Mechanosensitive channels in bacteria: signs of closure?

Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.     

Vitellogenin cleavage products as indicators for toxic stress in zebra fish embryos: a proteomic approach

Vitellogenins (Vtgs) are the major yolk proteins in all oviparous animals. Systematic and regulated processing of these during embryogenesis is crucial for embryonic development. In the present study, toxicant-induced disturbance of Vtg degradation processes during Danio rerio (DR) embryogenesis was analysed to establish a sensitive tool for monitoring toxic stress at the molecular level. A 2-DE-based proteomic approach for whole DR embryos was established to study Vtg cleavage products (lipovitellin (Lv) derivatives). Ethanol was chosen as a positive control for a toxicity related change in the proteome of whole zebra fish embryos. Protein extracts from embryos treated with two ethanol concentrations, 0.5 and 2% v/v, showing either no or very strong visible effects, like absent heartbeat and blood circulation, were examined. Significant changes in the Lv pattern were detected for both conditions. The results are interpreted as scope for the use of the high abundant Lv derivatives as sensitive stress indicators in zebra fish embryos reflecting the overall fitness of the intact organisms.     

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.     

Metabolomics of dietary Fatty Acid restriction in patients with phenylketonuria

Background

Patients with phenylketonuria (PKU) have to follow a lifelong phenylalanine restricted diet. This type of diet markedly reduces the intake of saturated and unsaturated fatty acids especially long chain polyunsaturated fatty acids (LC-PUFA). Long-chain saturated fatty acids are substrates of mitochondrial fatty acid oxidation for acetyl-CoA production. LC-PUFA are discussed to affect inflammatory and haemostaseological processes in health and disease. The influence of the long term PKU diet on fatty acid metabolism with a special focus on platelet eicosanoid metabolism has been investigated in the study presented here.

Methodology/Principal Findings

12 children with PKU under good metabolic control and 8 healthy controls were included. Activated fatty acids (acylcarnitines C6–C18) in dried blood and the cholesterol metabolism in serum were analyzed by liquid chromatographic tandem mass spectrometry (LC-MS/MS). Fatty acid composition of plasma glycerophospholipids was determined by gas chromatography. LC-PUFA metabolites were analyzed in supernatants by LC-MS/MS before and after platelet activation and aggregation using a standardized protocol. Patients with PKU had significantly lower free carnitine and lower activated fatty acids in dried blood compared to controls. Phytosterols as marker of cholesterol (re-) absorption were not influenced by the dietary fatty acid restriction. Fatty acid composition in glycerophospholipids was comparable to that of healthy controls. However, patients with PKU showed significantly increased concentrations of y-linolenic acid (C18:3n-6) a precursor of arachidonic acid. In the PKU patients significantly higher platelet counts were observed. After activation with collagen platelet aggregation and thromboxane B₂ and thromboxane B₃ release did not differ from that of healthy controls.

Conclusion/Significance

Long-term dietary fatty acid restriction influenced the intermediates of mitochondrial beta-oxidation. No functional influence on unsaturated fatty acid metabolism and platelet aggregation in patients with PKU was detected.     

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (Etf_Af) and butyryl-CoA dehydrogenase (Bcd_Af) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. Etf_Af contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The Etf_Af-NAD⁺ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH⁻ is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH⁻ by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD^⨪, which donates this electron further to Dh-FAD of Bcd_Af after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH^•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH⁻ that converts crotonyl-CoA to butyryl-CoA.     

The Link between Microbial Diversity and Nitrogen Cycling in Marine Sediments Is Modulated by Macrofaunal Bioturbation

Objectives

The marine benthic nitrogen cycle is affected by both the presence and activity of macrofauna and the diversity of N-cycling microbes. However, integrated research simultaneously investigating macrofauna, microbes and N-cycling is lacking. We investigated spatio-temporal patterns in microbial community composition and diversity, macrofaunal abundance and their sediment reworking activity, and N-cycling in seven subtidal stations in the Southern North Sea.

Spatio-Temporal Patterns of the Microbial Communities

Our results indicated that bacteria (total and β-AOB) showed more spatio-temporal variation than archaea (total and AOA) as sedimentation of organic matter and the subsequent changes in the environment had a stronger impact on their community composition and diversity indices in our study area. However, spatio-temporal patterns of total bacterial and β-AOB communities were different and related to the availability of ammonium for the autotrophic β-AOB. Highest bacterial richness and diversity were observed in June at the timing of the phytoplankton bloom deposition, while richness of β-AOB as well as AOA peaked in September. Total archaeal community showed no temporal variation in diversity indices.

Macrofauna, Microbes and the Benthic N-Cycle

Distance based linear models revealed that, independent from the effect of grain size and the quality and quantity of sediment organic matter, nitrification and N-mineralization were affected by respectively the diversity of metabolically active β-AOB and AOA, and the total bacteria, near the sediment-water interface. Separate models demonstrated a significant and independent effect of macrofaunal activities on community composition and richness of total bacteria, and diversity indices of metabolically active AOA. Diversity of β-AOB was significantly affected by macrofaunal abundance. Our results support the link between microbial biodiversity and ecosystem functioning in marine sediments, and provided broad correlative support for the hypothesis that this relationship is modulated by macrofaunal activity. We hypothesized that the latter effect can be explained by their bioturbating and bio-irrigating activities, increasing the spatial complexity of the biogeochemical environment.     

Purification and Properties of a Thermoactive Glucoamylase from Clostridium thermosaccharolyticum

A bacterial glucoamylase was purified from the anaerobic thermophilic bacterium Clostridium thermosaccharolyticum and characterized. The enzyme, which was purified 63-fold, with a yield of 36%, consisted of a single subunit with an apparent molecular mass of 75 kDa. The purified enzyme was able to attack α-1,4- and α-1,6-glycosidic linkages in various α-glucans, liberating glucose with a β-anomeric configuration. The purified glucoamylase, which was optimally active at 70°C and pH 5.0, attacked preferentially polysaccharides such as starch, glycogen, amylopectin, and maltodextrin. The velocity of oligosaccharide hydrolysis decreased with a decrease in the size of the substrate. The K_m values for starch and maltose were 18 mg/ml and 20 mM, respectively. Enzyme activity was not significantly influenced by Ca²⁺, EDTA, or α- or β-cyclodextrins.