Fine-Tuning Modulation of Oxidation-Mediated Posttranslational Control of Bradyrhizobium diazoefficiens FixK2 Transcription Factor.

FixK2 is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK2 gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK2 activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK2, we replaced C183 for aspartic acid. Purified C183D FixK2 protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK2, expression of a fixNOQP'-'lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK2 encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK2 gene expression levels and FixK2 protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK2 encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK2 in vivo.

Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors.

Nitrous oxide (N2 O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N2 O decomposition typically contain precious metals and/or operate at elevated temperatures driving a desire for more sustainable alternatives. Here we demonstrate self-assembly of liposomal microreactors enabling catalytic reduction of N2 O to the climate neutral product N2 . Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N2 O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N2 O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N2 O removal in ambient, aqueous conditions.

A bet-hedging strategy for denitrifying bacteria curtails their release of N2O.

When oxygen becomes limiting, denitrifying bacteria must prepare for anaerobic respiration by synthesizing the reductases NAR (NO3- → NO2-), NIR (NO2- → NO), NOR (2NO → N2O), and NOS (N2O → N2), either en bloc or sequentially, to avoid entrapment in anoxia without energy. Minimizing the metabolic burden of this precaution is a plausible fitness trait, and we show that the model denitrifier Paracoccus denitrificans achieves this by synthesizing NOS in all cells, while only a minority synthesize NIR. Phenotypic diversification with regards to NIR is ascribed to stochastic initiation of gene transcription, which becomes autocatalytic via NO production. Observed gas kinetics suggest that such bet hedging is widespread among denitrifying bacteria. Moreover, in response to oxygenation, P. denitrificans preserves NIR in the poles of nongrowing persister cells, ready to switch to anaerobic respiration in response to sudden anoxia. Our findings add dimensions to the regulatory biology of denitrification and identify regulatory traits that decrease N2O emissions.

A dual functional redox enzyme maturation protein for respiratory and assimilatory nitrate reductases in bacteria.

Nitrate is available to microbes in many environments due to sustained use of inorganic fertilizers on agricultural soils and many bacterial and archaeal lineages have the capacity to express respiratory (Nar) and assimilatory (Nas) nitrate reductases to utilize this abundant respiratory substrate and nutrient for growth. Here, we show that in the denitrifying bacterium Paracoccus denitrificans, NarJ serves as a chaperone for both the anaerobic respiratory nitrate reductase (NarG) and the assimilatory nitrate reductase (NasC), the latter of which is active during both aerobic and anaerobic nitrate assimilation. Bioinformatic analysis suggests that the potential for this previously unrecognized role for NarJ in functional maturation of other cytoplasmic molybdenum-dependent nitrate reductases may be phylogenetically widespread as many bacteria contain both Nar and Nas systems.

nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase.

Nitrous oxide (N2O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N2OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N2OR contains two important copper cofactors (CuA and CuZ centres) that are essential for activity, and in copper-limited environments, N2OR fails to function, contributing to rising levels of atmospheric N2O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A P. denitrificans ΔnosX deletion mutant failed to reduce N2O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N2OR activity. N2OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the CuA and CuZ centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N2OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from Pseudomonas stutzeri, which functions in the flavinylation of another N2OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N2OR and cellular N2O reduction.

Redox-dependent control of i-Motif DNA structure using copper cations.

Previous computational studies have shown that Cu+ can act as a substitute for H+ to support formation of cytosine (C) dimers with similar conformation to the hemi-protonated base pair found in i-motif DNA. Through a range of biophysical methods, we provide experimental evidence to support the hypothesis that Cu+ can mediate C-C base pairing in i-motif DNA and preserve i-motif structure. These effects can be reversed using a metal chelator, or exposure to ambient oxygen in the air that drives oxidation of Cu+ to Cu2+, a comparatively weak ligand. Herein, we present a dynamic and redox-sensitive system for conformational control of an i-motif forming DNA sequence in response to copper cations.

Destabilization of i-Motif DNA at Neutral pH by G-Quadruplex Ligands.

Numerous studies have been published stressing the importance of finding ligands that can bind specifically to DNA secondary structures. Several have identified ligands that are presented as having specific binding to the G-quadruplex; however, these were not originally tested on the complementary i-motif structure. The i-motif was overlooked and presumed to be irrelevant due to the belief that the hemiprotonated (cytosine+-cytosine) base pair at the core of the structure required acidic pH. The pathophysiological relevance of i-motifs has since been documented, as well as the discovery of several genomic sequences, which can form i-motif at neutral pH. Using different biophysical methodologies, we provide experimental evidence to show that widely used G-quadruplex ligands interact with i-motif structures at neutral pH, generally leading to their destabilization. Crucially, this has implications both for the search for quadruplex binding compounds as well as for the effects of compounds reported to have G-quadruplex specificity without examining their effects on i-motif.

The Paracoccus denitrificans NarK-like nitrate and nitrite transporters-probing nitrate uptake and nitrate/nitrite exchange mechanisms.

Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange.

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism.

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse(TM) platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

Tuning the modular Paracoccus denitrificans respirome to adapt from aerobic respiration to anaerobic denitrification.

Bacterial denitrification is a respiratory process that is a major source and sink of the potent greenhouse gas nitrous oxide. Many denitrifying bacteria can adjust to life in both oxic and anoxic environments through differential expression of their respiromes in response to environmental signals such as oxygen, nitrate and nitric oxide. We used steady-state oxic and anoxic chemostat cultures to demonstrate that the switch from aerobic to anaerobic metabolism is brought about by changes in the levels of expression of relatively few genes, but this is sufficient to adjust the configuration of the respirome to allow the organism to efficiently respire nitrate without the significant release of intermediates, such as nitrous oxide. The regulation of the denitrification respirome in strains deficient in the transcription factors FnrP, Nnr and NarR was explored and revealed that these have both inducer and repressor activities, possibly due to competitive binding at similar DNA binding sites. This may contribute to the fine tuning of expression of the denitrification respirome and so adds to the understanding of the regulation of nitrous oxide emission by denitrifying bacteria in response to different environmental signals.

An integrated biochemical system for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum.

Rhizobia are recognized to establish N2-fixing symbiotic interactions with legume plants. Bradyrhizobium japonicum, the symbiont of soybeans, can denitrify and grow under free-living conditions with nitrate (NO3 (-)) or nitrite (NO2 (-)) as sole nitrogen source. Unlike related bacteria that assimilate NO3 (-), genes encoding the assimilatory NO3 (-) reductase (nasC) and NO2 (-) reductase (nirA) in B. japonicum are located at distinct chromosomal loci. The nasC gene is located with genes encoding an ABC-type NO3 (-) transporter, a major facilitator family NO3 (-)/NO2 (-) transporter (NarK), flavoprotein (Flp) and single-domain haemoglobin (termed Bjgb). However, nirA clusters with genes for a NO3 (-)/NO2 (-)-responsive regulator (NasS-NasT). In the present study, we demonstrate NasC and NirA are both key for NO3 (-) assimilation and that growth with NO3 (-), but not NO2 (-) requires flp, implying Flp may function as electron donor to NasC. In addition, bjgb and flp encode a nitric oxide (NO) detoxification system that functions to mitigate cytotoxic NO formed as a by-product of NO3 (-) assimilation. Additional experiments reveal NasT is required for NO3 (-)-responsive expression of the narK-bjgb-flp-nasC transcriptional unit and the nirA gene and that NasS is also involved in the regulatory control of this novel bipartite assimilatory NO3 (-)/NO2 (-) reductase pathway.

Copper control of bacterial nitrous oxide emission and its impact on vitamin B12-dependent metabolism.

Global agricultural emissions of the greenhouse gas nitrous oxide (N2O) have increased by around 20% over the last 100 y, but regulation of these emissions and their impact on bacterial cellular metabolism are poorly understood. Denitrifying bacteria convert nitrate in soils to inert di-nitrogen gas (N2) via N2O and the biochemistry of this process has been studied extensively in Paracoccus denitrificans. Here we demonstrate that expression of the gene encoding the nitrous oxide reductase (NosZ), which converts N2O to N2, is regulated in response to the extracellular copper concentration. We show that elevated levels of N2O released as a consequence of decreased cellular NosZ activity lead to the bacterium switching from vitamin B12-dependent to vitamin B12-independent biosynthetic pathways, through the transcriptional modulation of genes controlled by vitamin B12 riboswitches. This inhibitory effect of N2O can be rescued by addition of exogenous vitamin B12.

Nitrogen oxyanion-dependent dissociation of a two-component complex that regulates bacterial nitrate assimilation.

Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.

Structure of a bacterial cell surface decaheme electron conduit.

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split ß-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

An S-methyltransferase that produces the climate-active gas dimethylsulfide is widespread across diverse marine bacteria.

Hydrogen sulfide (H2S), methanethiol (MeSH) and dimethylsulfide (DMS) are abundant sulfur gases with roles in biogeochemical cycling, chemotaxis and/or climate regulation. Catabolism of the marine osmolyte dimethylsulfoniopropionate (DMSP) is a major source of DMS and MeSH, but both also result from S-methylation of H2S via MddA, an H2S and MeSH S-methyltransferase whose gene is abundant in soil but scarce in marine environments. Here we identify the S-adenosine methionine (SAM)-dependent MeSH and H2S S-methyltransferase 'MddH', which is widespread in diverse marine bacteria and some freshwater and soil bacteria. mddH is predicted in up to ~5% and ~15% of seawater and coastal sediment bacteria, respectively, which is considerably higher than mddA. Furthermore, marine mddH transcript levels are similar to those for the most abundant DMSP lyase gene dddP. This study implies that the importance of H2S and MeSH S-methylation pathways in marine environments is significantly underestimated.

The 'porin-cytochrome' model for microbe-to-mineral electron transfer.

Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via 'porin-cytochrome' electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer-membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as 'nanowires', or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram-negative bacteria.

Electrode assemblies composed of redox cascades from microbial respiratory electron transfer chains.

Respiratory and photosynthetic electron transfer chains are dependent on vectorial electron transfer through a series of redox proteins. Examples include electron transfer from NapC to NapAB nitrate reductase in Paracoccus denitrificans and from CymA to Fcc3 (flavocytochrome c3) fumarate reductase in Shewanella oneidensis MR-1. In the present article, we demonstrate that graphite electrodes can serve as surfaces for the stepwise adsorption of NapC and NapAB, and the stepwise adsorption of CymA and Fcc3. Aspects of the catalytic properties of these assemblies are different from those of NapAB and Fcc3 adsorbed in isolation. We propose that this is due to the formation of NapC-NapAB and of CymA-Fcc3 complexes that are capable of supporting vectorial electron transfer.

First in-situ monitoring of sponge response and recovery to an industrial sedimentation event.

Assessment of risks to seabed habitats from industrial activities is based on the resilience and potential for recovery. Increased sedimentation, a key impact of many offshore industries, results in burial and smothering of benthic organisms. Sponges are particularly vulnerable to increases in suspended and deposited sediment, but response and recovery have not been observed in-situ. We quantified the impact of sedimentation from offshore hydrocarbon drilling over ∼5 days on a lamellate demosponge, and its recovery in-situ over ∼40 days using hourly time-lapse photographs with measurements of backscatter (a proxy of suspended sediment) and current speed. Sediment accumulated on the sponge then cleared largely gradually but occasionally sharply, though it did not return to the initial state. This partial recovery likely involved a combination of active and passive removal. We discuss the use of in-situ observing, which is critical to monitoring impacts in remote habitats, and need for calibration to laboratory conditions.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.