Lanthanide-dependent methanol dehydrogenase from the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium diazoefficiens strain USDA110.

The legume symbiotic nitrogen-fixing bacterium, B. diazoefficiens strain USDA110, utilizes methanol for growth in the presence of light lanthanides, such as La3+, Ce3+, Pr3+ or Nd3+, and its cells possess significant methanol dehydrogenase (MDH) activity. We purified MDH to homogeneity from B. diazoefficiens strain USDA110 grown in a methanol/Ce3+ medium; the protein was identified as XoxF5-type MDH (blr6213). The purified XoxF contained 0.58 cerium atoms per enzyme subunit. Moreover, the in-solution structure of XoxF was analyzed by small angle X-ray scattering (SAXS) analysis; the radius of gyration (Rg) and maximum particle dimension (Dmax) of XoxF were calculated to be 32.3 and 96.8 Å, respectively, suggesting that XoxF adopts a dimer structure in solution. These results show that B. diazoefficiens strain USDA110 has XoxF, a lanthanides-dependent MDH, which has methanol oxidation activity and is induced by methanol/lanthanaides, and that lanthanide is one of the important factors in methanol utilization by the strain.

Effects of Adding Chicken Blood Meal and Fishmeal to Sludge Biogas as White Oyster Mushroom Media.

BACKGROUND AND OBJECTIVE: The biogas sludge is generally not used optimally and people even pour it directly into the river that it cause environmental pollution. One of the ways to get the benefit from sludge and chicken blood meal or fishmeal is to use it as a substitute for bran in mushroom media. This study aimed to improve the nutrient content of biogas sludge with the addition of chicken blood meal (CBM) and fishmeal (FM) as substitute material for bran in white oyster mushroom media. MATERIALS AND METHODS: The biogas sludge was dried in the sun for 3 days until its form resembled the soil. The treatment consisted of dried biogas sludge without CBM (BP0), dried biogas sludge with 1% CBM (BP1) and dried biogas sludge with 3% CBM (BP2). Each was added to the media of white oyster mushroom as much as 15% as substitute material for bran. The other treatment consisted of dried biogas sludge without FM (FP0), dried biogas sludge with 2% FM (FP1) and dried biogas sludge with 4% FM (FP2). Each was added to the media of white oyster mushroom as much as 5% as substitute material for bran. BP0 and FP0 created from white oyster mushroom media were commonly used by farmers. Each treatment was analyzed of nutritional and biological contents. All the data were tested using completely randomized design one-way ANOVA. RESULTS: The results of the research on the use of chicken blood meal and fishmeal showed that the best treatments were BP2 and FP2. Their nutrient content increased, including the organic-C, organic matter, nitrogen, P2O5 and K2O. The productivity of the oyster mushroom increased, shown by the increase of fresh weight and diameter of caps in treatment BP2. The replacement of bran by biogas sludge with 4% FM addition (FP2) in oyster mushroom media increased the fresh weight, the number of caps and the length of oyster mushroom stalks. CONCLUSION: The best treatments for mushroom media were BP2 and FP2 to be used as substitute material for bran in white oyster mushroom media.

Molecular structure and gene analysis of Ce3+ -induced methanol dehydrogenase of Bradyrhizobium sp. MAFF211645.

The molecular structure and nucleotide sequence of Ce(3+)-induced methanol dehydrogenase (MDH) of Bradyrhizobium sp. MAFF211645 were investigated. The addition of 30 µM Ce(3+) to 1/10 nutrient broth containing 0.5% methanol remarkably increased MDH activity. Furthermore, La(3+) increased MDH activity, but other heavier rare earth and metal elements did not have the same effect. MDH increased by Ce(3+) was purified by sequential column chromatography, and the purified MDH migrated as a single band with an apparent molecular weight of 68 kDa on SDS-PAGE. The apparent molecular weight of native MDH was estimated to be 108,000 by gel chromatography. The MDH was comprised of two identical subunits. N-terminal 23-amino acid sequence, 1-NDELHKMAQNPKDWVMPAGDYAN-23, of the purified MDH exhibited 91.3% identity to that of the MDH large subunit-like protein encoded by mxaF' of Bradyrhizobium japonicum USDA110. Nucleotide sequencing of the MDH gene of strain MAFF211645 yielded a deduced amino acid sequence comprising 601 amino acid residues, an N-terminal signal peptide, and a mature MDH comprising 578 amino acid residues with a predicted molecular mass of 62,918 Da. Further analysis of the deduced amino acid sequence of mature MDH revealed that the functional amino acids in its active site, such as two adjacent Cys residues, and bacterial quinoprotein signatures 1 and 2 were conserved. These results indicate that Ce(3+)-induced MDH encoded by mxaF' may be involved in methanol metabolism in Bradyrhizobium sp. MAFF211645.

Ce³+-induced exopolysaccharide production by Bradyrhizobium sp. MAFF211645.

Ce³+, a rare earth element (REE), has been widely used in high-technology industries. Despite the importance of Ce³+ in the fields of chemistry and physics, the role of Ce³+ in biology has been ignored. To investigate physiological effects of Ce³+ on microorganisms, we screened microorganisms that showed peculiar growth in the presence of Ce³+. We isolated a free-living soil bacterium that produced exopolysaccharide (EPS) around its colonies on 1/100 nutrient agar with 30 µM CeCl3 or 1.0% D-mannitol. The bacterium was identified as Bradyrhizobium sp. by morphological, biochemical, and physiological tests as well as 16S rDNA sequence analysis. La³+, Pr³+, and Nd³+ also induced EPS production in large quantities, while Sm³+ did in small amounts. However, other heavier REEs from Eu³+ to Lu³+, and metals such as Na+, Al³+, K+, Ca²+, V³+, Cr³+, Co²+, Ni²+, Sr²+, Ba²+, and Pb²+ did not induce EPS production. The mean molecular weight of EPS was estimated to be approximately 1 x 106 by Sepharose CL-4B column chromatography. TLC revealed that EPS was composed of L-rhamnose. Quantitative analysis of alditol acetate derivatives of acid hydrolyzate of EPS by GLC revealed that EPS was composed of more than 95% L-rhamnose, indicating that this EPS was a rhamnan. The spectrum of FT-IR of the rhamnan demonstrated that L-rhamnose residues in the rhamnan were α-linked. GC/MS analysis of methylated alditol acetate derivatives of the rhamnan demonstrated that it was composed of main chain α-(1→4)-linked L-rhamnopyranosyl residues. From spectral analyses of ¹H-NMR and FT-IR, EPS produced in the presence of 1.0% D-mannitol was found to be structurally similar to rhamnans.

Distribution of ytterbium (Yb) in cells of Streptomyces sp. YB-1 which can accumulate Yb, and reusability of cells and cell membrane as bioadsorbent for Yb.

The cells of Streptomyces sp. YB-1 adsorbed 4-6 mg ytterbium (Yb) per g dry weight. The Yb contents of the cell wall fraction, cell-free extract, and cell membrane fraction were 11%, 2%, and 87%, respectively. The Yb content in the cell membrane fraction was 20-25 mg per g dry weight. The adsorbed Yb could be quantitatively desorbed by treating the cell membrane fraction with 1 mM EDTA and 1 M HCl at 37 degrees C for 4 h. Treatment with 1 M NaOH caused Yb desorption to some extent. Treatments with proteinase K, lysozyme, 0.5% Triton X-100, 0.4% sodium dodecyl sulfate, and 1 M NaCl did not cause Yb desorption. Elemental analysis of Yb-adsorbed materials after removal of proteins and then extraction of lipids from the membrane fraction revealed that the molar ratio of Yb and P in the materials was about 1:1. The cells and the membrane fraction could be used repeatedly as a bioadsorbent for Yb.