Chang Long An

Endophytic bacillus sp. isolated from the interior of balloon flower root.

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.

Analysis of bgl Operon Structure and Characterization of β-Glucosidase from Pectobacterium carotovorum subsp. carotovorum LY34

A putative bgl operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated. Sequence analysis of the 5,557 bp cloned DNA fragment (accession no. AY542524) showed three open reading frames (bglT, bglP, and bglB) predicted to encode 287, 633, and 468 amino acid proteins respectively. BglT and BglP ORFs show high similarity to that of the Pectobacterium chrysanthemi ArbG antiterminator and ArbF permease respectively. Also, the latter contains most residues important for phosphotransferase activity. The amino acid sequence of BglB showed high similarity to various β-glucosidases and is a member of glycosyl hydrolase family 1. The purified BglB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. The molecular weight of the enzyme was estimated to be 53,000 Da by SDS–PAGE. The purified β-glucosidase exhibited maximal activity at pH 7.0 and 40 °C, and its activity was enhanced in the presence of Mg2+. Two glutamate residues (Glu173 and Glu362) were found to be essential for enzyme activity.

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons of Pectobacterium carotovorum subsp. carotovorum LY34

A third bgl operon containing bglE, bglF, bglI, and bglA was isolated from Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34). The sequences of BglE, BglF, and Bgll were similar to those of the phosphotransferase system (PTS) components IIB, IIC, and IIA respectively. BglF contains important residues for the phosphotransferase system. The amino acid sequence of BglA showed high similarity to various 6-phospho-β-glucosidases and to a member of glycosyl hydrolase family 1. Sequence and structural analysis also revealed that these four genes were organized in a putative operon that differed from two operons previously isolated from Pcc LY34, bglTPB (accession no. AY542524) and ascGFB (accession no. AY622309). The transcription regulator for this operon was not found, and the EII complexes for PTS were encoded separately by three genes (bglE, bglF, and bglI). The BglA enzyme had a molecular weight estimated to be 57,350 Da by SDS–PAGE. The purified β-glucosidase hydrolyzed salicin, arbutin, ρNPG, ρNPβG6P, and MUG, exhibited maximal activity at pH 7.0 and 40 °C, and displayed enhanced activity in the presence of Mg2+ and Ca2+. Two glutamate residues (Glu178 and Glu378) were found to be essential for enzyme activity.

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35.

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.

Phylogenetic analysis of yeast in the rumen contents of cattle based on the 26S rDNA sequence

The composition of yeast communities in the rumen of cattle was investigated using comparative DNA sequence analysis of yeast 26S rDNA genes. 26S rDNA libraries were constructed from rumen fluid (FF), rumen solid (FS) and rumen epithelium (FE). A total of 97 clones, containing a partial 26S rDNA sequence of 0.6 kb length, were sequenced and subjected to an on-line similarity search. The 41 FF clones could be divided into five classes. The largest class was affiliated with Pezizomycotina class (85.4% of clones), and the remaining classes were related with the Urediniomycotina (2.4%), Hymenomycetes (4.9%), Ustilaginomycetes (4.9%) and Saccharomycotina (2.4%) classes. The 26 FE clones could be divided into three classes and the Saccharomycetes class (92.4 % of clones) was the largest group. The remaining classes were related with either Pezizomycotina (3.8%) or Ustilaginomycetes (3.8 %). The 30 FS clones were all affiliated with Saccharomycotina. Saccharomycotina were predominant in rumen epithelium and rumen solid while Pezizomycotina were predominant in rumen fluid. Yeast belonging to the Saccharomycotina class was predominant in the rumen as a whole (57%). One clone (FF34) had less than 90% similarity to any sequence in the database and was thus apparently unrelated to any previously described yeast.