The glycolate test is a method to discriminate N-acyl groups of muramyl residue in peptidoglycan of bacterial cell walls by color reaction without purification of the cell walls. The glycolyl residue presents red purple color by heating with 0.02% 2,7-dihydroxynaphthalene (DON) dissolved in concentrated H2SO4. Instead of the previous column methods for quantitative analysis, a qualitative method by solvent works was developed to simplify and to miniaturize the analysis. In this method, solvents played two roles, removal of interfering materials and extraction of glycolic acid from the cell hydrolysates. Of several solvent systems tested, diethyl ether was studied in detail on such properties as the efficiency of glycolic acid extraction under several conditions, the ability of removing various interfering compounds, and the advantage on evaporation procedure of the solvent from extracts. DON reaction of the second diethyl ether extract from cell hydrolysate of “Micromonospora nigra” JCM 3328 showed a clear red purple color of a strong absorbance at 530 nm, which is the same as that of authentic glycolic acid. The solvent method was applied to 20 strains of typical actinomycete species whose acyl types have already been known (Uchida and Seino, 1997). All glycolate test positive strains showed the clear red purple color mentioned above, whereas acetyl type strains revealed no apparent color by the same procedures. Additional experiments indicated that the glycolate test could be determined with less than 1 mg of actinomycete cells by using a smaller amount of DON reagent and ordinary polypropylene tubes. The new method was discussed for advantages in the identification of actinomycetes and for possible applications to other fields.
An endo-β-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector. The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate. The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr~41,014). Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen. The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295μmol of glucose min−1mg−1 protein when the substrates used were CMC, β-glucan, and xylan, respectively. A β-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate. To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.
A plasmid vector for fungal expression of an enhanced, red-shifted variant of the Aequoria victoriae green fluorescent protein was constructed by fusion of the EGFP gene to the highly expressed Aspergillus nidulansgpd promoter and the A. nidulanstrpC terminator. This construction was introduced by cotransformation, using benomyl selection, into Trichoderma harzianum strain 1051, a strain being evaluated for the biological control of witches'-broom disease of cocoa caused by Crinipellis perniciosa. Epifluorescence microscopy was used to monitor germination and attachment of stable transformant conidia on the surface of C. perniciosa hyphae.
In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes. The specific activities of these oxygenases were determined for C2 to C4 chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol·min−1·mg−1 of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol·min−1·mg−1 of dry cells by the cumene dioxygenase. The oxidation products were identified by gas chromatography-mass spectrometry. Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides. In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.
Aspergillus subgenus Clavati has four recognized species: A. clavatus (the type species), A. clavatonanicus, A. giganteus, and A. longivesica. These species are strictly anamorphic (mitotic) and defined by the morphological species concept. However, their genealogical relationships remain uncertain. In this study, we examined the genetic relatedness among the four species in this section, using electrophoretic comparison of enzymes, DNA base composition, and DNA-DNA hybridization. In a dendrogram based on the calculated similarity values of four enzymes, 10 strains in section Clavati, 3 strains in the xerophilic species, a strain in section Ornati, and a strain in section Cremei were separated into nine major clusters at a 60% similarity level. A. longivesica JCM 10186T had Q-10 in our analysis, but Kuraishi et al. (1990) reported A. longivesica JCM 1720T had Q-9 (49%) and Q-10 (46%). The G+C contents of the four species of section Clavati ranged from 48 to 50 mol%. The degree of the intraspecific reassociation among the DNAs from the strains of these species ranged from 77 to 99%, whereas the degrees of interspecific relative binding among strains of the four species ranged from 30 to 59%. Our data from enzyme patterns and DNA relatedness support the validity of the three species in section Clavati, except for A. longivesica.