Pseudochoricystis ellipsoidea is a recently isolated unicellular green alga, which is classified within the family Trebouxiophyceae. This alga has a unique ability to synthesize and accumulate intracellularly a significant amount of aliphatic hydrocarbons. To elucidate molecular mechanisms of the hydrocarbon production in this organism, the development of genetic methods including DNA transformation methods are important. Towards the goal, we constructed several plasmids in which neomycin phosphotransferase II-encoding G418-resistant gene (nptII) is flanked by a P. ellipsoidea-derived promoter and terminator. These plasmids were introduced into P. ellipsoidea cells through particle-gun bombardment, and transformants were screened among G418-resistant cells by PCR amplification of plasmid-borne genes. Southern blot analysis demonstrated that the exogenous DNA was integrated into the genome of the transformants. Furthermore, the expression of nptII was confirmed at the transcript and protein levels by RT-PCR and immunoblot analyses, respectively. These results clearly indicated that a genetic transformation system was successfully established for P. ellipsoidea.
Lactobacillus gasseri ATCC33323T expresses four enzymes showing phospho-β-galactosidase activity (LacG1, LacG2, Pbg1 and Pbg2). We previously reported the purification and characterization of two phospho-β-galactosidases (Pbg1 and Pbg2) from Lactobacillus gasseri JCM1031 cultured in lactose medium. Here we aimed to characterize LacG1 and LacG2, and classify the four enzymes into ‘phospho-β-galactosidase’ or ‘phospho-β-glucosidase.’ LacG1 and recombinant LacG2 (rLacG2), from Lb. gasseri ATCC33323T, were purified to homogeneity using column chromatography. Kinetic experiments were performed using sugar substrates, o-nitrophenyl-β-D-galactopyranoside 6-phosphate (ONPGal-6P) and o-nitrophenyl-β-D-glucopyranoside 6-phosphate (ONPGlc-6P), synthesized in our laboratory. LacG1 and rLacG2 exhibited high kcat/Km values for ONPGal-6P as compared with Pbg1 and Pbg2. The Vmax values for ONPGal-6P were higher than phospho-β-galactosidases previously purified and characterized from several lactic acid bacteria. A phylogenetic tree analysis showed that LacG1 and LacG2 belong to the phospho-β-galactosidase cluster and Pbg1 and Pbg2 belong to the phospho-β-glucosidase cluster. Our data suggest two phospho-β-galactosidase, LacG1 and LacG2, are the primary enzymes for lactose utilization in Lb. gasseri ATCC33323T. We propose a reclassification of Pbg1 and Pbg2 as phospho-β-glucosidase.
Boat lubricants are continuously released into the marine environment and thereby cause chronic oil pollution. This study aims to isolate lubricant-degrading microorganisms from Thai coastal areas as well as to apply a selected strain for removal of boat lubricants. Ten microorganisms in the genera of Gordonia, Microbacterium, Acinetobacter, Pseudomonas, Brucella, Enterococcus and Candida were initially isolated by crude oil enrichment culture techniques. The lubricant-removal activity of these isolates was investigated with mineral-based lubricants that had been manufactured for the 4-stroke diesel engines of fishing boats. Gordonia sp. JC11, the most effective strain was able to degrade 25-55% of 1,000 mg L-1 total hydrocarbons in six tested lubricants, while only 0-15% of the lubricants was abiotically removed. The bacterium had many characteristics that promoted lubricant degradation such as hydrocarbon utilization ability, emulsification activity and cell surface hydrophobicity. For bioaugmentation treatment of lubricant contaminated seawater, the inoculum of Gordonia sp. JC11 was prepared by immobilizing the bacterium on polyurethane foam (PUF). PUF-immobilized Gordonia sp. JC11 was able to remove 42-56% of 100-1,000 mg L-1 waste lubricant No. 2 within 5 days. This lubricant removal efficiency was higher than those of free cells and PUF without bacterial cells. The bioaugmentation treatment significantly increased the number of lubricant-degrading microorganisms in the fishery port seawater microcosm and resulted in rapid removal of waste lubricant No. 2.
Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.
The morphology of mitochondrial nucleoids (mt-nucleoids), mitochondria, and nuclei was investigated during meiosis and sporulation of the diploid cells of the ascosporogenic yeast Saccharomycodes ludwigii. The mt-nucleoids appeared as discrete dots uniformly distributed in stationary-phase cells as revealed by 4′,6-diamidino-2-phenylindole (DAPI) staining. Throughout first and second meiotic divisions, the mt-nucleoids moved to be located close to the dividing nuclei with the appearance of dots. On the other hand, mitochondria, which had tubular or fragmented forms in stationary-phase cells, increasingly fused with each other to form elongated mitochondria during meiotic prophase as revealed by 3,3′ -dihexyloxacarbocyanine iodide [DiOC6(3)] staining. Mitochondria assembled to be located close to dividing nuclei during first and second meiotic divisions, and were finally incorporated into spores. During the first meiotic division, nuclear division occurred in any direction parallel, diagonally, or perpendicular to the longitudinal axis of the cell. In contrast, the second meiotic division was exclusively parallel to the longitudinal axis of the cell. The behavior of dividing nuclei explains the formation of a pair of spores with opposite mating types at both ends of cells. In the course of this study, it was also found that ledges between two spores were specifically stained with DiOC6(3).