Kenji Nakajima

Pseudomonas alcaliphila sp. nov., a novel facultatively psychrophilic alkaliphile isolated from seawater

Facultatively psychrophilic alkaliphilic strains were isolated from seawater obtained off the coast of Rumoi, Hokkaido, Japan. They were Gram-negative, aerobic straight rods with polar flagella. The isolates were catalase- and oxidase-positive and able to grow at 4 degrees C, but not at 40 degrees C. They produced acid from D-glucose under aerobic conditions. The isolates reduced nitrate to nitrite and hydrolysed casein and gelatin, but not starch or DNA. NaCl was required for growth at pH 10 but was not required at neutral pH. The major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 62.3-63.2 mol%. The whole-cell fatty acids mainly consisted of C16:0, C16:1(9c) and C18:1(9c), with 3-OH C10:0 and 3-OH C12:0 as the hydroxyl fatty acids. A larger amount of trans-unsaturated fatty acid, C16:1(9t) was observed when the cells were grown at pH 7 compared to when cells were grown at pH 10. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacteria are members of the genus Pseudomonas. Analysis of DNA-DNA relatedness data with several close phylogenetic neighbours revealed a low level of hybridization (less than 61%). On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, it is concluded that these isolates represent a separate new species. Accordingly, the name Pseudomonas alcaliphila is proposed. The type strain is AL15-21T (= JCM 10630T = IAM 14884T).

Comparative study on cytochrome content of alkaliphilic Bacillus strains

Abstract The cytochrome content was estimated in a wide range of alkaliphilic Bacillus strains with DNA G + C contents from 34.4 to 44.3%. Among the strains tested, four facultative alkaliphiles, whose G + C contents were around 36%, and one obligate alkaliphile, with a G + C content of 41.4%, possessed relatively high amounts of total cytochrome (more than 0.3 nmol · mg cells −1 ). These five alkaliphiles contained high amounts of cytochromes b and c compared with the other strains tested, but their cytochrome a contents were not noticeably different. All the tested alkaliphilic strains had much higher amounts of cytochrome than B. subtilis (neutrophile), except for two facultative alkaliphiles with G + C contents around 40%. These results suggest that various bioenergetic strategies are used by alkaliphilic Bacillus for adaptation to a high alkaline environment, and that cytochromes b and c have a bioenergetically very important role in certain kinds of alkaliphiles.

Purification and characterization of o-hydroxyphenylacetate 5-hydroxylase, m-hydroxyphenylacetate 6-hydroxylase and p-hydroxyphenylacetate 1-hydroxylase from Rhodococcus erythropolis

Abstract The gram-positive Rhodococcus erythropolis strain S1 was found to utilize o -, m -, and p -hydroxyphenylacetic acids as sole carbon sources. Each isomer of monohydroxyphenylacetate was degraded via the homogentisate pathway, which contained a reduced glutathione independent-isomerase. Three monohydroxy-phenylacetate monooxygenases, o -hydroxyphenylacetate 5-hydroxylase, m -hydroxyphenylacetate 6-hydroxylase, and p -hydroxyphenylacetate 1-hydroxylase, were purified to homogeneity from strain S1. Each enzyme was a 45-kDa monomeric NADH-dependent monooxygenase containing FAD, and all three appeared to belong to the p -hydroxybenzoate hydroxylase-class as regards the flavin-containing aromatic compound monooxygenase family. However, the three enzymes differed greatly in terms of substrate specificity.

Location of flavoprotein-aromatic compound oxygenases from Rhodococcus erythropolis

Abstract Gram-positive Rhodococcus erythropolis strain S-1, grown in an aromatic carboxylic acid-salts medium, synthesized enzymes for the degradation of aromatic carboxylic acids, phthalate, monohydroxybenzoates, and dihydroxybenzoates. Flavoprotein-aromatic compound monooxygenases and dioxygenase from R. erythropolis, involved in the initial steps of the degradation pathways of aromatic carboxylic acids, were found in the membrane fraction. The enzymes in the membrane fraction were solubilized by Brij 58 or Tween 80 but denatured by Triton X-100. Solubilized and purified enzyme stability was conserved by Tween 80.

Temperature- and detergent-dependent oligomeric structures of flavoprotein monohydroxybenzoate hydroxylases from Rhodococcus erythropolis

The results of analytical gel filtration chromatography experiments under various conditions showed each of three flavoprotein monohydroxybenzoate hydroxylases, p-hydroxybenzoate 3-hydroxylase, m-hydroxybenzoate 6-hydroxylase, and salicylate 5-hydroxylase, from the gram-positive Rhodococcus erythropolis strain S1 to be present in solution mainly as a tetramer at 4°C and as a dimer at 30°C. In the presence of 1% Tween 80, the three flavoenzymes were each observed to be a monomer with activity about half that of the dimer.

Physicochemical and immunochemical characterization of salicylate 5-hydroxylase, m-hydroxybenzoate 6-hydroxylase and p-hydroxybenzoate 3-hydroxylase from Rhodococcus erythropolis

Salicylate 5-hydroxylase (SAL5H), m-hydroxybenzoate 6-hydroxylase (MHB6H), and p-hydroxybenzoate 3-hydroxylase (PHB3H) from Gram-positive Rhodococcus erythropolis strain S1 were characterized physicochemically and immunochemically. The subunit size and amino acid composition of SAL5H, MHB6H, and PHB3H from strain S1 showed properties similar to those of other flavin-containing aromatic compound monooxygenases such as p-hydroxybenzoate hydroxylase and salicylate 1-hydroxylase (SAL1H), belonging to p-hydroxybenzoate hydroxylase-class, except for homotetrameric structure and cofactor specficity. The N-terminal amino acid sequence of MHB6H from strain S1 indicated significant similarity of ADP-binding region in the N-terminal portion of the enzyme with that known for SAL1H from Pseudomonas putida. Immunochemical properties, determined while conducting serological experiments, showed SAL5H and MHB6H from strain S1 to be immunologically different from PHB3H from strain S1, while SAL5H and MHB6H to apparently share partial antigenic determinants.