Metabolite labelling reveals hierarchies in Clostridium acetobutylicum that selectively channel carbons from sugar mixtures towards biofuel precursors

Clostridial fermentation of cellulose and hemicellulose relies on the cellular physiology controlling the metabolism of the cellulosic hexose sugar (glucose) with respect to the hemicellulosic pentose sugars (xylose and arabinose) and the hemicellulosic hexose sugars (galactose and mannose). Here, liquid chromatography–mass spectrometry and stable isotope tracers in Clostridium acetobutylicum were applied to investigate the metabolic hierarchy of glucose relative to the different hemicellulosic sugars towards two important biofuel precursors, acetyl‐coenzyme A and butyryl‐coenzyme A. The findings revealed constitutive metabolic hierarchies in C. acetobutylicum that facilitate (i) selective investment of hemicellulosic pentoses towards ribonucleotide biosynthesis without substantial investment into biofuel production and (ii) selective contribution of hemicellulosic hexoses through the glycolytic pathway towards biofuel precursors. Long‐term isotopic enrichment demonstrated incorporation of both pentose sugars into pentose‐phosphates and ribonucleotides in the presence of glucose. Kinetic labelling data, however, showed that xylose was not routed towards the biofuel precursors but there was minor contribution from arabinose. Glucose hierarchy over the hemicellulosic hexoses was substrate‐dependent. Kinetic labelling of hexose‐phosphates and triose‐phosphates indicated that mannose was assimilated but not galactose. Labelling of both biofuel precursors confirmed this metabolic preference. These results highlight important metabolic considerations in the accounting of clostridial mixed‐sugar utilization.     

A conserved surface loop in type I dehydroquinate dehydratases positions an active site arginine and functions in substrate binding

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.     

Tracking dietary habits of cave arthropods associated with deposits of hematophagous bat guano: A study from a neotropical savanna

Carbon and nitrogen stable isotopes were used to track major dietary variations in arthropods found in a cave located in a neotropical savanna in Central Brazil. We determined the δ¹³C and δ¹⁵N for cave crickets, cockroaches, spiders, guano of a hematophagous bat, and leaf litter found on the ground near the cave entrance. The δ¹³C and δ¹⁵N for the cricket and cockroaches showed that bat guano was not the only food item for these arthropods. They had intermediate δ¹³C and δ¹⁵N between bat guano and leaf litter, which means that they consumed additional food resources other than guano in their diet, independent of distribution of guano deposits in the cave. The spiders, predators with great mobility, seemed to not have a preferential diet item since their isotope signals indicated they hunt both crickets and cockroaches. The δ¹³C of the bat guano (?15.4‰) indicated that the diet of these hematophagous bats relied on animals fed with C₄ plants. The conversion of native savanna vegetation of which the debris is mostly from C₃ plants, to pasture, based on C₄ African grass, a remarkably common land use transformation in the region, is indirectly influencing the diet of hematophagous bats, which basically relied on exotic fauna. The C and N stable isotope ratios showed that the arthropods inside the cave do not rely solely on bat guano for their diet, but interact directly with the external environment, through litter debris, indicating a significant exchange of energy and matter between the cave environment and the surrounding area.     

Crystal structure of scallop Myosin s1 in the pre-power stroke state to 2.6 a resolution: flexibility and function in the head

We have extended the X-ray structure determination of the complete scallop myosin head in the pre-power stroke state to 2.6 A resolution, allowing an atomic comparison of the three major (weak actin binding) states of various myosins. We can now account for conformational differences observed in crystal structures in the so-called "pliant region" at the motor domain-lever arm junction between scallop and vertebrate smooth muscle myosins. A hinge, which may contribute to the compliance of the myosin crossbridge, has also been identified for the first time within the regulatory light-chain domain of the lever arm. Analysis of temperature factors of key joints of the motor domain, especially the SH1 helix, provides crystallographic evidence for the existence of the "internally uncoupled" state in diverse isoforms. The agreement between structural and solution studies reinforces the view that the unwinding of the SH1 helix is a part of the cross-bridge cycle in many myosins.     

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H_I and H_IV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca²⁺ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca²⁺ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca²⁺ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca²⁺. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.     

Honey bees can disseminate a microbial control agent to more than one inflorescence pest of oilseed rape

Pollen beetles (Meligethes aeneus) and cabbage seed weevils (Ceutorhynchus assimilis) are major pests of oilseed rape (Brassica napus) throughout Europe. In field cage experiments in both winter and spring rape, honey bees (Apis mellifera) effectively transported the entomopathogenic fungus Metarhizium anisopliae to the flowers, causing infection and mortality of both adult and larval pollen beetles, as well as of adult seed weevils. External conidiation was observed on many of the dead pest insects. Although some external conidiation also occurred on dead honey bees, reduction in honey bee colony size during the experiments appeared unrelated to the fungus. The potential of this technique for integration into pest management strategies for the crop, particularly in association with a trap crop, is discussed.     

Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome

Speculation has long surrounded the question of whether past exposure to ionizing radiation leaves a unique permanent signature in the genome. Intrachromosomal rearrangements or deletions are produced much more efficiently by densely ionizing radiation than by chemical mutagens, x-rays, or endogenous aging processes. Until recently, such stable intrachromosomal aberrations have been very hard to detect, but a new chromosome band painting technique has made their detection practical. We report the detection and quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear-weapons workers who were exposed to plutonium many years ago. Even many years after occupational exposure, more than half the blood cells of the healthy plutonium workers contain large (>6 Mb) intrachromosomal rearrangements. The yield of these aberrations was highly correlated with plutonium dose to the bone marrow. The control groups contained very few such intrachromosomal aberrations. Quantification of this large-scale chromosomal damage in human populations exposed many years earlier will lead to new insights into the mechanisms and risks of cytogenetic damage.