Secondary metabolite-assisted protection of an aerobic bacterium during anoxic stress

All bacteria must overcome nutrient limitation in their environment; a consequence of natural variability and fluctuations within their specific niche. Despite the commonality of this challenge, the strategies bacteria use to survive nutrient limitation are understudied. This applies to methane-oxidizing bacteria (methanotrophs), which use methane as their only source of carbon and energy. Methanotrophs are obligate aerobes, meaning they require oxygen to survive. However, they must also survive periods of low oxygen to obtain methane created by anaerobic communities found deeper in sediments. Rising methane emissions are fueling the rapid warming of our planet, and it is critical that we identify ways to remove methane from our atmosphere. Methanotrophs are useful tools in bioremediation because they serve as methane sinks to sequester this potent greenhouse gas. We recently discovered that a methanotroph, Methylobacter tundripaludum strain 21/22 (21/22), produces a new secondary metabolite called tundrenone. This project investigates the role that tundrenone plays in the survival of 21/22 under anoxic stress. After subjecting cultures of 21/22 to periods of oxygen deprivation, we can assess the viability of the cultures. We found that wild-type 21/22 has increased cell viability when compared with a mutant strain that does not produce tundrenone. We now hypothesize that tundrenone acts as an ionophore or extracellular electron shuttle to support 21/22'2 survival in hypoxia. Understanding the mechanism by which 21/22 survives low-oxygen conditions may enable optimization of this organism, and others, as methane-sinks and other useful environmental tools.

SECONDARY METABOLITE-ASSISTED PROTECTION OF AN AEROBIC BACTERIUM DURING ANOXIC STRESS by Victoria Medvedeva A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Department of Chemistry Approved: _ _____________________________ Matthew Sigman Chair, Department of Chemistry _______________________________ Thomas Richmond Honors Faculty Liaison _____________________________ Monisha Pasupathi Dean, Honors College __ Aaron Puri Thesis Faculty Supervisor December 2023 Copyright © 2023 All Rights Reserved ABSTRACT All bacteria must overcome nutrient limitation in their environment; a consequence of natural variability and fluctuations within their specific niche. Despite the commonality of this challenge, the strategies bacteria use to survive nutrient limitation are understudied. This applies to methane-oxidizing bacteria (methanotrophs), which use methane as their only source of carbon and energy. Methanotrophs are obligate aerobes, meaning they require oxygen to survive. However, they must also survive periods of low oxygen to obtain methane created by anaerobic communities found deeper in sediments. Rising methane emissions are fueling the rapid warming of our planet, and it is critical that we identify ways to remove methane from our atmosphere. Methanotrophs are useful tools in bioremediation because they serve as methane sinks to sequester this potent greenhouse gas. We recently discovered that a methanotroph, Methylobacter tundripaludum strain 21/22 (21/22), produces a new secondary metabolite called tundrenone. This project investigates the role that tundrenone plays in the survival of 21/22 under anoxic stress. After subjecting cultures of 21/22 to periods of oxygen deprivation, we can assess the viability of the cultures. We found that wild-type 21/22 has increased cell viability when compared with a mutant strain that does not produce tundrenone. We now hypothesize that tundrenone acts as an ionophore RU H[WUDFHOOXODU HOHFWURQ VKXWWOH WR VXSSRUW ¶V VXUYLYDO in hypoxia. Understanding the mechanism by which 21/22 survives low-oxygen conditions may enable optimization of this organism, and others, as methane-sinks and other useful environmental tools. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 5 RESULTS 7 DISCUSSION 14 CONCLUSIONS 17 REFERENCES 18 iii 1 INTRODUCTION Over a 20-year period, methane traps heat in the atmosphere 80 times more efficiently than carbon dioxide.1 Methane emissions are a significant contributor to global warming, and it is crucial to investigate methods of removing methane from the atmosphere. Methane-oxidizing bacteria (methanotrophs) use methane as their sole source of carbon and energy but require oxygen to survive. Thus, methanotrophs serve as a promising means of methane sequestration. Methanotrophs oxidize methane into methanol, and as a consequence of this unique metabolism, they are often found in diverse bacterial communities.2 Methylobacter tundripaludum sp. 21/22 (21/22) is a methanotroph isolated from the sediment of Lake Washington (Seattle, Washington, USA). In the environment, Methylobacter is a robust genus, often found dominating sediment where oxygen is limited (Figure 1).3 Figure 1: Distribution of methanotrophs and non-methanotrophic methylotrophs in methane enrichments of varying oxygen concentration (Figure adapted from Hernandez et al, PeerJ, 2015).3 2 Given this environmental dominance, and because 21/22 requires both oxygen and methane to survive, it must have means of adaptation to nutrient limitation, specifically anoxic stress. Oxygen limitation is a widespread challenge encountered by many bacteria. Thus, there are several strategies commonly used by bacteria to cope with this environmental challenge.4 One way that bacteria are able to survive low oxygen conditions is by membrane depolarization. ATP production is driven by an electrochemical gradient across the membrane. As a result, when the membrane is depolarized, the production of ATP in cellular respiration is reduced. Because oxygen is the terminal electron acceptor in this process, when ATP production slows, oxygen consumption is also reduced, which can prolong cellular viability during periods of anoxic stress.5 A membrane can be depolarized by equilibrating ion concentrations across this boundary. Depolarization can be accomplished using secondary metabolites known as ionophores, which transport ions across the lipid membrane6. Research has shown that Bacillus subtilis uses the secondary metabolite surfactin as a K+ ionophore, allowing the membrane to depolarize during periods of anoxic stress.5 This ultimately leads to increased survival of B. subtilis during these periods. Another class of secondary metabolites known to aid in bacterial survival is extracellular electron shuttles (EES). Organisms use these shuttles to carry electrons to remote or limited substrates, such as oxygen. Thus, EESs serve as a valuable resource in nutrient limitation. Phenazines are a class of EES produced by Pseudomonas aeruginosa.7 In oxygen-limited environments, P. aeruginosa uses phenazines as electron acceptors, and these reduced phenazines then diffuse from the cell and reduce distant oxygen molecules. 4 electrospray ionization mass spectrometry method that detects metal-binding compounds.10 Additionally, we found that this mutant strain does not survive as well as the wild-type (WT) strain under low oxygen conditions. :KLOH WXQGUHQRQH¶V H[DFW molecular mechanism is unclear, we hypothesize that it helps 21/22 to survive periods of anoxic stress. We predict that tundrenone may play a similar biological role in 21/22 to surfactin in B. subtilis or phenazines in P. aeuruginosa. Specifically, tundrenone may act as an ionophore that depolarizes the cell membrane, or as an extracellular electron shuttle to FRQWULEXWH WR ¶V DELOLW\ to survive periods of anoxic stress. However, this hypothesis needs to be tested more systematically to confirm tundrHQRQH¶V PROHFXODU PHFKDQLVP RI action and biological function. 2QH PHWKRG RI GHWHUPLQLQJ FHOO YLDELOLW\ LV E\ H[DPLQLQJ D FHOO FXOWXUH¶V JURZWK patterns in a growth curve.11 In a growth curve, optical density at 600 nm (OD600) is measured and recorded over time (Figure 3) to track the cell density in a culture. Figure 3: Example growth curve11 In the first stage of growth, known as the lag phase, bacteria are preparing for replication, and the OD600 remains constant. When the cells begin replicating, they enter 5 exponential growth phase. This growth continues until stationary phase, where the growth curve plateaus. Here, cells are no longer replicating, but they remain metabolically active. The time to exit lag phase is an indicator of cell viability.11 If the cells in a bacterial culture are more viable, these cells will begin to replicate at an earlier time. A second method of tracking viability is through a colony-forming units-based approach. Here, cell viability is GHWHUPLQHG E\ D FHOO¶V DELOLW\ WR IRUP D FRORQ\ XQLW on an agar plate. Thus, a cell culture that is more viable will form a greater number of colony forming units.12 METHODS Cell viability was tracked using two methods: a colony forming units-based approach, and an optical-density based approach. Time course to track cell viability by colony forming units: From a mature plate culture of bacteria, a P 0 liquid culture was made in nitrate mineral salt (NMS) medium. After growth overnight, the P0 liquid culture was diluted by a factor of 10 to make a P1 culture. Finally, after this culture was allowed to grow overnight, the optical density of the culture at 600nm (OD600 ) of this P1 culture was measured. Based on this measurement, a P2 culture was made at an OD600 of 0.05. This series of dilutions ensured homogeneity and consistency across all cultures used in the experiment. The P2 cultures prepared as described above were monitored over time to understand the differences in viability between WT and ¨mbaI mutant 21/22 cells under low-oxygen conditions over time. By growing 21/22 in a 50% (v/v) methane atmosphere, oxygen is the limiting nutrient. After the culture has reached stationary phase (oxygen limitation), the cultures remained stationary for 0-5 days in this period of oxygen deprivation. The amount of oxygen present in the culture was regularly measured using an oxygen probe to confirm 6 that oxygen was being depleted. During this interval, cells were harvested to quantify cell viability via colony counts. From the liquid culture, the optical density was normalized, and serial dilutions were performed. Then, 7µl of each dilution was spotted onto an agar plate. The cell viability was measured by the ability of a cell to form a colony on NMSagar plate after oxygen depletion. This experiment was completed with both wild-type 21/22 DQG ¨mbaI mutant. Tracking cell viability using optical density: A P1 culture was made following the same procedure described above. A P 2 culture was made at an OD600 of 0.04. These cultures were allowed to reach stationary phase and then remained stagnant in oxygen limitation for eight days. After this period of anoxic stress, the OD600 of the P2 culture was measured. Based on this measurement, three new subcultures (P3) were made at a normalized OD600 of 0.04. The OD600 of growing P3 cultures was measured twice daily to track cell growth and viability. This experiment was completed with both wild-type DQG ¨tunJ mutant. Cell viability across differing media conditions: A separate time course was conducted to determine the influence of oxygen on the survival of 21/22 ¨tunJ mutant. After two days in shaking conditions, the headspace of the P2 cultures was flushed for one minute with either air or nitrogen gas as a control. These cultures were then placed in static conditions for seven days. After this period, a P 3 subculture was made, and its growth was tracked using OD600 measurements. Two additional time course experiments were conducted with different media conditions. The first was done to compare ammonium mineral salts (AMS) medium to NMS medium. The second time course experiment compared the normally used potassium nitrate mineral salts 7 (KNMS) medium with sodium nitrate mineral salts (NaNMS) medium. The same time course procedure was followed for both experiments with the exception that the cultures remained in anoxic stress for eight days. Electrochemistry to determine electrochemical environment of cell cultures: Potentiometry experiments were performed on P2 cultures that remained in anoxic stress for seven days in an anaerobic chamber to prevent interference from ambient oxygen. Open circuit potential was measured for 10 minutes using a potentiostat, a platinum disk working electrode, and a saturated calomel electrode for both the reference and counter electrode. Measurements were obtained in triplicate for wild-type 21/22 and ¨tunJ mutant. RESULTS To ensure a robust experimental design, we confirmed that 21/22 cultures reach an oxygen-limited state upon entry into stationary phase. We used an oxygen sensor to measure the oxygen in 21/22 liquid cultures at two time points. After the time required to reach stationary phase (48 hours), the 21/22 culture vials are depleted of oxygen, and the cells are oxygen limited (Figure 4). Thus, the cells are exposed to hypoxia throughout the period where the cultures are left static on the bench. 8 Time course to track cell viability by colony forming units: Figure 4: Oxygen depletion in culture vials after 48 hours We began by using colony forming units (CFUs) on an agar plate to assess how oxygen limitation impacts the viability of 21/22 cultures. Using this method, we found that ERWK WKH ZLOG W\SH DQG ¨mbaI initially maintain comparable viability (Figure 5). Figure 5: Cell viability (CFUs/ml) of wild-type (WT) DQG ¨PED, PXWDQW on agar plates after 0, 3, and 5 days of anoxic stress. As time in oxygen-limited conditions progresses, the viability of the wild-type and ¨mbaI begins to diverge (Figure 5). The viability of the wild-type 21/22 is greater than that RI ¨mbaI mutant after three days of anoxic stress. However, after five days of anoxic stress, 9 the viability of both strains is reduced in a similar manner. This experiment could not be replicated on agar plates due to stochastic growth patterns of 21/22 on solid media. We discovered that measuring optical density of cultures is a more robust method of tracking cell viability. Thus, moving forward, all experiments were conducted using this method. Tracking cell viability using optical density: The growth of 21/22 in liquid media was more consistent, which allowed for efficient screening of various growth conditions. Measuring the optical density of 21/22 over time yields a growth curve, as in Figure 6. From these curves, we can assess the amount of time that 21/22 cells spend in lag phase before transitioning into exponential growth. Using this method, we found that the viability of wild-type 21/22 and tundrenone deficient mutant did not diverge until eight days in oxygen-limited conditions compared to the three days originally indicated by the CFUs on agar plates. Figure 6: Logarithmic growth curve of wild-type (WT) 21/22 and ¨tunJ mutant subcultures after 8 days of anoxic stress in KNMS media. After eight days in oxygen limited conditions, the ¨tunJ mutant takes a significant amount of time to enter exponential growth compared to the wild type (Figure 6). Both 10 mutant and wild type reach a similar maximum cell density. One quantitative measure of cell viability is the time for the culture to exit lag phase. This value is equal to the time at which the line defining the logarithmic cell growth intersects with a line fit at the initial optical density of the culture (Figure 3).11 In each replicate, the wild-type culture did not KDYH D ODJ SKDVH WKXV WLPH WR H[LW ODJ SKDVH ZDV ]HUR KRXUV 2Q DYHUDJH ¨tunJ mutant took 28 hours to exit lag phase (Figure 7). Thus, there is a possibility that the wild-type FHOOV DUH PRUH YLDEOH WKDQ ¨tunJ mutant cells. Figure 7: Time to exit lag phase of wild-W\SH :7 DQG ¨WXQ- PXWDQW after 8 days of anoxic stress in KNMS media. To confirm that WXQGUHQRQH¶V DFWLYLW\ ZDV FRUUHODWHG ZLWK oxygen limitation, we flushed the headspace of 21/22 cultures with air after they reached oxygen limited stationary phase. The addition of oxygen to the bacterial cultures significantly increased YLDELOLW\ DQG GHFUHDVHG WLPH WR H[LW ODJ SKDVH IRU ERWK WKH ¨tunJ mutant and the wild type (Figure 6). As a negative control, we flushed the headspace of a separate pair of wild-type DQG ¨tunJ mutant cultures with nitrogen. Surprisingly, with the addition of nitrogen, the time to exit lag phase also decreased. Due to the inconsistent growth of 21/22, we conducted only one replicate of this experiment. 11 Figure 8: Time to exit lag phase after 7 days of anoxic stress after supplementing with air (O2), nitrogen (control), or no supplement. To better understand the biological function of tundrenone, we performed the time course experiment in different media conditions. All previous experiments were conducted using KNMS growth medium. Because it is known that bacteria can use nitrate as an alternative terminal electron acceptor, we wanted to understand if the observed phenotype was maintained without an available source of nitrate. We selected AMS growth medium as it is a similar growth medium to NMS, but with ammonium chloride replacing potassium nitrate. When the experiment was completed in AMS media, the previously observed difference in viability between the wild type and the mutant was lost, and both cultures took a similar amount of time to exit lag phase (Figure 9). Again, due to the inconsistent growth of 21/22, we conducted only one replicate of this experiment. 13 Figure 10: Time to exit lag phase after 8 days of anoxic stress in NaNMS and KNMS media. Electrochemical environment of cultures: Finally, we studied the electrochemical environment of 21/22 cultures to further LQYHVWLJDWH WXQGUHQRQH¶V SRWHQWLDO WR DFW DV DQ H[WUDFHOOXODU HOHFWURQ VKXWWOH :H hypothesize that if tundrenone is transporting electrons to molecules such as oxygen, there will be a difference in reducing SRWHQWLDO EHWZHHQ WKH ZLOG W\SH DQG ¨tunJ mutant cultures. To investigate this hypothesis, we measured the open circuit potential of 21/22 cultures after 7 days of anoxic stress. We found that the wild-type culture has a greater negative reducing potential compared to the mutant (Figure 11). 14 a. b. Figure 11: a) Open circuit potential trace of wild-type (WT) 21/22 DQG ¨WXQ- mutant cultures after 7 days of anoxic stress. b) Negative reducing potential of wild-type 21/22 DQG ¨WXQ- mutant cultures after 7 days of anoxic stress. DISCUSSION The goal of this work was to determine the biological function of the natural product tundrenone, and to better understand how bacteria overcome oxygen limitation. Preliminary data indicates that tundrenone may be involved in extending the viability of 21/22 during periods of oxygen limitation. We used two methods of tracking cell viability to investigate this hypothesis: colony forming units on agar plates, and optical density measurements of liquid cultures. On agar plates, we found that wild-type cells had more &)8¶V WKDQ WXQGUHQRQH GHILFLHQW ¨mbaI mutant after five days in oxygen limitation (Figure 5). Similarly, after eight days in oxygen limitation, wild-type cultures required less time to H[LW ODJ SKDVH WKDQ ¨tunJ mutant (Figure 7). In this particular experiment, the lag time of the wild type was zero hours. However, in other experiments, the lag time of the wild type is much greater. This result may be a limitation of the frequency of OD 600 measurements, as well as an element of stochasticity in 21/2 ¶V JURZWK 15 Both mutant and wild type reach a similar maximum cell density. This may indicate that tundrenone preserves a level of metabolic activity in 21/22 cells to allow for a prompt exit from lag phase following prolonged hypoxia. In both cases, this difference may be due to a larger population of non-viable cells in the tundrenone-deficient mutant cultures. This phenotypic difference in growth patterns is consistent with the hypothesis that tundrenone LV UHVSRQVLEOH IRU WKH ZLOG W\SH¶V LQFUHDVHG YLDELOLW\ LQ DQR[LF conditions. However, to be certain that this is the case, further experiments must be completed where D ¨tunJ mutant culture is exogenously complemented with tundrenone. If this complemented culture displays increased viability, then we can be more confident that tundrenone is responsible for this phenotype. Importantly, when 21/22 cultures are provided with air after they reach stationary phase, viability is restored or increased (Figure 8). This supports the hypothesis that ¨tunJ mutant exhibits decreased viability due to a lack of oxygen. In this experiment, the addition of nitrogen gas as a control did not change the viability of the wild-type culture in a VLJQLILFDQW PDQQHU +RZHYHU QLWURJHQ LQFUHDVHG WKH YLDELOLW\ RI WKH ¨tunJ mutant culture to the same extent as the addition of air, which may have been a result of air in the nitrogen line. Replicates of this experiment must be completed to determine the validity of the results. We leveraged the fact that 21/22 can grow in several different media conditions to better understand the specific mechanism of action of tundrenone. Specifically, we identified that when grown in AMS medium, both the wild-type and ¨tunJ mutant cultures take a significant amount of time to exit lag phase, and the differential recovery between the cultures is not pronounced (Figure 9). In NMS medium, however, the wild-type exits 16 lag phase more quickly compared WR ¨tunJ mutant. It is reasonable that the phenotypic difference is lost in AMS, because this medium lacks a source of nitrate. Methanotrophs can use nitrate as an alternate terminal electron acceptor in place of molecular oxygen in the electron transport chain.13 Thus, when oxygen is limited, nitrate becomes an important alternative. This is consistent with our finding that the wild-type 21/22 exhibits decreased viability in AMS media. Replicates of this experiment must be completed to confirm this result. Furthermore, in an additional experiment designed to better understand the ionbinding potential of tundrenone, we tracked the viability of the cultures in NaNMS and KNMS. As seen in Figure 10 WKH ZLOG W\SH KDV LQFUHDVHG YLDELOLW\ FRPSDUHG WR ¨tunJ mutant in both media. However, the time to exit lag phase is significantly greater for both cultures in NaNMS. Thus, potassium may play a role in extending the viability of 21/22 in hypoxic conditions. Importantly, the viability is increased in KNMS for both wild-type 21/22 DQG ¨tunJ mutant. This suggests that the potassium is equally impacting both cultures and is not interacting specifically with tundrenone. Finally, the electrochemical experiment demonstrates the difference in negative reducing potential between wild-type DQG ¨tunJ mutant (Figure 11). Specifically, the wild type has a greater reducing potential. This indicates that the wild-type culture contains molecules, such as tundrenone, that can be readily oxidized or have potential to reduce other molecules. This supports the hypothesis that tundrenone may act as an electron shuttle, similar to phenazines. Further experiments must be conducted to determine the significance of this result in the context RI WXQGUHQRQH¶V ELRORJLFDO PHFKDQLVP RI DFWLRQ 17 For example, we can test the impact of adding pure tundrenone to a culture during an open circuit voltammetry measurement. CONCLUSIONS The experiments conducted in this work support the hypothesis that Methylobacter tundripaludum 21/22 uses the secondary metabolite tundrenone to survive periods of anoxic stress. We were able to show that after 8 days of oxygen-limited conditions, the 21/22 ¨tunJ mutant has reduced viability compared to wild-type 21/22 that produces WXQGUHQRQH :H DOVR VKRZHG WKDW R[\JHQ LV WKH OLPLWLQJ IDFWRU LQ ¶V JURZWK DQG E\ supplementing 21/22 cultures with air, we can restore viability. 21/22 also relies on nitrate as an alternate electron acceptor, and viability decreases when 21/22 is grown in media that does not contain nitrate. Finally, we demonstrated that there is a difference in reducing potential between wild-type DQG ¨tunJ mutant. Many of these experiments must be repeated to validate these results. Moving forward, we plan to conduct experiments with pure tundrenone to confirm that it is the cause of wild-type 21/22¶V increased viability. In addition, RNA sequencing can provide insight into genes that are up or downregulated during anoxic stress. Finally, we hope to investigate the molecular mechanism of action of tundrenone with experiments such as isothermal titration calorimetry and further electrochemistry experiments. 7XQGUHQRQH¶V ELRORJLFDO IXQFWLRQ DQG PHFKDQLVP RI DFWLRQ can help us understand how bacteria overcome nutrient limitation in the environment. With this knowledge, we can optimize methanotroph growth for use in bioremediation to mitigate the impacts of global warming. 18 REFERENCES (1) Sperandio, V.; Burnham, C.-A.; Bruns, M. A.; Lennon, J. T.; Tiedje, J.; Zheng, J. Report on an American Academy of Microbiology (Academy) and The American Geophysical Union (AGU), Colloquium Held on May 31 & June 1, 2023. (2) Oshkin, I. Y.; Beck, D. A.; Lamb, A. E.; Tchesnokova, V.; Benuska, G.; McTaggart, T. L.; Kalyuzhnaya, M. G.; Dedysh, S. N.; Lidstrom, M. E.; Chistoserdova, L. Methane-Fed Microbial Microcosms Show Differential Community Dynamics and Pinpoint Taxa Involved in Communal Response. 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Microbiol. 2021, 12, 678057. https://doi.org/10.3389/fmicb.2021.678057. 21 Name of Candidate: Victoria Medvedeva Date of Submission: December 7, 2023