The Aer2 chemoreceptor from Vibrio vulnificus is a tri-PAS-heme oxygen sensor

The marine pathogen Vibrio vulnificus senses and responds to environmental stimuli via two chemosensory systems and 42–53 chemoreceptors. Here, we present an analysis of the V. vulnificus Aer2 chemoreceptor, VvAer2, which is the first V. vulnificus chemoreceptor to be characterized. VvAer2 is related to the Aer2 receptors of other gammaproteobacteria, but uncharacteristically contains three PAS domains (PAS1-3), rather than one or two. Using an E. coli chemotaxis hijack assay, we determined that VvAer2, like other Aer2 receptors, senses and responds to O₂. All three VvAer2 PAS domains bound pentacoordinate b-type heme and exhibited similar O₂ affinities. PAS2 and PAS3 both stabilized O₂ via conserved Iβ-Trp residues, but PAS1, which was easily oxidized in vitro, was unaffected by Iβ-Trp replacement. Our results support a model in which PAS1 is largely dispensable for O₂-mediated signaling, whereas PAS2 modulates PAS3 signaling, and PAS3 signals to the downstream domains. Each PAS domain appeared to be positionally optimized, because PAS swapping caused altered signaling properties, and neither PAS1 nor PAS2 could replace PAS3. Our findings strengthen previous conclusions that Aer2 receptors are O₂ sensors, but with distinct N-terminal domain arrangements that facilitate, modulate and tune responses based on environmental signals.     

Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane

MARTX (multifunctional autoprocessing repeats‐in‐toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram‐negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine‐rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ～ 1.4‐fold (from 21.8 to 16.1 kJ mol^?1). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi‐domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane‐penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins.     

Control of the bifunctional O2-sensor kinase NreB of Staphylococcus carnosus by the nitrate sensor NreA: Switching from kinase to phosphatase state

The NreB–NreC two-component system of Staphylococcus carnosus for O₂ sensing cooperates with the accessory nitrate sensor NreA in the NreA–NreB–NreC system for coordinated sensing and regulation of nitrate respiration by O₂ and nitrate. ApoNreA (NreA in the absence of nitrate) interacts with NreB and inhibits NreB autophosphorylation (and activation). NreB contains the phosphatase motif DxxxQ. The present study shows that NreB on its own was inactive for the dephosphorylation of the phosphorylated response regulator NreC (NreC-P), but co-incubation with NreB and NreA stimulated NreC-P dephosphorylation. Either the presence of instead of apoNreA or mutation of the phosphatase motif (D160 or Q164) of NreB abrogated phosphatase activity of NreB. Phosphatase activity was observed for anoxic (active) NreB as well as oxic NreB, therefore the functional state of NreB is not relevant for phosphatase activity. Thus, NreB is a bifunctional sensor kinase with an integral cryptic phosphatase activity. Activation of phosphatase activity and dephosphorylation of NreC-P requires NreA as a cofactor. Accordingly, NreA and nitrate have major and dual roles in NreA–NreB–NreC regulation by (i) inhibiting NreB phosphorylation and (ii) triggering a kinase/phosphatase switch of NreB when present as apoNreA.     

Delineating PAS‐HAMP interaction surfaces and signalling‐associated changes in the aerotaxis receptor Aer

The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer‐PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N‐terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signalling domains in the kinase‐off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS‐HAMP surfaces overlapped with a cluster of PAS kinase‐on lesions and with cysteine substitutions that crosslinked the PAS β ‐scaffold to the HAMP AS‐2 helix. A refined Aer PAS‐HAMP interaction model is presented. Compared to the kinase‐off state, the kinase‐on state increased the accessibility of HAMP residues (apparently relaxing PAS‐HAMP interactions), but decreased the accessibility of proximal signalling domain residues. These data are consistent with an alternating static‐dynamic model in which oxidized Aer‐PAS interacts directly with HAMP AS‐2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signalling domain, resulting in a kinase‐off output. When PAS‐FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signalling domain convey a kinase‐on output.     

The role of a cytosolic superoxide dismutase in barley–pathogen interactions

Reactive oxygen species (ROS), including superoxide ( / ) and hydrogen peroxide (H₂O₂), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi‐biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of / to H₂O₂, regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi‐biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up‐regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild‐type plants, suggesting that cytosolic / contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi‐biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.     

Cross‐protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv

Gram‐negative Vibrio species secrete multifunctional autoprocessing repeats‐in‐toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross‐reactivity and cross‐protectivity of mAbs against V. vulnificus RtxA1/MARTX_Vv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTX_Vc, inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins.     

The effect of O2 and pressure on thiosulfate oxidation by Thiomicrospira thermophila

Microbial sulfur cycling in marine sediments often occurs in environments characterized by transient chemical gradients that affect both the availability of nutrients and the activity of microbes. High turnover rates of intermediate valence sulfur compounds and the intermittent availability of oxygen in these systems greatly impact the activity of sulfur‐oxidizing micro‐organisms in particular. In this study, the thiosulfate‐oxidizing hydrothermal vent bacterium Thiomicrospira thermophila strain EPR85 was grown in continuous culture at a range of dissolved oxygen concentrations (0.04–1.9 mM) and high pressure (5–10 MPa) in medium buffered at pH 8. Thiosulfate oxidation under these conditions produced tetrathionate, sulfate, and elemental sulfur, in contrast to previous closed‐system experiments at ambient pressure during which thiosulfate was quantitatively oxidized to sulfate. The maximum observed specific growth rate at 5 MPa pressure under unlimited O₂ was 0.25 hr^?1. This is comparable to the μ_max (0.28 hr^?1) observed at low pH (<6) at ambient pressure when T. thermophila produces the same mix of sulfur species. The half‐saturation constant for O₂ () estimated from this study was 0.2 mM (at a cell density of 10⁵ cells/ml) and was robust at all pressures tested (0.4–10 MPa), consistent with piezotolerant behavior of this strain. The cell‐specific was determined to be 1.5 pmol O₂/cell. The concentrations of products formed were correlated with oxygen availability, with tetrathionate production in excess of sulfate production at all pressure conditions tested. This study provides evidence for transient sulfur storage during times when substrate concentration exceeds cell‐specific and subsequent consumption when oxygen dropped below that threshold. These results may be common among sulfur oxidizers in a variety of environments (e.g., deep marine sediments to photosynthetic microbial mats).     

High light‐induced hydrogen peroxide production in Chlamydomonas reinhardtii is increased by high CO2 availability

The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO₂ availability putatively includes enhanced ROS production in the so‐called Mehler reaction. Such conditions are thought to encourage O₂ to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical () and hydrogen peroxide (H₂O₂). In contrast, here it is shown in Chlamydomonas reinhardtii that CO₂ depletion under high light levels lowered cellular H₂O₂ production, and that elevated CO₂ levels increased H₂O₂ production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H₂O₂ production under high CO₂ availability. High light levels under low CO₂ availability induced photoprotective mechanisms called non‐photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE‐deficient mutant npq4 produced more H₂O₂ than wild‐type cells under high light levels, although less so under high CO₂ availability, whereas it demonstrated equal or greater enzymatic H₂O₂‐degrading capacity. The qT‐deficient mutant stt7‐9 produced the same H₂O₂ as wild‐type cells under high CO₂ availability. Physiological levels of H₂O₂ were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO₂ availability wild‐type cells behaved like stt7‐9 cells stuck in state 1.     

The crystal structure of Z‐(Aib)10‐OH at 0.65 Å resolution: three complete turns of 310‐helix

The synthetic peptide Z‐(Aib)₁₀‐OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection reflected synchrotron radiation to sub‐atomic resolution, where the bonding electron density becomes visible between the non‐hydrogen atoms. Crystals belong to the centrosymmetric space group P . Both molecules in the asymmetric unit form regular 3₁₀‐helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure determined to date of longer Aib‐homopeptides. These other peptides are C‐terminal protected by OtBu or OMe. In these cases, because of the missing ability of the C‐terminal protection group to form a hydrogen bond to the residue i‐3, the sense of the helix is reversed in the last residue. Here, the C‐terminal OH‐groups form hydrogen bonds to the residues i‐3, in part mediated by water molecules. This makes Z‐(Aib)₁₀‐OH an Aib‐homopeptide with three complete 3₁₀‐helical turns in spite of the shorter length it has compared with Z‐(Aib)₁₁‐OtBu, the only homopeptide to date with three complete turns.     

NMR‐based conformation and dynamics of a tetrasaccharide‐repeating sulfated fucan substituted by different counterions

The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [‐3)‐α‐l ‐Fucp‐4( )‐(1‐3)‐α‐l ‐Fucp‐2,4‐di( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐]_n. Conformation (of rings and chains) and dynamics of this tetrasaccharide‐repeating sulfated fucan substituted by Na⁺, Ca²⁺, and Li⁺ as counterions have been examined through experiments of liquid‐state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)‐based data have confirmed that all composing units occur as ¹C₄ chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin‐relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium‐based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well‐defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.     

Antarctic emerald rockcod have the capacity to compensate for warming when uncoupled from CO2‐acidification

Increases in atmospheric CO₂ levels and associated ocean changes are expected to have dramatic impacts on marine ecosystems. Although the Southern Ocean is experiencing some of the fastest rates of change, few studies have explored how Antarctic fishes may be affected by co‐occurring ocean changes, and even fewer have examined early life stages. To date, no studies have characterized potential trade‐offs in physiology and behavior in response to projected multiple climate change stressors (ocean acidification and warming) on Antarctic fishes. We exposed juvenile emerald rockcod Trematomus bernacchii to three PCO₂ treatments (~450, ~850, and ~1,200 μatm PCO₂) at two temperatures (?1 or 2°C). After 2, 7, 14, and 28 days, metrics of physiological performance including cardiorespiratory function (heart rate [f_H] and ventilation rate [f_V]), metabolic rate (), and cellular enzyme activity were measured. Behavioral responses, including scototaxis, activity, exploration, and escape response were assessed after 7 and 14 days. Elevated PCO₂ independently had little impact on either physiology or behavior in juvenile rockcod, whereas warming resulted in significant changes across acclimation time. After 14 days, f_H, f_V and significantly increased with warming, but not with elevated PCO₂. Increased physiological costs were accompanied by behavioral alterations including increased dark zone preference up to 14%, reduced activity by 12%, as well as reduced escape time suggesting potential trade‐offs in energetics. After 28 days, juvenile rockcod demonstrated a degree of temperature compensation as f_V, , and cellular metabolism significantly decreased following the peak at 14 days; however, temperature compensation was only evident in the absence of elevated PCO₂. Sustained increases in f_V and after 28 days exposure to elevated PCO₂ indicate additive (f_V) and synergistic () interactions occurred in combination with warming. Stressor‐induced energetic trade‐offs in physiology and behavior may be an important mechanism leading to vulnerability of Antarctic fishes to future ocean change.     

KVP2O7 as a Robust High‐Energy Cathode for Potassium‐Ion Batteries: Pinpointed by a Full Screening of the Inorganic Registry under Specific Search Conditions

Candidates for high‐energy cathodes in potassium‐ion batteries (KIBs) are selected by fully screening the inorganic compound structure database. The compounds that satisfy the specific conditions for plausible KIB cathodes are further subjected to theoretical and electrochemical verification, and KVP₂O₇ is finally pinpointed. KVP₂O₇ can reversibly desert/insert ≈60% of K⁺ (60 mA h g^?1) during either chemical or electrochemical oxidation/reduction. KVP₂O₇ shows an average discharge potential of ≈4.2 V versus K/K⁺, which corresponds to an energy density of 253 W h kg^?1 at 0.25 C. This high energy density characteristic of KVP₂O₇ is maintained both during fast charge/discharge (C/D) and prolonged redox cycles. The C/D of KVP₂O₇ is also accompanied by a phase transition between a monoclinic KVP₂O₇ (P2₁/c) and a triclinic K_1?xVP₂O₇. The structure interpretation of a new K_1?xVP₂O₇ phase indicates that K⁺‐extraction induces a conformational change of two tetrahedral PO₄ units in pyrophosphates. The phase of K_1?xVP₂O₇ (x ≈0.6) remains stable during the C/D process, although it returns to the inborn P2₁/c phase after thermal treatment. It is believed that the data‐mining protocol designed for this study will provide a new strategy for materials discovery and that the pinpointed KVP₂O₇ can be utilized as a reliable KIB cathode.     

Redox regulation of plant stem cell fate

Despite the importance of stem cells in plant and animal development, the common mechanisms of stem cell maintenance in both systems have remained elusive. Recently, the importance of hydrogen peroxide (H₂O₂) signaling in priming stem cell differentiation has been extensively studied in animals. Here, we show that different forms of reactive oxygen species (ROS) have antagonistic roles in plant stem cell regulation, which were established by distinct spatiotemporal patterns of ROS‐metabolizing enzymes. The superoxide anion () is markedly enriched in stem cells to activate WUSCHEL and maintain stemness, whereas H₂O₂ is more abundant in the differentiating peripheral zone to promote stem cell differentiation. Moreover, H₂O₂ negatively regulates biosynthesis in stem cells, and increasing H₂O₂ levels or scavenging leads to the termination of stem cells. Our results provide a mechanistic framework for ROS‐mediated control of plant stem cell fate and demonstrate that the balance between and H₂O₂ is key to stem cell maintenance and differentiation.     

Lower photorespiration in elevated CO2 reduces leaf N concentrations in mature Eucalyptus trees in the field

Rising atmospheric CO₂ concentrations is expected to stimulate photosynthesis and carbohydrate production, while inhibiting photorespiration. By contrast, nitrogen (N) concentrations in leaves generally tend to decline under elevated CO₂ (eCO₂), which may reduce the magnitude of photosynthetic enhancement. We tested two hypotheses as to why leaf N is reduced under eCO₂: (a) A “dilution effect” caused by increased concentration of leaf carbohydrates; and (b) inhibited nitrate assimilation caused by reduced supply of reductant from photorespiration under eCO₂. This second hypothesis is fully tested in the field for the first time here, using tall trees of a mature Eucalyptus forest exposed to Free‐Air CO₂ Enrichment (EucFACE) for five years. Fully expanded young and mature leaves were both measured for net photosynthesis, photorespiration, total leaf N, nitrate () concentrations, carbohydrates and reductase activity to test these hypotheses. Foliar N concentrations declined by 8% under eCO₂ in new leaves, while the fraction and total carbohydrate concentrations remained unchanged by CO₂ treatment for either new or mature leaves. Photorespiration decreased 31% under eCO₂ supplying less reductant, and in situ reductase activity was concurrently reduced (?34%) in eCO₂, especially in new leaves during summer periods. Hence, assimilation was inhibited in leaves of E. tereticornis and the evidence did not support a significant dilution effect as a contributor to the observed reductions in leaf N concentration. This finding suggests that the reduction of reductase activity due to lower photorespiration in eCO₂ can contribute to understanding how eCO₂‐induced photosynthetic enhancement may be lower than previously expected. We suggest that large‐scale vegetation models simulating effects of eCO₂ on N biogeochemistry include both mechanisms, especially where is major N source to the dominant vegetation and where leaf flushing and emergence occur in temperatures that promote high photorespiration rates.     

Nitrogen fixation by the diazotroph Cylindrospermopsis raciborskii (Cyanophyceae)

Nitrogen fixation has been proposed as a mechanism that allows the diazotrophic cyanobacterium, Cylindrospermopsis raciborskii, to bloom in nitrogen‐limited freshwater systems. However, it is unclear whether dinitrogen fixation (N₂ fixation) can supplement available dissolved inorganic nitrogen (DIN) for growth, or only provides minimum nitrogen (N) for cell maintenance under DIN deplete conditions. Additionally, the rate at which cells can switch between DIN use and N₂ fixation is unknown. This study investigated N₂ fixation under a range of nitrate concentrations. Cultures were grown with pretreatments of nitrate replete (single dose 941 μmol  · L^?1) and N‐free conditions and then either received a single dose of 941 μmol  · L^?1 (N941), 118 μmol  · L^?1 (N118) or 0 N. Heterocysts appeared from days 3 to 5 when treatments of high were transferred to N free media (N941:N0), and from day 5 in N941 transferred to N118 treatments. Conversely, transferring cells from N0 to N941 resulted in heterocysts being discarded from day 3 and day 5 for N0:N118. Heterocyst appearance correlated with a detectable rate of N₂ fixation and up‐regulation of nifH gene expression, the discard of heterocysts occurred after sequential reduction of nifH expression and N₂ fixation. Nitrate uptake rates were not affected by pretreatment, suggesting no regulation or saturation of this uptake pathway. These data demonstrate that for C. raciborskii, N₂ fixation is regulated by the production or discard of heterocysts. In conclusion, this study has shown that N₂ fixation only provides enough N to support relatively low growth under N‐limited conditions, and does not supplement available nitrate to increase growth rates.     

Gain‐of‐function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor,Aer

The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD‐containing PAS domain. Here, we randomly mutagenized the region encoding residues 14–119 of the PAS domain and found 72 aerotaxis‐defective mutants, 24 of which were gain‐of‐function, signal‐on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS–FAD domain in NifL from Azotobacter vinlandii. Signal‐on lesions clustered in the FAD binding pocket, the β‐scaffolding and in the N‐cap loop. We suggest that the signal‐on lesions mimic the ‘signal‐on’ state of the PAS domain, and therefore may be markers for the signal‐in and signal‐out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the β‐scaffolding and the N‐cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS β‐scaffold and residue Q248C in the HAMP AS‐2 helix.