Identification of the Gene Responsible for Lignin-Derived Low-Molecular-Weight Compound Catabolism in Pseudomonas sp. Strain LLC-1.

Pseudomonas sp. strain LLC-1 (NBRC 111237) is capable of degrading lignin-derived low-molecular-weight compounds (LLCs). The genes responsible for the catabolism of LLCs were characterized in this study using whole-genome sequencing. Despite the close phylogenetic relationship with Pseudomonas putida, strain LLC-1 lacked the genes usually found in the P. putida genome, which included fer, encoding an enzyme for ferulic acid catabolism, and vdh encoding an NAD+-dependent aldehyde dehydrogenase specific for its catabolic intermediate, vanillin. Cloning and expression of the 8.5 kb locus adjacent to the van operon involved in vanillic acid catabolism revealed the bzf gene cluster, which is involved in benzoylformic acid catabolism. One of the structural genes identified, bzfC, expresses the enzyme (BzfC) having the ability to transform vanillin and syringaldehyde to corresponding acids, indicating that BzfC is a multifunctional enzyme that initiates oxidization of LLCs in strain LLC-1. Benzoylformic acid is a catabolic intermediate of (R,S)-mandelic acid in P. putida. Strain LLC-1 did not possess the genes for mandelic acid racemization and oxidation, suggesting that the function of benzoylformic acid catabolic enzymes is different from that in P. putida. Genome-wide characterization identified the bzf gene responsible for benzoylformate and vanillin catabolism in strain LLC-1, exhibiting a unique mode of dissimilation for biomass-derived aromatic compounds by this strain.

Draft Genome Sequence of Pseudomonas sp. Strain LLC-1 (NBRC 111237), Capable of Metabolizing Lignin-Derived Low-Molecular-Weight Compounds.

Pseudomonas sp. strain LLC-1 (NBRC 111237), isolated from soil, metabolizes lignin-derived low-molecular-weight compounds and utilizes vanillin and vanillic acid as its sole sources of carbon. Here, we report the draft genome sequence of Pseudomonas sp. strain LLC-1.

Alkyl substituent effect on photosensitized inactivation of Escherichia coli by pyridinium-bonded P-porphyrins.

Activity of singlet oxygen sensitizers for photoinactivation of bacteria and photodynamic therapy of tumor cells has been evaluated using nonpathogenic model cells, such as Escherichia coli, Saccharomyces cerevisiae, and HeLa cells. Among them, E. coli, gram-negative bacterium, has complex membrane structures in the cell wall, resulting in an impermeable barrier to antimicrobial agents. Therefore, few singlet oxygen sensitizers have photoinactivation activities toward E. coli at low concentrations. Recently polycationic porphyrins have received much attention as a new type of singlet oxygen sensitizers because they have strong binding affinities for DNA and proteins. Here, we prepared 13 types of di- and tricationic P- and Sb-porphyrin sensitizers substituted with the N-alkylpyridinium (APy) group at the axial ligand or the meso position to examine their bactericidal activity toward E. coli under visible-light irradiation. Photobactericidal activities were evaluated using half-life (T1/2 in min) of E. coli and minimum effective concentrations of the porphyrin sensitizers. Di-cationic P-porphyrins containing the Apy group at meso position exhibited bactericidal activity under dark conditions. Tricationic porphyrins showed a higher bactericidal activity than monocationic porphyrins. It was found that the bactericidal activity depended on the alkyl chain length of APy. Tricationic porphyrin with N-heptylpyridinium in two axial ligands was the most reactive for the photoinactivation of E. coli.

Assistance of human serum albumin to photo-sensitized inactivation of Saccharomyces cerevisiae with axially pyridinio-bonded P-porphyrins.

As singlet-oxygen ((1)O2) sensitizer, water-soluble P-porphyrins (1) were prepared by the modification of axial ligands of tetraphenylporphyrinatophosphorus by N-alkyl-pyridinium group to give bis[3-(1-alkyl-4-pyridinio)propoxo]tetraphenylporphyrinatophosphorus(V) (alkyl=hexyl (1a) and butyl (1b)) and bis[5-(3-alkyl-1-pyridinio)-3-oxapentyloxo] tetraphenylporphyrinatophosphorus(V) (alkyl=hexyl (1c), butyl (1d), and ethyl (1e)). The quantum yields (ΦΔ) for the formation of (1)O2 were extremely high (e.g. ΦΔ=0.88 (1a) and 0.87 (1c)). The 1 could bind human serum albumin (HSA) with high binding constants to produce a complex of HSA with 1 (1/HSA). Here, inactivation of Saccharomyces cerevisiae (yeast) was examined using the complex of HSA (400nM) with 1 (20-50nM) under visible-light irradiation. The bactericidal activity of 1 was evaluated by half-life (T1/2 in min) which was time required to be reduced to one-half initial concentration of yeasts. Under irradiation in the presence of HSA, minimum concentrations ([P]) of 1 were adjusted as T1/2 became the values among 0-120min. The [P] values were determined to be 20nM for 1a, 30nM for 1b, and 50nM for 1c-1e. The 1/HSA sterilized yeast with 12-36min of T1/2 when the concentration of 1 was set to [P]. In the photo-activation in the absence of HSA at the concentration of [P] of 1, however, the T1/2 values of 1a, 1b, 1d, and 1e were >120min and T1/2 of 1c was 44min. Thus, the complexation of 1 with HSA apparently enhanced the bactericidal activity of 1. This is the first finding on the assistance of HSA to the photo-inactivation of yeast cell by porphyrins.

Enhancement of the antibody-dependent cellular cytotoxicity of low-fucose IgG1 Is independent of FcgammaRIIIa functional polymorphism.

PURPOSE: The most common polymorphic variant of Fcgamma receptor type IIIa (FcgammaRIIIa), FcgammaRIIIa-158F, has been associated with inferior clinical responses to anti-CD20 chimeric IgG1 rituximab compared with FcgammaRIIIa-158V. As we previously found that removal of fucose residues from the oligosaccharides of human IgG1 results in enhanced antibody-dependent cellular cytotoxicity, we compared the effects of the FcgammaRIIIa gene (FCGR3A) polymorphism on normal and low-fucose versions of rituximab on antibody-dependent cellular cytotoxicity. EXPERIMENTAL DESIGN: The polymorphism at position 158 of FcgammaRIIIa was determined for the peripheral blood mononuclear cells (PBMCs) of 20 healthy donors. The PBMCs were then used as effector cells to compare the antibody-dependent cellular cytotoxicity of rituximab and a low-fucose version, KM3065. The contributions of the different cell types within the PBMC to antibody-dependent cellular cytotoxicity were examined. RESULTS: We found KM3065-mediated antibody-dependent cellular cytotoxicity was increased 10 to 100-fold compared with rituximab for each of the 20 donors. In contrast to rituximab, KM3065 antibody-dependent cellular cytotoxicity enhancement was similar for both FCGR3A alleles and thus independent of genotype. In addition, antibody-dependent cellular cytotoxicity of both KM3065 and rituximab requires natural killer cells but not monocytes nor polymorphonuclear cells. The antibody-dependent cellular cytotoxicity (ADCC) of each of the 20 donors correlated with the natural killer cell numbers present in the PBMCs. Importantly, using KM3065, the ADCC mediated by effector cells bearing the lower affinity variant FcgammaRIIIa-158F was significantly increased compared with rituximab-mediated ADCC using effector cells bearing the higher affinity FcgammaRIIIa-158V receptors. CONCLUSIONS: The use of low-fucose antibodies might improve the therapeutic effects of anti-CD20 therapy for all patients independent of FcgammaRIIIa phenotype beyond that currently seen with even the most responsive patients.

Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores.

A strain of B. subtilis produced 1.8 times more laccase on sporulation medium than on non-sporulation medium. Spores oxidized mono- and di-methoxyphenols (0.1 mM) at 50 degrees C. The half-life of laccase bound to spores was about 2 d and the substrate was repeatedly removed by spores recovered from the reaction mixture.

Continuous isomerization of D-fructose to D-mannose by immobilized Agrobacterium radiobacter cells.

D-Fructose was isomerized to D-mannose using immobilized Agrobacterium radiobacter that produces a thermostable mannose isomerase. The cells were immobilized by adsorption on chitosan or by glutaraldehyde crosslinking in the presence of albumin. Optimum conditions for mannose isomerase activity were 60 degrees C and pH 7.5. Continuous reaction at 55 degrees C was achieved with immobilized cells packed in a column to produce D-mannose.