a unique β-amino acid, Adda, at position five, and an N-methyldehydroalanine at position seven (Rinehart et al., 1994; Puddick et al, 2014).

This metabolite is synthesized via the expression of a bi-directional mcy operon (Figure 2) using a central promoter between mcyD and mcyA (Tillet et al., 2000). Over 100 congeners of microcystins have been reported due to the variation in amino acids found at positions two and four, with some congeners having greater toxicity than others (Rinehart et al., 1994). Acute toxigenic effects of microcystins, initially called Fast-Death Factor (Bishop et al., 1959), are due to inhibition of protein phosphatase types 1 and 2 in hepatocytes. Microcystins are also characterized as tumor-promoters and thus possess chronic toxicity also (reviewed by Falconer and Humpage, 1996; Dietrich and Hoeger, 2005). Ohio Environmental Protection Agency guidelines suggest a Do Not Drink advisory at microcystin concentrations of 0.3 µg/L to children under 6 years of age and sensitive persons (pregnant, nursing, elderly, immune-compromised, etc.), and at concentrations of 1.6 µg/L for children older than 6 years and adults. Recreationally, a suggested Public Health Advisory to avoid contact is placed at concentrations of 6.0 µg/L, when concentrations reach 20 µg/L, it is suggested to avoid all contact with contaminated waters

(Environmental Protection Agency, 2016). 5

Figure 1. Reproduced from Tillet et al. 2000. General structure of microcystin. Variable amino acids denoted at positions X and Y.

Figure 2. Reproduced from Tillet et al. 2000. Mcy operon in Microcystis spp. Genes encoding nonribosomal peptide synthetases are indicated in dark blue, polyketide synthese genes in light blue. Putative tailoring fuction genes are indicated in black, and non- microcystin synthetase genes are shown in white.

Physiological functions and environmental stimuli for the production of toxins have yet to be identified. Research has studied the impact of factors such as temperature, light intensity, nutrient stress and species, defense mechanism against grazers, intraspecies signaling/ quorum sensing (Pineda-Mendoza et al., 2016; Harke et al., 2015b), but many studies have reported ambiguous and contradictory findings (Wood et al., 2011; Pearson et al., 2010). Genome 6 sequencing has shown toxic and non-toxin producing strains (genomes containing an incomplete mcy operon or none at all) of Microcystis spp. co-inhabit systems with seasonal succession of genotypes observed, though the factors influencing the shift in genotypes remain unclear

(Kardinaal, et al., 2007a; Kardinaal, et al., 2007b; Davis, et al., 2009). M. aeruginosa strains appear to be highly specific to their environments, indicating that influences of proliferation and toxin production will differ between systems (Pineda-Mendoza et al., 2016).

1.5 Lake Erie and eutrophication

Lake Erie is the smallest of the Great Lakes, and is divided into three separate basins. The western basin is the shallowest, with a maximum depth of 20.7 m. The eastern basin is the deepest reaching depths of >60 m (Lake Erie LAMP, 2011) and, along with the central basin

(~25 m), experience stratification from spring-fall. The shallow western basin is considered polymictic, meaning it is continuously mixed during the ice-free season and does not stratify. It is also more susceptible to eutrophication due to its shallow bathymetry and elevated nutrient loads from riverine inputs and sediment re-suspension (Schertzer et al., 1987). The western basin receives water from the Maumee River, which drains a watershed dominated by high agricultural production. As a result, high nutrient loading occurs in Lake Erie’s western basin. Globally, phosphorus (P) inputs have increased through fertilizer use, detergents, industrial waste, increased land clearing for urbanization, forestry, agriculture, and livestock farms (Vitousek, et al., 1997a, 1997b). Many of these vectors are also applicable to Lake Erie. Eutrophication occurs with the transport of nutrients from these sources. As P and/or nitrogen (N) accumulate in soils, they may be transported from land into surface waters, move into groundwater, or enter the atmosphere. Point sources of nutrient inputs can be localized, and are easily regulated. Such an example would be municipal and industrial wastewater effluent. Discharge and nutrient 7 concentrations can be measured regularly and controlled at the source, making point sources the lesser input of importance (National Research Council, 2000). Non-point sources disperse nutrients into the environment over large areas of land, through groundwater, or atmospherically, and are not easily recorded or managed. Run-off from agriculture, pastures, irrigation, septic systems, urbanized areas and atmospheric deposition over a water surface are common non-point sources of nutrient input (Carpenter, et al., 1998). These sources tend to vary seasonally due to agricultural practices and weather conditions. Large precipitation events during the spring and early summer accelerate loading of N and P into lakes (Michalak et al., 2013).

The acceleration of eutrophication in waters has been linked to the global increase in harmful algal bloom (HAB) formation (Anderson, et al., 2002). With respect to our study in

Lake Erie, the dominant bloom formers are cyanobacteria (Microcystis spp. and Planktothrix spp.), both of which produce the potent liver toxin microcystin. The flux in nutrient inputs increases the amount of essential macronutrients available which creates an environment favorable for high rates of primary production (Anderson, 2002). The classical remediation strategy to limit eutrophication is to target P inputs (Schindler, 1971; Schindler, 1977). Due to the absence of a natural P cycle, it is hypothesized that targeting reductions of P over time will gradually deplete the excess P within the water and sediments, allowing ecosystems to recover within a period of years or decades (Schindler and Hecky, 2009). The Great Lakes Water Quality

Agreement of 1972 required reductions in P inputs into Lake Erie, and consequently saw a reduction of cHABs in the lake. In the 1990s, algal blooms returned to Lake Erie despite nutrient metrics indicating the lake remained P-limited. Given the ability of some cyanobacteria to counter N limitations through nitrogen fixation, P load reductions have been the primary focus in effort to control eutrophication in freshwater systems (Schindler, 1977). Present research 8 promotes dual N/P reduction due to recent research indicating that increases in biomass occur in response to both N and P (Conley, et al., 2009).

There are many other factors acting in a system that contribute to HAB formation such as wind mixing and flushing, light availability, temperature, weather patterns and the heterotrophic microbial community within a bloom (Canfield and Bachmann, 1981;Paerl and Huisman, 2008;

Steffen et al., 2012). The combination of high nutrient inputs, warm temperatures, and the morphology of the western basin of Lake Erie allow for increased primary production leading to eutrophic conditions. A shift to total cyanobacterial dominance is considered the final phase of eutrophication (Smith et al., 2006; Watson et al., 1997).

1.6 Rationale for circadian regulation

Circadian oscillators are genetic regulators of expression operating at a period of about

24 hours. The circadian ‘clock’ functions as a way to temporally regulate and anticipate daily environmental changes that effect cell metabolism. Circadian rhythms function as a constant, entrained by cycles of light/dark, independent of temperature effects (Bünning, 1973; Sweeney,

1987; Johnson and Hastings, 1986; Pittendrigh, 1981). These functions had been observed and thus thought to occur only in eukaryotes (Konopka and Benzer, 1971) until recent discovery in the prokaryotic cyanobacteria, and now are known to be universal to cyanobacteria and most organisms (Bünning, 1973; Kondo et al.,1993; Lorne et al., 2000). The discovery of the kaiABC gene cluster within cyanobacteria has been shown to specifically control the rhythmicity of cell functions (Ishiura et al., 1998). The photosynthetic nature of cyanobacteria presumes the circadian pacemaker will initiate expression of some genes to anticipate presumptive dawn in order to maximize a daytime function, such as photosynthetic light harvesting. Several physiological functions have been found to cycle in sync with typical circadian regulation such 9 as peak expression at subjective midday (Kucho et al., 2005). Data from Liu et al. shows that the cyanobacteria clock controls global expression via regulating the activity of all promoters (Liu et al.,1995; Xu et al., 2003; Labiosa et al., 2006).

Several cellular functions within cyanobacteria have been found to be coupled to circadian rhythms, such as nutrient acquisition and assimilation, amino acid uptake, respiration, carbohydrate synthesis, replication and cellular division (Ishiura et al., 1998; Golden et al., 1997;

Chen et al., 1991; Kramer et al., 1996). The evidence of global transcription regulation via clock genes requires that interpretation of gene expression data be viewed with caution. The intent of this thesis is to not detect mechanisms of circadian control, but to identify any patterns of diel variability that may exist based on the evidence of previous diel bloom studies and the knowledge of expression regulation within strains studied in vitro. Through this study and subsequent efforts, expression patterns of circadian-controlled genes vs. environmentally regulated genes can ultimately be differentiated.

In certain species, the circadian clock is used as a strategy to allow for conflicting processes to occur within the cell as efficiently as possible. Such examples are described in nitrogen-fixing non-heterocystous species of cyanobacteria. These utilize temporal variation in expression (in lieu of heterocysts) as a strategy to maintain efficiency in photosynthesis in concert with the oxygen-labile nitrogen-fixation process (Mitsui et al., 1986; Shi et al., 2010;

Kumar et al., 2010). While fluctuations in environmental conditions (light, temperature, nutrient availability) may invoke stress responses, it is important to understand the mechanisms and range of circadian control that may mask or overlay expression resulting from transient stress. What diel patterns of expression are observed may be consequences of clock genes regulating, for example, nutrient acquisition genes not from nutrient demand but by time of day. 10

Studies that measure gene expression of stress responses under changing conditions (light, temp, nutrient starvation) do not interpret the natural responses of cyanobacteria to daily fluctuations within the environment. In respect to cHAB events, it is critical to understand the in situ, or at least, natural responses of cyanobacteria to daily fluctuations (Labiosa et al., 2006; Penn et al.,

2014).

CHAPTER II. MATERIALS AND METHODS

2.1 Sample collection

In late August of 2014, a 48-hour Lagrangian survey of a harmful cyanobacterial algal bloom was conducted in the western basin of Lake Erie. Three weeks prior to sampling, this bloom reached the Toledo, OH (USA) municipal water intake, causing unsafe levels of microcystins to enter the Toledo water supply (Figure 3). Therefore, this study intended to track the bloom relative to the Toledo water intake. This was done by deploying a buoy with GPS capabilities (coordinates received hourly) offshore from the intake crib to prevent projected winds from pushing the buoy onto the intake structure. The bloom was tracked ~1-3km offshore, with depths varying from 10-15m. Samples were to be collected at 6 h intervals beginning on the

26th of August at 22:00 h, producing two sets of triplicate samples from 22:00 h (1S, 5S), 04:00 h (2S), 10:00 h (3S, 6S), and 16:00 h (4S, 7S). At each sampling time point, triplicate water samples were collected with a Niskin bottle from a depth of 1m and a second depth of 5m.

Biomass from each water sample was collected onto Sterivex cartridge filters (0.22 µm; EMD

Millipore, Billerica MA) using a peristaltic pump. Filters were immediately stored in liquid nitrogen upon collection followed by transfer to -80 until extraction. 12

Figure 3. Photo courtesy of NASA. Satellite image of the western basin Lake Erie cyanobacterial harmful algal bloom on August 3rd, 2014.

2.2 Physico-chemical measurements

Chlorophyll-a (Chl a) biomass was measured by concentrating lake water on a glass fiber filter (Whatman GF/F, 47mm diameter) using low vacuum pressure. Samples were extracted with N, N-dimethylformamide under low light levels and analyzed with a 10AU fluorometer

(Turner Designs, Sunnyvale CA; Speziale et al., 1984).

Phycocyanin concentration was measured by concentrating lake water on a glass fiber filter (Whatman GF/F, 47mm diameter). Phosphate buffer (Ricca Chemical, pH 6.8) was added to the filter and phycocyanin was extracted using two freeze-thaw cycles followed by sonication.

Relative fluorescence was measured using a Turner Aquafluor and converted 13 to phycocyanin concentration using a series of dilutions of a commercial standard (Sigma-

Alderich; Horváth et al, 2013).

For total phosphorus samples, duplicate 50 mL aliquots of whole lake water were collected into acid-washed glass culture tubes and stored at 4 °C until analysis within one week. For dissolved nutrients, duplicate whole water samples were collected in a triple rinsed

(ultrapure water) 20 mL syringe and filtered through a 0.22 µm nylon filterinto a 15 mL collection tube and stored at -20 °C until analysis. Nutrient concentrations were determined using standard automated colorimetric procedures (APHA 1998) as modified by Davis and

Simmons (1979) on a Seal QuAAtro (SEAL Analytical Inc., Mequon, WI, USA ) according to methods detailed by the manufacturer and in compliance with EPA Methods 365.4, 350.1, and

353.1. NH4 was determined by the Bethelot reaction in which ammonium ions react with salicylate and free chlorine to form a blue-green colored complex. NO3+NO2 was determined by the cadmium reduction method whereby nitrate is reduced to nitrite in a cadmium column and then reacted under acidic conditions to form a reddish-purple azo dye. Soluble reactive phosphorus (SRP) was determined by the molybdate/ascorbic acid method based on the formation of the blue phosphor-molybdenum complex. Total phosphorus (TP) and total dissolved phosphorus (TDP) used the same analysis following a persulfate digestion adapted from Menzel and Corwin (1965). SiO2 was determined by the reduction of a silico molybdate in an ascorbic acid solution to molybdenum blue.

2.3 Microcystin measurement

Particulate microcystins (MCs) were measured by filtering whole lake water onto a 3 µm polycarbonate membrane and kept at -20 ºC until analysis. Particulate MCs were extracted from samples using a combination of physical and chemical lysis techniques. All samples were 14 resuspended in 1 mL molecular grade water (pH 7; Sigma-Aldrich, St. Louis, MO) and subjected to three freeze/thaw cycles before the addition of the QuikLyse reagents (Abraxis LLC;

Warminster, PA) as per the manufacturer’s instructions. The samples were then centrifuged for 5 min at 2 ×103 g to pellet cellular debris. The concentrations of microcystins (reported as microcystin-LR equivalents) were measured using a microcystin enzyme-linked immunosorbent assay (Abraxis LLC) following the methodologies of (Fischer et al., 2001). This assay is a congener-independent as it detects the ADDA moiety, which is found in almost all MCs. These analyses yielded a detection limit of 0.04 µg L-1.

2.4 Nucleic acid extraction and sequencing

DNA was extracted with the MO BIO Laboratories PowerWater® Sterivex DNA

Isolation Kit (MoBio, Carlsbad, CA, USA) according to manufacturer’s protocol. DNA concentrations and quality were analyzed using internal QA/QC NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA was extracted using the

MO BIO PowerWater® DNA Isolation Kit for Sterivex, modified for RNA using manufacturer’s protocols. Sterivex cartridges were vortexed for 5 min longer than recommended each time and all wash buffers were allowed to sit for one minute before being pulled through the binding column using a vacuum manifold. DNase treatment was performed as recommended in the protocol using the MO BIO On-Spin Column DNase kit. This protocol was optimized by allowing the DNase solution to sit for an extra 15 min than recommended. RNA was checked for

DNA contamination using universal 16S primers (27F and 1522R). Any additional DNase treatments needed were performed using Turbo DNase kit (Ambion, Austin, TX, USA). RNA was stored at -80 ºC until shipped for sequencing. DNA/RNA extractions were sent to the

Department of Energy Joint Genome Institute (DOE-JGI; Walnut Creek, CA) for secondary 15 quality filtering and sequencing at the DOE-JGI using Illumina HiSeq 2500 (Walnut Creek, CA) technologies (Mavromatis et al., 2009). Metatranscriptomes were accessed and downloaded through the Integrated Microbial Genomes platform (IMG) developed by JGI (Markowitz et al.,

2012; Markowitz et al., 2014).

2.5 Bioinformatics and statistical analysis

Assembly, analysis and visualization of data was performed using CLC Genomics

Workbench version 9.0 (CLC Bio, Cambridge, MA). Sequences were imported and screened for failed reads using default settings quality scores. Transcriptome sequences were assembled de novo into contigs with minimum contig length of 350 bp. Reads were mapped back to contigs using default parameters to determine how contigs related to small reads. To identify community function, consensus sequences from the mapped reads were then assembled to identify CDS regions using the Microbial Genomics Module and MetaGeneMark. Identified CDS regions were then annotated with Pfam Domains and GO Terms to build a functional profile of each sample.

Functional profiles portrayed community functions in GO Terms.

For specific analysis of Microcystis function, a custom BLAST database containing three

Lake Erie-specific Microcystis spp. genomes was used against consensus reads from each transcriptome using genetic code 11 (Bacterial, Archeal, and Plant Plastid) with default BLAST parameters. Reads mapped to the genomes were extracted using default parameters. Expression values of M. aeruginosa reads were analyzed using the Advanced RNA-Seq plugin. RNA-Seq analysis of reads were normalized and expression values measured as Reads Per Kilobase of transcript per Million mapped reads (RPKM) using default parameters and mapped to nucleotide gene sequences of three lower Great Lakes Microcystis spp. genomes. 16

Statistical analyses were performed using the Advanced RNA-Seq module. Principal

Component Analysis (PCA) was performed to assess sample relations with regards to expression and environmental data. Differential expression of genes was determined using a Generalized

Linear Model (GLM) which does not assume normal distribution (assumes read counts follow a

Negative Binomial distribution). Differential expression first grouped samples between Night

(1S, 2S, 5S) and Day (3S, 4S, 6S 7S). Expression values followed default parameters, with minimum fold change of expression set to |2|, p-value of 0.05, and following the statistics generated from the Day vs. Night differential expression. The same procedure was used to determine differential expression between the two day periods (3S, 6S vs. 4S, 7S), night periods

(1S, 5S vs. 2S), and transition between late afternoon and dark period (4S, 7S vs. 1S, 5S). Heat maps of each analysis were produced within the module to visualize results.

CHAPTER III. RESULTS

3.1 Lake physico-chemical properties

The bloom tracked a southwesterly course travelling nearly 2 km over the 2-day late

August survey (Figure 4). Whereas winds originated from S/SW leading up to buoy deployment and initial bloom tracking, wind direction switched in the early morning of August 27 and remained E/NE at 4-6 knots for the remainder of the survey (data from KPCW: Erie-Ottawa

International Airport; KTDZ: Toledo Executive Airport). Increases in Chl a biomass of 75% and more than a doubling of phycocyanin (PC) indicate the bloom was growing over the two day survey. Molar ratios of dissolved inorganic nitrogen to dissolved inorganic phosphorus

(DIN:DIP) showed only minor variation around Redfield stoichiometry (N:P = 16:1; dashed line) during the first 18 h of sampling. The system then shifted to P-deficient during day 2 of the survey. Particulate microcystin, while initially low (1-2 µg L-1), reached as high as 5 µg L-1 toward the end of the survey (Figure 5). Although light data were not collected during the 48-h period, archived weather reports from KPCW and KTDZ confirmed skies to be clear leading up to the start of the survey and switching to overcast for the remainder of the survey (Appendix E). 18

Figure 4. Physico-chemical water data.

Figure 5. Map of Maumee Bay, western basin of Lake Erie indicating movements of bloom during 48-hour survey. Bloom was tracked ~2 km. Inset shows bloom three weeks prior to sampling (photo courtesy of NASA).

3.2 Community composition of bloom

Taxonomic analysis derived from MG-RAST (M5NR) reported the domains Bacteria

(~70% of all reads), Eukaryota (~30%), Viridae and Archaea (<1%). Of the Bacteria reads,

Cyanobacteria (22-35%), Proteobacteria (16-37%), and Bacteroidetes (17-40%) were equally abundant in each metatranscriptomes (Figure 6a). The Bacteroidetes (Figure 6d) were dominated by the classes Cytophagia (~30% of Bacteroidetes reads), Flavobacteriia (~30%), and

Sphingobacteria (~25%), whereas β-Proteobacteria were most abundant of Proteobacterial reads (~40%) followed by α-Proteobacteria (~30%), γ-Proteobacteria (~15%), and δ-

Proteobacteria (~12%; Figure 6b). Of the Cyanobacteria, the order Chrooccocales dominated read counts, making up ~65% of reads. Microcystis spp. was the dominant genus, contributing to half of the Chrooccocales population. Classes Nostocales and Oscillatoriales contributed to a third of the Cyanobacteria, namely genera Nostoc and Anabaena within the Nostocales (Figure 20

6c). Of eukaryotes, phylum Arthropoda dominated, and was mainly comprised Daphniidae

(Daphnia zooplankton) and Culicidae (mosquito).

Figure 6. Taxonomic analysis of bloom community. 6a. Community composition by phyla. Analysis indicates three abundant phyla are Proteobacteria, Cyanobacteria, and Bacteroidetes. 6b. Abundances of Proteobacteria by Class. 6c. Abundances of Cyanobacteria by Order. 6d. Abundances of Bacteroidetes by Class.

3.3 Metatranscriptome analysis

Seven metatranscriptomes were produced from the Maumee Bay water samples (1.97

Gbp) with reads count ranging from 109,124 to 570,660 per sample. rRNA genes accounted for

~1% of the assembled metatranscriptomes. Fifty seven percent of protein-coding RNA were annotated as known proteins, the remaining ~40% encoded unknown proteins. Of the predicted 21 proteins, ~70% were assigned to functional categories. Less than 5% of reads (per transcriptome) failed quality control tests. A transcriptome for 4:00 h on the second day is missing from the data set due to inclement weather that precluded sampling.

3.4 Differential expression in Microcystis spp. transcripts

The goal of this thesis was to study the metabolic functions over diel cycles of a Lake

Erie cHAB. Three recently available Microcystis spp. genomes specific to Lake Erie and Lake

St. Clair were used to annotate metatranscriptomes to increase transcript coverage compared to publicly available Microcystis genomes mainly derived from Japan’s National Institute for

Environmental Studies (NIES).

3.4.1 Day group (10:00, 16:00) vs. night group (22:00, 04:00)

Initial analysis of differential expression partitioned the seven transcriptomes into two groups; a Day group (3S 10:00, 4S 16:00, 6S 10:00, 7S 16:00) and a Night group (1S 22:00, 2S

4:00, 5S 22:00), as shown in the Principal Component Analysis (Figure 7) of transcript abundance (RPKM). Variability within the day samples may suggest that daily conditions were not constant, resulting in changes in gene expression patterns (Figure 8). 22

Figure 7. Principal Component Analysis of Microcystis reads. Night group (blue) show distinct clustering. Day group (red) indicate separation of gene expression between morning (3, 6) and afternoon (4, 7) samples.

An initial analysis examined all the night transcripts collectively against the daytime samples. Night transcripts were lower in abundance and yielded fewer definitive functions. Of the eight transcripts representing differential night functions, five transcripts annotated as hypothetical proteins or putative proteins. Further analysis of each transcript against multiple databases (NCBI BLAST, InterPro, Cyanobase) returned hypothetical/unknown proteins. The four remaining transcripts include an uncharacterized NAD(P)/FAD-binding protein YdhS, an ion channel transporter (mscS), and a putative pathogenesis transcript RTX toxin-related Ca2+ binding protein.

Transcripts significantly expressed among day samples were 5-fold greater than night sample transcripts. Six transcripts annotated as hypothetical proteins. The remaining were 23 involved in photosynthesis, porphyrin and chlorophyll metabolism, carbohydrate metabolism, oxidative phosphorylation, amino acids synthesis, nitrogen metabolism, heat shock chaperones, cell division, transporters.

Photosynthesis-related transcripts encoded a protein related to Photosystem II (PS II) stability and assembly, and the D1 subunit of PSII (psbA). Transcripts categorized as related to pigment synthesis (porphyrin, bilin, and carotenoid) included vitamin B12 synthesis (cobN), carotenoid synthesis enzymes phytoene desaturase, cofactor synthesis dxs, a carotenoid cleavage dioxygenase involved in apocarotenoid synthesis, and a photoprotection (quenching) orange carotenoid protein.

Carbohydrate metabolism functions included those encoding a glucan-branching enzyme

(glgB), a putative glycogen synthesis regulating protein (tldD), and pyruvate metabolism and

CO2 fixation protein (ppsA). Photosynthetic energy metabolism oxidoreductases (ndh, ndhF), and a non-specific serine/threonine protein kinase were also day specific.

Amino acid and protein synthesis transcripts include a translation elongation factor, a histidine dehydrogenase (hisD), degradation of branched chain amino acids (mmsD) and lysine biosynthesis (dapF). Day nutrient metabolism transcripts primarily consisted of proteins involved in nitrogen assimilation and included nirA, urtA, glnA.

Heat shock chaperones consist of multiple copies of each dnaK, groL, clpB. Similarly, three division protein homologues ftsH were expressed by day. Eight transcripts encoding pumps and transporters were significantly expressed in day time samples including a bicarbonate transporter (ictB), multiple multidrug efflux pumps and antibiotic exporters, and sulfate permeases. Remaining transcripts included fatty acid metabolism, cell wall and extracellular 24 matrix functions, and a number of transcripts encoding obscure functions. A full list of diel transcripts can be found in Appendix A, and a comparison of functions (KEGG) in Appendix B.

Figure 8. Heat map visualization of differential expression between Night group (right) and Day group (left).

3.4.2 Morning (10:00) vs. afternoon (16:00)

Following analysis of differential expression between day and night time points, the two daytime samples (1000 and 1600) were analyzed for differentiation of functions during the two daylight sample times. As shown in the PCA plot and Figure 9, the expression patterns between and within the two day groups appeared to differ in abundance and magnitude of expression. 25

Morning functions of note were involved in amino acid synthesis, stress responses, oxidative phosphorylation, protein assembly and transport, and nitrogen metabolism.

Figure 9. Heat map visualization of differential expression between Morning samples (right) and Afternoon samples (left).

Morning samples contained no photosystem-specific functions, but contained a bilin lyase transcript encoding the enzyme attaching phycocyanobilin to the beta subunit of phycocyanin (cpeT/cpcT family). Amino acid synthesis transcripts included glycine, serine, threonine synthesis, cysteine and methionine synthesis (metX), tryptophan, tyrosine and phenylalanine synthesis (aroA), valine, leucine, isoleucine degradation (mmsB), and nucleotide sugar synthesis (wecB). Three transcripts, glutamate racemase (murI), colanic acid biosynthesis glycosyl transferase (wcaI), and cell shape regulation protein (rodZ) suggested cell wall formation during the morning. Lipid metabolism functions included a lipase transcript and putative flippase gtrA mRNA for lipid transport. Repair and stress-related transcripts included a nucleotide excision repair (uvrC) and dps, a protein capable of providing resistance to photosynthetic oxidative stress and nutrient starvation (Peña and Bullerjahn, 1995). Morning nitrogen metabolism transcripts included type III glutamine synthetase, hydroxylamine reductase, and cyanophycin synthesis (chpA). Two morning transporter mRNAs encoded a zinc uptake system and an L-arginine-L-histidine-L-lysine transporter. Regarding toxin (microcystin) synthesis, three mcy operon transcripts are present during the morning, two mcyD copies and a singule mcyA. Oxidative phosphorylation transcripts in the morning employed a variety of roles.

Cytochrome bd-type quinol oxidase (cydA), NADH dehydrogenase (ndh), and a ferredoxin confer electron transfer for energy metabolism, signal transduction, and light-dependent reactions. Toulene-degradation pathway dehydrogenase expression (pchF) was also present in the morning. Heat shock chaperones consisted of mRNAs from single paralods of dnaK and groL.

Afternoon functions were more numerous than those in the morning, with ~150 mRNAs differentially expressed. Photosynthesis genes psbA, psbU, psbO were highly transcribed in the 27

afternoon samples. Cobalamin (vitamin B12) synthesis (cobA, cobN, and cobQ), and a cobalamin binding protein radical SAM superfamily protein (ygiQ) were cofactor transcripts present in afternoon samples. Antennae transcripts included a phycocyanobilin synthesis heme oxygenase

(hmuO) and cpcB, encoding a phycocyanin beta subunit protein. Afternoon nutrient uptake and metabolism functions were limited to carbon (CO2) and nitrogen. Three nutrient transporter transcripts detected were those encoding a carbon dioxide hydration protein (cphX), a putative bicarbonate transporter (ictB), and an ammonia transport channel (amt). Specific nitrogen metabolism gene expression occurring in the afternoon was ascribed to transcripts encoding assimilatory nitrate reductase (nirA), glutamine synthetase type III (glnA), arginine metabolism ornithine carbamoyltransferase (argF), and glutathione metabolism enzymes NADPH- glutathione reductase and aminopeptidase N (pepN). Branched chain amino acid metabolism

(valine, leucine, isoleucine) transcripts included ilvJ, ilvA and degradation mmsB. Other major functions expressed at 1600 included mRNAs for thiamine synthesis (thiO), pyrimidine synthesis

(umpS, carA), purine synthesis (pucG), multiple tRNA synthetases, DNA-directed RNA polymerase gamma chain, and an antitermination factor (nusB).

Protein synthesis and turnover were prevalent, such as ribosome assembly protein rsgA, large ribosome subunit L16 (rplP), large subunit assembly (rlmI), a peptide chain release factor, ribosome quality control complex, and two proteases.

Central carbon metabolism included transcripts encoding glycogen/starch phosphorylase

(glgP), pentose phosphate pathway 6-phosphogluconate dehydrogenase (gntZ), and phosphopyruvate hydratase enolase (eno).

Fatty acid and lipid metabolism included glycerophospholipid metabolism enzyme

(cdsA), acyl-CoA synthetase, an acyl-CoA reductase, and a biotin synthesis enzyme (bioH). 28

Energy metabolism respiratory and oxidative phosphorylation transcripts in the afternoon included those encoding an NADH dehydrogenase (ndh), a cyanobacterial NADPH:quinone oxidoreductase (ndhH), succinate dehydrogenase/fumarate reductase (sdhB/frdB), and an NADP- dependent nickel-iron dyhydrogenase (hoxH), and ATP synthase (atpI). Cofactor and vitamin synthesis oxidoreductases genes expressed included a riboflavin kinase (ribF) and a nicotinate/nicotinamide specific oxidoreductase (pntB).

Afternoon transporters functions present included a multidrug tranport system, a putative ion (Mg/Co) transporter, calcium transport, putative iron export (fetB), and an efflux transporter.

Repair and stress response transcripts present were a base incision repair (ligA), dnaJ, site- specific DNA adenine methylase (dam), peroxiredoxin, exodeoxyribonuclease double strand break repair (sbcD), and an acyl-CoA dehydrogenase alkylination damage repair protein (aidB).

Restriction modification system transcripts include abortive infection proteins, an Uma2 family restriction endonuclease, methyltransferases.

Cell wall synthesis and extracellular matrix functions included transcripts encoding a cadherin domain protein, cna protein, CARDB, and a glycosyltransferase involved in cell-cell contact and adhesion.The remaining 20% of afternoon transcripts annotated as hypothetical and unknown proteins. A full list of transcripts is found in Appendix C.

3.4.3 Night (22:00) vs. pre-dawn (04:00)

Following the same approach as previously described, the two night groups were compared in order to detect variation in gene expression during the dark period (Figure 10).

Functions at 22:00 h included mRNAs for amino acid metabolism, restriction modification system methylases, extracellular matrix functions, with some low level expression of 29 photosynthesis and oxidative phosphorylation. The early morning 04:00 h functions were limited to microcystin synthesis and functions in preparation for photosynthesis.

Figure 10. Heat map visualization of differential expression between Night samples (right) and pre-dawn sample (left).

Night (22:00 h) transcripts mainly encoded amino acid metabolism functions (~30%) such as a carbamoyl phosphate synthase (carB), an initiation factor IF-2, alanine, aspartate and glutamate metabolism (glmS), valine, leucine, isoleucine metabolism (ilvA), and arginine metabolism (speA). Notable carbon metabolism nighttime functions expressed included glycogen/starch synthesis (glgA), and glycolysis/gluconeogenesis acetalaldehyde dehydrogenase 30

(adhE). Some day genes still expressed during the night samples, such as genes encoding a MarR family low O2 chlorophyll synthesis transcription regulator, a photosynthesis energy metabolism oxidoreductase (petH), and oxidative stress-related protease (clpAX). Remaining transcripts included those encoding an obscure secondary metabolite synthase, a sulfate permease (sulP), a chemotaxis response domain CBS, a cell wall synthesis glycosyltransferase and a cell-cell contact cadherin domain protein.

Pre-dawn transcripts were low in number with 15 genes differentially expressed compared to the night transcriptomes, yet these were significant functions in cyanobacterial metabolism. Six transcripts encoded enzymes dedicated to the synthesis of secondary metabolites, such as the mcy operon genes mcyB, mcyD, mcyE, mcyG. The remaining transcripts were likely to prime cells for daytime photosynthesis. Transcripts of magnesium-chelatase protein gene comM encode the first unique step in chlorophyll synthesis, and cobL encodes an intermediate in cofactor B12 synthesis. Expression of low-affinity CO2 transporter cphX, chaperone clpX, large ribosome subunit L5 rblE, and a narL/fixJ family DNA-binding response regulator indicated a circadian expression of photosynthesis-related genes. See Appendix D for a full list of transcripts.

CHAPTER IV. DISCUSSION

Seven metatranscriptomes were produced from a known toxic cyanobacterial bloom in

Maumee Bay (western Lake Erie) during August 2014. Maumee Bay waters appeared green in coloration consistent with 30-50 µg L-1 chl a biomass increasing from 30 to 50 µg L-1 over the course of sampling. Taxonomic analysis showed Microcystis spp. to dominate Cyanobacteria reads. Along with Cyanobacteria, co-dominant phyla included the Proteobacteria and

Bacteroidetes. The nearly equal abundances of the dominant phyla within the bloom community gives evidence to suggest the bloom is in a decline. Recent western Lake Erie studies having shown during September 2013, Microcystis spp. were dominant within the prokaryotic community outside of the mouth of the Maumee River (Harke et al., 2015a).

Cyanobacteria are known to regulate gene expression under circadian influences. The intent of this study was to identify cellular processes within a Microcystis bloom that show diel patterns of expression. Principal Component Analysis (PCA) of Microcystis transcriptomes reveal grouping of samples based upon time of collection (i.e. ‘Day’ and ‘Night’), indicating there was a relation of samples with regards to expression during specific times of day.

Variations within the relatedness of day samples as shown by PCA suggested circadian rhythms were not the sole determiner of expression, rather transient environmental changes may have contributed to differing transcript accumulation.

Three recently annotated genomes of Great Lakes cHAB isolates (Meyer et al., 2016) were used to map the Microcystis transcripts from our survey. Read mappings differed within time points due to mapping to the various paralogues. An example of this are the multiple copies of hydroxylamine reductase and gas vesicle proteins, in which copes exhibited a variation in 32 pattern of expression. Therefore, some transcripts did not identify as differentially expressed via initial genetic assignment, but following transcript mapping in total, patterns were detected.

Below diel functions are discussed taking into consideration the major physiological characteristics of cyanobacteria.

4.1 Photosynthesis

Photosynthesis-related functions are highly expressed at all time points, regardless of day or night. Transcripts encoding functions of photosystem II appeared to differ in regards to specific photosynthetic functions of Microcystis between morning and afternoon samples. The differential analysis indicates higher levels of expression of psbA, psbU and psbO during the afternoon. Conversely, psbC, encoding the CP43 internal chlorophyll antenna protein, was down regulated in the afternoon. Simply put, photosynthesis functions partition so that CO2 fixation occurs in the morning, and the PSII-dependent O2 evolution peaks in mid-afternoon, as has been observed in laboratory studies (Mackenzie & Morse, 2011; Garczarek et al. 2001). Expression of cpeT/cpcT, encoding a bilin lyase which contructs phycocyanin (Zhou et al., 2014; Zhao et al.,

2007) and CO2 hydration proteins during the early morning compliment this finding, as CO2 availability can limit electron transport (Golden, 1995). This separation of photosynthetic events may be a strategy employed by Microcystis, but may also be due to varying environmental conditions throughout the sampling period. Though light data were not recorded, weather reports from nearby airfields showed conditions were generally overcast during the sampling period and light irradiance is known to influence phycobilisome expression (Müller et al., 1993; Nomsawai et al., 1999). Pigment and porphyrin biosynthesis expression occurred during the afternoon due to detection of transcripts for photo-protective carotenoids, phycobilins, and cobalamin.

4.2 Biosynthesis of amino acids and proteins

As general cell processes are required during the course of a 24-h period, expression of most protein metabolism transcripts did not yield a diel pattern. This may be explained by the high expression of ammonium transporter during the first 18 h of the study. However, those functions that did, included those for the synthesis of amino acids histidine and lysine, both expressed during the light period. Histidine residues are important metal ion ligands for electron transport processes (e.g., Tang et al., 1994). Lysine metabolism pathways are necessary for peptidoglycan synthesis and subsequently cell wall synthesis (McCoy et al., 2006). Perhaps daytime demand for these amino acids is necessary for photosynthetic metabolism and cell division. In the early morning before the onset of the light period, the assembly of the large subunit indicates preparation for protein synthesis as a result of energy metabolism, as well as intracellular protein transport in the morning. Afternoon transcripts involved quality control of ribosomes.

4.3 Nutrient metabolism

Nitrogen metabolism exhibited strong preferential expression during the light period.

Nitrogen uptake genes present included those involved in nitrate, urea and ammonium assimilation. Transcript abundances indicated that transport of nitrate by Microcystis decreased during the onset of morning, and increased all through the day into the dark period. Ammonium transport appears to have been maintained throughout the entirety of the light period, and urea transport peaked in the afternoon. Along with uptake transcripts, transcripts related to nitrogen assimilation also occurred during the day via nirA (encoding nitrite reductase) and glnA

(encoding glutamine synthetase GS), indicating the flow of assimilation and synthesis of amino 34 acids in daylight (Flores and Herrero 2005; Mérida et al. 1991). Glutamine is an essential amino acid but it also is a major donor in biosynthetic pathways (Marzluf, 1997). Ammonium availability acts as a repressor of GS activity, except during periods of global nitrogen starvation, when GS activity is retained (Mérida et al., 1990).

Physicochemical data suggests the first day of sampling was nitrogen depleted, which may be reflected in the abundances of glnA in respect to the presence of amtB encoding ammonia transport (Obst and Steinbüchel 2006). The morning transcription of cphA required for the synthesis of cyanophycin granules suggested the cells exhibit luxury N uptake at this time

(Mackeras et al. 1990; Parnas and Cohen 1976; Loitenberg et al. 1996). In response to the nitrogen depletion during the first 18 hours, an increase in nitrogen assimilation genes, urea and ammonium uptake genes are highly expressed, as similarly reported by Harke et al. (2013) in western Lake Erie M. aeruginosa cultures exposed to low nitrogen conditions. Nitrogen metabolism appears to depend on environmental stimulus rather than circadian control.

Phosphate uptake transcripts pstBS and a phosphate/sulfate permease (annotated as

‘hypothetical’ via NCBI) were expressed with a distinct diel pattern, though were not included in the analysis due to read mappings. pstS and the permease oscillated with a 12 h period peaking morning (10:00 h) and the beginning of night (22:00 h). Physico-chemical data indicates a period of phosphorus limitation (nitrogen replete) beginning at the second 22:00 h through the remainder of the survey. Harke et al. (2013) showed conditions of low phosphorus increased expression of phosphorus uptake genes. Despite the constant phosphorus-limiting conditions, pstS expression continues to follow oscillating expression patterns, suggesting phosphorus metabolism in M. aeruginosa is dependent upon circadian control. 35

Cyanobacteria are known to contain multiple inorganic carbon (Ci) uptake genes and pathways, allowing variable responses to availability (Sandrini et al. 2014, Sandrini et al. 2015).

A growing concern in bloom formation and mitigation is the reponses of cHAB species to rises in atmospheric CO2 (Verspagen et al., 2014). Associated with this Microcystis bloom, uptake of carbon species varied by time of day. CO2 hydration oscillates around a 24 h cycle at dawn.

Genes encoding high affinity (cmpA) and constitutively (bicA) bicarbonate uptake transporters were present. cmpA is inducible under conditions of low CO2 (Omata et al. 1999), and in our study, exprssion peaked after the onset of light period, and again at the beginning of the dark period. bicA (Price et al. 2004), remained evenly expressed during the day and decreases at night. A third, putative, inorganic carbon transporter ictB was present during the afternoon and beginning of dark period, but the specific function is uncertain as to whether it is a transporter.

Nonetheless, evidence that it functions in Ci uptake stems from work showing inactivation of ictB removes all activity of the high affinity bicarbonate transporter (Bonfil et al. 1998; Ogawa

1991). Carbon concentrating mechanism transcripts did not appear to express distinct diel patterns, although RuBisCo transcripts rose before the light period and decreased until the onset of night, and peak expression of carboxysome functions occurred at 10:00 h. In concordance with peak carbon fixation in the morning, a zinc transporter was expressed in the morning, a required metal for carbonic anhydrases (Keilin and Mann, 1940). The carbon uptake of

Microcystis appeared to induce CO2 uptake prior to the onset of the light period, then expressing both high and low affinity transporters during the remainder of the day.

4.4 Heat shock, stress response, gas vesicle, replication & division

As expected, a relationship exists between chaperone proteins, gas vesicle formation, and cell division proteins. Synechococcus cells have shown a specific gating of cell division 36 independent of the circadian clock in avoidance of peak irradiance exposure (Mori et al. 1996).

The role of gas vesicles is partly to allow Microcystis cells to migrate down the water column away from stressful high light exposure, during which heat shock chaperones are involved in protective functions (Walsby, 1994). Transcription of the heat shock chaperones dnaK, groL, and clpB all peak within the morning in preparation as light exposure increases, decreasing steadily as the day progresses (Webb et al., 1990; Aoki and Ishiura, 1995). Gas vesicle transcripts show an inverse relationship to those of the chaperones, increasing prior to the onset of the light period, and steadily decreasing until night. This expression pattern suggests a strategy in which

Microcystis rises to surface waters to maximize photosynthesis, and then repair damage accumulated as a consequence of photo oxidative stress.

Transcripts for cell division proteins exhibit similar patterns, necessary to avoid radiation damage of newly synthesized DNA (Dong et al., 2010). Two transcripts, ftsZ and rodZ, are responsible for the contractile ring and septum formation of the dividing cells, both of which maintain increased expression during the dawn before the beginning of the light period. As the morning continues, we see these transcripts decrease and the ATP-binding ftsH division protein increase in the morning. Cell wall formation transcripts are highly expressed every 12 hours, including in the morning in synchronicity with division transcripts.

4.5 Toxin synthesis

Peak expression of toxin synthesis genes mcyABC and mcyDEFGHIJ occurred before the onset of the light period. Most transcripts from the operon were present excluding mcyHIJ, which were not detected. Expression by the 10:00 h sampling time decreased by half, indicating that certain steps in synthesis were constrained by that time of day. The final step in toxin synthesis, cyclization of the peptide chain, is modulated via mcyC (Rouhiainen et al, 2004). The 37 expression of mcyC increased in the pre-dawn during Day 1 (speculative for the second 04:00 h) and maintained transcript levels throughout the daylight period, albeit at levels below peak. This could be an indication of toxin production occurring throughout the entirety of the day following initial synthetic steps in the morning. mcyD transcription is shown to be correlated with ntcA expression (Pimentel & Giani, 2014), although in this bloom, ntcA was down regulated during periods of toxin synthesis. We have found microcystin production to be occurring before the onset of the light period, though other studies have indicated toxin production to occur earlier in the night (Penn et al, 2014) or as a day-time function (Straub et al., 2011). The pre-dawn production of toxin parallels the photosynthesis functions (cphX, cobL, comM) seen during the same time of day. Synthesis appears organized to anticipate the onset of light suggesting a potential role of microcystins in the diurnal metabolism of Microcystis. The increase in toxin concentration within the water at 16:00 h on the first day (from 1 µg L-1- 3 µg L-1) supports the possible requirement of microcystins in the cell. The second day of the study showed microcystin levels increasing earlier at 10:00 h (from ~1.5 µg L-1- 5 µg L-1 ), though the ratio of

DIN:DIP suggested the system became P limited. This could indicate the role(s) and concentration of microcystin in the cell are subject to environmental stressors, though further analysis will reveal whether other members of the environmental community, such as viruses, are responsible for increased cell death at that time of sampling which could explain increased levels of microcystin earlier during the second day.

CHAPTER V. CONCLUSION

Lake Erie has been experiencing annual cHAB events, and are these are predicted to keep occurring as Lake Erie eutrophication intensifies (Michalak et al., 2013). Despite the effect on potable water sources and human health, the factors influencing cHAB formation, proliferation, and maintenance remain inconsistent and poorly understood. In an effort to narrow the knowledge gap, our intent was to use metatranscriptomic evidence to investigate the daily metabolism of a Lake Erie Microcystis bloom. Laboratory studies have found the genomes of

Microcystis to be highly plastic and adaptable to the environment, increasing their competitive ability. Previous investigations (Golden et al. 1997; Kondo et al. 1993) report the presence of circadian regulation of gene expression in cyanobacteria. The circadian clock has been shown to enhance the fitness of the species within the microbial community. Paired with a highly adaptive genome, Microcystis has the potential to be very successful. Our analysis indicates that Lake Erie

Microcystis likely utilizes efficient organization of gene expression to maintain productivity, such as utilization a variety of nutrient species throughout a diel cycle (CO2, bicarbonate, nitrate, ammonium, urea), regulating buoyancy to migrate within the water column, cell division restriction, and the production of microcystins. The occurrence of toxin synthesis prior to the onset of light suggests a potential role in light-period metabolism of Microcystis. Our results suggest the metabolism of Microcystis is system-specific, though diel patterns exist, environmental cues may influence regulation, as supported by PCA analysis. Additionally, taxonomic evidence suggests this bloom is in decline. The next step of this analysis is to study the metabolism of the global microbial community. In doing so, those results paired with this analysis of Microcystis spp. will provide insights into factors leading to the natural mitigation of a bloom, and how the surrounding consortium of heterotrophs interact and influence a cHAB. 39

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APPENDIX A

Diel differentiation (Day groups compared against Night groups)

Night group transcripts

Gene number Transcript annotation Protein sequence

2606708435 Small-conductance mechanosensitive channel

MGRRQRGKIFNLVIQGLAGCLLVSIFVLNPQPSLGQNFLVPFASPTGTRI

2606714733 hypothetical protein

MTTFRISGQIIDLHTHQGLFAVRVEAWDKDLICSDLVGSAVTDSQGVFQI

2606713034 Ca2+-binding protein, RTX toxin-related

MTSAANSGNILMGLGLEAILAQFANSETYYQNLTAIFGNQYDTSKAETLR

2606711336 hypothetical protein

MTTLLVPVYLDALYLPTKTNVLEEMTDYSKLPYYKDTELQKRGKAYISET

2606715104 hypothetical protein

MTTNTKPDLKEELEKGLSTTSDDSPAKGPKITISLGEDIEFISEVKPDEK

2606706376 hypothetical protein

MSPRKLTDATKKEILKLYRETEATTSTLAERYGVSSSTVSRFLKNSFSSE

2606705887 TIGR03032 family protein

MDSLPPNTNQFSDTQEPAASPTQHQAVPVNYEYSLNLPELLNQLDLSVLI

2606704633 Uncharacterized NAD(P)/FAD-binding protein ydhS

MSYIDRNQFSSTFDIAIIGGGFSGSLVTANLLRDTGTPLSIALIDHRKPL

2606711703 indole-3-glycerol phosphate synthase (EC 4.1.1.48)

MQIRRKHPNPAVKVESLSYVVKVPEAQPQNILEEIVWHKEIEVDKLRERL

Day group transcripts

2606705273 Uncharacterized protein related to plant photosystem II stability/assembly factor

MINRQSLLRFAYKFNLILLIIAIVFCLGSSAAFAHRPHDVISQLEVSPDF

2606706912 1-deoxy-D-xylulose-5-phosphate synthase (EC 2.2.1.7)

MHLSEITHPNQLHGLSLRQLEDIARQIREKHLQTIAATGGHLGPGLGVVE

2606703684 Uncharacterized conserved protein

MTSTLLPSFPAVDDVLFNFAQSDGLSQANQVDSARNQRLTATVVFLDAGV

2606706259 Predicted purR-regulated permease perM

MTFGSSLGLVLLVFCLLILWQIRQILLLLLMAILLAQILNLAVTWWQRHH

2606709504 ATP-dependent metalloprotease ftsH

MKLSWKTILLWTLPALVVGFFLWQGTFATATSNIGNNTASTRMTYGRFLE

2606709838 hypothetical protein

MGKSINKVRGTALILGILAIVFAFFTRIVSIFEYVTFDIGPDPDQISGAY

2606711226 transposase, IS605 orfB family, central region

MFVLEYKVKPKPNQIEAINEAIRTTQFVRNKVLRYWMDNQGVGKTELFRY

2606711670 hypothetical protein

MKSRKGLISVKQAYLKILSVILVLAIIGLSVVGNYGITWDEPIEVDMVNH

2606707371 ATP-dependent chaperone clpB

MQPTNPQQFTEKAWEAIVKTPDIAKQNSQQQIESEHLMKALLEQEGLAGS

2606713626 ATP-dependent metalloprotease ftsH

VNKNGNNKKWRNAGLYALLLIVVLALASAFFDRQPAVQQTWKYSEFLQEV

2606707220 translation elongation factor TU

MARAKFERTKPHVNIGTIGHVDHGKTTLTAAITMTLAALGNAQAKKYDEI

2606712595 Sulfate permease or related transporter, MFS superfamily

MEITNRIHFDNLRGDIFGGLTAAIVSLPLALAFGVASGAGAISGLYGAVC

2606707846 glutamine synthetase, type I

MPETPQEVLKMIQDNNIKIIDLKFIDMPGIWQHCSFYYDQIDESSFSDGV

2606704120 Multidrug efflux pump subunit acrB

MSSIFYKNVRLLVLTILAITVWGLSSFFSLPRMEDPELTPRFAVINTQFP

2606709231 ABC-type bacteriocin/lantibiotic exporters, contain an N-terminal double-glycine peptidase domain

MLDTHSLVNPWPEFLSETQWRSLQKTAIALSPEAGTLPVQSGLYLVVGGK

2606707219 translation elongation factor EF-G

VARTIPLERVRNIGIAAHIDAGKTTTTERILFYTGVAHKLGEVHDGTAIT

2606707726 diaminopimelate epimerase (EC 5.1.1.7)

MSLSMRCSSSIIPVEQLLLTMKIPFTKYQGLGNDFILIDNRHSPEPLITA

2606714984 NAD(P)H dehydrogenase, subunit ndhF3 family

MFESFSQTIWLVPLYAFAGTLLALPWSPGIIRQTGPRPAGYLNLIMTCWA

2606711387 chaperone protein dnaK

MGKVVGIDLGTTNSCVAVMEGGKPTVIANAEGFRTTPSVVAYAKSGDRLV

2606714763 Trypsin-like peptidase domain/TPR repeat

VRIDSSSTANGSGVIIAKKGNIYTVLTADHVLCEKEKSQDAASKPCLNFT

2606704238 histidinol dehydrogenase

MLRIITEPWSARTELRRIRERFDDPEIREKESLVREIVEQVRLSGDRSLV

2606708869 Carotenoid cleavage dioxygenase or a related enzyme

MNMSIKDTVQNTVNVRNFSRSQLGQPDENNLYQAVATITEGHWPENLSGY

2606711568 precorrin-3B synthase

MVQAPEKDGTTLNKFEKFKAEKDGLAIKDELDHFAQIGWEAMDKTDLEHR

2606710071 cobaltochelatase CobN subunit (EC 6.6.1.2)

MFTHVKSTIRHIVPDGLNGRSLVKVVYVVLEPQYQSALSSAVREINANNP

2606710095 Predicted Zn-dependent protease or its inactivated homolog

MSPTLLISKELPTLKYNPSLERFDSSWAAPLSTLLGLGRAAGATFIEFFL

2606709429 Uncharacterized conserved protein

MVPEYPPLDEMTLRQLRRVASQYSVSRYSRMRKMQLIEAITQAMGVSGNF

2606708579 Fatty acid desaturase

MRVTFTENQGFRKELNKRVDAYFTENGIPTRDNFAMYLKTITILTWVIAA

2606712266 NB-ARC domain/Trypsin-like peptidase domain

MPQFQPDRALEIVAPKYGTAYRIGGRLLLTCRHLFDNSNDCQVRFRSASN

2606704578 Family of unknown function (DUF490)

MSNSPGDPSPRPNLFLSIGRFVRRPSTLIVGGITLLGITSLGYFTTRYLV

2606710734 Phosphoenolpyruvate synthase/pyruvate phosphate dikinase

MTEIWGSLFIFLVCPIIGGLPLIDWINYLVTGRKLSQLGTGNIGVTAAFY

2606704131 chaperone protein dnaK

MGRVVGIDLGTTNSVVAVMEGGKPVVIANSEGMRTTPSVVGFNKDGELVV

2606708131 chaperonin groL

MAKSIIYNEDARRALEKGMDLLAEAVAVTLGPKGRNVVLEKKFGAPQIVN

2606707606 Serine/threonine protein kinase

MPDLNAIPGITALRSHTQGDPRIKIAVLDGPIDLDRACFQGANITRLDPY

2606707481 Cytochrome P450

MKIIAKVKTPTFLQMAQWIINPVAFMENAARKHGDIFSTKVGLTVDNFIF

2606703921 Glycosyltransferase, catalytic subunit of cellulose synthase and poly-beta-1,6-N-

acetylglucosamine synthase

MSNKESIFPFPQKPVYSSVFKSILAIISVILIMLAVIGGLMMVSLSLSWP

2606712573 ATP-dependent metalloprotease ftsH

MSIKSKPPSQSRLIGNVLLAVGGLFLIVNLLFPQLFGPSVPKVPYSLFID

2606706098 ATP-dependent metalloprotease ftsH

MDQKSPLTVVRAKSAKNRGNRPVWKGIVTTWMILQTFGHVTPAWSQKNPN

2606704545 ATP-dependent chaperone clpB

MQPTNPQQFTEKAWEAIVKTPDIAKQNSQQQIESEHLMKALLEQEGLAGS

2606705439 Bacterial Ig-like domain

MIKTLQLQPIDKLAITLIVSLTLVIGGLIWLGNSCGNQCLLQTGPRVKEF

2606708416 urea-binding protein

MSNLGRRKFLLYGSAALGTSILLKACGGNQSQTPTASPSPAASPAPASGE

2606708382 cyanobacterial porin (TC 1.B.23)

MLRMFWKSLLVSPAILGATLVASANASSFDPAKSTSVSTEFNNLEIAQVQ

2606710808 Multidrug efflux pump subunit acrB

MAFNISNWSIKNPIPTILISLVMALMGYIAFLGLGIDRSPNIDIPAVIIT

2606705236 DNA-directed RNA polymerase subunit beta' (EC 2.7.7.6)

MFYNQTVDKGKLKKLIAWTYQNYGSARCSQMADELKNLGFRFATKAGVSI

2606714466 Predicted periplasmic protein (DUF2233)

MKLNYLTLSSICLTVTSLIIIGSPAISLADHRSPITLASRYLSNQQQGKK

2606708358 Iron-regulated ABC transporter membrane component sufB

MSATTVKNLVNQPYKYGFITDIESETIPRGLNEEIIRLISAKKKEPEFML

2606712392 Formylglycine-generating enzyme, required for sulfatase activity, contains

SUMF1/FGE domainMLDQMMKMLEGQQIGPYRLNKFLGAGGFGGVFHASEMVRNTSVQEVAIKV

2606708242 NADH dehydrogenase, FAD-containing subunit

MTEKQPRVVIIGGGFAGLYTAKALKNAPVHVTLIDKRNFHLFQPLLYQVA

2606704763 bacteriocin biosynthesis cyclodehydratase domain

VFNQLDWNPAYSIETLEPNTVFFLSERESICFQEPLYYRLVRLIDGQRNL

2606714502 glycine dehydrogenase (decarboxylating)

MLNRSITAEKTEEFAPADLQDLLDCTDSFVNRHIGPTETEIEKMLAVLGV

2606712942 folate/biopterin transporter

MLINRLGIDRGKKFLKETILFGNDPNPELIGILGVYFVQGILGLSRLAVS

2606714019 chaperonin groL

MAKSIIYNEDARRALEKGMDLLAEAVAVTLGPKGRNVVLEKKFGAPQIVN

2606706968 phytoene desaturase

MRVAIAGAGLAGLSCAKYLVDVGHTPIVLERRDVLGGKIAAWKDADGDWY

2606707859 Family of unknown function (DUF490)

MSNSPGDPSPRPNLFLSLGRFVRRPSTLIVGGITLLGITSLGYFTTRYLV

2606710287 photosystem II, DI subunit (also called Q(B))

MSTTIRTQDNLSAWERFGQWITSTNNRLYIGWFGVILIPTILTATICYII

APPENDIX B

NIGHT

KEGG KEGG ORTHOLOGY FUNCTION FOLD GENE ID ORTHOLOGY P-VALUE ENTRY CATEGORY CHANGE DEFINITION Ca2+-binding 2606713034 protein, RTX toxin- - - -2.624055297 0.006864716 related Small-conductance 2606708435 mechanosensitive K03442 transporter -7.277617284 0.004347273 channel TIGR03032 family 2606705887 - - -2.916757332 0.035921022 protein 2606706376 hypothetical protein - - -2.916458702 0.035933296

2606711336 hypothetical protein - - -3.803620273 0.011010872

2606714733 hypothetical protein - - -22.56623847 0.014402749

2606715104 hypothetical protein - - -4.904445207 0.030009364 Uncharacterized NAD(P)/FAD- 2606704633 - - -4.145702534 0.026884465 binding protein YdhS

DAY

KEGG Orthology KEGG Orthology Gene ID Function Category Fold change P-value definition Entry Uncharacterized 2606703684 - - 3.150695626 0.015643982 conserved protein Glycosyltransferase, catalytic subunit of cellulose synthase 2606703921 K00694 glycosyltransferase 5.943619934 0.011101028 and poly-beta-1,6-N- acetylglucosamine synthase Multidrug efflux 2606704120 - transporters 2.011303171 0.03690085 pump subunit AcrB chaperone protein 2606704131 K04043 rna degradation 2.605937884 0.045361386 DnaK histidinol histidine 2606704238 K00013 7.314225804 0.035085139 dehydrogenase metabolism ATP-dependent longevity regulating 2606704545 K03695 2.696181753 0.041175104 chaperone ClpB pathway bacteriocin biosynthesis 2606704763 K09136 - 3.113037941 0.031414113 cyclodehydratase domain Uncharacterized protein related to 2606705273 plant photosystem II - photosynthesis 3.93842961 0.02083972 stability/assembly factor Bacterial Ig-like 2606705439 - - 6.541459894 0.048933571 domain ATP-dependent cell division 2606706098 K03798 5.328785217 0.017877153 metalloprotease FtsH protease

Predicted PurR- 2606706259 regulated permease - transporters 4.22603802 0.043010692 PerM alpha-1,4- glucan:alpha-1,4- starch and sucrose 2606706311 K00700 4.440394406 0.035891259 glucan 6- metabolism glycosyltransferase 1-deoxy-D-xylulose- thiamine 2606706912 5-phosphate synthase K01662 4.086376309 0.017424117 metabolism (EC 2.2.1.7) carotenoid 2606706968 phytoene desaturase K02293 8.533657337 0.022575445 biosynthesis translation elongation elongation factor 2606707220 K02358 3.338112012 0.021211609 factor TU TU ATP-dependent longevity regulating 2606707371 K03696 2.848015153 0.007997204 chaperone ClpB pathway 2606707481 cytochrome P450 - - 4.656736032 0.030010766 Serine/threonine 2606707606 - - 2.888579562 0.044584954 protein kinase diaminopimelate 2606707726 epimerase (EC K01778 lysine biosynthesis 3.793359118 0.02538912 5.1.1.7) glutamine synthetase, nitrogen 2606707846 K01915 3.791030465 0.024965775 type I metabolism Family of unknown 2606707859 - - 2.309216053 0.035399415 function (DUF490) 2606708131 chaperonin GroL K04077 rna degradation 2.475403415 0.005857222 NADH dehydrogenase, oxidative 2606708242 K03885 3.649706767 0.036110061 FAD-containing phosphorylation subunit Iron-regulated ABC 2606708358 K09014 ABC transporters 4.676465886 0.008962149 transporter

membrane component SufB 2606708416 urea-binding protein K11959 ABC transporters 4.439652892 0.035913505 linoleic acid 2606708579 Fatty acid desaturase K00508 6.961773966 0.039907411 metabolism Carotenoid cleavage carotenoid 2606708869 dioxygenase or a - 9.205246781 0.018427495 biosynthesis related enzyme ABC-type bacteriocin/lantibiotic exporters, contain an 2606709231 K06147 ABC transporters 2.413787193 0.045836465 N-terminal double- glycine peptidase domain Orange carotenoid protein, N- carotenoid 2606709408 terminal/Nuclear - 6.567422878 0.046681965 biosynthesis transport factor 2 (NTF2) domain ATP-dependent cell division 2606709504 K03798 3.333094473 0.010941079 metalloprotease FtsH protease 2606709838 hypothetical protein - - 3.788749635 0.010314094 cobaltochelatase porphyrin and 2606710071 CobN subunit (EC K03403 chlorophyll 2.448221759 0.013559176 6.6.1.2) metabolism Predicted Zn- dependent protease or 2606710095 K03568 peptidase 3.000729908 0.036967261 its inactivated homolog photosystem II, DI 2606710287 subunit (also called K02703 photosynthesis 4.386260671 0.011187568 Q(B)) 2606710734 Phosphoenolpyruvate K01007 co2 fixation 2.642295637 0.027546056

synthase/pyruvate phosphate dikinase Multidrug efflux 2606710808 - - 2.887981761 0.044629654 pump subunit AcrB transposase, IS605 2606711226 OrfB family, central K07496 transposase 6.567510958 0.046975969 region chaperone protein 2606711387 K04043 rna degradation 2.144247177 0.00270568 DnaK nitrogen 2606711568 NirA K00366 6.802659705 0.006058593 metabolism 2606711670 hypothetical protein - - 6.96034101 0.039929779 NB-ARC 2606712266 domain/Trypsin-like - - 4.441815173 0.035848678 peptidase domain Formylglycine- generating enzyme, 2606712392 required for sulfatase - - 8.229311846 0.026053637 activity, contains SUMF1/FGE domain Sulfate permease or 2606712595 related transporter, - ABC transporters 2.794567682 0.018522588 MFS superfamily folate/biopterin 2606712942 K03321 transporters 3.495387397 0.036118678 transporter ATP-dependent cell division 2606713626 K03798 3.938679064 0.02083339 metalloprotease FtsH protease 2606714019 chaperonin GroL K04077 rna degradation 4.821275202 0.017794716 Predicted periplasmic 2606714466 - - 6.960228032 0.039931543 protein (DUF2233) Trypsin-like 2606714763 peptidase - - 5.416324725 0.0038479 domain/TPR repeat

NAD(P)H dehydrogenase, oxidative 2606714984 K05577 4.440757235 0.03588038 subunit NdhF3 phosphorylation family

APPENDIX C

Day groups differential expression (10:00 h compared to 16:00 h)

10:00 h transcripts

Gene number Transcript annotation Protein sequence

2606705403 homoserine dehydrogenase (EC 1.1.1.3)

VTFKIGLLGFGVVGTGTAKILLDPFGRHPVLKEITIGRVGVRFLDKPREI

2606712609 homoserine O-acetyltransferase (EC 2.3.1.31)

MDRLDLISPETRFYSPPRPFVLESGAVLERVRIAYRTWGILNNKRDNGVI

2606712277 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 2.5.1.54)

MIIVMKVGSPEIEIERVSAEFEAWGLSPEKIVGKHKVVIGLVGETRELDP

2606704493 3-hydroxyisobutyrate dehydrogenase or related beta-hydroxyacid dehydrogenase

MKVGLLGTGLMGTPMVERLLNQKIPVIAYNRTLEKLTPLQQLGAKTVSSP

2606707411 UDP-N-acetylglucosamine 2-epimerase

MSKSICITLGTRPEAIKLAPVIRAFQESEQFKTHVILTGQHKEMVAQVME

2606711972 glutamate racemase (EC 5.1.1.3)

MRESQLSPIGVFDSGVGGLTVLRELYRQLPQESILYFADTARLPYGTRSK

2606710968 capsular exopolysaccharide family

MMNNQQNGALKTHSQGNLPVFAQPAFIPSVTQEEEELNLLQVFSVIKHRW

2606708355 Glycosyltransferase involved in cell wall bisynthesis

MRILLYSYNYYPEPIGIAPLMTELAEGLVKRGHQVRVLTAFPWYPNSEID

2606707155 glutamate racemase (EC 5.1.1.3)

MRESQLSPIGVFDSGVGGLTVLRELYRQLPQESILYFADTARLPYGTRSK

2606704490 Archaeal fructose-1,6-bisphosphatase or related enzyme of inositol monophosphatase family

METFWSQVLDFCYQATGQVGDRLLADFGKLQATNKADGTLVTAADRWSDG

2606704642 glycogen debranching enzyme glgX

MTLKTDHGKSHPVGATVLADGVNFSLFSKYATAIELLLFNDANSPVPSRT

2606715376 Glycosyl transferases group 1

MRNALACKMQTIYFYAPGEIRQKNIKEDQTLWTGDHSNFTAWIAQTYFHL

2606710805 Cytoskeletal protein rodZ, contains xre-like HTH and DUF4115 domains

MFRLLQSQLDPQHKQRTKLLEIGVYLQKNRLEQSLSLEEIAQKTLIPRHR

2606708065 Putative flippase gtrA (transmembrane translocase of bactoprenol-linked glucose)

MKVQQSHLFLTPPTGDLQVPLSPPPPDDGNLEAEKNYPLSLSLVIPTYNE

2606714921 GDSL-like Lipase/Acylhydrolase family

MRWIRYETLSDHKLANSHLSVLLEIGIIRLSLIILGVLFLLGLLLEAILR

2606708025 chaperone protein DnaK

MQLRLELAKGRRSQTRQKARTQLWDNFCRQSYPINYLYYQIVMGKVIGID

2606715065 chaperonin GroL

MSKIIAFKDESRRALERGVNALADAVKITLGPKGRNVLLEKKYGAPQIVN

2606709424 Uncharacterized membrane protein dedA, SNARE-associated domain

MTEWITNTMTSMGYLGIALLMFLENLFPPIPSELIMPLAGFTVHEGQMEF

2606709736 Tetratricopeptide repeat/TPR repeat

MFLRPFSILITLCLGLGFLSLPGPVQSLPLNALAKVNAASTALDFFNQGV

2606703651 hypothetical protein

MKRGIYITANDRVIEQAIALMNSIRLYDPDSTVILIPYDNNYQKIADLLS

2606708765 hypothetical protein

MSKGKELTENLVKRESIDSLTRWPEKTKSFLFLFWYRYQHRFTWKVWAAL

2606708930 cyanophycin synthetase

MKILRTQTLRGPNYWSIRRDKLIVMRLDLEDLAEKPSNEIPGFYEGLIDV

2606712948 Glutamine synthetase type III

MSYGTRVQAISQVTDRKPLPSKIPQRLEALWATDVFTLSKMQASLPKDVF

2606708808 hydroxylamine reductase

MFCEQCEQTASGNGCHQWGACGKSPEVNAVQDLLVYCLRGLALVVLKAKE

2606711829 cpeT/cpcT family (DUF1001)

MLKTSIALGFFLTQSLPLNPQVQGVANHLIGVMDTREQAQTNPRIAKVQM

2606706367 cysteinyl-tRNA synthetase (EC 6.1.1.16)

MTLILYNTLSRCEEPFTPLIPGIVSMYCCGITVYDYCHLGHARTCVVWDV

2606711918 WD-40 repeat-containing protein

MKFRFTRGEKLVGAIVVSVLTTLTIGQHQLSHAQNPIAPPKPAAESGEKT

2606703337 PEP-CTERM protein-sorting domain

MITNISRQLTTGALAIAGSVAAFGFAGAAPVQAQAECLPQSFNNFKTNPC

2606705461 HEAT repeats

MIKPNFLLLLAIGVSSWGHNFQTLPTANAQTPPRLEQPQTVATYDQLMKA

2606709614 Ribosomal protein L5

MSQRLKTTYQETIVPKLKEQFGYTNIHQVPKVIKITVNRGLGEASQNAKA

2606708597 excinuclease ABC subunit C

MFGNNLNSTLIQDLDRLENRLREIPLEPGVYFLRDQNGEILYIGKSKKLR

2606712892 DNA-binding ferritin-like protein dps

MVAIPVKTKTTGQYYATRIDLALDIRKAIVEILSQTLAASLDLKTQVKQA

2606706950 Serine/threonine protein phosphatase prpC

MWRTLCQSVVGSFHIQAGLPCQDYGAYQVLGQDVLIGAVADGAGSAKHSD

2606712738 Cytochrome bd-type quinol oxidase, subunit 1

MDLLANPVALSRIQFAITAIFHMLWPVLTTGMGIYLVIVEGLWLKTRNPD

2606706387 FAD binding domain

MDSLIVSENIISDNLTDALQEWENLLGSEYVSTDNKKRLEAETATYETHA

2606713038 NADH dehydrogenase, FAD-containing subunit

MTDSIPKICILGGGFGGLYTALRLSQLPWSDQHPPQITLIDKSDHFLFSP

2606709273 (2Fe-2S) ferredoxin

MEEADTRDILGENVAKLGLAQIQRHLFLCCDQTKPKCCDKEEGLEVWDYL

2606713911 Adenine-specific DNA methylase, contains a Zn-ribbon domain

MTYRKKLIEVALPLEAINVESAREKSIRHGHPSTLHLWWSRKPLATTRAV

2606708196 Endonuclease, Uma2 family (restriction endonuclease fold)

MTTFPIIDNNGFCADEVKFPPRDIWSEEPPLASYAHLQQILILLQCLEWL

2606707096 DNA-methyltransferase (dcm)

MNRQQKRVLSLFSGCGGMDLGLEGGFWVHQDCVNENIHQDWIVERREPWL

2606703693 Acyl transferase domain in polyketide synthase (PKS) enzymes

MDFQDKKNLSENEQSIQTRVFKALSEAKEKLKAFESQQSEPLAIVGLGCR

2606711579 Acyl transferase domain in polyketide synthase (PKS) enzymes

MDFQDKKNLSENDQSIQTRVFKALSEAKEKLKAFESQQREPLAIVGLGCR

2606711578 non-ribosomal peptide synthase domain TIGR01720/amino acid adenylation domain

MICTNLLGSVVSTVKIFCQNILGNVKICKSIWRCAECRLVCYNVEAQNNF

2606714006 ABC-type Zn uptake system znuABC, Zn-binding component znuA

MGKNFQLTAIALTLTIASLTACSGPSVKEEEAKTPLQVTVSIVPQEYFVK

2606704077 L-arginine ABC transporter membrane protein/L-arginine-binding protein/L- glutamine ABC transporter membrane protein/L-glutamine-binding protein/L-histidine ABC transporter membrane protein/L-histidine-binding protein/L-lysine ABC transporter membrane protein/L-lysine-binding protein

MIKFWRGRAVLRGLFALLGLVLAIGLSVIPAFSQTPPNPFRVATEATFPP

16:00 h transcripts

2606712359 tRNA:m(5)U-54 methyltransferase

MIDRPIHVIGGGLAGTEAAWQIAQAGIPVILYEMRPIRSSPAHHSQFLAE

2606710612 Uncharacterized membrane protein yccC

MLLQRFTFLLRPSGDIEIERGIRTLLAIAVPIIVGDILDRSKLGLMVGLA

2606714446 ribonuclease Z

VEITFLGTSSGVPTRSRNVSSIALRLPQRAEIWLFDCGEGTQHQLLRSDL

2606708458 Archaeal aspartate aminotransferase or a related aminotransferase, includes purine catabolism protein PucG

MENKSMFMIPGPTPVPEAVLLATAKAPIGHRTGAFSKVIAELTENLKWLH

2606706370 orotate phosphoribosyltransferase (EC 2.4.2.10)/orotidine-5'-phosphate decarboxylase (EC 4.1.1.23)

MNFFQKLNHAISQNQSLLVLGLDANPEMMPSTPGELIVNLEQWLKFIIDE

2606715428 Acetolactate synthase large subunit or other thiamine pyrophosphate-requiring enzyme

MGELNTAELLVKCLENEGVEYIFGLPGEENLDVLEALKNSSIKFITTRHE

2606706377 ABC-type branched-chain amino acid transport system, periplasmic component

MQENGTTETIYKFSSTLSPSDPSYVDRRADLELYQALMNSQYCYIFNSRK

2606703711 threonyl-tRNA synthetase (EC 6.1.1.3)

MDNQAPIKLPKTSESDHLKRIRHTTSHVMAMAVQKLFPKAQVTIGPWTET

2606711987 thiazole synthase [EC:2.8.1.10]

MSLNQESLAFSHGRVKNSTSEILIVGGGIIGLAIAVDLQLRGAKVTLLVR

2606707485 aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B (EC 6.3.5.-)

MSATEYEAIIGLETHCQLNTKSKIFCSCPTNFDSPPNTNVCPVCLGYPGV

2606709643 NusB antitermination factor

MPPRQQPRRIARELALLSLSQIKGNPENLEQQTLNDLILVAVRTLTLEIQ

2606707518 3-hydroxyisobutyrate dehydrogenase or related beta-hydroxyacid dehydrogenase

MKVGILGTGLMGTPMGERLLNQKIPVIAYNRTLEKLAPLQQLGAKTVSSP

2606709979 seryl-tRNA synthetase (EC 6.1.1.11)

MLDLKQIRENPEEVQQRLDSRGGSYDIAPILQLNQQQKALELERSSLQAR

2606705680 DNA-directed RNA polymerase gamma chain (EC 2.7.7.6)

MKTPTDQRFDYVKIGLASPERIRQWGERTLPNGQVVGEVTKPETINYRTL

2606713728 ADP-ribose pyrophosphatase yjhB, NUDIX family

MSESPQLLQQRLFYQGRVFQFEVSKLRLPNGVEGEWECVRHPGGALAVPI

2606714392 carbamoyl-phosphate synthase, small subunit

MISSAASQKALLVLADGTVYEGYSFGSKGTTVGEVVFNTGMTGYQEVLTD

2606706991 Cadherin domain

MAIIYVKSGSTGNGSAWNNAYGSLQSAITAAPAGDEIWVAAGTYKPTTGT

2606703194 Cna protein B-type domain/Subtilase family/Calx-beta domain

QDAAGNTSNTTLSLFADISQPVLTQFLNVPTSSANAINSIDVQFSEQINI

2606709085 Glycosyltransferase involved in cell wall bisynthesis

VSQATLSLQSNLKQWLRSSAPSLFQIKQRLSARRWPKKSIVIYTEKYRPM

2606704905 Mannosyltransferase putative

MDGICTLANDRVYDQLVALLNSIEVNGGKDLPVCVYPYDDNTERIKAEIA

2606706963 diguanylate cyclase (GGDEF) domain

MMIKFSAEDCLVLVVDDVGKNLQLAVGILDSAGYATSFASSVKQAIERVK

2606712469 nucleotide sugar dehydrogenase

MRVCVIGTGYVGLVTGVCLAHIGHHVICIDNNEEKVKLMKSGQSPIYEPG

2606710025 Glycosyltransferase, catalytic subunit of cellulose synthase and poly-beta-1,6-N- acetylglucosamine synthase

MAENYWSREDDNEELDPIGSLLSDWSDPEDEEDEFRSEIFQGRSGRRQKA

2606704163 CARDB

VTIPAGANSVNFNLNAVDDTLIELPKNYTIIATSQGFVSGSDTLAVIDND

2606710210 Dolichyl-phosphate-mannose-protein mannosyltransferase

LTKILSDRIAVYLLIGGLLFRIIIALGLYPGYDEAYYYVYSHNLDWSYFD

2606712036 Long-chain acyl-CoA synthetase (AMP-forming)

MTTLIDYSNIQSLPEVWSIVAQRFPNIIALHDPHSKPEVILTYRELYQQI

2606709184 glycolate oxidase, subunit glcD

MLLKNPPKISPNLIKQLEAILGKDGVIRRKDELLTYECDGITGYRQRPAL

2606704644 phosphopyruvate hydratase

MLDKIEVPIEAIAAREILDSRGRPTIEAEVLLESGAMGLAQVPSGASTGS

2606707262 glycogen/starch/alpha-glucan phosphorylases

METTSTLADGREALQCPTLIEDDRTGMSPETLKRAFLDNLFYLQGVNRTN

2606707554 6-phosphogluconate dehydrogenase (decarboxylating)

MTKRTFGVIGLAVMGENLALNVESRGFPIAVYNRTASKTKEFMETRAVGK

2606710342 4-amino-4-deoxy-L-arabinose transferase or related glycosyltransferase of PMT family

LKRSLDGRQGLIIVSIIWILGILVDRFWFSLDHTVPAWDQADYLNGGIIY

2606703308 Dynamin family

MGQKSYEVFQAVENKANLLAGYWGDFKTEIIQHLPESYHGEIHELSSNLE

2606707404 small GTP-binding protein domain

MKLPRLATLIIGLSFIFGLMLWLVNSIYRLYIQIAFTAPILANILLLLII

2606712984 His Kinase A (phospho-acceptor) domain/Histidine kinase-, DNA gyrase B-, and

HSP90-like ATPase

MTIFAFIFGVVLGLSICYWQVSRLNRELKRTLSALSDSVDLSASFPVTSL

2606708868 PAS domain S-box

MIVLKGYDILAQVYESVNSEVYRAIRTVDNQPVILKILKQEYPTTQELTH

2606704814 Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase/MASE1/GAF domain

MLRISSDRSKIVLVALVYLGVGWLGYLCLNLGSRPAPIWMSAGIGLTAVF

2606707868 Signal transduction histidine kinase

MNASPAYVCCQVLSSTHLEQIRSWWGQLAIQVTGPRISLSERDIPPQTGE

2606709925 Calx-beta domain/Subtilase family

PHVAGAAALLWSQNPTWTAQQVKNTLMNTGDSIAALAGKTVSGKRLNINN

2606711835 Protein-tyrosine-phosphatase

MPYNWQFSDDSTSQFMPYKLLFVCLGNICRSPAAENIMTYLIEQEGLGNK

2606705753 CDP-diglyceride synthetase

MPPNFSGLTMPWPRIVSAIFAIAFALGIIIVGGWYFTLGIGVIVFLGQLE

2606711466 L-amino acid N-acyltransferase yncA

MTTSPVPQGDDFSDRPIRPVQYRDLEDIEALTLDDFDEDIDRRAQNFRTQ

2606708901 Acyl-CoA synthetase (NDP forming)

MLNPILEPRGGEHQTIRAFFEPKTIAVVGFNRQNPQLDRTLLNNLYHNPG

2606705438 Acyl-CoA reductase or other NAD-dependent aldehyde dehydrogenase

MLSNSEKLAKLRQYFASGATRSYQFRRQQLEKLKQAIIKYEAEIYQALYS

2606707735 Pimeloyl-ACP methyl ester carboxylesterase

MSLSIRVIILVATTVYQTLSVWLEEQKAPPGNLINLGQYRLHFCLAGEAS

2606714794 Peptidyl-prolyl cis-trans isomerase (rotamase) - cyclophilin family EC 5.2.1.8

MMKIRKSGWLSVVCVFLITTTTLFGCSSPEANSTPDNSSITQTSQPASSG

2606703528 hypothetical protein

MPNNNNQPDINKAIDVCMEEYKTLRTEILQTVQNRTNVIVFGGALVLTLL

2606704883 hypothetical protein

MTEINPEINLMQIATILNQYRFEMRGYKAQELIERWLPIYSLNWIRMAIL

2606710070 hypothetical protein

MKNNFWGLIWSSFLGIQSLVLGFLGFVASLLAWAFAGQTPIPLVVLFVII

2606712420 hypothetical protein

MLIGKYLTLEELSTCSQTYQKYAAQIDPYPKNPATITAWQNLAHFIIDPI

2606707857 Domain of unknown function (DUF3854)

MKYLEEWRDSGVDAELIDLNVTSLAGLSPSEYLLYSQELPRRNDGRVRDD

2606707111 hypothetical protein

MSNKSTIKLLIAFNDPNLEPEERDKQVQVLLTELEQADEVESVKRILDPN

2606713825 Domain of unknown function (DUF1995)

MILPNSLEETILQAKAATQLALESGARRIQVELVIPEIALQAQALALDFA

2606711676 hypothetical protein

MEQTALNLIAITVFSITLSVFVTPLLSISPFVPALATFSLLGLVTVDTLT

2606710677 Protein of unknown function (DUF3769)

MSFFLLAVEPPALINTVAEPETEERQVSENSPASVIPLPVTPAPLEAKKP

2606707859 Family of unknown function (DUF490)

MSNSPGDPSPRPNLFLSLGRFVRRPSTLIVGGITLLGITSLGYFTTRYLV

2606708943 Domain of unknown function (DUF4168)

MVIYCYPLADLGQSLRRSFIVTFLAVIAILGGLIPEFSWTTEVVSFQSSA

2606714992 Protein of unknown function (DUF3153)

MNQLTTEKIGKTRFILPFLCGIFLFLTGCVRYDVGINFPHQHHGEIIQHI

2606704360 hypothetical protein

MKKMILTLLLAWLLVANMNSQGVLAVTIDFQWLGNQGYTAKGSFSYDAKT

2606708507 Protein of unknown function (DUF455)

MSARDIMKRVISDREIHLVTLNRYRYNEQRSCKDLTELIETLDGQPQELI

2606705504 hypohetical protein

MVRNLDSPLILEDNNRPPVSQLLEFLLTKEKNTKKTKENLTFEALVGTWR

2606706220 hypothetical protein

MTAATEKDLKRLEDLIIGIANGQKAIENRLTAMESRLITMESRLITMENG

2606710266 hypothetical protein

MNLLPRTTQSRLKKIPQIPSVWEGDRRALSLSERMPRATLEPNQESGGEC

2606710545 hypothetical protein

MSGYKDSSSMRYFLAGLKPFAQPLFWLPLGIFSLALMAFWVYQQNPQWLG

2606713295 Uncharacterized conserved protein ycbX, contains MOSC and Fe-S domains

MQVTDLYIYPIKSCRGIQLSQAEVTSKGLLWDREMMLVDGKGKFITQREY

2606707880 hypothetical protein

MNTFTPHQIVSLFCKNRTLYGEVIQVIESRSALWVRPLWLIVSDNFETID

2606714815 domain of unknown function

MTVRLGKPILVTGIGITIAFTLGETLNSTLSEISSWGIFGAIALGAGIWF

2606703796 hypothetical protein

MVLQVSRVGLVCAGNKIREKSPKSPFIAVSFILSLSFALVTVAKAEEPAI

2606704043 von Willebrand factor type A domain

MTSLRQKRTRKISKPLLFGLWGAGGCLIASLFGEIFLHFALPPAITPVTP

2606705900 hypothetical protein

MDSPSENIHEETESFPKIDSSKVESKNVSPFFTTLPEEPEILENRIDYEA

2606711590 AAA domain

MIKDIEISNFRCFEHTKIEGFERVNLIGGKNNSGKTALLEAIFLYSYPYP

2606705439 Bacterial Ig-like domain

MIKTLQLQPIDKLAITLIVSLTLVIGGLIWLGNSCGNQCLLQTGPRVKEF

2606704811 Pentapeptide repeats (8 copies)

MRLLAAFDRYPDSVSLTLEPVATDSQKFDLYLTLHLQAQIQSLLGGEMKW

2606710554 GDSL-like Lipase/Acylhydrolase family

VLLEIGIIRLSLIILGVLFALGLLLEAILRIVFGFGNPLIYKADAEIGYL

2606711995 Calcineurin-like phosphoesterase

MQHSSYLLATMNKEQLLTEPFLQLPTETSIQVIWFTEFFGTGHQVRYGEK

2606713790 Predicted dehydrogenase

MSTGIAILGVGRWGVHYVRHFSQHPSAQVVAIVDPSPEKLRLCRDQFNLD

2606712075 Sulfotransferase family

MSSLFHPLCGSNLSTLLRLFLTNGGIDRPNLAPATLALAVTLARLPFSTL

2606710110 ammonium transporter (TC 1.A.11)

MKRIARKSGELVGLLPKINPVWLACVPLSAIIFVVWNTAVQAQDAKPLTP

2606710844 Glutamine synthetase type III

MSYGTRVQAISQVTDRKPLPSKIPQRLEALWATDVFTLSKMQASLPKDVF

2606710310 Aminopeptidase N

VGLLLARKGTTDRVNLSLPDKNQQVRANMSHLTIDTENNQRQPFELPGAK

2606706307 ornithine carbamoyltransferase

MVATLKGRDVLRITDMSSEEISALLNLSAQLKAGTLNPSCKKVLGLLFYK

2606708772 CO2 hydration protein

MVTIPVKSSSHPLASYIDRLTAGEALLKDTPQNLIEVVGILKSYGVVLDA

2606703600 precorrin-3B synthase

VNWLVEANACPGLFYGTAAQDGFLIRLRTPGGWLNSPQGKAIATWLEEWQ

2606706782 Manganese-stabilising protein PsbO

MRFRALLVAFLALCLGVLTACSDAPSAVLNRNELTYDEILNTGLANKCPQ

2606710287 photosystem II, DI subunit

MSTTIRTQDNLSAWERFGQWITSTNNRLYIGWFGVILIPTILTATICYII

2606704362 Photosystem II 12 kDa extrinsic protein (PsbU)

MKNLVRLLAVIALIIGSFWGKVPAQALNLTSIAFPSLPVAVLNAADAKLT

2606707913 uroporphyrin-III C-methyltransferase

MNQSESCGKVYLVGAGPGDPGLLTLKAKVLLENADIVLYDALVSPSILAM

2606707304 Radical SAM superfamily enzyme ygiQ, UPF0313 family

MRALLIYPVFPPTFWSYNKILELVDRKVLLPPLGMVTVAAILPQEWEFKL

2606706257 Heme oxygenase

MSVNLSTQLREGTKKSHTMAENVGFVKCFLKGVVEKTSYRKLVANLYFVY

2606714563 adenosylcobyric acid synthase (glutamine-hydrolysing) (EC 6.3.5.10)

MVVGTTSHAGKSFLTAALCRLFNRKGWQVTPFKGQNMALNAYVTAGGEEM

2606715092 cobaltochelatase cobN subunit (EC 6.6.1.2)

MHRIAPQAGGWTADTEGVIIIDQNPAPIVLLTMADTDIQTLAAAIDHLPD

2606711935 glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.41)

MVTTSLSLPEIARETRQASRQLAILTNEERNEALEAIAEALTANADKILE

2606714743 phycocyanin, beta subunit

MFDAFTRVVSQADARGEYLSSSQLDALSAMVADSNKRMDSVNRITSNAST

2606709879 PEP-CTERM protein-sorting domain

VLTDERFTVILVGVGYTPTLIFLTTVYKMKKIRTYTLPSAAALAVAAGAG

2606703705 Predicted component of the ribosome quality control (RQC) complex, yloA/Tae2 family, contains fibronectin-binding (FbpA) and DUF814 domains

MQSVDFTTLSATCAEIAAVWLPARLEQVYQIDRQTIALYLRTFDRKGWLI

2606706965 Tetratricopeptide repeat/TPR repeat

MEDTLPLIYVSGLLIFVIGLGIFVIVQIFKTRRVEGTFNKLQKKLQKEKG

2606709619 ribosomal protein L16, bacterial/organelle

MLSPKRTKFRKQQRGRMRGLATGGNTINFGDFALQATEPCWITSRQIEAA

2606709684 protein-(glutamine-N5) methyltransferase, release factor-specific

VSASISGQELSQWRQQAITDLEQAQLSPKEVDIFLQAVTDLDTLSLRLQS

2606710221 methionine aminopeptidase, type I

MNILTKLLFPETKTPEQVGSPRVQKSRRGIETKSAAEIVIMRQAGKIAAT

2606708916 Transglutaminase-like enzyme, putative cysteine protease

MRYHIRHQTNYFYNQPVILSPHLLRLRPKSDGGQTLREFTLKINPEPLTI

2606709127 23S rRNA G2069 N7-methylase rlmK or C1962 C5-methylase rlmI

MPDLAKIILRNHKIDAVKRFHPWIFSGAIKEMEGEVKEGAIVEVYSDRGE

2606714502 glycine dehydrogenase (decarboxylating)

MLNRSITAEKTEEFAPADLQDLLDCTDSFVNRHIGPTETEIEKMLAVLGV

2606708028 ribosome small subunit-dependent GTPase A

MVDAQLTDLYGTVVAVQANFYQVRLDSPVMGENLLCTRRARLQKIGQSVM

2606713490 Zn-dependent protease (includes SpoIVFB)

MPERFAVLLQLLTEVNGVIDNLIFLALVPNNWRRYKMNGNIRVGNLFGIP

2606705376 Serine protease, subtilisin family

MKKLLLLTLFIIGLGFALFNFTGLANRGEYQSILIDFKDDIPVSVLDEQL

2606708586 Predicted nucleotidyltransferase

MISAFVSLAIIPLKSENRSLITGDREVKKLKIELPQQIISEFCQKWQISE

2606704840 hypothetical protein

MTLRLKQIRLTNWKCYPSQNITFNLHPDRKIQIIFGNNGHGKTSLMTAIL

2606712025 dnaJ domain/TPR repeat

MKNYYEILQIPRNASNNQIKAAFRRLARQYHPDYNPNDPEAVTKFREIEQ

2606710914 Site-specific DNA-adenine methylase

MRHNNPSAANCCKPQLCGLGDQQPNQTVASQLVNLMSKIKSPLRYPGGKS

2606712921 acetaldehyde dehydrogenase (EC 1.2.1.10)/alcohol dehydrogenase AdhE (EC

1.1.1.1)

MTVTNRQELEELIQQVKKAQQQFANYSQEQVDLIFKKAALAANNARIPLA

2606707703 Predicted peroxiredoxin

MLLKSLALGSLVGLSLVTSIPVLANPSLSPVQTNEAIIAVGQQKSDLFVN

2606705787 DNA ligase, NAD-dependent

MTIPLEIQQRCQQLKTELQRASYAYYVLDDPFIPDSVYDQLYRELEDIEK

2606709279 Acyl-CoA dehydrogenase related to the alkylation response protein aidB

MKKLKQYWTAEALEKDLGDPLNPDNPLSYKRVIEIDESEEFPHEEIRWLY

2606714276 Exodeoxyribonuclease I subunit D (EC 3.1.11.1)

MIKILHLSDIHMGSGFSHGRLNPKTGLNTRFEDFMGSLSLCIDRAIATPV

2606705508 NADPH-glutathione reductase (EC 1.8.1.7)

MSYDFDLLVIGGGSGGIATARRAAEYGAKVGLAEYDRLGGTCVNRGCIPK

2606715000 Dynamin family

MTELLPQCSNLAGNVNSIIELLKGESSLRNQQDTSKIEIALQKAISPKFE

2606711191 Superfamily II DNA or RNA helicase, SNF2 family

MTTLHGNWLIGSQDSVFFLWGEQWQGKELSETKENHPFCLESGALAQFIQ

2606705714 DNA gyrase, B subunit

MTSNYGAEQIQVLEGLEPVRKRPGMYIGTTGPRGLHHLVYEVVDNSIDEA

2606713026 ATP synthase I chain

LSDPSIESPPTPETNPPSDPMREYYQLQNTLLITTLILSGLIFIPVWLFY

2606707211 NAD/NADP transhydrogenase beta subunit

MNNALTTGIELSYLLAAILFIIGLKQLSSPATARQGNFLAAIGMLIAIIA

2606709363 NAD(P)-dependent nickel-iron dehydrogenase catalytic subunit

MTKTVVIDPVTRIEGHAKISIFLDDGGEVDDVRFHVVEYRGFEKFCEGRP

2606710245 succinate dehydrogenase and fumarate reductase iron-sulfur protein

MEVYFKILRQRPNDTPYLENFTLEVEAGNTILDCLNRIKWELDGTLAFRK

2606708639 Predicted unusual protein kinase regulating ubiquinone biosynthesis, aarF/ABC1/ubiB family

VFSLPQTSQRQKEILEIVLGNGWDYMRGVLTLGKTENPQIPTPEVLKKIL

2606706636 riboflavin kinase/FMN adenylyltransferase

VWIVDSPNTRILAPSAIALGNFDGLHLGHQTVLQPIFQDDPAYPTVVSFT

2606708242 NADH dehydrogenase, FAD-containing subunit

MTEKQPRVVIIGGGFAGLYTAKALKNAPVHVTLIDKRNFHLFQPLLYQVA

2606707073 Cytochrome b5-like Heme/Steroid binding domain

MNSEQEISSVQNVQTPVPSPNQSGMMPSLPPVDANRGAPHNQGGVSPFMP

2606711797 NADH dehydrogenase subunit D (EC 1.6.5.3)

MAKIETRTEPMVLNMGPHHPSMHGVLRLIVTLDGEDVIDCEPVIGYLHRG

2606709360 DNA methylase

MQLELFDFNNLEGFNRGTPRLVSFRDLVPEINSTSYLTHGIYYYPAKFIP

2606712405 CAAX protease self-immunity

MNKRLRVIATYPVPLRLGIFILLLLFLWLPIALPLYHFFATNANLVSILT

2606713518 DNA-methyltransferase (dcm)

MGKSAIFKPSLFGLKHSNRDFSQKETWGKNQFNSSFPASLCAYLDGKGMK

2606706649 sagB-type dehydrogenase domain

MPDQPISIAEYYHERTKYDPQTIASKSKGLDWSQQPYPFKEYKIGQTFDL

2606711379 Endonuclease, Uma2 family (restriction endonuclease fold)

MTTSLTESLTLEDFLKLPYLEDSPAWEYLHGVAIQKPMPKTRHSILQKRL

2606707524 AAA domain

MLIGFSVGNYRSFKDVVTLSMVAAEDACGNDELDKNNVFKVNQQFSLLKS

2606706408 Methyltransferase domain

LEIGVSSGGTFIRVKVPKKIAVDPCFKDNVHKKYANENSIFYEITSDSFF

2606707461 TaqI-like C-terminal specificity domain/Eco57I restriction-modification methylase

MIVNKRELKQTLNRVYLKIKPDTETIQVFQANLTRLLEQCDSKKSEEFNK

2606705091 Branched-chain amino acid aminotransferase/4-amino-4-deoxychorismate lyase

MMYWYNGELIEDERISLGIDDVGLLYGATIFTTLRIYQQSLDHPLTHWQE

2606712218 ABC-type multidrug transport system, ATPase and permease component

MASLRDILSYYRNYQKAAFLSIAASSVFEIIDLMVPYAVGQILNVLSDQP

2606710624 putative bicarbonate transporter, ictB family

MVTTATKNPMNALWKQITLLNFSPASWSKYSYLHRLVGLFSQWRQGSRFV

2606714580 pilus retraction protein pilT

MELMIEDLMEQLVEMGGSDMHIQSGAPVYFRISGKLSPINEESLTPQDCQ

2606706245 Hemolysin or related protein, contains CBS domains

MDNQDLLTRLILVFLLIVLNAFFVAAEFSLVSVRRTRVSQLVAAGDVAAQ

2606708873 ATPase, P-type (transporting), HAD superfamily, subfamily IC

VALAGAHFVACLKLCFCLYHLLLFKMRIAEIRESALTGEANSVNKSASID

2606708848 TIGR00245 family protein

MDGLVELNLVDLGWALGMMGICLLLSRWSNLALEGQLLLATGRSILQLLV

2606708541 RND family efflux transporter, MFP subunit

MASGFLAVGVVSYRLLQTPPPALELAKMTVPAQRETLAVEIKASGRVEPI

APPENDIX D

Night groups differential expression (04:00 h compared against 22:00 h)

04:00 h transcripts

Gene number Transcript annotation Protein sequence

2606708195 CO2 hydration protein

MTGTLIKAKIPPSTHEFANIIHRLEAGGSMLPDTPENLMQIIGIYKAYAV

2606703696 Acyl transferase domain in polyketide synthase (PKS) enzymes

MSKHSISFGGQLEDNWRTLSEVLESATKHNNHGITYIKNDATEYFQSYQD

2606706583 RNase H-fold protein, predicted Holliday junction resolvase in Firmicutes and mycoplasms, involved in anti-termination at Rho-dependent terminators

MCILGFDPGKDKCGIAVRSSTGKIYTHEVIASSQAIERLASLCQQYPIEL

2606708277 Mg chelatase-related protein

MLARVWSAAIIGIDAIKIGVEVDVSGGLPGIAVVGLPDTAVQESKERVKA

2606708305 hypothetical protein

MTRQIHDQFAKEYLEELLASLGTIKKSKKVKSEVQEIDVWFEPASSASRT

2606708500 precorrin-6Y C5,15-methyltransferase (decarboxylating) (EC 2.1.1.132)

MADRNCKSKWLSIIGIGEDGLEGLSSVGRSLLSLASVIVGGERHLAMLPP

2606713180 hypothetical protein

MGVFADKFAKIMNFWAKSLIFTFFLPLPYLMPATVTGDRCSWLAQGSDVQ

2606713854 Ribosomal protein L5

MSQKLKTTYQETIVPKLKEQFGYTNIHQVPKVIKITVNRGLGEASQNAKA

2606714424 DNA-binding response regulator, narL/fixJ family, contains REC and HTH

domains

MFRNGSNNPKAKIIESSPSSAQPGKNMDKVRVIVIEDHDLTRMGLVAALQ

2606703693 Acyl transferase domain in polyketide synthase (PKS) enzymes

MDFQDKKNLSENEQSIQTRVFKALSEAKEKLKAFESQQSEPLAIVGLGCR

2606714371 Acyl transferase domain in polyketide synthase (PKS) enzymes

MLKELQKSVVEEKMLCLSVESKSKGEIFQDISTFVDILNHRALQQAEQTA

2606711580 amino acid adenylation domain

MVESIDYKKLMATTLSKMEVMQARINELETRQSESIAVVGMACRFPGGIN

2606711577 amino acid adenylation domain

MADTKNQPAKNVESIYPLSPMQEGMLFHSLYTPDSGIYCSQTLITLEGEI

2606714399 Acyl transferase domain in polyketide synthase (PKS) enzymes

MNQQKQAFLEQYKHLDAMVKAQTEPIAIVGMGCRFPAGVNDSASYWQLLV

22:00 h transcripts

2606705999 carbamoyl-phosphate synthase, large subunit

MPRRDDLQKILILGAGPIVIGQACEFDYSGTQACKALREEGYEVILVNSN

2606709974 Superfamily II RNA helicase

VKESLNPTSLDLKTIFPFKLDDFQQSAIASLAAGKSVVVCAPTGSGKTLI

2606710823 threonine ammonia-lyase, biosynthetic, long form

MADRIFIANRTDDDLFWILARLANPHRQKRIMYCDYLVQILTARVYDVAQ

2606709786 translation initiation factor IF-2

MSNAKVRIYDLSKELNLDNKDILDICDQLNIEYKSHSSTISEEDAQRIKA

2606703546 arginine decarboxylase (EC 4.1.1.19)

MAGKTQLKKQEKQLSPVDLTPSDGNNLEKVAPVKHWSIEDSENLYRIQGW

2606708199 ribonuclease, rne/rng family

MPKQIIIAEQHHIAAVFWEDQIQELVVATGNQQVADIYLGLVENVIPGID

2606704205 hypothetical protein

MISQKIPAEVTLVLDKVKKKLPGNYLLLLCLLLLLIVGGILPNLISGQWS

2606709780 valyl-tRNA synthetase (EC 6.1.1.9)

MTSELSKQYDPKITETKWQQYWENQEIFTANPEKGGETYCIVIPPPNVTG

2606708048 glucosamine--fructose-6-phosphate aminotransferase (isomerizing)

MCGIVGYIGTQTAIDILLAGLERLEYRGYDSAGIATILEGELHRVRAQGK

2606704293 Glycosyltransferase involved in cell wall bisynthesis

MNILMISSTFPYPPSKGGTQGRTFNLLKYLSKNHDITLMVQRTADVSDEE

2606706991 Cadherin domain

MAIIYVKSGSTGNGSAWNNAYGSLQSAITAAPAGDEIWVAAGTYKPTTGT

2606705506 glycogen/starch synthase, ADP-glucose type

MYIVQIASECAPVIKAGGLGDVVYGLSRELEIRGHCVELILPMYDCMRYD

2606708124 CBS domain

MSLDTPLSWAPDLEKAIDRQPLTVPPTTSLADAIAMIGQAHSRLCLLTDD

2606714400 Acyl transferase domain in polyketide synthase (PKS) enzymes

MTQIPAKTDQMSLIKDALNQIKVLKTKLHTLEKAKNEPIAIIGMSCRFPQ

2606710271 BNR repeat-like domain/Repeat domain in Vibrio, Colwellia, Bradyrhizobium

and Shewanella/Calx-beta domain/FG-GAP repeat

EAIAFTGTEPVTSLNLVASGQLIDSSNPGLAVVWQQGNLNDSDFFYTAAQ

2606712602 hypothetical protein

MFLLLPKLKSDSDVRPSDQAGKWDAQDFKTFGDVASSLDYKSPGQVKSVS

2606711195 Predicted periplasmic protein

MIMRRFIPILSLLLPWVTAAAVLAEIPTLAQEIRPKTAEQVIEQLCANLT

2606708699 Uncharacterized conserved protein, contains CHAT domain

MNEQRTQTYLNLINQLLSCNDGDEPRILQENQELLDEGLVQVMVAVAQQY

2606711803 HDIG domain

MNHFLHSFTVFPDSARKSRSYSLGKLRRPLIFLFTVGCLTSVVGYRFYNQ

2606712253 SPFH domain / Band 7 family

MNSQNNSYLIQINPNQYDNNNNNSAGNLLLNSVPVALFIFLGIGAVWFIN

2606708012 marR family

MTQNKIEGKFYALKPEEWLQVTKELRYAEVRVLYYLRTIDPFGGRDLRVK

2606708670 FO synthase subunit 2 (EC 2.5.1.77)

VTNSSITAPVTDILAKARSGANLSAKEAIILLETTDNRLIAQIRETADFL

2606705205 ATP-dependent clp protease ATP-binding subunit clpA

MFERFTEKAIKVIMLAQEEARRLGHNFVGTEQILLGLIGEGTGVAAKVLK

2606708222 preprotein translocase, secA subunit

MLKALLGDPNARKIKKFQPLVTEINLLEEDIKNLSDEELRSKTSEFKERL

2606709261 endopeptidase clp ATP-binding regulatory subunit (clpX)

MSKYDSHLKCSFCGKSQEQVRKLIAGPGVYICDECVELCNEILDEELMEP

2606709667 acetaldehyde dehydrogenase (EC 1.2.1.10)/alcohol dehydrogenase adhE (EC

1.1.1.1)

MIVTNRQELEELIQQVKKAQQQFANYNQEQVDLIFKKAALAANNARIPLA

2606711912 Serine/threonine protein kinase

MSAESRHRRLLANRYQLVELIGSGAMGQVYRAEDKLLGGVTVAVKFLSQT

2606711864 Oxidoreductase NAD-binding domain/cpcD/allophycocyanin linker domain

MYSPSTLAGNRLFVYEVAGLNQNDNTDSLDYSIRQSGSVFFTVPYSRMNQ

2606709715 Formylglycine-generating enzyme, required for sulfatase activity, contains

SUMF1/FGE domain

MKKSDIQIFLAHASEDKPAVLALHERLKQAGYKPWLDEKDLIPGQIWRDE

2606715358 Methyltransferase domain

MPLSWNEIKNRAIAFQKEWEGETSEKAESQSFWNDFFNVFGISRRRVASF

2606712462 N-6 DNA Methylase

MMTANSAYELLESAYNELDFSQGDLVSSKVSLSEITLEEWIEKGDWLELA

2606707788 Acyl transferase domain in polyketide synthase (PKS) enzymes

MDKFNCFSLSPIELEHPGLAIASIRAGGIGILDREFCQPENLERAISNLE

2606712595 Sulfate permease or related transporter, MFS superfamily MEITNRIHFDNLRGDIFGGLTAAIVSLPLALAFGVASGAGAISGLYGAVC

APPENDIX E

August 26 Light period Temperature Wind (mph) sunset 20:16 mean 80 speed 6 [SW] Visible light 14h 20m max 89 max speed 15 min 71 max gust 22

Hourly temp

SEA DEW WIND GUST TIME TEMP LEVEL VISIBILITY WIND PRECIP. WIND DATE POINT HUMIDITY SPEED SPEED CONDITIONS EDT F PRESSURE MPH DIRECTION EVENTS DIR UTC F MPH MPH IN 12:53 73.0 69.1 87 30.06 9.0 SSW 4.6 - N/A Clear 200 2014- AM 08-26 04:53:00 1:53 73.9 69.1 85 30.06 10.0 SSW 4.6 - N/A Clear 210 2014- AM 08-26 05:53:00 2:53 71.1 68.0 90 30.05 10.0 South 4.6 - N/A Clear 180 2014- AM 08-26 06:53:00 3:53 71.1 68.0 90 30.05 9.0 SSW 4.6 - N/A Clear 200 2014- AM 08-26 07:53:00 5:53 71.1 68.0 90 30.05 10.0 SSW 6.9 - N/A Clear 200 2014- AM 08-26 09:53:00 6:53 71.1 66.9 87 30.07 9.0 SSW 8.1 - N/A Clear 210 2014- AM 08-26 10:53:00 8:53 73.9 69.1 85 30.09 10.0 SW 8.1 - N/A Clear 220 2014- AM 08-26 12:53:00 9:53 78.1 69.1 74 30.10 10.0 WSW 11.5 - N/A Clear 250 2014- AM 08-26 13:53:00

11:53 84.0 70.0 63 30.10 10.0 SW 9.2 - N/A Partly Cloudy 230 2014- AM 08-26 15:53:00 12:53 86.0 68.0 55 30.08 10.0 NW 4.6 - N/A Clear 310 2014- PM 08-26 16:53:00 1:53 87.1 68.0 53 30.06 10.0 SSW 6.9 - N/A Clear 200 2014- PM 08-26 17:53:00 2:53 89.1 69.1 52 30.06 10.0 SSW 10.4 - N/A Clear 210 2014- PM 08-26 18:53:00 3:53 89.1 70.0 53 30.05 10.0 SSW 6.9 - N/A Clear 210 2014- PM 08-26 19:53:00 4:53 89.1 69.1 52 30.05 10.0 West 15.0 21.9 N/A Clear 260 2014- PM 08-26 20:53:00 5:53 84.0 68.0 58 30.05 10.0 WNW 12.7 - N/A Clear 300 2014- PM 08-26 21:53:00 6:53 82.0 70.0 67 30.04 10.0 WNW 4.6 - N/A Clear 300 2014- PM 08-26 22:53:00 7:53 80.1 69.1 69 30.03 10.0 SE 5.8 - N/A Clear 140 2014- PM 08-26 23:53:00 8:53 78.1 69.1 74 30.04 10.0 SSE 9.2 - N/A Clear 160 2014- PM 08-27 00:53:00 9:53 75.9 68.0 76 30.06 10.0 Calm Calm - N/A Clear 0 2014- PM 08-27 01:53:00 10:53 73.9 68.0 82 30.05 10.0 SSE 4.6 - N/A Clear 160 2014- PM 08-27 02:53:00 11:53 75.0 69.1 82 30.06 10.0 WSW 4.6 - N/A Clear 240 2014- PM 08-27 03:53:00

August 27 Light period temperature wind Sunset 20:15 mean 74 speed 5 Visible light 14h 17m max 81 max speed 9 min 69 max gust -

Hourly temp SEA WIND GUST WIN TIM TEM DEW LEVEL VISIBILIT WIND PRECIP HUMIDIT SPEE SPEE CONDITION D DATE E P POIN PRESSUR Y DIRECTIO . Y D D S DIR UTC EDT F T F E MPH N IN MPH MPH IN 1:53 73.0 68.0 84 30.07 10.0 NNW 5.8 - N/A Clear 340 2014- AM 08-27 05:53:0 0 2:53 72.0 66.9 84 30.07 10.0 Calm Calm - N/A Clear 0 2014- AM 08-27 06:53:0 0 3:53 71.1 66.0 84 30.07 10.0 North 3.5 - N/A Clear 350 2014- AM 08-27 07:53:0 0 4:53 70.0 64.9 84 30.07 10.0 NNW 3.5 - N/A Mostly Cloudy 330 2014- AM 08-27 08:53:0 0 5:53 70.0 64.9 84 30.08 10.0 NNW 3.5 - N/A Mostly Cloudy 340 2014- AM 08-27 09:53:0 0 7:53 71.1 66.0 84 30.09 10.0 NW 4.6 - N/A Overcast 320 2014- AM 08-27 11:53:0

0 8:53 73.0 66.9 81 30.10 10.0 North 3.5 - N/A Mostly Cloudy 360 2014- AM 08-27 12:53:0 0 9:53 73.0 66.0 79 30.10 10.0 NE 8.1 - N/A Overcast 40 2014- AM 08-27 13:53:0 0 11:53 75.9 64.9 69 30.11 10.0 NE 8.1 - N/A Mostly Cloudy 50 2014- AM 08-27 15:53:0 0 12:53 77.0 64.9 66 30.11 10.0 Calm Calm - N/A Mostly Cloudy 0 2014- PM 08-27 16:53:0 0 1:53 79.0 64.9 62 30.09 10.0 East 5.8 - N/A Scattered 90 2014- PM Clouds 08-27 17:53:0 0 3:53 79.0 64.0 60 30.07 10.0 NNE 6.9 - N/A Clear 20 2014- PM 08-27 19:53:0 0 4:53 79.0 64.0 60 30.06 10.0 ENE 8.1 - N/A Clear 60 2014- PM 08-27 20:53:0 0 5:53 78.1 64.0 62 30.06 10.0 ENE 6.9 - N/A Partly Cloudy 60 2014- PM 08-27 21:53:0 0 6:53 75.0 64.0 69 30.06 10.0 ENE 3.5 - N/A Clear 70 2014- PM 08-27 22:53:0 0 7:53 73.9 63.0 68 30.06 10.0 NNE 4.6 - N/A Clear 30 2014- PM 08-27 23:53:0

0 8:53 72.0 63.0 73 30.08 10.0 NE 3.5 - N/A Clear 50 2014- PM 08-28 00:53:0 0 9:53 72.0 60.1 66 30.09 10.0 Calm Calm - N/A Clear 0 2014- PM 08-28 01:53:0 0 10:53 71.1 60.1 68 30.09 10.0 North 4.6 - N/A Partly Cloudy 10 2014- PM 08-28 02:53:0 0 11:53 71.1 60.1 68 30.10 10.0 NNE 4.6 - N/A Mostly Cloudy 30 2014- PM 08-28 03:53:0 0

August 28 Light period Temperature Wind Sunset 20:13 mean 68 speed 6 Visible light 14h 15m max 75 max speed 9 min 59 max gust -

Hourly temp SEA DEW WIND GUST TIME TEMP LEVEL VISIBILITY WIND PRECIP WIND DATE POINT HUMIDITY SPEED SPEED CONDITIONS EDT F PRESSURE MPH DIRECTION IN DIR UTC F MPH MPH IN 12:53 68.0 62.1 81 30.10 10.0 ENE 4.6 - N/A Partly Cloudy 60 2014-08- AM 28 04:53:00 1:53 66.9 61.0 81 30.10 10.0 NE 4.6 - N/A Clear 50 2014-08- AM 28 05:53:00 2:53 66.0 59.0 78 30.09 10.0 NNE 4.6 - N/A Clear 30 2014-08- AM 28 06:53:00 3:53 64.9 57.9 78 30.10 10.0 NNE 4.6 - N/A Scattered 30 2014-08- AM Clouds 28

07:53:00 4:53 64.9 57.9 78 30.12 10.0 NE 5.8 - N/A Mostly Cloudy 40 2014-08- AM 28 08:53:00 5:53 64.9 57.0 75 30.13 10.0 NE 4.6 - N/A Overcast 50 2014-08- AM 28 09:53:00 6:53 64.9 55.9 73 30.14 10.0 NE 5.8 - N/A Overcast 50 2014-08- AM 28 10:53:00 7:53 64.9 55.9 73 30.15 10.0 NE 5.8 - N/A Overcast 50 2014-08- AM 28 11:53:00 8:53 68.0 55.9 65 30.15 10.0 NE 8.1 - N/A Partly Cloudy 40 2014-08- AM 28 12:53:00 9:53 68.0 57.0 68 30.16 10.0 NE 6.9 - N/A Mostly Cloudy 40 2014-08- AM 28 13:53:00 10:53 69.1 55.9 63 30.16 10.0 ENE 8.1 - N/A Mostly Cloudy 70 2014-08- AM 28 14:53:00 11:53 69.1 55.9 63 30.16 10.0 ENE 6.9 - N/A Overcast 70 2014-08- AM 28 15:53:00 12:53 72.0 57.0 59 30.16 10.0 ENE 9.2 - N/A Overcast 70 2014-08- PM 28 16:53:00 1:53 71.1 55.9 59 30.14 10.0 NNE 5.8 - N/A Mostly Cloudy 30 2014-08- PM 28 17:53:00 2:53 73.0 55.9 55 30.14 10.0 East 3.5 - N/A Partly Cloudy 90 2014-08- PM 28 18:53:00 3:53 75.0 55.9 51 30.12 10.0 Variable 4.6 - N/A Clear 0 2014-08- PM 28 19:53:00 4:53 73.9 55.9 53 30.12 10.0 NE 8.1 - N/A Clear 50 2014-08- PM 28 20:53:00 5:53 72.0 55.0 55 30.11 10.0 East 6.9 - N/A Clear 90 2014-08- PM 28 21:53:00 6:53 70.0 55.0 59 30.11 10.0 East 6.9 - N/A Clear 80 2014-08- PM 28

22:53:00 7:53 66.9 57.0 70 30.11 10.0 East 5.8 - N/A Clear 90 2014-08- PM 28 23:53:00 8:53 64.9 55.9 73 30.13 10.0 East 5.8 - N/A Clear 80 2014-08- PM 29 00:53:00 9:53 61.0 55.0 81 30.14 10.0 ESE 5.8 - N/A Clear 110 2014-08- PM 29 01:53:00 10:53 60.1 54.0 80 30.14 10.0 SE 5.8 - N/A Clear 130 2014-08- PM 29 02:53:00 11:53 59.0 52.0 78 30.15 10.0 ESE 4.6 - N/A Clear 110 2014-08- PM 29 03:53:00