A p-nitrophenol (PNP)- and phenol-mineralizing bacterium (strain NSP41) was isolated from an industrial wastewater and identified as a member of the genus Nocardioides. PNP was degraded via a hydroquinone pathway, and phenol was degraded through a catechol pathway in strain NSP41. Both enzyme systems for the degradation of PNP and phenol were induced simultaneously in the presence of both compounds. Although both enzyme systems were induced at the same time, PNP and phenol were degraded by the hydroquinone and catechol pathway, respectively. However, during the simultaneous degradation in the low phenol concentration, after the exhaustion of phenol, some PNP was transformed by the catechol pathway and 4-nitrocatechol was transiently accumulated. Kinetically, the addition of phenol greatly enhanced the apparent PNP degradation rate, which may be due to the increased cell mass by the assimilation of phenol.
A total of 199 microorganisms were isolated from the intestinal contents of flounder (Paralichthys olivaceus) in a fish farm in Seoul, Korea. Among these strains, DS-12 was selected as a candidate for flounder probiotics because of its excellent exhibition of antimicrobial activity against fish pathogens such as edwardsiella, pasteurella, aeromonas, and vibrio, and initiate growth in 10% NaCl, 10% bile, and in broth at pH 3 for 90 min. This strain was Gram-positive, and catalase-negative coccoid rods that produced gas from glucose and formed more than 90% of lactate as the D(−) isomer. This organism is positioned at a cluster in the genus Weissella on the phylogenetic tree based on 16S rRNA sequences, which were assigned to Weissella hellenica on the basis of DNA-DNA relatedness. However, the type strain of W. hellenica JCM 10103T had no antibacterial activity against the fish pathogenic bacteria and was found to be quite different from the DS-12 strain in some sugar fermentation patterns of α-methyl-D-glucoside, esculine, cellobiose, melibiose, D-raffinose, and D-turanose, being especially unable to grow at 15 and 35°C in 7% NaCl and 10% bile. The results obtained in the present study demonstrated that the type strain of W. hellenica had no probiotic characteristics, but the strain DS-12 could be used as a specific probiotic for flounder.
According to sequence analysis, the spoIV-locus of Bacillus megaterium DSM319 is 1,185 bp long; it is the second gene of a sporulation operon, which altogether contains three open reading frames. The ORF preceding spoIV encodes a putative polypeptide with 94 amino acids; the 3rd ORF of the operon has 972 bp corresponding to 324 amino acids. The operon is flanked on both sides by palindromic sequences, probably representing Rho-independent terminators. A primer extension analysis revealed that mRNA synthesis starts immediately downstream of a promoter, which is similar to the consensus sequence of Bacillus subtilis σE dependent promoters. Both the −35 and the −10 region are within the terminator region of the preceding operon. Gene knockout experiments and reporter gene assays with a newly developed system based on the heterologous Paenibacillus macerans glucanase gene (bgl) confirmed σE-dependent transcription. Two open reading frames of a further upstream operon were also identified. Northern analysis revealed that transcription of these ORFs comes about in late sporulation phases. The genetic organization of the spoIV comprising operon and adjacent loci clearly resembles that of the B. subtilisyqfa-phoH gene cluster. Thus our findings are of general significance for endospore-forming bacteria.
A chromosomal DNA fragment with a length of 2,025 bp, carrying the structural gene coding for glucoamylase in Thermoanaerobacterium thermosaccharolyticum, was cloned and sequenced. It coded for 695 amino acids, representing a polypeptide with a predicted molecular mass of 77.5 kDa. The deduced amino acid sequence exhibited high homologies with the glucoamylase sequence of another bacterial glucoamylase (Clostridium sp. G0005) and with fungal glucoamylases. The catalytic domain (amino acids 271 to 695) of the T. thermosaccharolyticum enzyme shared a high degree of similarity (five conserved regions) with the catalytic domain of Aspergillus awamori glucoamylase. By comparing the secondary structure of the sequence of the catalytic domain of the T. thermosaccharolyticum enzyme with that of glucoamylase from A. awamori, and on the basis of X-ray crystallographic data available for the A. awamori enzyme, it turned out that, most probably, both enzymes have a catalytic domain organized into an “(α/α)6-barrel” and an overall size and shape that is very similar. These findings confirm and extend our working model for the macromolecular architecture of the T. thermosaccharolyticum glucoamylase obtained, in earlier experiments, by electron microscopy of negatively stained isolated enzyme molecules. Antibodies for an enzyme-specific peptide located near the active site were successfully applied for inhibition studies of enzyme activity and for electron microscopic epitope mapping. A study comparing the site of attachment of this kind of antibody to the T. thermosaccharolyticum glucoamylase molecule with the expected attachment site as deduced from the A. awamori enzyme structure confirmed the close similarity of both glucoamylases regarding the macromolecular architecture of that part of the enzyme carrying the catalytic center, though helices H9, H10, and H11 in peripheral parts of the A. awamori enzyme are missing in the T. thermosaccharolyticum enzyme.
Twenty-six species of ammonia fungi comprising 71 strains were screened for ligninolytic activity using agar plate tests. The tests comprised a wood powder plate test, the Bavendamm reactions, and a Remazol Brilliant blue R (RBBR) decolorization test. The wood powder plate test detected phenol oxidases of Coprinus spp., whereas this method obviously detected no activities from facultative mycorrhizal fungi, such as Hebeloma radicosoides and ectomycorrhiza: H. spoliatum and H. vinosophyllum. With quantitative assays of ligninolytic activity, Coprinus phlyctidosporus, C. echinosporus, Lyophyllum tylicolor, Lepista nuda, L. tarda, Calocybe leucocephala, and Crucispora rhombisperma, which grow on oak-leaf litter, the major phenol-oxidizing enzyme was a laccase. The concentration of urea affected laccase activity; however, urea was not the obligate nitrogen source for the laccase production.
A nonflocculent industrial polyploid yeast strain, Saccharomyces cerevisiae 396-9-6V, was converted to a flocculent one by introducing a functional FLO1 gene at the URA3 locus. The flocculent strain FSC27 obtained was a so-called self-cloned strain, having no bacterial DNA. FSC27 cells could be easily recovered for reuse from fermentation mash without any physical energy. The strain produced a concentration of alcohol as high as 396-9-6V, although the fermentation rate of FSC27 was slightly lower than that of 396-9-6V. When uracil was added to the medium or when URA3 was reintroduced into FSC27 (named FSCU-L18), the fermentation rate and the growth rate increased, and the ethanol concentration produced was higher than that produced by the parent strain. The stable flocculation and high ethanol productivity were observed by using FSCU-L18 during 10 cycles of repeated-batch fermentation test.