Rediscovery of 4-Trehalosamine as a Biologically Stable, Mass-Producible, and Chemically Modifiable Trehalose Analog.

Nonreducing disaccharide trehalose is used as a stabilizer and humectant in various products and is a potential medicinal drug, showing curative effects on the animal models of various diseases. However, its use is limited as it is hydrolyzed by trehalase, a widely expressed enzyme in multiple organisms. Several trehalose analogs are prepared, including a microbial metabolite 4-trehalosamine, and their high biological stability is confirmed. For further analysis, 4-trehalosamine is selected as it shows high producibility. Compared with trehalose, 4-trehalosamine exhibits better or comparable protective activities and a high buffer capacity around the neutral pH. Another advantage of 4-trehalosamine is its chemical modifiability: simple reactions produce its various derivatives. Labeled probes and detergents are synthesized in one-pot reactions to exemplify the feasibility of their production, and their utility is confirmed for their respective applications. The labeled probes are used for mycobacterial staining. Although the derivative detergents can be effectively used in membrane protein research, long-chain detergents show 1000-3000-fold stronger autophagy-inducing activity in cultured cells than trehalose and are expected to become a drug lead and research reagent. These results indicate that 4-trehalosamine is a useful trehalose substitute for various purposes and a material to produce new useful derivative substances.

Proteolytic Processing, Maturation, and Unique Synteny of the Streptomyces Hemagglutinin SHA.

SHA is an l-rhamnose- and d-galactose-binding lectin that agglutinates human group B erythrocytes and was first purified almost 50 years ago. Although the original SHA-producing Streptomyces strain was lost, the primary structure of SHA was more recently solved by mass spectrometry of the archived protein, which matched it to a similar sequence in the Streptomyces lavendulae genome. Using genomic and protein biochemical analyses, this study aimed to identify SHA-secreting Streptomyces strains to further investigate the expression and binding activities of these putative proteins. Of 67 strains genetically related to S. lavendulae, 17 secreted pro-SHAs in culture. Seven SHA homologues were purified to homogeneity and then subjected to liquid chromatography-high-resolution multistage mass spectrometry (LC-MS/MS) and hemagglutination (HA) assays. Processing of pro-SHAs occurred during and after purification, indicating that associated proteases converted pro-SHAs into mature SHAs with molecular masses and HA activities similar to that of the archived SHA. Previously, the SHA monomer was shown to have two carbohydrate binding sites. The present study, however, found no HA activity in pro-SHAs, suggesting that pro-SHAs have only one binding site. Genetically, the SHA gene resides in conserved syntenic regions. The published genomes of 1,234 Streptomyces strains were analyzed, revealing 18 strains with SHA genes, 16 of which localized to a unique syntenic region. The SHA syntenic region consists of ∼17 open reading frames (ORFs) and is specific to S. lavendulae-related strains. Notably, a lipoprotein gene excludes SHA from the synteny in some strains, suggesting that horizontal gene transfer events during the course of evolution shaped the distribution of SHA genes. IMPORTANCE Lectins are extremely useful molecules for the study of glycans and carbohydrates. Here, we show that homologous genes encoding the l-rhamnose- and d-galactose-binding lectins, SHAs, are present in multiple bacterial strains, genetically related to Streptomyces lavendulae. SHA genes are expressed as precursor pro-SHA proteins that are truncated and mature into fully active lectins with two carbohydrate binding sites, which exhibit hemagglutination activity for type B red blood cells. The SHA gene is located within a conserved syntenic region, hinting at specific but yet-to-be-discovered biological roles of this carbohydrate-binding protein for its soil-dwelling microbial producer.

Saccharobipyrimicin, a new antibiotic from the leaf-litter actinomycete Saccharothrix sp. MM696L-181F4.

In the course of screening for new antimicrobial compounds, a new antibiotic substance named saccharobipyrimicin was isolated from the leaf-litter actinomycete Saccharothrix sp. MM696L-181F4. The structure of saccharobipyrimicin was elucidated by various spectral methods, mainly single-crystal X-ray analysis and chemical degradation. It revealed that saccharobipyrimicin contained a 2,2'-bipyridine skeletal structure. Saccharobipyrimicin showed moderate and broad-spectrum antimicrobial activity. Two chemical derivatives of saccharobipyrimicin showed weaker antimicrobial activities than that of saccharobipyrimicin against most test microorganisms except two tolC mutants of Escherichia coli and Neisseria gonorrhoeae.

Fradiamine A, a new siderophore from the deep-sea actinomycete Streptomyces fradiae MM456M-mF7.

New bioactive substances were identified from several marine actinomycetes strains by LC-HRESI-MS based non-targeted metabolomics. A new siderophore and its derivative, named fradiamines A and B, were isolated from the extract of the deep-sea actinomycetes Streptomyces fradiae MM456M-mF7 by Diaion CHP-20P, Sephadex LH-20 column chromatography and HPLC. Fradiamine A was a new compound, but fradiamine B was previously patented as a sweetness enhancer. Their structures were determined by NMR and LC-HRESI-MS/MS analysis. Fradiamines A and B contained two alkyl amines asymmetrically bonded to citrate, a type of structure derived from actinomycetes and other bacteria and rarely observed in siderophores. Fradiamines A and B showed moderate antibiotic activity against Clostridium difficile with IC50 values of 32 and 8 µg ml-1, respectively.

Ultrastructural Changes Associated with Reversible Stiffening in Catch Connective Tissue of Sea Cucumbers.

The dermis of sea cucumbers is a catch connective tissue or a mutable collagenous tissue that shows rapid, large and reversible stiffness changes in response to stimulation. The main component of the dermis is the extracellular material composed of collagen fibrils embedded in a hydrogel of proteoglycans. The stiffness of the extracellular material determines that of the dermis. The dermis has three mechanical states: soft (Sa), standard (Sb) and stiff (Sc). We studied the ultrastructural changes associated with the stiffness changes. Transverse sections of collagen fibrils in the dermis showed irregular perimeters with electron-dense protrusions or arms that cross-bridged between fibrils. The number of cross-bridges increased in stiffer dermis. The distance between the fibrils was shorter in Sc than that in other states, which was in accord with the previous report that water exuded from the tissue in the transition Sb→Sc. The ultrastructure of collagen fibrils that had been isolated from the dermis was also studied. Fibrils aggregated by tensilin, which causes the transition Sa→Sb possibly through an increase in cohesive forces between fibrils, had larger diameter than those dispersed by softenin, which antagonizes the effect of tensilin. No cross-bridges were found in isolated collagen fibrils. From the present ultrastructural study we propose that three different mechanisms work together to increase the dermal stiffness. 1.Tensilin makes collagen fibrils stronger and stiffer in Sa→Sb through an increase in cohesive forces between subfibrils that constituted fibrils; 2. Cross-bridging by arms caused the fibrils to be a continuous network of bundles both in Sa→Sb and in Sb→Sc; 3. The matrix embedding the fibril network became stiffer in Sb→Sc, which was produced by bonding associated with water exudation.

Softenin, a novel protein that softens the connective tissue of sea cucumbers through inhibiting interaction between collagen fibrils.

The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.