Energy flux couples sulfur isotope fractionation to proteomic and metabolite profiles in Desulfovibrio vulgaris.

Microbial sulfate reduction is central to the global carbon cycle and the redox evolution of Earth's surface. Tracking the activity of sulfate reducing microorganisms over space and time relies on a nuanced understanding of stable sulfur isotope fractionation in the context of the biochemical machinery of the metabolism. Here, we link the magnitude of stable sulfur isotopic fractionation to proteomic and metabolite profiles under different cellular energetic regimes. When energy availability is limited, cell-specific sulfate respiration rates and net sulfur isotope fractionation inversely covary. Beyond net S isotope fractionation values, we also quantified shifts in protein expression, abundances and isotopic composition of intracellular S metabolites, and lipid structures and lipid/water H isotope fractionation values. These coupled approaches reveal which protein abundances shift directly as a function of energy flux, those that vary minimally, and those that may vary independent of energy flux and likely do not contribute to shifts in S-isotope fractionation. By coupling the bulk S-isotope observations with quantitative proteomics, we provide novel constraints for metabolic isotope models. Together, these results lay the foundation for more predictive metabolic fractionation models, alongside interpretations of environmental sulfur and sulfate reducer lipid-H isotope data.

Spatial Heterogeneity in Particle-Associated, Light-Independent Superoxide Production Within Productive Coastal Waters.

In the marine environment, the reactive oxygen species (ROS) superoxide is produced through a diverse array of light-dependent and light-independent reactions, the latter of which is thought to be primarily controlled by microorganisms. Marine superoxide production influences organic matter remineralization, metal redox cycling, and dissolved oxygen concentrations, yet the relative contributions of different sources to total superoxide production remain poorly constrained. Here we investigate the production, steady-state concentration, and particle-associated nature of light-independent superoxide in productive waters off the northeast coast of North America. We find exceptionally high levels of light-independent superoxide in the marine water column, with concentrations ranging from 10 pM to in excess of 2,000 pM. The highest superoxide concentrations were particle associated in surface seawater and in aphotic seawater collected meters off the seafloor. Filtration of seawater overlying the continental shelf lowered the light-independent, steady-state superoxide concentration by an average of 84%. We identify eukaryotic phytoplankton as the dominant particle-associated source of superoxide to these coastal waters. We contrast these measurements with those collected at an off-shelf station, where superoxide concentrations did not exceed 100 pM, and particles account for an average of 40% of the steady-state superoxide concentration. This study demonstrates the primary role of particles in the production of superoxide in seawater overlying the continental shelf and highlights the importance of light-independent, dissolved-phase reactions in marine ROS production.

Dynamic Regulation of Extracellular Superoxide Production by the Coccolithophore Emiliania huxleyi (CCMP 374).

In marine waters, ubiquitous reactive oxygen species (ROS) drive biogeochemical cycling of metals and carbon. Marine phytoplankton produce the ROS superoxide (O2 -) extracellularly and can be a dominant source of O2 - in natural aquatic systems. However, the cellular regulation, biological functioning, and broader ecological impacts of extracellular O2 - production by marine phytoplankton remain mysterious. Here, we explored the regulation and potential roles of extracellular O2 - production by a noncalcifying strain of the cosmopolitan coccolithophorid Emiliania huxleyi, a key species of marine phytoplankton that has not been examined for extracellular O2 - production previously. Cell-normalized extracellular O2 - production was the highest under presumably low-stress conditions during active proliferation and inversely related to cell density during exponential growth phase. Removal of extracellular O2 - through addition of the O2 - scavenger superoxide dismutase (SOD), however, increased growth rates, growth yields, cell biovolume, and photosynthetic efficiency (Fv/Fm ) indicating an overall physiological improvement. Thus, the presence of extracellular O2 - does not directly stimulate E. huxleyi proliferation, as previously suggested for other phytoplankton, bacteria, fungi, and protists. Extracellular O2 - production decreased in the dark, suggesting a connection with photosynthetic processes. Taken together, the tight regulation of this stress independent production of extracellular O2 - by E. huxleyi suggests that it could be involved in fundamental photophysiological processes.

NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica.

Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.

Tight Regulation of Extracellular Superoxide Points to Its Vital Role in the Physiology of the Globally Relevant Roseobacter Clade.

There is a growing appreciation within animal and plant physiology that the reactive oxygen species (ROS) superoxide is not only detrimental but also essential for life. Yet, despite widespread production of extracellular superoxide by healthy bacteria and phytoplankton, this molecule remains associated with stress and death. Here, we quantify extracellular superoxide production by seven ecologically diverse bacteria within the Roseobacter clade and specifically target the link between extracellular superoxide and physiology for two species. We reveal for all species a strong inverse relationship between cell-normalized superoxide production rates and cell number. For exponentially growing cells of Ruegeria pomeroyi DSS-3 and Roseobacter sp. strain AzwK-3b, we show that superoxide levels are regulated in response to cell density through rapid modulation of gross production and not decay. Over a life cycle of batch cultures, extracellular superoxide levels are tightly regulated through a balance of both production and decay processes allowing for nearly constant levels of superoxide during active growth and minimal levels upon entering stationary phase. Further, removal of superoxide through the addition of exogenous superoxide dismutase during growth leads to significant growth inhibition. Overall, these results point to tight regulation of extracellular superoxide in representative members of the Roseobacter clade, consistent with a role for superoxide in growth regulation as widely acknowledged in fungal, animal, and plant physiology.IMPORTANCE Formation of reactive oxygen species (ROS) through partial reduction of molecular oxygen is widely associated with stress within microbial and marine systems. Nevertheless, widespread observations of the production of the ROS superoxide by healthy and actively growing marine bacteria and phytoplankton call into question the role of superoxide in the health and physiology of marine microbes. Here, we show that superoxide is produced by several marine bacteria within the widespread and abundant Roseobacter clade. Superoxide levels outside the cell are controlled via a tightly regulated balance of production and decay processes in response to cell density and life stage in batch culture. Removal of extracellular superoxide leads to substantial growth inhibition. These findings point to an essential role for superoxide in the health and growth of this ubiquitous group of microbes, and likely beyond.

Production of extracellular reactive oxygen species by phytoplankton: past and future directions.

In aquatic environments, phytoplankton represent a major source of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide. Many phytoplankton taxa also produce extracellular ROS under optimal growth conditions in culture. However, the physiological purpose of extracellular ROS production by phytoplankton and its wider significance to ecosystem-scale trophic interactions and biogeochemistry remain unclear. Here, we review the rates, taxonomic diversity, subcellular mechanisms and functions of extracellular superoxide and hydrogen peroxide production by phytoplankton with a view towards future research directions. Model eukaryotic phytoplankton and cyanobacteria produce extracellular superoxide and hydrogen peroxide at cell-normalized rates that span several orders of magnitude, both within and between taxa. The potential ecophysiological roles of extracellular ROS production are versatile and appear to be shared among diverse phytoplankton species, including ichthyotoxicity, allelopathy, growth promotion, and iron acquisition. Whereas extracellular hydrogen peroxide likely arises from a combination of intracellular and cell surface production mechanisms, extracellular superoxide is predominantly generated by specialized systems for transplasma membrane electron transport. Future insights into the molecular-level basis of extracellular ROS production, combined with existing high-sensitivity geochemical techniques for the direct quantification of ROS dynamics, will help unveil the ecophysiological and biogeochemical significance of phytoplankton-derived ROS in natural aquatic systems.

Production of extracellular superoxide and hydrogen peroxide by five marine species of harmful bloom-forming algae.

Harmful bloom-forming algae include some of the most prolific microbial producers of extracellular reactive oxygen species (ROS). However, the taxonomic diversity of ROS production, the underlying physiological mechanisms and ecophysiological roles of ROS cycling are not completely characterized among phytoplankton taxa that form harmful algal blooms (HABs). This study examines the extracellular production of the ROS superoxide and hydrogen peroxide by five marine HAB species: Chattonella marina, Heterosigma akashiwo, Karenia brevis, Pseudo-nitzschia sp. and Aureococcus anophagefferens. All species produced extracellular superoxide and hydrogen peroxide. Rates of ROS production per cell spanned several orders of magnitude and varied inversely with cell density, suggesting a potential signaling role for extracellular ROS. ROS production was also detected in the spent media of all cultures except K. brevis, indicating the presence of cell-free ROS-generating constituents, such as enzymes or metabolites, which could be further investigated as molecular targets for tracking ROS production in laboratory and field settings. Finally, ratios of superoxide to hydrogen peroxide production could not be accounted for by superoxide dismutation alone, except in the case of K. brevis, indicating a diversity of ROS production and degradation pathways that may ultimately help illuminate the functions of HAB-derived ROS.

Pharmacokinetic studies of a three-component complex that repurposes the front line antibiotic isoniazid against Mycobacterium tuberculosis.

The frontline tuberculosis (Tb) antibiotic isoniazid has been repurposed using a three component complex aimed at increasing the delivery efficiency and adding new avenues to its mechanism of action. This study focuses on pharmacokinetic studies of the isoniazid-sucrose-copper (II)-PEG-3350 complex. The assays include the Plasma Protein Binding Assay (85.8%), Caco-2 Permeability Assay (B→APapp, 0.13 × 10-6 cm/s), Cytochrome P450 Inhibition Assay (i.e. CYP2B6, IC50 = 7.26 µM), In vitro microsomal Stability Assay (t1/2 NADPH-Dependent > 240 min), and HepG2 Cytotoxicity (no toxicity). The National Cancer Institute's 60 cell line panel is used to measure activity against cancer cells. The percent growth values averaged over all 60 cell lines indicates the complex has no anti-cancer activity, which also suggests a lack of general toxicity. It also provides data for the complexes specificity against Mycobacterium tuberculosis.

Cell line studies and analytical measurements of three paclitaxel complex variations.

The copper(II) cation, sucrose, and hydroxychloroquine were complexed with the chemotherapy agent paclitaxel and studied for medicinal activity. Data (GI50, LD50) from single dose and five dose National Cancer Institute sixty cell line panels are presented. Analytical measurements of different complexes were made using Nuclear Magnetic Resonance (1H NMR), Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) and Fourier Transform-Ion Cyclotron Resonance (FT-ICR). Molecular modeling is utilized to better understand the impact that species could have on physical parameters associated with Lipinski's Rule of Five, such as logP and TPSA. On average, Cu(II) and hydroxychloroquine decreased GI50 values, while sucrose increased GI50 values of paclitaxel.

Isolation, analytical measurements, and cell line studies of the iron-bryostatin-1 complex.

Bryostatin-1 is a marine natural product that has demonstrated medicinal activity in pre-clinical and clinical trials for the treatment of cancer, Alzheimer's disease, effects of stroke, and HIV. In this study, iron-bryostatin-1 was obtained using a pharmaceutical aquaculture technique developed by our lab that cultivates marine bacteria for marine natural product extraction. Analytical measurements (1)H and (13)C NMR, mass spectrometry, and flame atomic absorption were utilized to confirm the presence of an iron-bryostatin-1 complex. The iron-bryostatin-1 complex produced was then tested against the National Cancer Institute's 60 cell line panel. Adding iron to bryostatin-1 lowered the anti-cancer efficacy of the compound.

Development of a three component complex to increase isoniazid efficacy against isoniazid resistant and nonresistant Mycobacterium tuberculosis.

The bacterium responsible for causing tuberculosis has evolved resistance to antibiotics used to treat the disease, resulting in new multidrug resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug resistant M. tuberculosis (XDR-TB) strains. Analytical techniques (1)H and (13)C Nuclear Magnetic Resonance (NMR), Fourier Transform-Ion Cyclotron Resonance with Electrospray Ionization (FT-ICR/ESI), and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-TOF-MS) were used to study different aspects of the Cu(II)-polyethylene glycol (PEG-3350)-sucrose-isoniazid and Cu(II)-polyethylene glycol (PEG3350)-glucose-isoniazid complexes. The Cu(II) cation, sucrose or glucose, and the aggregate formed by PEG primarily serve as a composite drug delivery agent for the frontline antibiotic, however the improvement in MIC values produced with the CU-PEG-SUC-INH complex suggest an additional effect. Several Cu-PEG-SUC-INH complex variations were tested against INH resistant and nonresistant strains of M. tuberculosis.