Shohei Sakuda

Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis

Biosynthesis of gelatinase, a virulence factor of Enterococcus faecalis, was found to be regulated in a cell density‐dependent fashion in which its production is active in late log to early stationary phase. Addition of early stationary phase culture filtrate to medium shifted the onset of gelatinase production to that of mid‐log phase, suggesting that E. faecalis secretes a gelatinase biosynthesis‐activating pheromone (GBAP). GBAP was isolated from culture supernatant of E. faecalis OG1S‐P. Structural analysis suggested GBAP to be an 11‐residue cyclic peptide containing a lactone structure, in which the α‐carboxyl group of the C‐terminal amino acid is linked to a hydroxyl group of the serine of the third residue. A synthetic peptide possessing the deduced structure showed GBAP activity at nanomolar concentrations as did natural GBAP. Database searches revealed that GBAP corresponds to a C‐terminal part of a 242‐residue FsrB protein. Northern analysis showed that GBAP slowly induces the transcription of two operons, fsrB‐fsrC encoding FsrB and a putative histidine kinase FsrC and gelE‐sprE encoding gelatinase GelE and serine protease SprE. Strains with an insertion mutation in either fsrC or a putative response regulator gene fsrA failed to respond to GBAP, suggesting that the GBAP signal is transduced by a two‐component regulatory system.

The structure of allosamidin, a novel insect chitinase inhibitor, produced by Streptomyces Sp.

Allosamidin(1), a novel insect chitinase inhibitor, was isolated from the mycelium of Streptomyces sp. and characterized as 1, which was a basic pseudotrisaccharide consisting of 2-acetamido-2-deoxy-D-allose(N-acetyl-D-allosamine) and a novel aminocyclitol derivative(3), termed allosamizoline.

Family 19 chitinases of Streptomyces species: characterization and distribution.

Chitinase C from Streptomyces griseus HUT6037, described in 1997, is the first family 19 chitinase found in an organism other than higher plants. In this study, some properties of chitinase C were compared with those of family 18 bacterial chitinases, and the distribution of family 19 chitinases in Streptomyces species was investigated. The specific hydrolysing activity of chitinase C against soluble and insoluble chitinous substrates was markedly higher than those of bacterial family 18 chitinases. Chitinase C exhibited marked antifungal activity, whereas the other bacterial chitinases examined had no antifungal activity. Chitinase C was insensitive to allosamidin, whereas the family 18 bacterial chitinases were sensitive. Taking advantage of this insensitivity to allosamidin, a search was made for family 19 chitinases in various Streptomyces species. Chitinases insensitive to allosamidin were detected in the culture supernatants of all tested Streptomyces species. Southern hybridization analysis using a labelled DNA fragment corresponding to the catalytic domain of chitinase C strongly suggested that these species have genes similar to the chiC gene of S. griseus HUT6037. DNA fragments corresponding to the major part of the catalytic domains were amplified by PCR. The amplified fragments encoded amino acid sequences very similar to that of the corresponding region of chitinase C. Therefore, it was concluded that Streptomyces species generally possess family 19 chitinases which are very similar to chitinase C. Comparison of their amino acid sequences with those of plant family 19 chitinases revealed that Streptomyces family 19 chitinases are class IV type in terms of the presence and positions of deletions of amino acid sequences which are characteristic of plant class IV chitinases.

Inhibitory effects of Satureja hortensis L. essential oil on growth and aflatoxin production by Aspergillus parasiticus

In an effort to screen the essential oils of some Iranian medicinal plants for novel aflatoxin (AF) inhibitors, Satureja hortensis L. was found as a potent inhibitor of aflatoxins B1 (AFB1) and G1(AFG1) production by Aspergillus parasiticus NRRL 2999. Fungal growth was also inhibited in a dose-dependent manner. Separation of the plant inhibitory substance(s) was achieved using initial fractionation of its effective part (leaf essential oil; LEO) by silica gel column chromatography and further separation by reverse phase-high performance liquid chromatography (RP-HPLC). These substances were finally identified as carvacrol and thymol, based on the interpretation of 1H and 13C NMR spectra. Microbioassay (MBA) on cell culture microplates contained potato-dextrose broth (PDB) medium (4 days at 28 degrees C) and subsequent analysis of cultures with HPLC technique revealed that both carvacrol and thymol were able to effectively inhibit fungal growth, AFB1 and AFG1 production in a dose-dependent manner at all two-fold concentrations from 0.041 to 1.32 mM. The IC50 values for growth inhibition were calculated as 0.79 and 0.86 mM for carvacrol and thymol, while for AFB1 and AFG1, it was reported as 0.50 and 0.06 mM for carvacrol and 0.69 and 0.55 mM for thymol. The results obtained in this study clearly show a new biological activity for S. hortensis L. as strong inhibition of aflatoxin production by A. parasiticus. Carvacrol and thymol, the effective constituents of S. hortensis L., may be useful to control aflatoxin contamination of susceptible crops in the field.

Isolation and structure of a new butyrolactone autoregulator from Streptomyces sp. FRI-5

A hormone-like factor, which triggers blue pigment production in Streptomyces sp. FRI-5, was purified 170,000-fold from a culture broth of the strain with 14% activity recovery. The compound, named IM-2, was effective at a concentration of 0.6 ng/ml in initiating blue pigment-production. The structure was determined to be 2, 3-trans-2-(1′-hydroxybutyl)-3-(hydroxymethyl)butanolide (10) on the basis of spectral data and chemical synthesis.

Identification of Chitin in the Prismatic Layer of the Shell and a Chitin Synthase Gene from the Japanese Pearl Oyster, Pinctada fucata

The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is β-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.

Glycolytic intermediates induce amorphous calcium carbonate formation in crustaceans

It has been thought that phosphorus in biominerals made of amorphous calcium carbonate (ACC) might be related to ACC formation, but no such phosphorus-containing compounds have ever been identified. Crustaceans use ACC biominerals in exoskeleton and gastroliths so that they will have easy access to calcium carbonate inside the body before and after molting. We have identified phosphoenolpyruvate and 3-phosphoglycerate, intermediates of the glycolytic pathway, in exoskeleton and gastroliths and found them important for stabilizing ACC.

Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes

The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, l-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn2+ ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C3 and C4 primary metabolites.

Chitinase Gene Expression in Response to Environmental Stresses in Arabidopsis thaliana: Chitinase Inhibitor Allosamidin Enhances Stress Tolerance

The expression levels of three chitinase genes in Arabidopsis thaliana, AtChiA (class III), AtChiB (class I), and AtChiV (class IV), were examined under various stress conditions by semi-quantitative RT-PCR. Under normal growth conditions, the AtChiB and AtChiV genes were expressed in most organs of Arabidopsis plants at all growth stages, whereas the AtChiA gene was not expressed at all. The class III AtChiA gene was expressed exclusively when the plants were exposed to environmental stresses, especially to salt and wound stresses. Treatment of Arabidopsis plants with allosamidin, which inhibits class III chitinases, did not affect the growth rate. Surprisingly, however, the plants treated with allosamidin were more tolerant of abiotic stresses (cold, freezing, heat, and strong light) than the control plants. It also appeared that allosamidin enhances AtChiA and AtChiB expression under heat and strong light stresses. Allosamidin is likely to enhance abiotic stress tolerance, probably through crosstalk between the two signaling pathways for biotic and abiotic stress responses.

Mycothiol, 1-O-(2′-[N-acetyl-L-cysteinyl]amido-2′-deoxy-α-D-glucopyranosyl)-D-myo-inositol, is the factor of NAD/factor-dependent formaldehyde dehydrogenase

Two different NAD/coenzyme‐dependent formaldehyde dehydrogenases exist, the well‐known NAD/GSH‐dependent (EC 1.2.1.1) and the more recently discovered NAD/Factor‐dependent enzyme. The GSH‐dependent one has been found in eukaryotes and Gram‐negative bacteria, the Factor‐dependent one in two different Gram‐positive bacteria. Previous work also showed that Factor and GSH are not interchangeable in the enzymatic reactions. Here it is revealed that the Factor is identical to mycothiol (MySH), 1‐O‐(2′‐[N‐acetyl‐L‐cysteinyl]amido‐2′‐deoxy‐α‐D‐glucopyranosyl)‐D‐myo‐inositol, a thiol compound which has recently been detected in Actinomycetes. Thus, MySH is GSHs companion as it is the coenzyme for the enzyme which henceforth can be indicated as NAD/MySH‐dependent formaldehyde dehydrogenase.