The c‐ring ion binding site of the ATP synthase from Bacillus pseudofirmus OF4 is adapted to alkaliphilic lifestyle

In the c‐ring rotor of ATP synthases ions are shuttled across the membrane during ATP synthesis by a unique rotary mechanism. We investigated characteristics of the c‐ring from the alkaliphile Bacillus pseudofirmus OF4 with respect to evolutionary adaptations to operate with protons at high environmental pH. The X‐ray structures of the wild‐type c₁₃ ring at pH 9.0 and a ‘neutralophile‐like’ mutant (P51A) at pH 4.4, at 2.4 and 2.8 Å resolution, respectively, reveal a dependency of the conformation and protonation state of the proton‐binding glutamate (E⁵⁴) on environmental hydrophobicity. Faster labelling kinetics with the inhibitor dicyclohexylcarbodiimide (DCCD) demonstrate a greater flexibility of E⁵⁴ in the mutant due to reduced water occupancy within the H⁺ binding site. A second ‘neutralophile‐like’ mutant (V21N) shows reduced growth at high pH, which is explained by restricted conformational freedom of the mutant's E⁵⁴ carboxylate. The study directly connects subtle structural adaptations of the c‐ring ion binding site to in vivo effects of alkaliphile cell physiology.     

Direct observation of stepped proteolipid ring rotation in E. coli F₀F₁-ATP synthase

Although single‐molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F_oF₁‐ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F_o has been particularly hampered by signal‐to‐noise and time resolution. Here, we show the presence of a transient dwell between F_o subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli F_oF₁ embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μs to form and 175 μs to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c‐ring to a limit of ～36° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F_o motor where c‐ring rotational diffusion is limited to 36°.     

RpoS and oxidative stress conditions regulate succinyl‐CoA: 3‐ketoacid‐coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei

Burkholderia pseudomallei, a pathogenic gram‐negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild‐type B. pseudomallei following exposure to H₂O₂ and between wild‐type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down‐regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT‐PCR that these genes are indeed co‐transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down‐regulation of SCOT expression in response to oxidative stress in B. pseudomallei.     

Inter‐subunit interaction of gastric H+,K+‐ATPase prevents reverse reaction of the transport cycle

The gastric H⁺,K⁺‐ATPase is an ATP‐driven proton pump responsible for generating a million‐fold proton gradient across the gastric membrane. We present the structure of gastric H⁺,K⁺‐ATPase at 6.5 Å resolution as determined by electron crystallography of two‐dimensional crystals. The structure shows the catalytic α‐subunit and the non‐catalytic β‐subunit in a pseudo‐E₂P conformation. Different from Na⁺,K⁺‐ATPase, the N‐terminal tail of the β‐subunit is in direct contact with the phosphorylation domain of the α‐subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β‐subunit N‐terminus allowed the reverse reaction to occur. These results suggest that the β‐subunit N‐terminus prevents the reverse reaction from E₂P to E₁P, which is likely to be relevant for the generation of a large H⁺ gradient in vivo situation.     

Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free‐living micro‐oxic and symbiotic conditions

Aim

In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free‐living and symbiotic conditions have been carried out.

Methods and Results

Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD‐dependent oxidase activity when cells were incubated under micro‐oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane‐bound c‐type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb₃ oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N‐free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation.

Conclusions

The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro‐oxic conditions as well as in the expression of the FixO and FixP components of the cbb₃ oxidase present in free‐living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis.

Significance and Impact of the Study

It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb₃ oxidase operating in free‐living micro‐oxic cultures and in bacteroids of E. meliloti have been identified.     

Conserved V‐ATPase c subunit plays a role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking

In plant cells, the vacuolar‐type H⁺‐ATPases (V‐ATPase) are localized in the tonoplast, Golgi, trans‐Golgi network and endosome. However, little is known about how V‐ATPase influences plant growth, particularly with regard to the V‐ATPase c subunit (VHA‐c). Here, we characterized the function of a VHA‐c gene from Puccinellia tenuiflora (PutVHA‐c) in plant growth. Compared to the wild‐type, transgenic plants overexpressing PutVHA‐c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V‐ATPase activity. Consistently, the Arabidopsis atvha‐c5 mutant shows reduced V‐ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA‐c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V‐ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA‐c‐GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling‐1 (AtRGS1). These findings suggest that the decrease in V‐ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA‐c plays an important role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking.     

Structures of reduced and ligand‐bound nitric oxide reductase provide insights into functional differences in respiratory enzymes

Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N₂O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non‐heme Fe center. We report herein on the structures of the reduced and ligand‐bound forms of cytochrome c‐dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3–2.7 Å, to elucidate structure‐function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO‐bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH₃‐CH=N‐OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ～0.5 Å increase in the heme/non‐heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton‐pumping activity in cNOR, because redox‐coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non‐heme Fe distance even in the bulky ligand‐bound form of cNOR (～4.5 Å) than the heme/Cu distance in CCO (～5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N‐N coupling to produce a hyponitrite intermediate for the generation of N₂O. Proteins 2014; 82:1258–1271. © 2013 Wiley Periodicals, Inc.     

A triazole‐based fluorescence probe for detecting Hg2+ ions and its biological application

A new compound, ethyl 5‐phenyl‐2‐(p‐tolyl)‐2H‐1,2,3‐triazole‐4‐carboxylate was successfully introduced and synthesized as a novel rhodamine B derivative named REPPC, and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high resolution mass spectrometry (HRMS). It showed an obvious fluorescence and UV–visible light absorption enhancement towards Hg²⁺ ion without interference from common metal ions in N,N‐dimethylformamide–H₂O (pH 7.4). The spirolactam ring moiety of rhodamine in REPPC was converted to the open‐ring form generating a 1:1 complex with the intervention of a mercury ion, verified by electrospray ionization‐mass spectroscopy testing and density functional theory calculation. REPPC was used to visualize the level of mercury ions in living HeLa cells with encouraging results.     

TRANSPARENT TESTA 13 is a tonoplast P3A‐ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P‐type H⁺‐ATPases in the plasma membrane, and multimeric vacuolar‐type H⁺‐ATPases (V‐ATPases) and vacuolar H⁺‐pyrophosphatases (H⁺‐PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P_3A‐ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA‐filled central vacuoles as observed in the wild‐type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P‐type H⁺‐ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H⁺‐PPase VHP1. Our findings indicate that the P_3A‐ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12‐mediated transport of PA precursors to the vacuole.     

Improving Nelumbo nucifera genome assemblies using high‐resolution genetic maps and BioNano genome mapping reveals ancient chromosome rearrangements

Genetic and physical maps are powerful tools to anchor fragmented draft genome assemblies generated from next‐generation sequencing. Currently, two draft assemblies of Nelumbo nucifera, the genomes of ‘China Antique’ and ‘Chinese Tai‐zi’, have been released. However, there is presently no information on how the sequences are assembled into chromosomes in N. nucifera. The lack of physical maps and inadequate resolution of available genetic maps hindered the assembly of N. nucifera chromosomes. Here, a linkage map of N. nucifera containing 2371 bin markers [217 577 single nucleotide polymorphisms (SNPs)] was constructed using restriction‐site associated DNA sequencing data of 181 F₂ individuals and validated by adding 197 simple sequence repeat (SSR) markers. Additionally, a BioNano optical map covering 86.20% of the ‘Chinese Tai‐zi’ genome was constructed. The draft assembly of ‘Chinese Tai‐zi’ was improved based on the BioNano optical map, showing an increase of the scaffold N50 from 0.989 to 1.48 Mb. Using a combination of multiple maps, 97.9% of the scaffolds in the ‘Chinese Tai‐zi’ draft assembly and 97.6% of the scaffolds in the ‘China Antique’ draft assembly were anchored into pseudo‐chromosomes, and the centromere regions along the pseudo‐chromosomes were identified. An evolutionary scenario was proposed to reach the modern N. nucifera karyotype from the seven ancestral eudicot chromosomes. The present study provides the highest‐resolution linkage map, the optical map and chromosome level genome assemblies for N. nucifera, which are valuable for the breeding and cultivation of N. nucifera and future studies of comparative and evolutionary genomics in angiosperms.     

36° step size of proton‐driven c‐ring rotation in FoF1‐ATP synthase

Synthesis of adenosine triphosphate ATP, the ‘biological energy currency’, is accomplished by F_oF₁‐ATP synthase. In the plasma membrane of Escherichia coli, proton‐driven rotation of a ring of 10 c subunits in the F_o motor powers catalysis in the F₁ motor. Although F₁ uses 120° stepping during ATP synthesis, models of F_o predict either an incremental rotation of c subunits in 36° steps or larger step sizes comprising several fast substeps. Using single‐molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36° sequential stepping mode of the c‐ring during ATP synthesis.     

Assembly of the Escherichia coli F1F0 ATPase, a large multimeric membrane-bound enzyme

The F₁F₀ proton translocating ATPase of Escherichia coli is a large membrane-bound enzyme complex consisting of more than 20 polypeptides that are encoded by the unc operon. Besides being a system for analysing the enzymology of ATP synthesis and energy coupling, the ATPase is a model system for determining how large oligomeric membrane-bound proteins are synthesized and assembled. The assembly of the ATPase involves differential gene expression and assembly of the subunits within the membrane and with each other. This review discusses the influence of F₁ subunits on the assembly and proton permeability of the F₀ proton channel, and the possible advantages to assembly of the particular arrangement of genes in the unc operon.     

Measuring carbon and N2 fixation in field populations of colonial and free‐living unicellular cyanobacteria using nanometer‐scale secondary ion mass spectrometry1

Unicellular cyanobacteria are now recognized as important to the marine N and C cycles in open ocean gyres, yet there are few direct in situ measurements of their activities. Using a high‐resolution nanometer scale secondary ion mass spectrometer (nanoSIMS), single cell N₂ and C fixation rates were estimated for unicellular cyanobacteria resembling N₂ fixer Crocosphaera watsonii. Crocosphaera watsonii‐like cells were observed in the subtropical North Pacific gyre (22°45′ N, 158°0′ W) as 2 different phenotypes: colonial and free‐living. Colonies containing 3–242 cells per colony were observed and cell density in colonies increased with incubation time. Estimated C fixation rates were similarly high in both phenotypes and unexpectedly for unicellular cyanobacteria 85% of the colonial cells incubated during midday were also enriched in ¹⁵N above natural abundance. Highest ¹⁵N enrichment and N₂ fixation rates were found in cells incubated overnight where up to 64% of the total daily fixed N in the upper surface waters was attributed to both phenotypes. The colonial cells retained newly fixed C in a sulfur‐rich matrix surrounding the cells and often cells of both phenotypes possessed areas (<1 nm) of enriched ¹⁵N and ¹³C resembling storage granules. The nanoSIMS imaging of the colonial cells also showed evidence for a division of N₂ and C fixation activity across the colony where few individual cells (<34%) in a given colony were enriched in both ¹⁵N and ¹³C above the colony average. Our results provide new insights into the ecophysiology of unicellular cyanobacteria.     

Inter‐subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1‐ATPase

V‐type ATPases (V‐ATPases) are categorized as rotary ATP synthase/ATPase complexes. The V‐ATPases are distinct from F‐ATPases in terms of their rotation scheme, architecture and subunit composition. However, there is no detailed structural information on V‐ATPases despite the abundant biochemical and biophysical research. Here, we report a crystallographic study of V₁‐ATPase, from Thermus thermophilus, which is a soluble component consisting of A, B, D and F subunits. The structure at 4.5 Å resolution reveals inter‐subunit interactions and nucleotide binding. In particular, the structure of the central stalk composed of D and F subunits was shown to be characteristic of V₁‐ATPases. Small conformational changes of respective subunits and significant rearrangement of the quaternary structure observed in the three AB pairs were related to the interaction with the straight central stalk. The rotation mechanism is discussed based on a structural comparison between V₁‐ATPases and F₁‐ATPases.     

Breaking up and making up: The secret life of the vacuolar H+‐ATPase

The vacuolar ATPase (V‐ATPase; V₁V_o‐ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin‐coated vesicles. V‐ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V‐ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V‐ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high‐resolution structure determination by cryo electron microscopy and X‐ray crystallography together with sophisticated single‐molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V‐ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.     

Active‐Site Imprinting: Preparation of Fe–N–C Catalysts from Zinc Ion–Templated Ionothermal Nitrogen‐Doped Carbons

Atomically dispersed Fe–N–C catalysts are considered the most promising precious‐metal‐free alternative to state‐of‐the‐art Pt‐based oxygen reduction electrocatalysts for proton‐exchange membrane fuel cells. The exceptional progress in the field of research in the last ≈30 years is currently limited by the moderate active site density that can be obtained. Behind this stands the dilemma of metastability of the desired FeN₄ sites at the high temperatures that are believed to be a requirement for their formation. It is herein shown that Zn²⁺ ions can be utilized in the novel concept of active‐site imprinting based on a pyrolytic template ion reaction throughout the formation of nitrogen‐doped carbons. As obtained atomically dispersed Zn–N–Cs comprising ZnN₄ sites as well as metal‐free N₄ sites can be utilized for the coordination of Fe²⁺ and Fe³⁺ ions to form atomically dispersed Fe–N–C with Fe loadings as high as 3.12 wt%. The Fe–N–Cs are active electocatalysts for the oxygen reduction reaction in acidic media with an onset potential of E₀ = 0.85 V versus RHE in 0.1 m HClO₄. Identical location atomic resolution transmission electron microscopy imaging, as well as in situ electrochemical flow cell coupled to inductively coupled plasma mass spectrometry measurements, is employed to directly prove the concept of the active‐site imprinting approach.     

Nucleotide recognition by CopA,a Cu+‐transporting P‐type ATPase

Heavy metal pumps constitute a large subgroup in P‐type ion‐transporting ATPases. One of the outstanding features is that the nucleotide binding N‐domain lacks residues critical for ATP binding in other well‐studied P‐type ATPases. Instead, they possess an HP‐motif and a Gly‐rich sequence in the N‐domain, and their mutations impair ATP binding. Here, we describe 1.85 Å resolution crystal structures of the P‐ and N‐domains of CopA, an archaeal Cu⁺‐transporting ATPase, with bound nucleotides. These crystal structures show that CopA recognises the adenine ring completely differently from other P‐type ATPases. The crystal structure of the His462Gln mutant, in the HP‐motif, a disease‐causing mutation in human Cu⁺‐ATPases, shows that the Gln side chain mimics the imidazole ring, but only partially, explaining the reduction in ATPase activity. These crystal structures lead us to propose a role of the His and a mechanism for removing Mg²⁺ from ATP before phosphoryl transfer.     

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.