Bamboos are common and useful plants in Japan but little information is available about their endophytes. In this study, 257 fungal strains were isolated from bamboo tissues, and 71 representative ...strains were characterized by 18S rRNA gene and internal transcriber spacer region sequence analysis. Phylogenetic analysis showed that the fungal isolates were located in Sordariomycetes and Dothideomycetes. Xylariales was the dominant group within bamboos. Several rRNA gene sequences were not similar to any current sequence in the database and might be a novel species or genera that could represent sources of novel biological compounds. These findings reveal that bamboos are a huge reservoir of microorganisms that should be extensively investigated.
1 Department of Microbiology, Faculty of Science, Kasetsart University, 50 Paholyothin Rd, Bangkok 10900, Thailand
2 Department of Botany, University of East Yangon, Thanyin Township, Myanmar
3 The ...International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita-City, Osaka 565-0871, Japan
Correspondence Savitree Limtong fscistl{at}ku.ac.th
Two strains (S-34 T and S-35) of a novel ascomycetous yeast species belonging to the genus Kazachstania were isolated from soil from a mixed deciduous forest in Amphoe Wang Nam Khiao, Nakhon Ratchasima Province, Thailand. The D1/D2 domains of the large-subunit rDNA sequences of the two strains were identical and also indicated a close relationship with respect to Kazachstania aquatica , Kazachstania unispora , Kazachstania aerobia , Kazachstania servazzii and Kazachstania solicola . The most closely related species, K. aquatica , has 14 nucleotide substitutions and three gaps in 566 nt. The phenotypic characteristics of the two strains were typical of those of members of the genus Kazachstania . These characteristics include the formation of a single globose ascospore in an unconjugated and persistent ascus, multilateral budding, the absence of arthrospores and ballistospores, the fermentation of glucose, the inability to assimilate nitrate, negative diazonium blue B and urease reactions, and the presence of ubiquinone Q-6. The novel strains can be distinguished from K. aquatica on the basis of a number of phenotypic characteristics and represent a novel species in the genus Kazachstania , for which the name Kazachstania siamensis sp. nov. is proposed. The type strain is S-34 T (=CBS 10361 T =NBRC 101968 T =BCC 21230 T ).
Abbreviations: LSU, large subunit; SSU, small subunit
The GenBank/EMBL/DDBJ accession numbers for the D1/D2 domains of the LSU rDNA sequences of strains S-34 T and S-35 are AB258462 and AB258463 , respectively.
A neighbour-joining phylogenetic tree based on SSU rDNA sequences is available as a supplementary figure in IJSEM Online.
A polyphasic study was performed to determine the taxonomic position of strain EK05
T
isolated from a root-outgrowth of Entada koshunensis, a legume available in Okinawa, Japan. Phylogenetic analysis ...of the 16S rRNA gene showed that the strain belongs to the genus Bradyrhizobium. Subsequent multilocus sequence analysis with ITS, glnII, recA, gyrB, and atpD sequences revealed that the isolate represents a distinct evolutionary lineage within the genus Bradyrhizobium. DNA-DNA hybridization indicated that strain EK05
T
shares <61% DNA relatedness with the type strains of all six recognized species of Bradyrhizobium, confirming that this strain is a novel species within the genus. Phylogenetic trees based on symbiotic loci, nifH and nodC, also placed strain EK05
T
clearly in a novel branch. On the basis of its phylogenetic distinctiveness, we propose Bradyrhizobium iriomotense sp. nov. for strain EK05
T
. The type strain is EK05
T
(= NBRC 102520
T
= LMG 24129
T
).
Plant cells have no β1,4-galactosylated and sialylated glycan, which plays important roles in biological functions in animal cells. Previously, we generated transgenic tobacco BY2 suspension-cultured ...cells that produced human β1,4-galactosyltransferase N.Q. Palacpac, S. Yoshida, H. Sakai, Y. Kimura, K. Fujiyama, T. Yoshida, T. Seki, Stable expression of human β1,4-galactosyltransferase in plant cells modifies N-linked glycosylation pattern, Proc. Natl. Acad. Sci. USA 96 (1999) 4692–4697. In this study, we introduced two critical genes encoding human CMP-
N-acetylneuraminic acid synthetase and CMP-sialic acid transporter into tobacco suspension-cultured cell to pave a route for sialic biosynthetic pathway. The recombinant human proteins showed their biological activities. These results show that the plant cell can be a useful bioreactor for the production of mammalian glycoproteins.
1 Department of Microbiology, Faculty of Science, Kasetsart University, 50 Paholyothin Rd, Bangkok 10900, Thailand
2 The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, ...Suita-City, Osaka 565-0871, Japan
Correspondence Savitree Limtong fscistl{at}ku.ac.th
Four yeast strains (TM1-01 T , TM1-07, TM3-47 and TM3-49) were isolated by membrane filtration from estuarine water collected from a mangrove forest in Phang-Nga province, southern Thailand. Analysis of the D1/D2 domains of the large-subunit rDNA sequence revealed that the sequences of the four strains were identical. The closest recognized species in terms of pairwise sequence similarity was Pichia deserticola , but the level of nucleotide substitution (4.8 %) was sufficient to justify the description of a separate species. Phylogenetic analysis demonstrated that the four strains occupy a basal position with respect to Pichia membranifaciens and close relatives. The four strains showed identical phenotypic characteristics, including proliferation by multilateral budding, absence of ascospores, arthrospores and ballistospores and negative reactions for Diazonium blue B and urease. The major ubiquinone was Q-7. On the basis of the above findings, these four strains were assigned to a single novel species of the genus Candida , for which the name Candida thaimueangensis sp. nov. is proposed. The type strain is TM1-01 T (=CBS 10360 T =NBRC 101967 T =BCC 21229 T ).
The GenBank/EMBL/DDBJ accession number for the D1/D2 domain of the LSU rDNA sequence of TM1-01 T is AB264009 .
β1,4-Galactosyltransferase (UDP galactose: β- N -acetylglucosaminide: β1,4-galactosyltransferase; EC 2.4.1.22 ) catalyzes the transfer of galactose from UDP-Gal to N -acetylglucosamine in the ...penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blotting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligosaccharide structures shows that the galactosylated N -glycans account for 47.3% of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the transformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glycoproteins.
The gene encoding sucrose isomerase (
palI NK33) was cloned from
Klebsiella pneumoniae strain NK33-98-8. The gene was over-expressed in
Escherichia coli BL21 (DE3) pLysS and its enzyme product (PalI ...NK33) was purified and characterized. PalI NK33 converts sucrose to 76.8% palatinose, 21.2% trehalulose and 1% each of glucose and fructose. The purified PalI NK33 showed the very high specific activity at 2362
U/mg and
K
m for sucrose was 42.7
±
0.75
mM (at pH 6.0 and 30
°C). The enzyme activity was completely inhibited by 1
mM concentration of either Hg
2+ or SDS. Ca
2+, Li
2+ and Mg
2+ at 1
mM slightly enhanced enzyme activity. Mutations on Asp140, located within the conserved sequence region I to either glutamic acid, glycine or asparagine had drastically reduced enzyme activity. The change of amino acid residues in the sequence
325RLDRD
329 to
325RYDRA
329 reduced enzyme activity 24-fold and did not affect ratio of palatinose and trehalulose formation.
The production of antibodies using plants as bioreactors has been realized. Because sugar chain structures on recombinant proteins are a cause of concern, remodeling technology is highly promising. ...Localizing recombinant proteins in the endoplasmic reticulum (ER) affects the glycosylation pattern in transgenic plants. Previously, a mouse antibody produced by suspension-cultured tobacco BY2 cells has sugar chains with possible glycoepitopes as the predominant structures. In this study, we extended the Lys-Asp-Glu-Leu (KDEL) ER retention signal sequence over the heavy (H) and light (L) chains of the antibody and expressed the altered antibody in tobacco BY2 cells to study the effect of the KDEL sequence on glycosylation. For the antibody with the KDEL-extended H-chains, glycans with β(1,2)-xylose or α(1,3)-fucose residues accounted for 49% of the total glycans. Meanwhile, for the antibody with the KDEL-extended H- and L-chains, glycans with xylose or fucose accounted for 38% of the total glycans. Although the addition of an ER retention signal shifted the dominant glycan structures of the KDEL-extended antibody to high-mannose-type structures, some of the antibodies escaped the retrieval system during intracellular traffic and were then modified by xylosylation or fucosylation.
Beta-N-Acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in ...insects. A search using the Bombyx mori cDNA database revealed the existence of two putative beta-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a beta-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda beta-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the alpha-3 and alpha-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.
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