David Kaftan

A novel tumor necrosis factor–mediated mechanism of direct epithelial sodium channel activation

RATIONALE Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Characterization of the microaerophilic, bacteriochlorophyll a-containing bacterium Gemmatimonas phototrophica sp. nov., and emended descriptions of the genus Gemmatimonas and Gemmatimonas aurantiaca

A red-pigmented, bacteriochlorophyll (BChl) a-producing strain, AP64T, was isolated previously from the freshwater Swan Lake located in the western Gobi Desert. Based on its 16S rRNA gene sequence identity (96.1%) to the type strain Gemmatimonas aurantiaca T-27T, the new isolate was tentatively classified as a member of the bacterial phylum Gemmatimonadetes. Here, we report its formal description and polyphasic characterization. Strain AP64T grew best on agar media under 9.8-15.2% atmospheric oxygen. The cells were rods, dividing by symmetrical or asymmetrical binary fission. Budding structures were also observed. Its genomic DNA G+C content was 64.4% (from the draft genome sequence). Phylogenetic analysis based on the 16S rRNA gene sequence clearly separated AP64T from related species. Its genotypic differentiation from phylogenetically close relatives was further supported by performing in silico DNA-DNA hybridization and calculating average nucleotide identity, whereas the high percentage (67.3%) of shared conserved proteins between strain AP64T and Gemmatimonas aurantiaca T-27T supports the classification of the two strains into the same genus. Strain AP64T contained C16 : 1, C14 : 1 and C18 : 1ω9c as predominant fatty acids. The main respiratory quinone was menaquinone 8 (MK-8). The most distinctive feature of strain AP64T was the presence of fully functional purple bacterial photosynthetic reaction centres. The main CO2-fixation pathways were absent. Strain AP64T was capable of growth and BChl production in constant darkness. Thus, strain AP64T is a facultatively photoheterotrophic organism. It represents a novel species of the genus Gemmatimonas, for which the name Gemmatimonasphototrophica sp. nov. is proposed. The type strain is AP64T ( = DSM 29774T = MCCC 1K00454T). Emended descriptions of the genus Gemmatimonas and Gemmatimonas aurantiaca are also provided.

Second harmonic atomic force microscopy imaging of live and fixed mammalian cells.

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip-sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase-distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever-sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.

Raman Spectroscopy Adds Complementary Detail to the High-Resolution X-Ray Crystal Structure of Photosynthetic Psbp from Spinacia Oleracea.

Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally “dry,” has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.

Temperature dependence of photosynthesis and thylakoid lipid composition in the red snow alga Chlamydomonas cf. nivalis (Chlorophyceae)

Here, we report an effect of short acclimation to a wide span of temperatures on photosynthetic electron transfer, lipid and fatty acid composition in the snow alga Chlamydomonas cf. nivalis. The growth and oxygen evolution capacity were low at 2 °C yet progressively enhanced at 10 °C and were significantly higher at temperatures from 5 to 15 °C in comparison with the mesophilic control Chlamydomonas reinhardtii. In search of the molecular mechanisms responsible for the adaptation of photosynthesis to low temperatures, we have found unprecedented high rates of QA to QB electron transfer. The thermodynamics of the process revealed the existence of an increased structural flexibility that we explain with the amino acid changes in the D1 protein combined with the physico-chemical characteristics of the thylakoid membrane composed of > 80% negatively charged phosphatidylglycerol.

Carotenoid charge transfer states and their role in energy transfer processes in LH1-RC complexes from aerobic anoxygenic phototrophs.

Light-harvesting complexes ensure necessary flow of excitation energy into photosynthetic reaction centers. In the present work, transient absorption measurements were performed on LH1-RC complexes isolated from two aerobic anoxygenic phototrophs (AAPs), Roseobacter sp. COL2P containing the carotenoid spheroidenone, and Erythrobacter sp. NAP1 which contains the carotenoids zeaxanthin and bacteriorubixanthinal. We show that the spectroscopic data from the LH1-RC complex of Roseobacter sp. COL2P are very similar to those previously reported for Rhodobacter sphaeroides, including the transient absorption spectrum originating from the intramolecular charge-transfer (ICT) state of spheroidenone. Although the ICT state is also populated in LH1-RC complexes of Erythrobacter sp. NAP1, its appearance is probably related to the polarity of the bacteriorubixanthinal environment rather than to the specific configuration of the carotenoid, which we hypothesize is responsible for populating the ICT state of spheroidenone in LH1-RC of Roseobacter sp. COL2P. The population of the ICT state enables efficient S1/ICT-to-bacteriochlorophyll (BChl) energy transfer which would otherwise be largely inhibited for spheroidenone and bacteriorubixanthinal due to their low energy S1 states. In addition, the triplet states of these carotenoids appear well-tuned for efficient quenching of singlet oxygen or BChl-a triplets, which is of vital importance for oxygen-dependent organisms such as AAPs.

Double Mutation in Photosystem II Reaction Centers and Elevated CO2 Grant Thermotolerance to Mesophilic Cyanobacterium

Photosynthetic biomass production rapidly declines in mesophilic cyanobacteria grown above their physiological temperatures largely due to the imbalance between degradation and repair of the D1 protein subunit of the heat susceptible Photosystem II reaction centers (PSIIRC). Here we show that simultaneous replacement of two conserved residues in the D1 protein of the mesophilic Synechocystis sp. PCC 6803, by the analogue residues present in the thermophilic Thermosynechococcus elongatus, enables photosynthetic growth, extensive biomass production and markedly enhanced stability and repair rate of PSIIRC for seven days even at 43°C but only at elevated CO2 (1%). Under the same conditions, the Synechocystis control strain initially presented very slow growth followed by a decline after 3 days. Change in the thylakoid membrane lipids, namely the saturation of the fatty acids is observed upon incubation for the different strains, but only the double mutant shows a concomitant major change of the enthalpy and entropy for the light activated QA −→QB electron transfer, rendering them similar to those of the thermophilic strain. Following these findings, computational chemistry and protein dynamics simulations we propose that the D1 double mutation increases the folding stability of the PSIIRC at elevated temperatures. This, together with the decreased impairment of D1 protein repair under increased CO2 concentrations result in the observed photothermal tolerance of the photosynthetic machinery in the double mutant

Nonlinear effect of irradiance on photoheterotrophic activity and growth of the aerobic anoxygenic phototrophic bacterium Dinoroseobacter shibae : Photoheterotrophic activity of Dinoroseobacter shibae

Aerobic anoxygenic photosynthetic bacteria are an important component of marine microbial communities. They produce energy in light using bacteriochlorophyll a containing photosystems. This extra energy provides an advantage over purely heterotrophic bacteria. One of the most intensively studied AAP bacteria is Dinoroseobacter shibae, a member of the environmentally important Roseobacter clade. Light stimulates its growth and metabolism, but the effect of light intensity remains unclear. Here, we show that an increase in biomass along an irradiance gradient followed the exponential rise to the maximum curve, with saturation at about 300 µmol photons m-2 s-1 , without any inhibition at light intensities up to 600 µmol photons m-2 s-1 . The cells adapted to higher irradiance by reducing pigmentation and increasing the electron transfer rate. This additional energy allowed D. shibae to redirect the metabolism of organic carbon sources such as glucose, leucine, glutamate, acetate and pyruvate toward anabolism, resulting in a twofold increase of their assimilation rates. We provide equations that can be feasibly incorporated into the existing model of D. shibae metabolism to further advance our understanding of the role of photoheterotrophy in the ocean.

Unique double concentric ring organization of light harvesting complexes in Gemmatimonas phototrophica

The majority of life on Earth depends directly or indirectly on the sun as a source of energy. The initial step of photosynthesis is facilitated by light-harvesting complexes, which capture and transfer light energy into the reaction centers (RCs). Here, we analyzed the organization of photosynthetic (PS) complexes in the bacterium G. phototrophica, which so far is the only phototrophic representative of the bacterial phylum Gemmatimonadetes. The isolated complex has a molecular weight of about 800 ± 100 kDa, which is approximately 2 times larger than the core complex of Rhodospirillum rubrum. The complex contains 62.4 ± 4.7 bacteriochlorophyll (BChl) a molecules absorbing in 2 distinct infrared absorption bands with maxima at 816 and 868 nm. Using femtosecond transient absorption spectroscopy, we determined the energy transfer time between these spectral bands as 2 ps. Single particle analyses of the purified complexes showed that they were circular structures with an outer diameter of approximately 18 nm and a thickness of 7 nm. Based on the obtained, we propose that the light-harvesting complexes in G. phototrophica form 2 concentric rings surrounding the type 2 RC. The inner ring (corresponding to the B868 absorption band) is composed of 15 subunits and is analogous to the inner light-harvesting complex 1 (LH1) in purple bacteria. The outer ring is composed of 15 more distant BChl dimers with no or slow energy transfer between them, resulting in the B816 absorption band. This completely unique and elegant organization offers good structural stability, as well as high efficiency of light harvesting. Our results reveal that while the PS apparatus of Gemmatimonadetes was acquired via horizontal gene transfer from purple bacteria, it later evolved along its own pathway, devising a new arrangement of its light harvesting complexes.