Bulbimidazoles A-C, Antimicrobial and Cytotoxic Alkanoyl Imidazoles from a Marine Gammaproteobacterium Microbulbifer Species.

Three new alkanoyl imidazoles, designated bulbimidazoles A-C (1-3), were found from the culture extract of the gammaproteobacterium Microbulbifer sp. DC3-6 isolated from a stony coral of the genus Tubastraea. The absolute configuration of the anteiso-methyl substitution in 1 was established to be a mixture of (R)- and (S)-configurations in a ratio of 9:91 by applying the Ohrui-Akasaka method. Compounds 1-3 displayed unique broad-spectrum antimicrobial activity against Gram-positive and -negative bacteria and fungi with MICs ranging from 0.78 to 12.5 µg/mL. They also exhibited cytotoxicity toward P388 murine leukemia cells with IC50 in the micromolar range.

Immobilization of cadmium by immobilized Alishewanella sp. WH16-1 with alginate-lotus seed pods in pot experiments of Cd-contaminated paddy soil.

This study prepared immobilized Alishewanella sp. WH16-1 using alginate and lotus seed pods as a matrix and investigated the effects of its immobilization on Cd2+ in a culture solution and in soil. Compared with the free WH16-1 strain, the immobilized WH16-1 strain possessed greater stability for long-term use and storage and higher removal ability for Cd2+ in the culture solution. A model of Cd2+ removal by the immobilized WH16-1 strain was proposed. The immobilized WH16-1 strain was incubated in the pot experiments of Cd-contaminated paddy soil for 120 days, and the pot experiments of Cd-contaminated paddy soil without the immobilized WH16-1 strain were used as a control. Compared with the control, the exchangeable and carbonate-bound Cd in the paddy soil incubated with the immobilized WH16-1 strain significantly decreased by 33.6% (P < 0.05) and 17.36%, respectively, and the Cd concentrations in the rice significantly decreased by 78.31% (P < 0.05). The results indicate that alginate-lotus seed pods can be used as excellent cost-effective cell carriers for the immobilization of Alishewanella sp. WH16-1 and that the immobilized WH16-1 strain may be applicable for the biological stabilization of Cd in Cd-contaminated paddy soil.

High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug.

l-Methionine γ-lyase (MGL), a pyridoxal 5'-phosphate-dependent enzyme, possesses anti-tumor activity. However, the low activity of MGL blocks the anti-tumor effect. This study describes an efficient production process for the recombinant MGL (rMGL) from Idiomarina constructed using the overexpression plasmid in Escherichia coli BL21 (DE3), purification, and large-scale production. The enzyme produced by the transformants accounted for 53% of the total proteins and accumulated at 1.95 mg/mL using a 500 L fermentor. The enzyme was purified to approximately 99% purity using a high-pressure mechanical homogenizer and nickel (Ni) Sepharose 6 Fast Flow (FF) chromatography. Then, the enzyme was polished by gel filtration, the endotoxins were removed using diethyl-aminoethanol (DEAE) Sepharose FF, and the final product was lyophilized with a vacuum freeze dryer at -35 °C. The specific activity of rMGL in the lyophilized powder was up to 108 U/mg. Compared to the control, the enzyme significantly inhibited cellular proliferation in a concentration-dependent manner as tested using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and induced cellular apoptosis as analyzed by Annexin V-fluorescein isothiocyanate (FITC) with fluorescence-activated cell sorting (FACS) in leukemia cells. This paper demonstrated the cloning, overexpression, and large-scale production protocols for rMGL, which enabled rMGL to be used as a novel anti-leukemic drug.

Molecular characterization of a new N-acetylneuraminate synthase (NeuB1) from Idiomarina loihiensis.

N-Acetylneuraminate lyase synthase (NeuB; E.C. 2.5.1.56) is a key enzyme in pathogenic microorganisms for producing N-acetylneuraminic acid through the irreversible condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). However, nothing is known about this enzyme in non-pathogenic bacteria. This paper describes, for the first time, one of the two putative N-acetylneuraminate synthases from the halophilic non-pathogenic gamma-proteobacterium Idiomarina loihiensis NeuB1 (IlNeuB1). The obtained 95-kDa dimeric enzyme showed maximal activity at pH 7.0 and 40°C and was more stable at pH 8.0 (8 h half-life) than the previously described NeuB. Its catalytic efficiency towards ManNAc and PEP was 10- and 40-fold higher, respectively, than that determined for Campylobacter jejuni NeuB, but only half that found for Neisseria meningitidis NeuB towards PEP. The phylogenetic and structural analyses of NeuB enzymes revealed the new domain architecture 4 has no cystathionine-ß-synthase domain (cystathionine-ß-synthetase domain), unlike domain architecture 3. In addition, 10 conserved blocks (I-X) were found, and surprisingly, this study showed that the arginine essential for catalysis that is present in antifreeze-like domain (block X) was not fully conserved in NeuB, but is replaced by a serine in a long sequence (>700 residues) NeuB, such as that existing in domain architectures 3 and 4.

Marinobacterium marisflavi sp. nov., isolated from a costal seawater.

A marine bacterium designated strain IMCC4074(T) was isolated from surface seawater collected off Incheon Port, the Yellow Sea, and subjected to a polyphasic taxonomy. The strain was Gram-negative, chemoheterotrophic, slightly halophilic, strictly aerobic, and motile rods. Based on 16S rRNA gene sequence comparisons, the strain was most closely related to Marinobacterium litorale KCTC 12756(T) (93.9%) and shared low 16S rRNA gene sequence similarities with members of the genus Marinobacterium (91.8-93.9%) and the genus Neptunomonas (93.4%) in the order Oceanospirillales. Phylogenetic analyses showed that this marine isolate formed an independent phyletic line within the genus Marinobacterium clade. The DNA G+C composition of the strain was 56.0 mol% and the predominant constituents of the cellular fatty acids were C(16:0) (28.0%), C(16:1 )omega7c and/or iso-C(15:0) 2-OH (19.3%), C(18:1 )omega7c (17.8%), and C(17:1) cyclo (12.5%), which differentiated the strain from other Marinobacterium species. Based on the taxonomic data collected in this study, only a distant relationship could be found between strain IMCC4074(T) and other members of the genus Marinobacterium, thus the strain represents a novel species of the genus Marinobacterium, for which the name Marinobacterium marisflavi sp. nov. is proposed. The type strain of Marinobacterium marisflavi is IMCC4074(T) (= KCTC 12757(T) = LMG 23873(T)).

Microbulbifer celer sp. nov., isolated from a marine solar saltern of the Yellow Sea in Korea.

A Gram-negative, non-motile, rod-shaped, Microbulbifer-like bacterial strain, ISL-39(T), was isolated from a marine solar saltern of the Yellow Sea in Korea and was subjected to a polyphasic taxonomic investigation. Strain ISL-39(T) grew optimally at pH 7.0-8.0 and 37 degrees C. It contained Q-8 as the predominant ubiquinone and iso-C(15 : 0), C(16 : 0) and iso-C(17 : 0) as the major fatty acids. The DNA G+C content was 57.7 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ISL-39(T) belonged to the genus Microbulbifer. Strain ISL-39(T) exhibited 16S rRNA gene sequence similarity values of 94.7-97.5 % with respect to the type strains of four recognized Microbulbifer species. DNA-DNA relatedness data and the differential phenotypic properties and phylogenetic distinctiveness of ISL-39(T) make this strain distinguishable from the recognized Microbulbifer species. On the basis of the phenotypic, phylogenetic and genetic data, strain ISL-39(T) represents a novel species of the genus Microbulbifer, for which the name Microbulbifer celer sp. nov. is proposed. The type strain is ISL-39(T) (=KCTC 12973(T)=CCUG 54356(T)).