The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR): Physiological role, structure and function of a redox-driven, molecular machine.

Many bacterial processes are powered by the sodium motive force (smf) and in case of pathogens, the smf contributes to virulence. Vibrio cholerae, the causative agent of Cholera disease, possesses a Na+-translocating NADH:quinone oxidoreductase (NQR), a six-subunit membrane protein assembly. The 3D structure of NQR revealed the arrangement of the six subunits NqrABCDEF, the position of all redox cofactors (four flavins, two [2Fe-2S] centers) and the binding sites for the substrates NADH (in NqrF) and ubiquinone (in NqrB). Upon oxidation of NADH, electrons are shuttled twice across the membrane, starting with cytoplasmic FADNqrF and electron transfer to the [2Fe2S] clusterNqrF and from there to an intra-membranous [2Fe-2S] clusterNqrDE, periplasmic FMNNqrC, FMNNqrB and from there to riboflavinNqrB. This riboflavin is located at the cytoplasmic entry site of the sodium channel in NqrB, and it donates electrons to ubiquinone-8 positioned at the cytoplasmic side of NqrB. Targeting the substrate binding sites of NQR is a promising strategy to identify new inhibitors against many bacterial pathogens. Detailed structural information on the binding mode of natural inhibitors and small molecules in the active sites of NQR is now available, paving the way for the development of new antibiotics. The NQR shows different conformations as revealed in recent cryo-EM and crystallographic studies combined with spectroscopic analyses. These conformations represent distinct steps in the catalytic cycle. Considering the structural and functional data available, we propose a mechanism of Na+-NQR based on conformational coupling of electron transfer and Na+ translocation reaction steps.

Conditional essentiality of the 11-subunit complex I-like enzyme in strict anaerobes: the case of Desulfitobacterium hafniense strain DCB-2.

In oxidative phosphorylation, respiratory complex I serves as an entry point in the electron transport chain for electrons generated in catabolic processes in the form of NADH. An ancestral version of the complex, lacking the NADH-oxidising module, is encoded in a significant number of bacterial genomes. Amongst them is Desulfitobacterium hafniense, a strict anaerobe capable of conserving energy via organohalide respiration. This study investigates the role of the complex I-like enzyme in D. hafniense energy metabolism using rotenone as a specific complex I inhibitor under different growth conditions. The investigation revealed that the complex I-like enzyme was essential for growth with lactate and pyruvate but not in conditions involving H2 as an electron donor. In addition, a previously published proteomic dataset of strain DCB-2 was analysed to reveal the predominance of the complex under different growth conditions and to identify potential redox partners. This approach revealed seven candidates with expression patterns similar to Nuo homologues, suggesting the use of diverse electron sources. Based on these results, we propose a model where the complex I-like enzyme serves as an electron entry point into the respiratory chain for substrates delivering electrons within the cytoplasm, such as lactate or pyruvate, with ferredoxins shuttling electrons to the complex.

Benzene Amide Ether Scaffold is Active against Non-replicating and Intracellular Mycobacterium tuberculosis.

New drugs to treat tuberculosis which target intractable bacterial populations are required to develop shorter and more effective treatment regimens. The benzene amide ether scaffold has activity against intracellular Mycobacterium tuberculosis, but low activity against extracellular, actively replicating M. tuberculosis. We determined that these molecules have bactericidal activity against non-replicating M. tuberculosis but not actively replicating bacteria. Exposure to compounds depleted ATP levels in non-replicating bacteria and increased the oxygen consumption rate; a subset of molecules led to the accumulation of intrabacterial reactive oxygen species. A comprehensive screen of M. tuberculosis strains identified a number of under-expressing strains as more sensitive to compounds under replicating conditions including QcrA and QcrB hypomorphs. We determined the global gene expression profile after compound treatment for both replicating and nutrient-starved M. tuberculosis. We saw compound-dependent changes in the expression of genes involved in energy metabolism under both conditions. Taken together, our data suggest that the scaffold targets respiration in M. tuberculosis.

Nanoarchitectonics of nitric oxide releasing supramolecular structures for enhanced antibacterial efficacy under visible light irradiation.

Light-controlled therapies offer a promising strategy to prevent and suppress infections caused by numerous bacterial pathogens. Excitation of exogenously supplied photosensitizers (PS) at specific wavelengths elicits levels of reactive oxygen intermediates toxic to bacteria. Porphyrin-based supramolecular nanostructure frameworks (SNF) are effective PS with unique physicochemical properties that have led to their widespread use in photomedicine. Herein, we developed a nitric oxide (NO) releasing, biocompatible, and stable porphyrin-based SNF (SNF-NO), which was achieved through a confined noncovalent self-assembly process based on π-π stacking. Characterization of the SNFs via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis showed the formation of three-dimensional, well-defined octahedral structures. These SNF-NO were shown to exhibit a red shift due to the noncovalent self-assembly of porphyrins, which also show extended light absorption to broadly cover the entire visible light spectrum to enhance photodynamic therapy (PDT). Under visible light irradiation (46 J cm-2), the SNF generates high yields of singlet oxygen (1O2) radicals, hydroxyl radicals (HO), superoxide radicals (O2), and peroxynitrite (ONOO-) radicals that have shown potential to enhance antimicrobial photodynamic therapy (APDT) against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli (E. coli). The resulting SNFs also exhibit significant biofilm dispersion and a decrease in biomass production. The combination of robust photosensitizer SNFs with nitric oxide-releasing capabilities is dynamic in its ability to target pathogenic infections while remaining nontoxic to mammalian cells. The engineered SNFs have enormous potential for treating and managing microbial infections.

Bacteroides fragilis Maintains Concurrent Capability for Anaerobic and Nanaerobic Respiration.

Bacteroides species can use fumarate and oxygen as terminal electron acceptors during cellular respiration. In the human gut, oxygen diffuses from intestinal epithelial cells supplying "nanaerobic" oxygen levels. Many components of the anaerobic respiratory pathway have been determined, but such analyses have not been performed for nanaerobic respiration. Here, we present genetic, biochemical, enzymatic, and mass spectrometry analyses to elucidate the nanaerobic respiratory pathway in Bacteroides fragilis. Under anaerobic conditions, the transfer of electrons from NADH to the quinone pool has been shown to be contributed by two enzymes, NQR and NDH2. We find that the activity contributed by each under nanaerobic conditions is 77 and 23%, respectively, similar to the activity levels under anaerobic conditions. Using mass spectrometry, we show that the quinone pool also does not differ under these two conditions and consists of a mixture of menaquinone-8 to menaquinone-11, with menaquinone-10 predominant under both conditions. Analysis of fumarate reductase showed that it is synthesized and active under anaerobic and nanaerobic conditions. Previous RNA sequencing data and new transcription reporter assays show that expression of the cytochrome bd oxidase gene does not change under these conditions. Under nanaerobic conditions, we find both increased CydA protein and increased cytochrome bd activity. Reduced-minus-oxidized spectra of membranes showed the presence of heme d when the bacteria were grown in the presence of protoporphyrin IX and iron under both anaerobic and nanaerobic conditions, suggesting that the active oxidase can be assembled with or without oxygen. IMPORTANCE By performing a comprehensive analysis of nanaerobic respiration in Bacteroides fragilis, we show that this organism maintains capabilities for anaerobic respiration on fumarate and nanaerobic respiration on oxygen simultaneously. The contribution of the two NADH:quinone oxidoreductases and the composition of the quinone pool are the same under both conditions. Fumarate reductase and cytochrome bd are both present, and which of these terminal enzymes is active in electron transfer depends on the availability of the final electron acceptor: fumarate or oxygen. The synthesis of cytochrome bd and fumarate reductase under both conditions serves as an adaptation to an environment with low oxygen concentrations so that the bacteria can maximize energy conservation during fluctuating environmental conditions or occupation of different spatial niches.

Unveiling the membrane bound dihydroorotate: Quinone oxidoreductase from Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen and one of the most frequent causes for community acquired and nosocomial bacterial infections. Even so, its energy metabolism is still under explored and its respiratory enzymes have been vastly overlooked. In this work, we unveil the dihydroorotate:quinone oxidoreductase (DHOQO) from S. aureus, the first example of a DHOQO from a Gram-positive organism. This protein was shown to be a FMN containing menaquinone reducing enzyme, presenting a Michaelis-Menten behaviour towards the two substrates, which was inhibited by Brequinar, Leflunomide, Lapachol, HQNO, Atovaquone and TFFA with different degrees of effectiveness. Deletion of the DHOQO coding gene (Δdhoqo) led to lower bacterial growth rates, and effected in cell morphology and metabolism, most importantly in the pyrimidine biosynthesis, here systematized for S. aureus MW2 for the first time. This work unveils the existence of a functional DHOQO in the respiratory chain of the pathogenic bacterium S. aureus, enlarging the understanding of its energy metabolism.

Effects of physiologic activities of plankton on CO2 flux in the Three Gorges Reservoir after rainfall during algal blooms.

The nutrient supply to the freshwater system may be changed by rainfall, which also encourages the cyclic succession of microorganisms. However, in a highly dynamic land-water reservoir, the microbial metabolic changes brought on by the changes of water nutrients following rainfall are not clearly documented. The study selected the Three Gorges Reservoir (TGR) backwater region during algal bloom seasons as the study area and time, and used the Biolog-EcoPlates technique to examine the heterotrophic metabolism conditions of the water before and after rain. The field monitoring assessed how biotic and abiotic variables affected CO2 flux at the water-air interface. The tests conducted in the laboratory investigated the water-integrated metabolic process was affected by post-rainfall environmental changes. The results showed that the average flux of CO2 at the water-air interface before rainfall was -489.17 ± 506.66 mg·(m2·d)-1, while the average CO2 flux reached 393.35 ± 793.49 mg·(m2·d)-1 after rainfall. This is mostly explained by the heterotrophic metabolic variability of plankton in response to changes in the aqueous environment brought on by precipitation. These discoveries help us better understand how biological metabolisms after rain affect the CO2 flux at the water-air interface and reservoir greenhouse gas (GHG) emission equivalents can be evaluated more accurately.

Making comparable measurements of bacterial respiration and production in the subtropical coastal waters.

Accurate estimates of bacterial carbon metabolic rates are indispensable for understanding the regulation of carbon fluxes in aquatic environments. Here, changes in bacterial growth, production, and cell volume in both pre-filtered and unfiltered seawater during 24 h incubation were monitored. The methodological artifacts during Winkler bacterial respiration (BR) measurements in subtropical Hong Kong coastal waters were assessed. Bacterial abundance increased by 3- and 1.8-fold in the pre-filtered and unfiltered seawater after incubation, respectively. Bacterial production (BP) and cell volume also showed significant enhancement. Compared with the BR measurements obtained by the Winkler method, the instantaneous free-living BR measurements, after correction, decreased by ~ 70%. The time-integrated free-living BR and BP during 24 h incubation in the pre-filtered sample provided an improved estimate of bacterial growth efficiency, which increased by ~ 52% compared to the common estimations using the noncomparable measurements of integrated free-living BR and instantaneous total BP. The overestimation of BR also exaggerated the contribution of bacteria to community respiration, affecting the understanding on the metabolic state of the marine ecosystems. Furthermore, the BR estimates by the Winkler method may be more biased in environments with a higher bacterial growth rate and tightly coupled grazing mortality, as well as in those with higher nutrient concentrations. These results reveal obvious problems associated with the BR methodology and raise a warning for caution when comparing BP and BR, as well as when making estimations of carbon flow through the complex microbial networks in aquatic ecosystems. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00133-2.

A Novel Signaling Pathway Connects Thiamine Biosynthesis, Bacterial Respiration, and Production of the Exopolysaccharide Amylovoran in Erwinia amylovora.

Erwinia amylovora is a plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. This bacterium colonizes host vascular tissues via the production of exopolysaccharides (EPSs) including amylovoran. It is well-established that the nearly ubiquitous plasmid pEA29 of E. amylovora is an essential virulence factor, but the underlying mechanism remains uncharacterized. Here, we demonstrated that pEA29 was required for E. amylovora to produce amylovoran and to form a biofilm, and this regulation was dependent on the thiamine biosynthesis operon thiOSGF. We then conducted carbohydrate and genetic analyses demonstrating that the thiamine-mediated effect on amylovoran production was indirect, as cells lacking thiOSGF produced an EPS that did not contain glucuronic acid, one of the key components of amylovoran, whereas the transcriptional activity and RNA levels of the amylovoran biosynthesis genes were not altered. Alternatively, addition of exogenous thiamine restored amylovoran production in the pEA29-cured strain of E. amylovora and positively impacted amylovoran production in a dose-dependent manner. Individual deletion of several chromosomal thiamine biosynthesis genes also affected amylovoran production, implying that a complete thiamine biosynthesis pathway is required for the thiamine-mediated effect on amylovoran production in E. amylovora. Finally, we determined that an imbalanced tricarboxylic acid cycle negatively affected amylovoran production, which was restored by addition of exogenous thiamine or overexpression of the thiOSGF operon. In summary, our report revealed a novel signaling pathway that impacts E. amylovora virulence in which thiamine biosynthesis enhances bacterial respiration that provides energetic requirements for the biosynthesis of EPS amylovoran.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The impact of eutrophication towards selected bacterial process rates in tropical coastal waters.

The dissolved organic nutrient conditions and bacterial process rates at two tropical coastal sites in Peninsular Malaysia (Port Klang and Port Dickson) were initially studied in 2004-2005 period and later revisited in 2010-2011. We observed that dissolved organic nitrogen (DON) increased about two- and ten-fold at Port Klang and Port Dickson, respectively and resulted in a significant change in DOC:DON ratio (t ≥ 2.077, p < 0.05). Among the bacterial processes measured, bacterial respiration (BR) was lower in the 2010-2011 period at both stations (t ≥ 3.390, p < 0.01). BR also correlated to the DOC:DON ratio (R2 ≥ 0.259, p < 0.01). The increase in substrate quality enabled the bacteria to respire less in the dissolved organic matter degradation. As a result, the average bacterial growth efficiency increased slightly in the 2010-2011 period.

Inhibition of Tumor Progression through the Coupling of Bacterial Respiration with Tumor Metabolism.

By leveraging the ability of Shewanella oneidensis MR-1 (S. oneidensis MR-1) to anaerobically catabolize lactate through the transfer of electrons to metal minerals for respiration, a lactate-fueled biohybrid (Bac@MnO2 ) was constructed by modifying manganese dioxide (MnO2 ) nanoflowers on the S. oneidensis MR-1 surface. The biohybrid Bac@MnO2 uses decorated MnO2 nanoflowers as electron receptor and the tumor metabolite lactate as electron donor to make a complete bacterial respiration pathway at the tumor sites, which results in the continuous catabolism of intercellular lactate. Additionally, decorated MnO2 nanoflowers can also catalyze the conversion of endogenous hydrogen peroxide (H2 O2 ) into generate oxygen (O2 ), which could prevent lactate production by downregulating hypoxia-inducible factor-1α (HIF-1α) expression. As lactate plays a critical role in tumor development, the biohybrid Bac@MnO2 could significantly inhibit tumor progression by coupling bacteria respiration with tumor metabolism.

INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity.

Plankton community respiration (R) is a major component of the carbon flux in aquatic ecosystems. However, current methods to measure actual respiration from oxygen consumption at relevant spatial scales are not sensitive enough in oligotrophic environments where respiration rates are very low. To overcome this drawback, more sensitive indirect enzymatic approaches are commonly used as R proxies. The in vivo electron transport system (ETSvivo) assay, which measures the reduction of (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride salt, INT) to INT-formazan in the presence of natural substrate levels, was recently proposed as an indirect reliable estimation of R for natural plankton communities. However, under in vivo conditions, formazan salts could be toxic to the cells. Here, we test the toxicity of 0.2 mM of final INT concentration, widely used for ETSvivo assays, on natural bacterial assemblages collected in coastal and oceanic waters off Gran Canaria (Canary Islands, subtropical North Atlantic), in eight independent experiments. After 0.5 h of incubation, a significant but variable decline in cell viability (14-49%) was observed in all samples inoculated with INT. Moreover, INT also inhibited leucine uptake in less than 90 min of incubation. In the light of these results, we argue that enzymatic respiratory rates obtained with the ETSvivo method need to be interpreted with caution to derive R in oceanic regions where bacteria largely contribute to community respiration. Moreover, the variable toxicity on bacterial assemblages observed in our experiments questions the use of a single R/ETSvivo relationship as a universal proxy for regional studies.

Analysis of the strategy to intervene the progress of inflammatory bowel disease by targeting intestinal bacterial respiration and energy metabolism / 药学学报

Gut microbiota dysbiosis is closely related to a variety of host diseases. Recently, targeting the metabolic pathways of gut microbiota for the prevention and treatment of host diseases has become a frontier strategy and research hotspot. Inflammatory bowel disease (IBD) is a group of chronic progressive intestinal inflammatory diseases of unknown etiology. The relationship between IBD and gut microbiota disorders and bacterial respiratory/energy metabolism has been confirmed in recent research. This article will introduce the relationship among them, and propose a new treatment strategy to alleviate host gut inflammation by regulating gut microbiota respiration and energy metabolism based on the latest research progress. In the progression of IBD, the gut microbiota homeostasis is disturbed. The main reasons include two aspects: on the one hand, when the intestinal inflammation of the host occurs, with increasing of oxygen concentration in the intestinal cavity, facultative anaerobic bacteria, especially Enterobacteriaceae bacteria would proliferate abnormally; while the growth of absolute anaerobic bacteria such as Firmicutes is inhibited. On the other hand, intestinal inflammation by-products also support the expansion of facultative anaerobic bacteria, which ultimately exacerbates the imbalance of gut microbiota. Dysregulated intestinal flora will further disturb intestinal immune homeostasis and exacerbate intestinal inflammation. The latest research proposed the possibility that IBD can be alleviated by interfering with the respiration of bacteria, inhibiting the abnormal proliferation of bacteria, or increasing the level of "beneficial" metabolites of gut microbiota. The above studies suggest that alleviating host intestinal inflammation can be explored by focusing on the metabolic pathways of gut microbiota and regulating the intestinal bacterial respiration and energy metabolism, which is of great significance for the clinical treatment of IBD and the research of innovative drugs.

Response of Bacterial Metabolic Activity to the River Discharge in the Pearl River Estuary: Implication for CO2 Degassing Fluxes.

Bacterial production (BP), respiration (BR) and growth efficiency (BGE) were simultaneously determined along an environmental gradient in the Pearl River Estuary (PRE) in the wet season (May 2015) and the dry season (January 2016), in order to examine bacterial responses to the riverine dissolved organic carbon (DOC) in the PRE. The Pearl River discharge delivered labile dissolved organic matters (DOM) with low DOC:DON ratio, resulting in a clear gradient in DOC concentrations and DOC:DON ratios. BP (3.93-144 µg C L-1 d-1) was more variable than BR (64.6-567 µg C L-1 d-1) in terms of the percentage, along an environmental gradient in the PRE. In response to riverine DOC input, BP and the cell-specific BP increased; in contrast, the cell-specific bacterial respiration declined, likely because labile riverine DOC mitigated energetic cost for cell maintenance. Consequently, an increase in bacterial respiration was less than expected. Our findings implied that the input of highly bioavailable riverine DOC altered the carbon portioning between anabolic and catabolic pathways, consequently decreasing the fraction of DOC that bacterioplankton utilized for bacterial respiration. This might be one of the underlying mechanisms for the low CO2 degassing in the PRE receiving large amounts of sewage DOC.

Indirect link between riverine dissolved organic matter and bacterioplankton respiration in a boreal estuary.

Increasing loading of terrestrially derived dissolved organic matter tends to enhance bacterioplankton respiration (BR) in boreal estuaries, but knowledge on the mechanisms behind this effect is not complete. We determined the stable isotopic signature of the reactive estuarine dissolved organic carbon (DOC) in the Öre estuary (Baltic Sea) by using the Keeling plot method. The δ13C ratio of the estuarine labile DOC varied from -26.0‰ to -18.7‰ with most values resembling those typical for DOC of coastal phytoplanktonic origin (-18 to -24‰), while being distinctly higher than those of DOC from ter-res-trial sources (-28‰ to -27‰). Furthermore, the δ13C of the respired carbon was positively correlated to DOC concentrations, indicating that carbon of marine origin increasingly dominated the reactive substrates when input of organic matter into the estuary became higher. This suggests that riverine organic matter mainly affects BR indirectly, by providing nutrients that stimulate the production of phytoplankton-derived reactive DOC in the estuary. Thus, riverine derived DOC per se may not be as important for coastal CO2 emissions as previously thought.

Atmospheric Electricity Influencing Biogeochemical Processes in Soils and Sediments.

The Earth's subsurface represents a complex electrochemical environment that contains many electro-active chemical compounds that are relevant for a wide array of biologically driven ecosystem processes. Concentrations of many of these electro-active compounds within Earth's subsurface environments fluctuate during the day and over seasons. This has been observed for surface waters, sediments and continental soils. This variability can affect particularly small, relatively immobile organisms living in these environments. While various drivers have been identified, a comprehensive understanding of the causes and consequences of spatio-temporal variability in subsurface electrochemistry is still lacking. Here we propose that variations in atmospheric electricity (AE) can influence the electrochemical environments of soils, water bodies and their sediments, with implications that are likely relevant for a wide range of organisms and ecosystem processes. We tested this hypothesis in field and laboratory case studies. Based on measurements of subsurface redox conditions in soils and sediment, we found evidence for both local and global variation in AE with corresponding patterns in subsurface redox conditions. In the laboratory, bacterial respiratory responses, electron transport activity and H2S production were observed to be causally linked to changes in atmospheric cation concentrations. We argue that such patterns are part of an overlooked phenomenon. This recognition widens our conceptual understanding of chemical and biological processes in the Earth's subsurface and their interactions with the atmosphere and the physical environment.

Different pathways of nitrogen and phosphorus regeneration mediated by extracellular enzymes in temperate lakes under various trophic state.

Several Italian and Chinese temperate lakes with soluble reactive phosphorus concentrations < 0.015 mg L-1 were studied to estimate nitrogen and phosphorus regeneration mediated by microbial decomposition and possible different mechanisms driven by prevailing oligo- or eutrophic conditions. Leucine aminopeptidase (LAP), beta-glucosidase (GLU) and alkaline phosphatase (AP), algal, and bacterial biomass were related to trophic and environmental variables. In the eutrophic lakes, high algal and particulate organic carbon concentrations stimulated bacterial respiration (> 20 µg C L-1 h-1) and could favor the release of inorganic phosphorus. High extracellular enzyme activities and phosphorus solubilizing bacteria abundance in sediments accelerated nutrient regeneration. In these conditions, the positive GLU-AP relationship suggested the coupling of carbon and phosphorus regeneration; an efficient phosphorus regeneration and high nitrogen levels (up to 0.067 and 0.059 mg L-1 NH4 and NO3 in Italy; 0.631 and 1.496 mg L-1 NH4 and NO3 in China) led to chlorophyll a peaks of 14.9 and 258.4 µg L-1 in Italy and China, respectively, and a typical algal composition. Conversely, in the oligo-mesotrophic lakes, very low nitrogen levels (in Italy, 0.001 and 0.005 mg L-1 NH4 and NO3, respectively, versus 0.053 and 0.371 mg L-1 in China) induced high LAP, while low phosphorus (33.6 and 46.3 µg L-1 total P in Italy and China, respectively) led to high AP. In these lakes, nitrogen and phosphorus regeneration were coupled, as shown by positive LAP-AP relationship; however, the nutrient demand could not be completely met without the supply from sediments, due to low enzymatic activity and phosphorus solubilizing bacteria found in this compartment.

Effects of increasing atmospheric CO2 on the marine phytoplankton and bacterial metabolism during a bloom: A coastal mesocosm study.

Increases of atmospheric CO2 concentrations due to human activity and associated effects on aquatic ecosystems are recognized as an environmental issue at a global scale. Growing attention is being paid to CO2 enrichment effects under multiple stresses or fluctuating environmental conditions in order to extrapolate from laboratory-scale experiments to natural systems. We carried out a mesocosm experiment in coastal water with an assemblage of three model phytoplankton species and their associated bacteria under the influence of elevated CO2 concentrations. Net community production and the metabolic characteristics of the phytoplankton and bacteria were monitored to elucidate how these organisms responded to CO2 enrichment during the course of the algal bloom. We found that CO2 enrichment (1000µatm) significantly enhanced gross primary production and the ratio of photosynthesis to chlorophyll a by approximately 38% and 39%, respectively, during the early stationary phase of the algal bloom. Although there were few effects on bulk bacterial production, a significant decrease of bulk bacterial respiration (up to 31%) at elevated CO2 resulted in an increase of bacterial growth efficiency. The implication is that an elevation of CO2 concentrations leads to a reduction of bacterial carbon demand and enhances carbon transfer efficiency through the microbial loop, with a greater proportion of fixed carbon being allocated to bacterial biomass and less being lost as CO2. The contemporaneous responses of phytoplankton and bacterial metabolism to CO2 enrichment increased net community production by about 45%, an increase that would have profound implications for the carbon cycle in coastal marine ecosystems.

Regulation of the mechanism of Type-II NADH: Quinone oxidoreductase from S. aureus.

Type-II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and the only enzymes with NADH:quinone oxidoreductase activity expressed in Staphylococcus aureus (S. aureus), one of the most common causes of clinical infections. NDH-2s are members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, having a flavin adenine dinucleotide, FAD, as prosthetic group and NAD(P)H as substrate. The establishment of a Charge-Transfer Complex (CTC) between the isoalloxazine ring of the reduced flavin and the nicotinamide ring of NAD+ in NDH-2 was described, and in this work we explored its role in the kinetic mechanism using different electron donors and electron acceptors. We observed that CTC slows down the rate of the second half reaction (quinone reduction) and determines the effect of HQNO, an inhibitor. Also, protonation equilibrium simulations clearly indicate that the protonation probability of an important residue for proton transfer to the active site (D302) is influenced by the presence of the CTC. We propose that CTC is critical for the overall mechanism of NDH-2 and possibly relevant to keep a low quinol/quinone ratio and avoid excessive ROS production in vivo.

Bacterioplankton Responses to Increased Organic Carbon and Nutrient Loading in a Boreal Estuary-Separate and Interactive Effects on Growth and Respiration.

Increases in the terrestrial export of dissolved organic carbon (C) to rivers may be associated with additional loading of organic nitrogen (N) and phosphorus (P) to the coastal zone. However, little is known about how these resources interact in the regulation of heterotrophic bacterioplankton metabolism in boreal coastal ecosystems. Here, we measured changes in bacterioplankton production (BP) and respiration (BR) in response to full-factorial (C, N, and P) enrichment experiments at two sites within the Öre estuary, northern Sweden. The BR was stimulated by single C additions and further enhanced by combined additions of C and other nutrients. Single addition of N or P had no effect on BR rates. In contrast, BP was primarily limited by P at the site close to the river mouth and did not respond to C or N additions. However, at the site further away from the near the river mouth, BP was slightly stimulated by single additions of C. Possibly, the natural inflow of riverine bioavailable dissolved organic carbon induced local P limitation of BP near the river mouth, which was then exhausted and resulted in C-limited BP further away from the river mouth. We observed positive interactions between all elements on all responses except for BP at the site close to the river mouth, where P showed an independent effect. In light of predicted increases in terrestrial P and C deliveries, we expect future increases in BP and increases of BR of terrestrially delivered C substrates at the Öre estuary and similar areas.