High-Density 3D Single Molecular Analysis Based on Compressed Sensing

Single molecule fitting-based superresolution microscopy achieves sub-diffraction-limit image resolution but suffers from a need for long acquisition times to gather enough molecules. Several methods have recently been developed that analyze high molecule density images but most are only applicable to two dimensions. In this study, we implemented a high-density superresolution localization algorithm based on compressed sensing and a biplane approach that provides three-dimensional information about molecules, achieving super-resolution imaging at higher molecule densities than those achieved using the conventional single molecule fitting method.     

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore''s nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore''s inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa''s amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.     

A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments

Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination resulted in up to 98% specificity, and the recovered anammox 16S rRNA gene sequences were >95% sequence identical to published sequences from anammox bacteria in the Candidatus “Scalindua” group. Furthermore, four primer sets used in detecting the hzo gene of anammox bacteria were highly specific (up to 92%) and efficient, and the newly designed primer set in this study amplified longer hzo gene segments suitable for phylogenetic analysis. The optimized primer set for the 16S rRNA gene and the newly designed primer set for the hzo gene were successfully applied to identify anammox bacteria from marine sediments of aquaculture zone, coastal wetland, and deep ocean where the three ecosystems form a gradient of anthropogenic impact. Results indicated a broad distribution of anammox bacteria with high niche-specific community structure within each marine ecosystem.     

Growth condition and bacterial community for maximum hydrolysis of suspended organic materials in anaerobic digestion of food waste-recycling wastewater

This paper reports the effects of changing pH (5–7) and temperature (T, 40–60 °C) on the efficiencies of bacterial hydrolysis of suspended organic matter (SOM) in wastewater from food waste recycling (FWR) and the changes in the bacterial community responsible for this hydrolysis. Maximum hydrolysis efficiency (i.e., 50.5% reduction of volatile suspended solids) was predicted to occur at pH 5.7 and T = 44.5 °C. Changes in short-chain volatile organic acid profiles and in acidogenic bacterial communities were investigated under these conditions. Propionic and butyric acids concentrations increased rapidly during the first 2 days of incubation. Several band sequences consistent with Clostridium spp. were detected using denaturing gel gradient electrophoresis. Clostridium thermopalmarium and Clostridium novyi seemed to contribute to butyric acid production during the first 1.5 days of acidification of FWR wastewater, and C. thermopalmarium was a major butyric acid producer afterward. C. novyi was an important propionic acid producer. These two species appear to be important contributors to hydrolysis of SOM in the wastewater. Other acidogenic anaerobes, Aeromonas sharmana, Bacillus coagulans, and Pseudomonas plecoglossicida, were also indentified.     

Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions

WF146 protease, a thermophilic subtilase from thermophile Bacillus sp. WF146, suffers excessive autolysis in the presence of reducing agents. In this report, two autolytic sites of WF146 protease were modified by site-directed mutagenesis. The introduction of prolines into the autolytic sites increased the autolysis resistance of the enzyme under reducing conditions. The double mutant N63P/A66P displayed a 2.8-fold longer half-life at 80°C and higher hydrolytic activities than wild-type enzyme toward soluble (casein) and insoluble (keratin azure) substrates at high temperatures. In the presence of reducing agents, N63P/A66P was able to degrade feather at 80°C (∼3 h), with hydrolysis efficiency comparable to that of proteinase K at 50°C (∼24 h). Meanwhile, the mutant N63P/A66P had the ability to hydrolyze PrP^Sc-like prion protein at high temperatures. In virtue of these properties, N63P/A66P is of great interest to be used in recycling of keratinous wastes, such as feather, and disinfection of medical apparatus. In addition, our study may provide useful information needed to explore keratinolytic potential of thermophilic subtilases, even if they are produced by non-keratinolytic microorganisms.     

Direct dynamin-actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.