Mina Yasumoto-Hirose

Isolation and functional analysis of cytochrome P450 CYP153A genes from various environments.

The cytochrome P450 CYP153 family is thought to mediate the terminal hydroxylation reactions of n-alkanes. We isolated 16 new P450 CYP153A genes (central region) from various environments such as petroleum-contaminated soil and groundwater, as well as one from the n-alkane-degrading bacterium Alcanivorax borkumensis SK2 (designated P450balk). The sequences of the new P450 genes were extended by PCR to generate full-length chimeric P450 genes, using the N- and C-terminal domains of P450balk. A differential CO-reduced P450 spectral analysis indicated that 8 P450 genes among the 16 chimeric genes were expressed in Escherichia coli to generate a soluble and functional enzyme. The several functional chimeric P450s and P450balk were further fused to the reductase domain of the self-sufficient P450 monooxygenase (P450RhF) at the C-terminus. E. coli cells expressing these self-sufficient P450 chimeric genes converted n-alkanes, cyclohexane, 1-octene, n-butylbenzene, and 4-phenyl-1-butene into 1-alkanols, cyclohexanol, 1,2-epoxyoctane, 1-phenyl-4-butanol, and 2-phenethyl-oxirane, respectively.

Rare carotenoids, (3R)-saproxanthin and (3R,2′S)-myxol, isolated from novel marine bacteria (Flavobacteriaceae) and their antioxidative activities

We isolated three orange or yellow pigment-producing marine bacteria, strains 04OKA-13-27 (MBIC08261), 04OKA-17-12 (MBIC08260), and YM6-073 (MBIC06409), off the coast of Okinawa Prefecture in Japan. These strains were classified as novel species of the family Flavobacteriaceae based on their 16S rRNA gene sequence. They were cultured, and the major carotenoids produced were purified by chromatographic methods. Their structures were determined by spectral data to be (3R)-saproxanthin (strain 04OKA-13-27), (3R,2′S)-myxol (strain YM6-073), and (3R,3′R)-zeaxanthin (strains YM6-073 and 04OKA-17-12). Saproxanthin and myxol, which are monocyclic carotenoids rarely found in nature, demonstrated significant antioxidative activities against lipid peroxidation in the rat brain homogenate model and a neuro-protective effect from l-glutamate toxicity.

Frondibacter mangrovi sp. nov., a member of the family Flavobacteriaceae isolated from seawater by in situ cultivation, and emended description of Frondibacter aureus

A Gram-stain-negative, rod-shaped, non-motile, strictly aerobic, chemoheterotrophic, light-yellow-pigmented bacterium, designated strain 02OK1/10-76T, was isolated from a mangrove estuary in Japan by use of an in situ cultivation technique. Preliminary analysis based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (96.9 %) to Frondibacter aureus A5Q-67T. The DNA G+C content of strain 02OK1/10-76T was 35 mol%; MK-6 was the only menaquinone; and iso-C15 : 0, iso-C17 : 0 3-OH and iso-C16 : 0 3-OH were the major (>10 %) cellular fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, three unidentified aminolipids, an unidentified aminophospholipid and an unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain represents a novel species of the genus Frondibacter, for which the name Frondibacter mangrovi sp. nov. is proposed. The type strain is 02OK1/10-76T (= KCTC 52666T = NBRC 112695T). An emended description of F. aureus is also provided.

Isolation of Marine Bacteria by In Situ Culture on Media-Supplemented Polyurethane Foam

Polyurethane foam (PUF) supplemented with various agar media was used in situ to trap marine bacteria and it consequently provided a substrate on which they could be cultivated while exposed to natural seawater in the coral reef area. The bacterial population on the PUF blocks was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. Changing the composition of the cultivation medium in the PUF blocks and selecting different sampling sites resulted in different bacteria being detected on the PUF blocks. For example, iron-utilizing (IU) bacteria, siderophore-producing (SP) bacteria, and petroleum-degrading (PD) bacteria were isolated from PUF blocks and it was discovered that IU and SP contained iron and PD contained hydrocarbon. This method opens up the possibility for isolating novel and useful marine bacteria.

Okicamelliaside, an extraordinarily potent anti-degranulation glucoside isolated from leaves of Camellia japonica.

Guided by anti-degranulation assays, we isolated from leaves of Camellia japonica an ellagic acid glucoside named okicamelliaside. The structure was elucidated as 3,4-dioxoloellagic acid 4′-O-β-D-glucopyranoside by spectroscopic and chemical methods. Okicamelliaside was 12,000 times more potent than the antihistaminic drug, ketotifen fumarate, in inhibiting the degranulation of RBL-2H3 cells.

Psychromonas agarivorans sp. nov., a novel agarolytic bacterium

Two heterotrophic, agarolytic bacteria were isolated from marine environments. A 16S rRNA gene sequence analysis showed the isolates (designated strains J42-3A(T) and 04OZ-AS15-7A) to be positioned in a separate lineage within the genus Psychromonas. Members of the genus Psychromonas are recognized as being psychrophilic or psychrotolerant, whereas none of the currently established Psychromonas species is known to be agarolytic. The DNA G+C contents of the isolates were about 42 mol% and the predominant cellular fatty acids were 16 : 1omega7c and 16 : 0. Based on the results of the phylogenetic and phenotypic analyses and DNA-DNA hybridization data, the isolates represent a novel species, for which the name Psychromonas agarivorans sp. nov. is proposed. The type strain is J42-3A(T) (=NBRC 104585(T)=KCTC 22285(T)).

Leptobacterium flavescens gen. nov., sp. nov., a marine member of the family Flavobacteriaceae, isolated from marine sponge and seawater.

Six obligately aerobic, Gram-negative, non-motile, pale-yellow-pigmented, rod-shaped bacterial strains, designated YM3-301(T), HG868, 04PA2 Co4-8B, 04PA2 Co4-99A, 04PA2 018SW-3 and 04PA2 018SW-18, were isolated from a marine sponge and seawater and were subjected to a polyphasic taxonomic investigation. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the novel isolates were affiliated with the family Flavobacteriaceae (phylum Bacteroidetes) and that they showed the highest sequence similarity (90.3-90.9 %) to members of the genus Leeuwenhoekiella and to Zhouia amylotica. The 16S rRNA gene sequence similarities with respect to members of other related genera were only 86.3-89.1 %. In contrast, the six isolates shared high levels of 16S rRNA gene sequence similarity (99.7-100 %) and DNA-DNA relatedness (72-94 %) with each other. The novel isolates were phenotypically and physiologically different from members of related genera. The G+C content of the DNA was 41.8-43.5 mol%, MK-6 was the major menaquinone and i15 : 0, i15 : 1 and i17 : 0 3-OH were the major fatty acids. On the basis of the data from the taxonomic studies, it was concluded that these six novel strains represent a novel genus and species of the family Flavobacteriaceae, for which the name Leptobacterium flavescens gen. nov., sp. nov. is proposed. The type strain of Leptobacterium flavescens is YM3-301(T) (=KCTC 22160(T)=MBIC 06275(T)=NBRC 104141(T)).

Psychromonas heitensis sp. nov., a psychrotolerant bacterium isolated from seawater in Japan

Four bacterial strains, 04HE-4-40, A4I-9, A4I-21 and AK15-027(T), were isolated from Heita Bay off Kamaishi in Japan. Based on 16S rRNA gene sequence analysis, the isolates were placed as a new lineage within the genus Psychromonas. The 16S rRNA gene sequence similarity values between the isolates and type strains of recognized Psychromonas species were less than 97 %. The DNA G+C contents of the isolates were about 38 mol%. The major respiratory quinone was Q-8, and the predominant cellular fatty acids were 16 : 1omega7c and 16 : 0. The isolates were able to grow at 30 degrees C, unlike other Psychromonas species. Based on the results of the phylogenetic analysis and phenotypic characteristics and DNA-DNA hybridization data, the isolates represent a novel species, for which the name Psychromonas heitensis sp. nov. is proposed. The type strain is AK15-027(T) (=MBIC 06898(T)=NCIMB 14416(T)).