2
Acknowledgements
I would like to thank multiple people for their indispensible contributions to this project:
The examiner, Dr. Filipe Branco dos Santos for his motivational enthusiasm and valuable guidance as well as willingness to accept me into this research group for my project.
The daily supervisor, Hugo Pineda Hernández for teaching me countless skills essential for experimental design, performing experiments, and data processing, as well as helping me stay focused and think practically about my progress by giving me imperative advice.
The secondary examiner, Prof. Dr. Leendert Hamoen for his willingness to help asses this project.
Vera Ladislau Benavente of Systems Bioinformatics at the Vrije Universiteit Amsterdam for spending many hours helping me obtain, understand, and process HPLC data as well as for permission to use this instrument.
Henk Dekker of Mass Spectrometry of Biomacromolecules in the Swammerdam Institute of Life Sciences at the University of Amsterdam for answering my many questions, helping gather and process UV/Visible spectrometry data, as well as for permission to use the required instrument.
Dr. rer. nat. Andreas Ehlers of the Van 't Hoff Institute for Molecular Sciences at the University of Amsterdam, for considerable help in performing H1-NMR experiments as well as explanations about and permission to use this instrument.
Prof. Dr. Mark Post of the Physiology Department in the Faculty of Health, Medicine, and Life Sciences at Maastricht University for allowing me to perform experiments as a side project in his lab to attempt to optimize the growth of mammalian cells in Synechocystis hydrolysate.
Anon van Essen for spending many hours explaining in vitro cell culture and helping me perform these experiments. Eugenie Troia for performing HPLC analysis as well as for vital wisdom about HPLC data interpretation and processing. Dr. Joost Teixeira de Mattos for hours of insightful conversation and for helping inspire me to perform this research project.
Prof. Dr. Klaas Hellingwerf for answering many questions with useful insights about experimental design and data processing.
Dennis Rijnsburger for continually being available to help make my research possible in practice.
In no particular order: Théo Veaudor, Wei Du, Joeri Jongbloets, Mara Vincelli, Max Guillaume, Luna Meister, Wicher Otten, and all other members of MMP and Photanol who have spent their valuable time helping me find things and for answering questions about experimental design, data processing, microbiology, and data interpretation.
Edith Lengkeek for wise insights and reading material on microbiology.
Hood Chatham for hours of entertaining and insightful conversation about the behavior of my cultures.
Carmen de Jong for countless crucial observations and inspiring conversations, support and encouragement, as well as willingness to read and give critical feedback on my drafts.
3
Effects of Extracellular Amino Acid Supplementation on Growth, Bleaching, and
Glycogen Accumulation in the Absence of Extracellular Mineral Nitrogen Sources
in Synechocystis sp. PCC 6803
Renate Mols*
*Chemistry Master thesis, Molecular Microbial Physiology, Swammerdam Institute of Life Sciences, University of Amsterdam, Amsterdam, the Netherlands
Abstract
Synechocystis sp. PCC 6803, hereafter Synechocystis, is a non-diazotrophic species of cyanobacteria. Hydrolyzed Synechocystis cells are theorized to be applicable for use as the primary biomass source in an inexpensive and
sustainable in vitro mammalian cell culture medium. This application would require increasing intracellular glycogen to protein ratios, for example through nitrogen deprivation. Gaining a better understanding of the mechanisms driving glycogen accumulation during the nitrogen deprivation response in Synechocystis can help to achieve this. It was theorized that intracellular amino acid pool concentrations may help signal nitrogen status and thus influence the nitrogen deprivation response. The 20 encoded amino acids are all known to be taken up at various rates by this species. The presence of extracellular amino acids was therefore hypothesized to prevent intracellular amino acid pool depletion. To gain an indication of whether intracellular amino acid pool depletion may help signal nitrogen deprivation in Synechocystis, cells were provided amino acids in otherwise nitrogen limiting conditions. Batch cultures were deprived of mineral nitrogen sources both abruptly as well as gradually over time in nitrogen limiting media containing 0 and 3.53 mM nitrate, respectively, as well as all 20 encoded amino acids. In cultures grown without amino acids, ceased growth, glycogen accumulation, and bleaching were quickly observed upon measured medium nitrate depletion. Cells inoculated in medium without nitrate and with amino acids did not grow for longer than 48 hours, which was presumably when they had depleted intracellular nitrogen reservoirs or when cells were unable to continue growing due to amino acid toxicity. Those grown with amino acids and limiting nitrate concentrations of 3.53 mM ceased growth within 40-50 hours following inoculation. Approximately 150 hours later, growth resumed. It was theorized that this was a result of glutamine toxicity and emergence of a resistant mutant, possibly a neutral amino acid transporter (Nat) loss-of-function mutant and/or CS 141, as described previously in literature. After growth resumed, the putative mutant took up all amino acids for which medium concentrations over time could be reliably quantified by HPLC (alanine, asparagine, glycine, histidine, leucine, lysine, methionine, threonine, tyrosine, and valine), with the exceptions of glutamine and arginine. Cultures inoculated into media containing nitrate and amino acids continued to take up and metabolize a hitherto undetermined subset of the added amino acids as well as starting to accumulate glycogen once medium nitrate levels were depleted. However, the cultures did not visibly bleach and growth did not cease. This indicates that intracellular concentrations of one or multiple amino acids may influence regulation of phycocyanin degradation in
Synechocystis. Repetition of these experiments with extracellular amino acids supplemented separately and in non-toxic
concentrations is recommended. Transcriptome and proteome analysis in combination with intracellular free amino acid concentration determination of cultures showing irregular physiological responses to nitrate depletion could then be used to help determine the mechanism through which intracellular amino acids may help regulate nitrogen metabolism and phycocyanin degradation.
4
Introduction and Background
Cyanobacteria are photoautotrophic prokaryotes widely studied for their potential to convert carbon dioxide into organic chemicals (Vermaas, 1996; Ruffing, 2011; Wang et al., 2012; Yu et al., 2013; Wilson, 2018; Sebesta et al., 2019). To this end, they can be used as a more sustainable and potentially less expensive alternative for more common sources such as animal biomass or crude oil. Synechocystis is a widely studied non-diazotrophic model species of cyanobacteria. These cells can be hydrolyzed and subsequently used as part of a relatively inexpensive and sustainable culture medium for in vitro mammalian cell culture. This has been demonstrated specifically for bovine satellite as well as Chinese hamster ovary (CHO) cells (Tuomisto & Teixeira de Mattos, 2015; Sharma, 2013; Klaas Hellingwerf, personal communication, 2019). In vitro mammalian cell culture is widely used in the field of medical research and is also studied for its potential to yield edible meat for human consumption (Post, 2012). Mammalian cells are most often cultured in a defined basal medium with the addition of up to 20% serum, often from bovine calf (Post, 2012). Basal medium provides amino acids, vitamins, salts, and glucose (Schwartz, 2015; Aldrich Co. LCC., 2017a; Sigma-Aldrich Co. LCC., 2017b). Serum is used because many mammalian cell types, including bovine satellite cells for cultured meat production, require the addition of growth factors (proteins essential for rapid cell proliferation) and other potentially unknown serum components to grow at a workable rate (Post, 2012). One of the most commonly used mammalian cell culture media is Dulbecco’s Modified Eagle Medium (DMEM) combined with fetal bovine serum (FBS). Serum-free variants such as Essential 8 (E8), containing basal medium and a select few added components such as growth factors, are also available. Growth rates are generally significantly lower in serum-free media than in those containing serum (Post, 2012; Sharma, 2013). Both variants are quite expensive, which prevents more widespread use in medical research and hampers the potential for scale-up of in vitro meat production. Finding a less expensive and more sustainable alternative could increase the use of in vitro cell culture
in the medical field as an animal-cruelty-free and more sustainable alternative for animal testing. Synechocystis hydrolysate has been used together with the addition of small quantities of only a few additional defined components to replace FBS in the culturing of Chinese hamster ovary as well as bovine satellite cells in vitro (Sharma, 2013; Joost Teixeira de Mattos, personal communication, 2019). Growth rates up to 73% of those reached in DMEM with added FBS were achieved (Sharma, 2013; Personal communication, 2019).
Synechocystis hydrolysate is expected to be able to
provide most basal medium and serum components typically used for mammalian cell culture and thus act as a replacement for the basal medium as well (Tuomisto & Teixeira de Mattos, 2015; Schwartz, 2015; Sigma-Aldrich Co. LCC., 2017a; Sigma-Aldrich Co. LCC., 2017b).
In the supplementary information (Sup) provided for this document, the components present in serum-free culture medium E8 are listed. Those components also expected to be present in Synechocystis cells, and therefore potentially also in hydrolyzed Synechocystis cells to be used as a medium replacement, are also listed [Sup I] (Schwartz, 2015; Sigma-Aldrich Co. LCC., 2017a; Sigma-Aldrich Co. LCC., 2017b; Maarleveld et al., 2014). These include amino acids or small peptides, vitamins, salts, and glucose. This entails that the proteins as well as glycogen in the Synechocystis cells must be sufficiently hydrolyzed for uptake and metabolism by mammalian cells. Synechocystis hydrolysate can provide most necessary medium components. However, the intracellular molar ratio of protein to glycogen is typically too high (3.8-6.8 : 1) (Zavřel et al., 2017; Joseph et al., 2014) in comparison to the amino acid to glucose ratio required for an effective mammalian cell culture medium such as E8 (1.15 : 1) (Schwartz, 2015; Sigma-Aldrich Co. LCC., 2017a; Sigma-Aldrich Co. LCC., 2017b). When using the hydrolysate as a medium replacement, this problem can be solved by adding glucose from an external source. However, it may also be possible and potentially more sustainable and economically efficient, to provide sufficient glucose through an increase in the glucose content in Synechocystis hydrolysate. This can be
5 achieved through an increase in intracellular glycogen, for example through manipulation of cell genetics or a change in culturing conditions. The latter is more practical to achieve, particularly on a large scale, because maintaining genetic stability of wild-type cells over time is more easily achieved.
Synechocystis can easily be stimulated to produce a
much greater quantity of intracellular glycogen through medium nitrogen limitation (Ball & Morell, 2003; Osanai et al., 2007; Suzuki et al., 2010; Gründel et al., 2012; Xu et al., 2013; Díaz-Troya et al., 2014; Koch et al., 2019; Arisaka et al., 2019). This has been reported to drastically increase cell glycogen levels up to 70 times within 48 hours after the onset of nitrogen deprivation, or up to a maximum of 44% of the cellular dry weight (Gründel et al., 2012; Monshupanee & Incharoensakdi, 2013; Joseph et al., 2014; Klotz & Forchhammer, 2017). Since cell dry weight was shown to be approximately 0.148 mg/mL per OD730, this equates to about 0.065 mg/mL of glycogen per OD730 (Du et al., 2016). Understanding the mechanism through which
Synechocystis accumulates glycogen during nitrogen
deprivation can be of aid in manipulating intracellular glycogen levels in this manner for production purposes. A significant quantity of research has already been performed with the aim of understanding glycogen accumulation in Synechocystis and other cyanobacteria during nitrogen deprivation (Lehmann and Wöber, 1978; Osanai et al., 2007; Krasikov et al., 2012; Gründel et al., 2012; Iijima et al., 2014; Díaz-Troya et al., 2014; Damrow et al., 2016; Nakajima et al., 2017; Koch et al., 2019; Arisaka et al., 2019). Synechocystis continually and sensitively determines and responds to changes in external nitrogen availability and internal nitrogen status. It harbors an extensive nitrogen deprivation response, only in part represented by the regulation of central metabolic flows, as shown in Figure 1 (Giner-Lamia, 2017). In addition to the accumulation of glycogen, deprivation of a significant duration causes the cells to actively and rapidly degrade the majority of the protein in their phycobilisomes (PBS), chiefly phycobiliproteins allophycocyanin and phycocyanin, for use as a nitrogen source (Stevens & Paone, 1981;
Grossman et al., 1993; Baier et al., 2001; Richaud et al., 2001; Baier et al., 2014). This is referred to as chlorosis and causes cultures to visibly change in color or bleach from green or green-blue to yellow. Changes in cell phycocyanin content can be monitored through absorbance measurements at 634 nm, in a similar manner to that through which cell chlorophyll a content can be determined through absorbance measurements at 685 nm (Krasikov et al., 2012; Baier et al, 2014; Du et al., 2016; Du et al., 2018). Over a longer period of deprivation, even more compact carbon storage is achieved as glycogen is converted to poly-β--hydroxybutyrate (PHB), and cells enter a dormant state from which they can awaken rapidly upon reintroduction of a usable nitrogen source (Monshupanee & Incharoensakdi, 2013; Klotz et al., 2016; Velmurugan & Incharoensakdi, 2018; Koch et al., 2019; Arisaka et al., 2019).
Synechocystis takes up and readily metabolizes nitrate,
nitrite, ammonium, and urea as primary mineral nitrogen sources (Herrero et al., 2001; Muro-Pastor et al., 2005). All non-ammonium sources are first reduced by the cells to ammonium, which is subsequently assimilated into cellular metabolic flows by means of the glutamine synthetase/ glutamine oxoglutarate aminotransferase (GS/GOGAT) cycle, as shown in Figure 1 (Flores & Herrero, 2005; Giner-Lamia et al., 2017). The tricarboxylic acid cycle (TCA) metabolite 2-oxoglutarate (2OG) is used by Synechocystis primarily for the GS/GOGAT cycle to provide carbon for amino acid and subsequent protein production (Flores & Herrero, 2005). If no extracellular nitrogen source is present, cell ammonium levels drop and 2OG accumulates due to lack of GS/GOGAT cycle activity (Flores & Herrero, 2005). 2OG levels are known to signal nitrogen status to the cells, in large part through its ability to bind to and activate NtcA (Muro-Pastor et al., 2001). NtcA is the central regulator steering the cell response to nitrogen status, also upon nitrogen deprivation (Giner-Lamia et al., 2017). Part of its regulatory activity is displayed in Figure 1 (Giner-Lamia et al., 2017). NtcA expression is upregulated and its activity increases when intracellular 2OG accumulates (Muro-Pastor et al., 2001). This regulator is in part responsible for activation of genes
6 which encode proteins that break down phycocyanin as well as inactivation of those which encode phycocyanin production (Richaud et al., 2001; Sendersky et al., 2015). Expression of another gene, sll1961, which has not been linked to NtcA regulation, is also required for phycocyanin degradation to occur upon nitrogen
deprivation (Sato et al., 2008). NtcA is also known to aid in regulation of glycogen accumulation, for example by increasing the expression of glycogen anabolic genes (Joseph et al., 2014; Giner-Lamia et al., 2017). Thus, cell bleaching and glycogen accumulation are both at least in part regulated through 2OG accumulation.
Figure 1. This figure, taken from Giner-Lamia et al., 2017, illustrates NtcA target genes found to be involved in central carbon metabolism and nitrogen assimilation of Synechocystis. A number of genes required for the GS/GOGAT cycle, glycogen anabolism and catabolism, and the TCA cycle are all shown to be regulated by NtcA.
7 It was postulated that one or multiple intracellular amino acids may also help to signal nitrogen status, as proteins built from the amino acids are the primary product for which cells need nitrogen. It is expected that absence of such an amino acid would initiate a more prominent physiological nitrogen deprivation response and/or that its presence would decrease the extent of the response or even prevent it entirely from being initiated. Synechocystis has been shown to contain several permeases, which together allow for uptake of all 20 encoded amino acids (Labarre et al., 1987). Aspartate uptake rates are however notably slow (Labarre et al., 1987). Arginine has also been shown to be metabolized as a primary nitrogen source in the absence of more easily metabolized sources (Quintero et al., 2000). Since uptake mechanisms are in place, it is not unlikely that other amino acids can also be metabolized by the cells after consumption. It is theorized that extracellular supplementation of culture medium with amino acids can prevent free amino acid levels in the cells from being depleted despite the absence of another extracellular nitrogen source. It is as of yet unclear and certainly not trivial how medium supplementation with extracellular amino acids would affect intracellular 2OG levels.
Synechocystis is typically cultured in Blue-Green
Medium (BG-11). In batch cultures at atmospheric carbon dioxide levels, BG-11 is initially carbon limiting (Joeri Jongbloets, personal communication, 2019). This is because carbon is provided to cells in the form of bicarbonate, which is in the medium only as a result of carbon dioxide from the atmosphere gradually being dissolved to retain equilibrium with atmospheric levels as cells take it up. Over time, cells grown in these conditions become light limited due to shading by other cells as culture density increases. BG-11-PC was developed by van Alphen et al. and is a sulfate limiting variant of BG-11 in which batch cultures of
Synechocystis consistently reach cell densities with an
optical density at 730 nm (OD730) of 16 (van Alphen et al., 2018). In this medium, nitrogen is provided in the form of 17.4 mM sodium nitrate. The amount of sodium nitrate added can be reduced to make this medium nitrogen limiting even after substantial culture densities
are reached, as might be desirable during large-scale production.
Several amino acids - glutamine, histidine, and lysine - are known to be toxic to Synechocystis in relatively low concentrations (on solid agar plates, 1 mM, 0.64 mM, and 33 µM, respectively). This has only explicitly been shown for cultures grown in BG-11 together with an organic buffer such as TES (2-[(2-Hydroxy-1,1--bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid) or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Labarre et al., 1987; Flores & Muro-Pastor, 1990; Quintero et al., 2001). This toxicity is marked by ceased cell growth and cell death. The mechanisms driving amino acid toxicity are as of yet unknown. Several permease loss-of-function and altered function mutants of Synechocystis incapable of or slower at toxic amino acid uptake have been found to emerge from wild-type cultures following extracellular introduction of toxic quantities of lysine or glutamine (Labarre et al., 1987; Flores & Muro-Pastor, 1990; Quintero et al., 2001). For example, Can1 mutants are devoid of active transport of the basic amino acids arginine, histidine, and lysine as well as experiencing decreased glutamine uptake (Labarre et al., 1987). Their Can1 permease loss-of-function mutation was found to prevent histidine, lysine and glutamine toxicity (Labarre et al., 1987). Another transport system, Nat, has been found to facilitate glutamine transport at high throughput rates, as well as histidine transport at low rates (Quintero et al., 2001). It has been theorized that a Nat mutation could prevent glutamine toxicity at higher concentrations, as this protein is responsible for the bulk of glutamine transport whilst the Can1 protein only exhibits low rates of glutamine throughput (Quintero et al., 2001). Flores & Muro-Pastor propose a more complex network of amino acid uptake systems. 35 different naturally emerging mutants were isolated and sequenced following growth on solid BG-11 plates with 10 mM bicarbonate supplemented with either 2 mM glutamine or 2 mM lysine (Flores & Muro-Pastor, 1990). One such mutant, potentially relevant to the findings in this report, was termed CS 141 (Flores & Muro-Pastor, 1990). CS 141 showed severely inhibited glutamine and arginine uptake as well as higher lysine tolerance (at
8 least 10 mM lysine) than wild type (10-33 µM lysine) (Labarre et al., 1987; Flores & Muro-Pastor, 1990). Organic buffers such as those used during previous research demonstrating amino acid toxicity, including Tris-type buffers such as TES, have been shown to interact with amino acids in some cases (Burcham et al., 2003; Ferreira et al., 2015; Kabadi et al., 2016). Therefore, it was speculated that a product of this interaction rather than extracellular amino acid presence alone may cause ceased cell growth or cell death. One proposed mechanism of such an interaction is spontaneous TES aziridine derivative formation and the reaction of such an aziridine with amino acid functional groups. A small number of examples of this type of reaction occurring at very slow rates in practice have been illustrated (Noort et al., 2002; Burcham et al., 2003; Kabadi et al., 2016). For such a reaction to occur, highly reactive and electrophilic aziridine is first formed spontaneously in small amounts as a TES derivative in neutral or mildly acidic aqueous conditions (Kabadi et al., 2016). This electrophilic aziridine would react readily with nucleophilic groups in amino acids, forming a covalent bond in the process (Kabadi et al., 2016). The amines and carboxyl groups found in all free amino acids are nucleophilic and have the potential to react in this manner.
The research described in this report aims to give insight into the nitrogen deprivation response of
Synechocystis sp. PCC 6803. It aims particularly to
further illuminate the complex mechanisms driving glycogen accumulation upon nitrogen deprivation. It was theorized that free intracellular amino acid levels may help signal nitrogen status to affect regulation of mechanisms steering glycogen accumulation and chlorosis. Synechocystis was deprived of extracellular nitrogen sources with the exception of amino acids both immediately and gradually over time. Cells were inoculated in batch in media containing no sodium nitrate (BG-11NoN) as well as limiting nitrate (BG-11N) concentrations both with (BG-11A and BG-11NA, respectively) and without all twenty amino acids, and grown for up to 485 hours. Growth, onset of chlorosis, and cell glycogen levels were monitored. Extracellular
nitrate and amino acid concentrations were also monitored over time. All data describing OD measurements as well as nitrate and amino acid concentrations was adjusted for medium evaporation. A period of ceased nitrate uptake and ceased cell growth was observed in medium containing amino acids and abundant residual nitrate. Amino acid toxicity was presumed to be the cause. This occurred in the presence of 10 mM TES or 10 mM bicarbonate buffer and not in the presence of 50 mM bicarbonate buffer. To enable appropriate interpretation of results, it was deemed desirable to determine how resumed growth was achieved by the cells. Toxic amino acid degradation may have occurred chemically or biologically. Cell adaptation could have occurred through mutation or a change in metabolic flows. Cells were reinoculated after resumed growth in fresh medium containing amino acids to establish whether cell adaptation had occurred. Furthermore, to determine whether the presence of an organic buffer (TES) and amino acids or the amino acids alone caused temporarily ceased growth, cells were inoculated in amino acid containing BG-11 without a buffer. Reactivity between TES and a model amino acid lysine was screened for using H1-NMR (nuclear magnetic resonance) analysis.
Amino acids were found to be toxic in the concentrations added regardless of organic buffer presence. Results indicate that the most likely amino acid responsible for medium toxicity was glutamine and that resumed growth was a result of the emergence of a resistant mutant: possibly a neutral amino acid transporter (Nat) loss-of-function mutant and/or CS 141, as described previously in literature. After growth resumed, the putative mutant took up all amino acids for which medium concentrations over time could be reliably quantified by HPLC (alanine, asparagine, glycine, histidine, leucine, lysine, methionine, threonine, tyrosine, and valine), with the exceptions of glutamine and arginine. Results indicate that these cultures were likely able to metabolize a hitherto undetermined but substantial subset of the amino acids both before and after medium nitrate levels were depleted. Upon nitrate depletion, they were also found to start accumulating glycogen without visible bleaching or growth
9 retardation. This suggests that intracellular concentrations of one or multiple amino acids or related intracellular derivatives may influence the regulation of PBS degradation in Synechocystis.
Methodology
Nitrogen Limiting Medium Definition
Two experiments were run to determine how much sodium nitrate should be added to otherwise regular BG-11 such that the medium would be nitrogen limiting after growth to an OD730 between 5 and 10. Cultures were inoculated in 100 mL of medium in 300 mL Erlenmeyer flasks. During the first experiment, BG-11 was mixed with BG-11NoN such that six nitrate concentrations which were 0, 20, 40, 60, 80, and 100% of the concentration found in BG-11 were obtained. These media were termed BG-11NoN, BG-1120N, BG-1140N, BG-1160N, BG-1180N, and BG-11100N, respectively. 10 mM TES buffer was also added. Ceased cell growth, determined using OD730 measurements plotted over time, and visible chlorosis were designated as indicators of nitrogen deprivation. One biological and technical sample was used for each condition and data point. To determine whether cultures could reach a higher density before nitrogen limitation occurred in medium containing 30% of regular nitrate levels, the first experiment was repeated. Several adjustments to the methodology of the first experiment were made. This time, cultures were inoculated in triplo in 25 mL of medium in 100 mL Erlenmeyer flasks. Also, the growth media tested contained 30, 50, and 100% of nitrate levels present in regular BG-11 and are hereafter termed BG-1130N, BG-1150N, and BG-11100N, respectively. Limiting Nitrate and Concurrent Addition of Extracellular Amino Acids
Synechocystis cells were incubated in BG-11, BG-11NoN, BG-11A, BG-11N, and BG-11NA, three experiments were conducted using variants of these media. The only exception to this is omission of BG-11NoN during the first experiment, hereafter experiment 1. All three experiments were conducted using cultures inoculated
in duplo in 25 mL of medium in 100 mL Erlenmeyer flasks. In the first experiment, all media were buffered using 50 mM bicarbonate buffer. In the second and third experiments, hereafter experiments 2 and 3, 10 mM TES buffer was used. Unlike during the first experiment, cultures were washed thrice aseptically prior to inoculation in BG-11NoN prior to inoculation. Growth and chlorosis were monitored almost daily using visual observation of culture color as well as OD measurements. OD730 was used to determine change in population size over time. During experiments 2 and 3, OD685 and OD634 were measured to determine, respectively, changes in the cells’ chlorophyll a levels and phycocyanin content over time. Medium nitrate concentrations were also determined daily, and samples were taken for HPLC analysis of medium amino acid content and for glycogen assays at various points in time. During experiment 2, two replicates containing BG-11NA without cells were added to the other samples to screen for any possible chemical amino acid degradation in incubator conditions. For experiment 3, a culture which had grown in BG-11NA during experiment 2 was washed thrice aseptically with BG-11NoN and reinoculated into BG-11N and BG-11NA (with likely omission of arginine). Reinoculated cultures are also referred to as OC (old cultures).
Growth in Amino Acid Containing Media with Various Buffers
Synechocystis was inoculated in duplo in BG-11N, BG--11A, or BG-11NA containing 10 mM TES, 10 mM bicarbonate, or no buffer. The slight volume increase resulting from buffer addition was compensated for in media without a buffer through the addition of MQ (MilliQ) water. Growth was monitored through OD730 measurements almost daily and visual screening for chromophore degradation was performed.
Preinoculation Methodology, Incubator Conditions, and Screening for Contamination
During all culturing experiments, non-motile and glucose tolerant Synechocystis descended from the Williams GT Synechocystis strain was used, from the Bhaya Lab at Carnegie’s Department of Plant Biology in
10 Stanford, USA. To reduce mutations in the strain, cultures were restreaked regularly from glycerol stocks preserved at -80°C onto agar plates containing BG-11. Plates were kept outside of an incubator at room temperature behind a dark cloth, with one exception. For the experiment during which the effects of various buffers on growth was determined, plates with starter cultures were kept in a Panasonic Versatile Environmental Test Chamber MLR-350H. This incubator, kept at 30°C, was humidified and contained 2% v/v pCO2. Fluorescent white light at 50 μM photons m-2 s-1 was provided. Synechocystis was inoculated in regular BG-11 so that a substantial and zealously growing preculture could develop. Within three to ten days after inoculation, preculture cells were reinoculated into the media used in the various experiments. For several experiments, as noted in the results section, cells were washed before inoculation in BG-11NoN. Preculture was
added aseptically to 25 or 100 mL of medium in, respectively, 100 or 300 mL flasks (Thermo Fisher Scientific) with paper stoppers (VWR Stopper Cellulose). The amount of preculture added varied based on preculture density and culture medium volume, and was calculated based on the intent of achieving a starting OD730 of 0.050 after reinoculation. Variations in preculture density prior to inoculation, and therefore growth phase and rate, may have influenced initial growth after inoculation. Synechocystis was cultured in precultures as well as for experiments in a New Brunswick Scientific Innova 44 batch incubator under orange-red (632 nm) and blue (451 nm) light in a 10:1 ratio, with shaking at 120 rpm, at 30 °C, and with atmospheric carbon dioxide concentrations. During all culturing experiments, contamination of the media was regularly screened for through aseptic inoculation of 25-100 μL of cultures in Petri dishes containing LB and Agar incubated at 37 °C in the dark in an Inventum Salvis incubator.
OD Measurements
All OD measurements were performed using an Isogen Lightwave II diode array UV/Visible spectrophotometer. OD730 measurements were used to indicate population density. OD685 and OD634 measurements were used to
indicate chlorophyll a and phycocyanin content of the cells, respectively. Samples were diluted prior to measurement to obtain OD values between 0.025 and 0.800 in order for data to fit within the reliable linear portion of the absorbance calibration curve.
Medium Preparation
The formulations of the four stock solutions of BG-11 are given in Table 1. The final medium contains 17.65 mM nitrate and was made by adding 2.5 mL, each, of Stocks 1-3 to 1 L of MQ water.
Table 1. BG-11 stock solution concentrations
Stock Compound (g/L) 1 NaNO3 CaCl2·2H2O 600 14.4 2 MgSO4·7H2O FeCl3·6H2O EDTA Na2·2H2O Stock 4 30.0 1.62 2.24 400 mL 3 K2HPO4 EDTA Na2·2H2O 16.0 5.2 4 H3BO3 MnCl2·4H2O ZnSO4·7H2O Na2MoO4·2H2O CuSO4·5H2O Co(NO3)2·6H2O 2.86 1.81 0.222 0.391 0.079 0.049 (van Alphen et al., 2018)
BG-11NoN was prepared in the same fashion as BG-11, with the exception that no sodium nitrate was added to Stock 1. It should be noted that there is a minuscule amount of nitrate in this medium at a concentration of 168.4 nM due to the addition of Co(NO3)2 · 6H2O. It is not expected that this will significantly affect results, because this low amount will likely be taken up rapidly by the cells after inoculation. It should be noted that medium osmolarity is slightly lower than that of regular BG-11 as a result of sodium nitrate removal. However, the effect of this difference is expected to be insignificant, because the concentration of sodium chloride is much higher in both media.
BG-11 with various concentrations of nitrate was prepared through the volumetric combination of BG-11 with BG-11NoN. Final nitrate concentrations are given in
11 Table 2. BG-11N was prepared through the volumetric combination of BG-11 and BG-11NoN such that the final nitrate concentration in the medium was 3.53 mM, 20% of that in regular BG-11.
Table 2. BG-11 with varying nitrate concentrations Percentage of BG-11 Nitrate Concentration, % Final Nitrate Concentration, mM 0 0 20 (BG-11N) 3.53 30 5.30 40 7.06 50 8.83 60 10.59 80 14.12 100 17.65
BG-11A and BG-11NA were prepared starting with BG--11NoN or BG-11N, respectively, and adding two additional amino acid stock solutions. In the first, L-glutamic acid was dissolved in BG-11NoN, and the pH was adjusted using 6M NaOH in MQ water to 6.5, the approximate pH of BG-11 before buffer addition (van Alphen et al., 2018). In the second stock, the L-isomers of the other 19 encoded amino acids were dissolved in BG-11NoN and the pH was again adjusted to 6.5 in the same fashion. L--lysine was added in the form of L-Lysine·HCl. In this report, amino acid names are always used to describe L--isomers only. The concentrations of the various amino acids relative to one another were based on data in literature indicating typical intracellular free amino acid levels of Synechocystis (Kiyota et al., 2014). This was done because extracellular amino acids were added with the intention of replenishing intracellular free amino acids. The same total amount of nitrogen as is present in BG-11N was added in the form of amino acids. This way, growth in BG-11N and BG-11A could be more accurately compared if necessary, and sufficient extra nitrogen would be present in the media with amino acids to achieve a measurable difference in growth between cultures inoculated in 11N and BG-11NA. Amino acid concentrations in these stock solutions as well as BG-11A and BG-11NA are given in Table 3. Final media were prepared by volumetrically combining 50% of the glutamic acid stock and 20% of the stock
containing the other amino acids with 30% of BG-11NoN for BG-11A or with 20% of BG-11 and 10% of BG-11NoN for BG-11NA.
Table 3. Amino acid concentrations in stocks and media
Amino Acid Concentrations
in Stock, mM Concentrations in Media, mM L-Alanine 1.723 0.345 L-Arginine 0.259 0.052 L-Asparagine 0.174 0.035 L-Aspartic acid 2.589 0.518 L-Cysteine 0.001 0.0002 L-Glutamic acid 22.570 11.285 L-Glutamine 2.155 0.430 L-Glycine 0.433 0.087 L-Histidine 0.052 0.010 L-Isoleucine 0.088 0.018 L-Leucine 0.218 0.044 L-Lysine 0.431 0.086 L-Methionine 0.171 0.034 L-Phenylalanine 0.173 0.035 L-Proline 0.083 0.017 L-Serine 0.348 0.070 L-Threonine 0.302 0.060 L-Tryptophan 0.086 0.017 L-Tyrosine 0.431 0.086 L-Valine 0.346 0.069
UV/Visible Spectrometry to Determine Nitrate Concentrations
At least 0.1 mL of culture medium extracted from the flasks aseptically by pipette was filtered through a 0.22 µm 4mm syringe PES filter from BGB Analytik. The samples were then diluted in MQ water such that the nitrate concentration was between 10 and 50 mg/mL. For example, before considerable amounts of nitrate were taken up from the media by cells, BG-11 was diluted 50x for nitrate determination and BG-11N was diluted 25x. Dilutions were adjusted accordingly during growth and uptake. A calibration curve was made each time measurements were taken using standard samples with sodium nitrate concentrations ranging from 0 to 50 mg/L. Absorbance of the standards and samples, transferred into glass cuvettes, was measured at 220 nm (Carvalho et al., 1998) using a Varian Cary-50 dual beam UV/Visible spectrometer. Linear regression of the
12 calibration curve was performed in order to calculate nitrate concentrations of samples using on the generated regression equation. R2 values of the calibration curves ranged between 0.997 and 0.999. Glycogen Assay
The following sample preparation methodology was based on Parrou & Francois, 1997. Enough culture volume to dilute samples and obtain 5 mL with an OD730 of 1 was obtained aseptically at each sampling time point, with equal volume removal across all cultures per time point (for samples with an OD730 below 1, 5 mL was taken). Samples were centrifuged for 10 minutes at 3500 rpm (Eppendorf Centrifuge 5810 R) and supernatant was removed. 200 µL of 30% KOH was added and the cell pellet was resuspended by vortexing (Bioblock Scientific Top-Mix). Samples were hydrolyzed for 90 minutes at 100 °C under constant agitation (Grant Bio PHMT Thermoshaker PSC24N) and then cooled on ice. Glycogen was precipitated out of the solutions by adding 600 µL of cold absolute ethanol and samples were kept on ice for 2 hours. Samples were centrifuged for 5 minutes at 15,000 rpm at 4°C (VWR CT 15AE). Ethanol washing was repeated two times, and after the last washing, remaining ethanol was evaporated at 90 °C (Electrolux) for 15 minutes. The pellets were resuspended in 300 µL acetate buffer with a pH of 5.2. Then, 50 µL of amyloglucosidase dissolved in the same acetate buffer was added to each sample. Samples were mixed very gently and incubated overnight at 55 °C under constant agitation (Grant Bio PHMT Thermoshaker PSC24N). After this, they were centrifuged for 5 minutes at 21,400 g at 4 °C and stored at -20 °C until assays would be performed.
The Megazyme D-fructose and D-Glucose Microplate Assay Procedure; Residual Sugars was followed using 96-well plates (SpectraPlate). Standards and samples were diluted in buffered MQ water, as prescribed in this procedure. The only exception to this was that more MQ water volume was replaced with sample volume for samples with an OD730 below 1, at appropriate ratios to account for this. This way, the final amount of glucose added to each well was representative of glycogen in 5 mL of sample at an OD730 of 1. After sample dilution,
adenosine-5’-triphosphate (ATP) was added to phosphorylate D-glucose into glucose-6-phosphate (G-6-P), thereby turning into adenosine-5’-diphosphate (ADP). This is catalyzed by the enzyme hexokinase. Subsequently, G-6-P is oxidized to form gluconate-6--phosphate by nicotinamide-adenine dinucleotide phosphate (NADP+), which is reduced through addition of a proton and two electrons to NADPH. During this reaction, a proton is also released into solution, thereby increasing the acidity and necessitating buffer addition during sample preparation. NADPH formation increases stoichiometrically and thus linearly with the increase in glucose concentration. NADPH absorbs at 340 nm. Thus, glucose concentrations of the samples and standards can be measured through absorbance measurements at 340 nm (BMG Labtech SPECTROstar Nano plate reader). This description of the methodology was based on ‘Principle’ in the handbook Megazyme D-fructose and D-Glucose Microplate Assay Procedure; Residual Sugars provided with the Megazyme D-Fructose and D-Glucose (K-FRUGL) Assay kit. R2 values were always above 0.997. By using a linear calibration curve made using data obtained using standards and taking into account sample dilution, sample glycogen content in mg/mL per OD730 was determined.
HPLC to Determine Amino Acid Concentrations
HPLC methodology was based on Price et al., 2019. Culture samples were filtered through a 0.22 µm 4mm syringe PES filter (BGB Analytik). HPLC vials were filled with 875 μL MQ water, 25 μL of 0.1 M borate buffer at a pH of 10.2, 25 μL of 1 mM norvaline as an internal standard, and 25 μL of sample, of standard, or of MQ water for the blank. Samples and standards were derivatized for 3 min using 3 μL of fluorescent detection reagent o-phtaldialdehyde (OPA) to enable detection, by fluorescent detector RF-10AXL, of all amino acids except proline. The excitation wavelength was 350 nm and the emission wavelength was 450 nm. After this, 5 µL was injected into the Rezex (Phenomenex) column at a temperature between 40 and 85 °C with eluent containing 1.42 g/L of Na2HPO4, 3.81 g/L of Na2B4O7 · 10 H2O, and 325 mg/L of sodium azide with a pH of 8.2. The HPLC system components are listed in Table 4.
13 Table 4. Shimadzu HPLC system components
Module Type Serial number Version
Software LabSolutions v5 Degasser DGU-14A Pump A LC-20AD L20104473038 1.07 Pump B L20104473039 1.07 Autosampler SIL-10ADvp C21053970957 5.32 Oven CTO-10ASvp Fluorescence detector RF-10AXL Controller SCL-10Avp C21014071741 5.42
Peaks were automatically assigned as well as manually checked and reassigned as appropriate. Proline could not be measured by HPLC due to its lack of ability to react with the fluorescent marker OPA. Glutamate and cysteine levels in the medium could not be analyzed due to concentrations which were too high or too low respectively to be accurately quantified. Isoleucine, phenylalanine, and tryptophan concentrations could not be accurately determined due to HPLC column contamination which generated peaks at the same retention times as these amino acids. Arginine levels measured for experiment 2 were all approximately 50 mM higher than expected, also in media such as BG-11 where no amino acids were added. This was also true for BG-11NA inoculated without a culture, indicating that peak interference from an excreted compound can be ruled out as a potential explanation for this. No viable potential explanation was found. In interpreting results, it is assumed that 50 mM arginine is the approximate baseline equivalent to 0 mM of amino acid in other graphs. Lysine peaks interfered with those of column contaminants at times, thus causing lysine graphs to be less reliable.
Amino acid concentrations were derived using standard curves after data was corrected using internal standard norvaline. The standard curves were made using data
obtained by running samples in which no cells were grown, frozen immediately in -80 °C after medium formulation. Errors were calculated using the distance from the calibration curves of control measurements taken between sets of samples. The control samples were repeat injections of the standard solution used for the midway point on the calibration curve. Compensation for OPA degradation and vial refreshment was attempted by correcting data using the multiple control measurements performed throughout the experiment. Each set of data values following a control measurement was adjusted based on the difference between the peak size of the standard in the calibration curve and that of the same standard measured before the set of data following that control. Because of the high degree of uncertainty propagation generated over the various steps of this methodology, only severe changes in amino acid concentrations over time were considered during interpretation of results. Compensating for Medium Evaporation
In order to accurately determine changes in medium nitrate and amino acid concentrations as well as OD values, calculations were performed across all such data presented to compensate for medium evaporation. This was done by determining changes in the TES buffer concentration over time in a batch flask containing BG--11NA and a starting concentration of 10 mM TES, incubated under the same conditions as the cells for 473 hours. As TES buffer concentrations are expected to be stable otherwise, an increase in its concentration over time is expected to be indicative of medium evaporation. TES concentrations of the samples were determined, after addition of 5 mM PIPPS buffer as an internal standard, using an Agilent 1100 system HPLC with a multi-wave detector set to 210 nm and an Aminex HPX-87H separation column (300 x 7.8 mm) (Carpine et al., 2017). TES peak height was divided by PIPPS peak height and graphed over time after flask incubation. Evaporation rates were expected to be approximately linear because liquid surface area and incubation conditions were approximately constant. Relative peak heights were divided by the starting point peak height to determine the percentage increase in
14 TES concentration over time. These values were multiplied by 10 mM, the starting concentration, to determine the TES concentrations at the various time points. A linear regression of TES concentrations over time with the y-intercept set to be 10.0 was found to have an R2 value of 0.9328. All nitrate and amino acid concentration data, as well OD data was divided by the appropriate factor based on the corresponding time since inoculation to compensate for this increase in concentration over time due to evaporation.
Screening for TES and Lysine Reactivity using H1-NMR
Reactivity between TES buffer and lysine was screened for by determining whether new bonds would form between the compounds. This was done with lysine because it contains numerous nucleophilic groups which could react with a TES aziridine derivative and because it was the only amino acid added to growth media in concentrations reported in literature to be toxic. TES buffer and L-Lysine·HCl (hereafter lysine when describing this experiment) were dissolved individually in D2O, and H1-NMR was performed at 500 MHz using a Bruker Avance Neo. D2O was used as the lock nucleus. TES was also dissolved in combination with lysine approximately 18 hours prior to analysis. All samples were dissolved at the highest achievable concentrations after 2-3 minutes of vortexing (W & co. Vortex-Genie). Sample concentrations are given in Table 5. Individual sample spectra were compared with the combined spectrum to determine whether any detectable additional bonds were present. Such bonds would indicate reactivity between TES and lysine.
Table 5. H1-NMR Sample Concentrations
Sample Concentration, % w/v
TES L-Lysine·HCl
TES 50 -
Lysine - 50
TES and Lysine 25 25
Results
The first experiments aimed to determine which medium nitrate concentration would facilitate gradual
nitrogen deprivation over time, as nitrate is not limiting in BG-11. Hence, BG-11N was defined as containing 3.53 mM sodium nitrate. During three experiments, cells were inoculated in regular BG-11, BG-11NoN, BG-11N, BG—11A and BG-11NA. However, HPLC results indicate that the addition of arginine was likely forgotten in the third of these experiments. In experiment 1, 50 mM bicarbonate was added to each sample as a buffer. Due to expected bicarbonate uptake and dissipation, the experiment was repeated using 10 mM TES buffer. Cells grown in BG-11N and BG-11NA with 50 mM bicarbonate initially grew similarly to the positive control in BG-11 with 50 mM bicarbonate. Contrarily to this, cultures grown in BG-11NA with TES buffer were found to cease growth and turn yellow in color approximately 48 hours after inoculation. Cells resumed growth several days later. To confirm or invalidate culture adaptation to a toxin, cells which had resumed growth in BG-11NA were reinoculated in BG-11NA and BG-11N in parallel with fresh precultures. Lack of ceased growth in BG-11NA with 50 mM bicarbonate led to the design of another experiment in which growth was monitored in BG-11N, BG-11A, and BG-11NA with 10 mM bicarbonate or TES, or no buffer. An experiment was also conducted in which covalent interaction between TES buffer and lysine was screened for using H1-NMR.
Nitrogen Limiting Medium Definition
Cell growth during both experiments is indicated in Figure 2. Nitrate became limiting only in BG-11NoN and BG-1120N. In BG-11NoN, deprivation occurred at an OD730 of 0.89 approximately 139 hours after inoculation. In BG-1120N, deprivation occurred at an OD730 of 7.1, 264 hours after inoculation.
Only BG-11 with 30% of regular nitrate levels, or 5.30 mM nitrate, was nitrogen limiting. In this medium, deprivation likely occurred at an OD730 of 8.8, after 354 hours following inoculation, as indicated by a culture color change to yellow and slowed – not ceased – growth. BG-1120N, thereafter referred to as BG-11N, was chosen to be used in subsequent experiments as a nitrogen limited variant of BG-11, because it was most clearly nitrogen limiting after a substantial culture density was reached.
15
Figure 2. Growth of Synechocystis in variants of BG-11 with 10 mM TES buffer is plotted over time, in various nitrate concentrations. Percentage values given in the legend indicate what percent of the nitrate concentration present in BG-11 was added to each sample type. Concentrations corresponding to these percentage values are found in the methodology section of this report. Error bars in the top graph indicate machine error. Those in the bottom graph represent the sum of machine error and the standard deviation of data from all three replicates.
16 Limiting Nitrate and Concurrent Addition of Extracellular Amino Acids
Amino Acid Supplementation during Nitrate Limitation in Bicarbonate Buffer
Change in population density and medium sodium nitrate concentrations over time are shown in Figure 3. Cultures grown in BG-11A grew slightly during the first 48 hours of the experiment. After this, these cultures did not grow. Cultures grown in 11, 11N, and BG--11NA all started growing steadily after inoculation. Cultures grown in BG-11NA continued to grow steadily up to an OD730 of approximately 6 whilst those in
BG--11N ceased to grow after reaching an OD730 between 4 and 5, which was when medium nitrate levels had been depleted. At this point, cultures were visibly bleached. During the first 48 hours of the experiment, cultures grown in BG-11NA did not measurably take up nitrate as the cultures grown in BG-11N did. However, population size growth between the two was approximately equal. The positive controls in BG-11 stopped growing before reaching an OD730 of 4 whilst they are expected to grow up to an OD730 of 16. Results, particularly at later time points in the experiment, were thus deemed unreliable and the experiment was repeated using TES buffer in order to determine longer term behavior of
17
Figure 3. Culture density (OD730) and sodium nitrate concentration (g/L) over time (h) in BG-11, BG-11NoN, BG-11A and BG-11NA, all with 50 mM bicarbonate buffer. Error bars represent the sum of machine error and the standard deviations of averages of the data taken for biological replicates.
18
Amino Acid Supplementation and Nitrogen Deprivation with TES Buffer
Growth, Visible Bleaching, and Glycogen Content
During experiment 2, all cultures grew steadily starting after inoculation. Growth ceased after a maximum of 48 hours in BG-11NoN, BG-11A, and BG-11NA. Growth in BG--11 continued steadily without any visible chlorosis as medium nitrate was consumed and cell glycogen content increased gradually. Cultures grown in BG-11N stopped growing and showed bleaching when medium nitrate was depleted. After this point, they started to accumulate glycogen. They accumulated up to approximately 5 times the glycogen content found at the same time point in cultures grown in BG-11 (0.026±0.009 and 0.005±0.003 mg/mL per OD730, respectively), within 120 hours of medium nitrate depletion and the start of ceased growth. Cultures in BG-11NoN bleached rapidly and stopped growing within 40 hours following inoculation. Within 122 hours after they stopped growing, these cultures accumulated up to approximately 8.5 times the amount of glycogen found in those grown in regular BG-11 (0.017±0.000 and 0.002±0.001 mg/mL per OD730, respectively). Cultures grown in BG-11A ceased growth and showed bleaching within 40 hours and did not grow during the remainder of the experiment. They accumulated up to about 6 times the glycogen content of cultures grown in regular BG-11 within 69 hours after they stopped growing (0.006±0.002 and 0.001±0.000 mg/mL per OD730, respectively). Cultures grown in BG-11NA ceased growth within 40 hours following inoculation. They gradually resumed growth starting approximately 185 hours into the experiment and were growing steadily within 120 hours after this. Once growth resumed, medium nitrate was consumed at rates similar to those seen during growth in BG-11N. Once medium nitrate levels ran out,
cells continued to grow and showed no bleaching. The uncontaminated biological sample started accumulating glycogen at rates similar to those seen in BG-11N starting within 49 hours before medium nitrate depletion. By the end of the experiment and 119 hours after medium nitrate depletion, accumulation of about 1.6 times that seen in regular BG-11 had occurred (0.016 and 0.010±0.002, respectively). Data for the last five time points for cultures grown in BG-11NA during experiment 2 represent only one biological replicate due to contamination found in the other replicate. During experiment 3, contamination was found in a replicate of BG-11A as well as BG-11NA early on. The positive control in BG-11 did not grow steadily to an OD730 as is expected. Also, arginine was likely omitted from amino acid stocks added to the media. Therefore, only results describing reinoculated cultures are tentatively presented, as more reliable results for all other media were obtained during experiment 2. Cells which were taken from one of the cultures grown in BG--11NA and reinoculated into fresh BGBG--11NA (likely without arginine) did not experience a period of ceased growth, unlike fresh precultures inoculated into this medium in parallel. Cells reinoculated from BG-11NA into BG-11N immediately started to grow steadily to a slightly higher OD730 before nitrate depletion than fresh cultures inoculated into the same medium.
Cells reinoculated from BG-11NA into BG-11N showed the highest levels of glycogen accumulation observed (0.035 mg/mL per OD730) by the end of the 458 hour experiment, approximately 200 hours after medium nitrate depletion. This result only represents one biological replicate. Cells reinoculated into BG-11NA had accumulated 0.009±0.005 mg/mL per OD730 of glycogen by the time of the first measurement, 266 hours into the start of the experiment. No further accumulation was observed after this.
19
Figure 4. Culture density (OD730), medium nitrate concentration (g/L sodium nitrate), and cell glycogen content (mg/mL per OD730) are graphed over time (h) for experiments 2 and 3. Error bars indicating standard deviation are shown for all data representing more than one uncontaminated biological sample. For experiment 2, this excludes the last five time points for BG-11NA. For experiment 3, this excludes all data for BG-11A and BG-11NA as well as the second time point for all cultures in the glycogen assay.
20
Amino Acid Uptake over Time
The concentrations of all quantifiable amino acids are plotted over time in Figure 5. Only glutamine showed a decrease in medium concentration over time when incubated without cultures. In BG-11A, no considerable measurable decrease in amino acid concentrations was observed during experiment 2, when compared with the negative control where no cells were added, with the exception of glutamine. Glutamine depletion in this medium happened approximately at the same speed as in medium incubated without cells. However, large increases in medium concentrations of arginine and glycine presented themselves starting within 66 hours after inoculation. Levels of these amino acids once again decreased to quantities approximately equal to those initially added to the media within 185 hours following inoculation.
In BG-11NA to which cultures were added, all quantifiable amino acids with the exceptions of aspartate, glutamine, and arginine showed concentration decreases over time. No notable decrease in the medium aspartate concentration was
seen. Glutamine levels were depleted by the end of the experiment at similar rates to those seen in the negative control without cells. Concentration decreases of amino acids which showed decreases started around the time when culture growth resumed. All of these amino acids were entirely depleted by the end of the experiment. No serious increase in amino acid concentrations was seen in BG-11NA.
BG-11A used in experiment 3 appears to contain no added amino acids. Cells freshly inoculated in BG-11NA show uptake similar to that seen for all amino acids (except omitted arginine) in experiment 2. Within the first 100 hours following inoculation, concentrations of amino acids which were depleted from BG-11NA by the end of experiment 2 started to decrease, whereas concentrations in media containing freshly inoculated precultures did not decrease notably until after resumed growth. Glutamine levels also decreased slightly more quickly in media containing reinoculated cells than in those containing freshly inoculated cells.
21
Figure 5. Medium amino acid concentrations over time for experiments 2 and 3 have been plotted. Measurements taken for alanine, arginine, asparagine, aspartate, glutamine, glycine, histidine, leucine, lysine, methionine, threonine, tyrosine, and valine were deemed quantifiable and are shown. Error bars are too large in the graphs for aspartate to confirm any concentration change reliably. All data represents only one biological sample. Error bar calculations were performed in an attempt to estimate the extent of uncertainty propagation for each amino acid, in a manner described in the methodology section of this report.
24
Chromophore Degradation
Change in chromophore content of the cultures over time is shown in Figure 6. Due to the lack of reliability of experiment 3, only results for experiment 2 are discussed. Both chromophores show a sharp decrease in cell content within 24 hours after inoculation.
Chlorophyll a levels in cultures grown in 11 and BG--11N decreased slightly over time as culture density increased. Levels for cultures grown in BG-11N start to decrease more rapidly following medium nitrate depletion. Cultures inoculated with amino acids show decreased chlorophyll a content during ceased growth. Once growth resumed in cultures grown in BG-11NA, chlorophyll a levels rose suddenly to levels similar to those in the positive control in BG-11. When nitrate in this medium ran out, levels dropped until reaching lower levels similar to those seen for cultures grown in BG-11N. Levels in cultures grown in BG-11NoN and BG-11A are similar to those seen for cultures during stalled growth in BG-11NA and fluctuated somewhat over the
course of the experiment. These fluctuations could not be linked to any change in culture density or medium nitrate content as those parameters did not change after the first 40 hours of the experiment. However, levels reached a peak during which they were closer to positive control levels between 17 and 117 hours after inoculation.
Phycocyanin content of the cultures grown in BG-11NA and in BG-11N decreased slowly over time at similar speeds. Cultures grown in BG-11A and BG-11NA contained levels that were about half of the amounts seen in BG-11 and BG-11N grown cultures during the first 150 hours of the experiment, after which content increased gradually to levels similar to those in the positive control cultures for the remainder of the experiment. Cultures in BG-11NoN contained phycocyanin content similar to those grown in BG-11A and BG-11NA for the majority of the experiment. However, levels reached a peak during which they were similar to positive control levels between 17 and 117 hours after inoculation.
25
Figure 6. Chlorophyll a and phycocyanin content of the cells ((OD685-OD730)/OD730 and (OD634-OD730)/OD730, respectively) are plotted over time (h). Error bars indicating standard deviation are shown for all data representing more than one uncontaminated biological sample. For experiment 2, this excludes the last five time points for BG-11NA. For experiment 3, this excludes all data for BG-11A and BG-11NA as well as the second time point for all cultures in the glycogen assay.
26 Growth in Amino Acid Containing Media with Various Buffers
Growth over time is shown in Figure 7. All cells grown in BG-11N grew until reaching OD730 values around 5 or 6. At this point, cultures stopped growing and turned yellow in color. Cultures grown in BG-11A all ceased growth within 67 hours of inoculation and did not resume growth during the length of the experiment. Cultures grown in BG-11NA with TES or no buffer
experienced a period of stalled growth starting approximately 67 hours into the experiment. This lasted approximately 144 hours, after which steady growth resumed within 23 hours. Population size increased more within the first 67 hours after inoculation in BG--11NA with bicarbonate than with TES or no buffer. After this, population size decreased slowly over time until a very slight increase in population size occurred between the last two sampling points for these cultures.
Figure 7. Culture density (OD730) is plotted over time (h) for cultures grown in BG-11N, BG-11A, and BG-11NA, with 10 mM bicarbonate, 10 mM TES, or no buffer. Error bars represent standard deviation after averaging data from the various biological replicates.
27
H1-NMR to Determine TES and Nucleophile Reactivity
The spectra derived are shown and interpreted in Figure 8. All peaks found in the separate spectra of TES and
lysine were also visible in the spectrum of the combined compounds. No new peaks were found.
28
Discussion
Multiple experiments were performed with the intention of gaining an indication of whether intracellular amino acid pool depletion may help signal nitrogen deprivation in Synechocystis. Cells were provided with all 20 encoded amino acids in otherwise nitrogen limiting conditions. Several known physiological effects of the nitrogen deprivation response in Synechocystis – ceased growth, chlorosis, and glycogen accumulation – were monitored to determine whether these aspects of the deprivation response were influenced by the presence of extracellular amino acids, which were expected to be taken up to replenish intracellular pools.
During the first experiments, performed to determine which nitrate concentration would be limiting in BG-11, modified BG-11 was found to be nitrogen limiting with nitrate concentrations up to 5.30 mM, or 30% of the concentration in regular BG-11. Cultures grown in this medium reached an OD730 of 8.8 around 354 hours after inoculation. However, limitation at this concentration was marked by bleaching and only a gradual decrease in growth. Clear nitrogen limitation was observed in nitrate concentrations up to 20% of the concentration in regular BG-11, or 3.53 mM nitrate (BG-11N). The culture grown in this medium exhibited bleaching and a sudden halt in growth upon achieving an OD730 of 7.1, 264 hours after inoculation. The culture grown in BG-11 without any added sodium nitrate (BG-11NoN) grew to an OD730 of 0.89, 139 hours after inoculation. These cells were likely able to grow for some time using residual intracellular nitrate and other nitrogen reservoirs. It should also be noted that some more nitrate than is reported was present in the media because precultures were not washed prior to transfer during this experiment. To gain more accurate and reproducible results, it would be advisable to perform a repeat experiment using three replicates to test each medium variant and to wash precultures thrice aseptically in BG-11NoN prior to inoculation.
The nitrogen starvation response was observed, as indicated by depleted medium nitrate levels, ceased
growth, and visible bleaching of cultures, within 48 hours of medium nitrogen depletion, during multiple experiments in which extracellular nitrogen was limited. This was the case for all cultures grown in both BG-11N and BG-11NoN, regardless of which buffer was used or whether the media were buffered. These results are in agreement with a significant body of existing literature (Baier, 2001; Richaud, 2001; Sato, 2008; Krasikov, 2012; Klotz et al., 2016).
In all experiments, growth stopped and did not resume after the first 40-50 hours following inoculation in BG-11A, possibly after nitrate sometimes transferred from preculture medium as well as in intracellular reservoirs was depleted. This appears to indicate that cells cannot take up and metabolize the amino acids as a primary source of nitrogen. However, literature states that
Synechocystis can take up all 20 amino acids and that
they can at least metabolize arginine (Labarre et al., 1987; Flores & Muro-Pastor, 1990; Quintero et al., 2001). This information points to the existence of a more likely explanation for the lack of growth – perhaps amino acid toxicity, for example due to a disruption of metabolic flows.
When 50 mM bicarbonate was used as a buffer, cell growth in BG-11NA was steady for over 200 hours, and nitrate uptake was halted until 48 hours after inoculation. The results for this experiment are not particularly reliable because medium buffering capacity is expected to have decreased over time as bicarbonate dissipated into the atmosphere and was taken up by cells. The positive control did not reach OD730 values of 4 despite being able to grow to an OD730 of 16 in BG-11, according to literature (van Alphen et al., 2018). This may have been a result of uninhabitable alkalinity by the end of the experiment. Another possibility is that cells adapted to high carbon availability and quickly consumed all bicarbonate, which dissolves rather slowly from the atmosphere. It is possible that cells stopped growing because they were unable to adapt quickly to lower carbon availability and that carbon was limited due to prior rapid uptake. The cultures grown in BG--11NA with 50 mM bicarbonate grew to an OD730 of 6. This may be because amino acids, present at a total
