Energy conservation by Rhodothermus marinus respiratory complex I

A sodium ion efflux, together with a proton influx and an inside-positive ΔΨ, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by ²³Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.     

Subunit composition of Rhodothermus marinus respiratory complex I

The basic structural characterization of complex I is still not trivial due to its complexity, not only in the number of its protein constituents but especially because of the different properties of the several subunits. Bacterial complex I generally contains 14 subunits: 7 hydrophilic proteins located in the peripheral arm and 7 hydrophobic proteins present in the membrane arm. It is the identification of the hydrophobic proteins that makes the characterization of complex I, and of membrane proteins in general, very difficult. In this article, we report the identification of the subunits of complex I from Rhodothermus marinus. The original approach, presented here, combined several protein and peptides separation strategies (different reversed phase materials, high-performance liquid chromatography, and gel electrophoresis) with different identification methods (electrospray ionization-tandem mass spectrometry, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and Edman degradation analysis) and represents a step forward in the characterization of membrane proteins that studies are still technically highly challenging. The combination of the different methodologies allowed the identification of complex I canonical subunits and also a possible novel subunit, namely a pterin-4α-carbinolamine dehydratase (PCD). This was the first time that a PCD was suggested to be part of complex I, and its possible regulatory role is discussed.     

Role of guanidinium group in the insertion of l-arginine in DMPE and DMPC lipid interphases

l-Arginine (Arg) is a positively charged amino acid constituent of peptides and proteins, participating in diverse mechanisms of protein-membrane interaction. The effect of Arg on phosphatidylcholine (PC) membranes has been previously related to water structure changes and to the presence of water defects in the hydrocarbon region. However, no information is available with regard to phosphatidylethanolamine (PE), another important component of lipid membranes. For this reason, the aim of this study is to determine the effect of Arg on DMPE membranes and partially methylated PEs in comparison to DMPC. The adsorption of the amino acid onto the lipid membranes was followed by determining the changes in the surface potential as a function of the bulk amino acid concentrations. The effects of Arg on the surface properties were also measured by changes in the surface pressure and the dipole potential. The onset of the transition temperature was measured with a fluorophore anchored at the membrane interphase. The results provide a new insight on amino acid—PE interactions, which can be ascribed to specific perturbations in the head group region induced by the guanidinium residue.     

Inter‐conversion of catalytic abilities in a bifunctional carboxyl/feruloyl‐esterase from earthworm gut metagenome

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k_cat/K_m)]_CE/[(k_cat/K_m)]_FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys²⁸¹, Asp²⁸², Asn³¹⁶ and Lys³¹⁷) situated close to the catalytic core (Ser¹⁴³/Asp²⁷³/His³⁰⁵) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k_cat/K_m values) and enzymes with inverted specificity ((k_cat/K_m)_CE/(k_cat/K_m)_FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol⁻¹), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol⁻¹) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.     

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic (¹⁵N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (δ¹⁵N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (±0.07 SE) and 49.6% (±0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian–bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.     

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH₂ (RGE) and IVYYPDRGETGL-NH₂ (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED₅₀ 0.16% and LD₅₀ 0.09%), this being even more effective than the native protein.     

mEosFP-based green-to-red photoconvertible subcellular probes for plants

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.     

Mangrove dynamics in the Cispata lagoon system (Colombian Caribbean) during last 900 years

The lagoon complex of Cispatá (old Sinú river delta) located at the Northwestern coast of the Colombian Caribbean, encloses one of the biggest mangrove areas in this region. This area has changed during the last 330 years because of several environmental and climatic causes, mainly changes in the position of the delta (Sinú River), which is the main freshwater source in this area, and sea level rise. We hypothesized that the climatic and geomorphologic dynamics has caused changes in the extension and composition of mangrove vegetation, especially during last 150 years. The dynamics of mangroves during the last 900 years was reconstructed based on the changes in the stratigraphy, pollen record, calcite concentrations (CaCO3) and C/N ratio, along two sediment cores from La Flotante and Navio lagoons, located in Cispatá complex. The age model was built based on lineal interpolation of 210Pb ages and changes in granulometry. Establishment and expansion of mangrove forests during the last 900 years were related to fluviomarine dynamics in the area and the lagoon formation. During the period encompassed between 1064 and 1762 A.D., the Mestizos spit was formed when marine conditions predominated in the surroundings of La Flotante Lagoon. At the site of Navío, a river dominated lagoon, terrigenous conditions dominated since 1830. Although the colonization of herbaceous pioneer vegetation started between 1142 and 1331 A.D., mangrove colonization only took place since 1717 A.D. Mangrove colonization was a result of the delta progradation. In 1849 A.D. the Sinú river delta migrated to the Cispatá bay. The eustatic sea level rise, the increase in river discharges and sedimentation rates produced the establishment of mangrove forests dominated by Rhizophora since 1849. Since 1900 a marine intrusion was recorded in both lagoons. In 1938, the migration of the delta toward its actual location in Tinajones gave place to the formation of the present lagoon system and to the expansion of mangrove forests, which reflects the balance between the high alluvial sediment input and the current sea level rise as has been recorded in similar ecosystems.     

Centrocestus formosanus (Opisthorchiida: Heterophyidae) as a cause of death in gray tilapia fry Oreochromis niloticus (Perciforme: Cichlidae) in the dry Pacific of Costa Rica

Centrocestusformosanus is a zoonotic trematode from Asia and has been mainly associated as cause of death of cultured fish. To identify pathogen trematode species in tilapia fry (Oreochromis niloticus) and to determine mollusks hosting these parasites, freshwater mollusks were collected from tilapia cultured ponds and experimental infections were carried out with tilapia fries and different mollusk species. A total of 907 freshwater mollusks were obtained from tilapia ponds and were identified to species level, four gastropods and one bivalve were determined: Melania tuberculata, Melanoides turricula, Pomacea flagellata, Haitia cubensis and Anodontiles luteola. For the first time, the presence of M. turricula and H. cubensis are reported in Costa Rica. Seven morphotypes of cercariae (Xifiodiocercaria, Equinostoma, Oftalmocercaria, Parapleurolofocercus, Cistocerca, Furcocercaria and Leptocercaria) parasitizing all five species of mollusks were found, all of distome type. Experimental exposure of tilapia fry to M. tuberculata demonstrated that the parapleurolofocercus morphotype found in the mollusk is in accordance with the finding of C. formosanus in tilapia fry. An abundance and mean intensity of 1018-1027 digeneans per gill in each exposed fish was determined. Centrocestus formosanus is reported for the first time in Costa Rica, for which the primary and secondary intermediate hosts were also determined.