Kouta Hatayama

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5′ RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59–72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F2 population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

Spirosoma fluviale sp. nov., isolated from river water.

A bacterial strain, designated MSd3T, was isolated from a freshwater sample collected from the Hosoda River in Japan. The cells of strain MSd3T were Gram-stain-negative, non-spore-forming, aerobic, non-motile, curved rods forming rings, coils and undulating filaments. The 16S rRNA gene sequence of strain MSd3T showed closest similarity to that of Spirosoma linguale DSM 74T (97.6 % similarity) and similarity to other members of the genus Spirosoma ranged from 90.3 to 95.9 %. Strain MSd3T contained menaquinone 7 as the sole respiratory quinone. The major cellular fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and C16 : 1ω5c. The polar lipids were phosphatidylethanolamine, three unidentified aminophospholipids and three unidentified polar lipids. The DNA G+C content was 53.3 mol%. The DNA-DNA relatedness between strain MSd3T and S. linguale DSM 74T was 19 % or 25 % (reciprocal value). From the chemotaxonomic and physiological data and the levels of DNA-DNA relatedness, strain MSd3T should be classified as the representative of a novel species of the genus Spirosoma, for which the name Spirosoma fluviale sp. nov. (type strain MSd3T = JCM 30659T = DSM 29961T) is proposed.

Cellulomonas soli sp. nov. and Cellulomonas oligotrophica sp. nov., isolated from soil.

Two novel bacterial strains, designated Kc1(T) and Kc5(T), were isolated from soil in Japan. Cells of the novel strains were Gram-reaction-positive, aerobic or facultatively anaerobic, motile rods. Phylogenetic analyses based on 16S rRNA gene sequences indicated that both strains belonged to the genus Cellulomonas. The 16S rRNA gene sequences of strains Kc1(T) and Kc5(T) showed closest similarity to that of Cellulomonas terrae DB5(T) (98.1 % and 98.4 % similarity, respectively), and the 16S rRNA gene similarity between the two novel strains was 97.8 %. In both strains, the major menaquinone was MK-9(H(4)), the predominant polar lipids were diphosphatidylglycerol and phosphatidylinositol mannosides, and the peptidoglycan contained ornithine and glutamic acid. Cell-wall sugars were identified as rhamnose, galactose and mannose in strain Kc1(T) and rhamnose and glucose in strain Kc5(T). The DNA G+C contents of strains Kc1(T) and Kc5(T) were 73.6 mol% and 75.8 mol%, respectively. Based on the chemotaxonomic and physiological data and the results of DNA-DNA hybridizations, the two strains represent two novel species within the genus Cellulomonas, for which the names Cellulomonas soli sp. nov. (type strain Kc1(T) =DSM 24484(T) =JCM 17535(T)) and Cellulomonas oligotrophica sp. nov. (type strain Kc5(T) =DSM 24482(T) =JCM 17534(T)) are proposed.

Engineering of erythropoietin receptor for use as an affinity ligand

Recombinant human erythropoietin receptor (rhEPOR) has applicability as an affinity ligand for purification of recombinant human erythropoietin (rHuEPO) because of its specific binding to rHuEPO. For application of rhEPOR as a ligand for purification of rHuEPO, soluble rhEPOR was expressed in the periplasm of Escherichia coli and engineered by directed evolution through random mutagenesis and integration of mutations. From the screening of random mutagenesis, we identified an amino acid mutation (H114Y) contributing to rHuEPO binding and four amino acid mutations (R76S, A132D, A162D, and C181Y) contributing to expression of soluble rhEPOR. However, the rHuEPO that binds to engineered rhEPOR having H114Y mutation is difficult to dissociate from the engineered rhEPOR. Therefore, H114Y mutation was not suitable for the construction of the rhEPOR ligand. As a rhEPOR ligand, engineered rhEPOR containing four amino acid mutations (EPORm4L) was constructed by integration of mutations except for H114Y. The expression of EPORm4L (127mgl(-1) of culture medium) was markedly increased in comparison with wild-type rhEPOR (2mgl(-1) of culture medium). Small-scale affinity chromatography demonstrated that EPORm4L worked as an affinity ligand for purification of rHuEPO.

Comamonas humi sp. nov., isolated from soil

A bacterial strain, designated GAU11(T), was isolated from soil in Japan. Cells of the strain were Gram-stain-negative, aerobic, non-motile rods. The 16S rRNA gene sequence of strain GAU11(T) showed high similarity to those of Comamonas zonglianii BF-3(T) (98.8 %), Pseudacidovorax intermedius CC21(T) (96.4 %), Acidovorax caeni R-24608(T) (96.2 %), Alicycliphilus denitrificans K601(T) (96.2 %), Pseudorhodoferax soli TBEA3(T) (95.9 %) and Comamonas terrigena LMG 1253(T) (95.9 %). Strain GAU11(T) contained ubiquinone 8 as the sole ubiquinone and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol as major polar lipids. Its major cellular fatty acids were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The DNA G+C content of strain GAU11(T) was 68.2 mol%. The DNA-DNA relatedness between strain GAU11(T) and C. zonglianii DSM 22523(T) was 52 or 68 % (reciprocal value). Phenotypic characterization indicated that strain GAU11(T) represents a member of the genus Comamonas, but at the same time distinguished it from C. zonglianii DSM 22523(T). From polyphasic characterization, this strain should be classified as representing a novel species of the genus Comamonas, for which the name Comamonas humi sp. nov. (type strain GAU11(T) = JCM 19903(T) = DSM 28451(T)) is proposed.

Croceifilum oryzae gen. nov., sp. nov., isolated from rice paddy soil.

A mesophilic, aerobic, Gram-stain-positive, filamentous bacterial strain, designated ZYf1a3T, was isolated from rice paddy soil in Japan. This strain grew on a solid medium with formation of substrate mycelium; endospores were produced singly along the mycelium. Formation of aerial mycelium was not observed on any of the media tested. This strain produced a characteristic saffron yellow soluble pigment. Cloned 16S rRNA gene sequences of strain ZYf1a3T yielded three different copies (similarity between the three sequences: 99.8-99.9 %). One of these sequences had one base deletion. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ZYf1a3T belongs to an independent phylogenetic lineage of the family Thermoactinomycetaceae. The cell wall of strain ZYf1a3T contained meso-diaminopimelic acid, alanine and glutamic acid, but no characteristic sugars. It contained menaquinone 7 as the sole menaquinone. The major cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0.The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidyl-N-methylethanolamine and unidentified aminophospholipids. The DNA G+C content was 42.5 mol%. From phylogenetic analysis based on 16S rRNA gene sequences and phenotypic characteristics, this strain is considered to represent a novel species in a new genus, for which the name Croceifilum oryzae gen. nov., sp. nov. is proposed. The type strain of Croceifilum oryzae is ZYf1a3T ( = JCM 30426T = CCUG 66446T = DSM 46876T).

Flavobacterium aquicola sp. nov., isolated from river water.

A bacterial strain, designated TMd3a3T, was isolated from a freshwater sample collected from the Tamagawa River in Japan. The cells of strain TMd3a3T were facultatively anaerobic, Gram-stain-negative, non-spore-forming rods that showed gliding motility. This strain was capable of denitrification and anaerobic growth with nitrate. Cloned 16S rRNA gene sequences of strain TMd3a3T yielded three different sequences (similarity between the three sequences: 98.9-99.7 %). The 16S rRNA gene sequences of strain TMd3a3T showed high similarity to those of Flavobacterium tructae 435-08T (97.2-97.4 % similarity), F. resistens BD-b365T (96.7-97.4 %), F. maotaiense T9T (97.0-97.3 %), F. limicola ST-82T (96.5-97.3 %), F. aquidurense WB 1.1-56T (96.9-97.2 %), F. spartansii T16T (96.9-97.2 %) and F. psychrolimnae LMG 22018T (96.4-97.0 %). Strain TMd3a3T contained menaquinone 6 as the sole respiratory quinone. The major cellular fatty acids were iso-C15 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids were phosphatidylethanolamine, five unidentified aminolipids and five unidentified polar lipids. The DNA G+C content was 36.5 mol %. The DNA-DNA relatedness values of strain TMd3a3Twith F. tructae CCUG 60100T, F. resistens DSM 19382T, F. maotaiense JCM 19927T, F. limicola DSM 15094T, F. aquidurense DSM 18293T, F. spartansii ATCC BAA-2541T and F. psychrolimnae DSM 16141T were below 13 %. From the chemotaxonomic and physiological data and the levels of DNA-DNA relatedness, strain TMd3a3T should be classified as the representative of a novel species of the genus Flavobacterium, for which the name Flavobacterium aquicola sp. nov. (type strain TMd3a3T=JCM 30987T=DSM 100880T) is proposed.

Brevibacillus fulvus sp. nov., isolated from a compost pile.

Two strains, designated K2814(T) and K282, were isolated from a compost pile in Japan. These strains were Gram-stain-variable, aerobic, motile and endospore-forming rods. The strains produced a characteristic brown non-diffusible pigment. The 16S rRNA gene sequences of the strains were 100% identical and had high similarity to that of Brevibacillus levickii LMG 22481(T) (97.3%). Phylogenetic analyses based on 16S rRNA gene sequences revealed that these strains belong to the genus Brevibacillus. Strains K2814(T) and K282 contained meso-diaminopimelic acid in their cell walls. Strains K2814(T) and K282 contained MK-7 (96.0 and 97.2%, respectively) and MK-8 (4.0 and 2.8%, respectively) as the major and minor menaquinones, respectively. Their major cellular fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(15 : 0) and iso-C(17 : 0). The DNA G+C contents of strains K2814(T) and K282 were 48.8 and 49.8 mol%, respectively. Polar lipids of strain K2814(T) were composed of phosphatidyl-N-methylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, three unidentified polar lipids, an unidentified aminophospholipid and an unidentified aminolipid. The level of DNA-DNA relatedness between strains K2814(T) and K282 was 99 or 100%, and levels between strain K2814(T) and the type strains of seven related species of the genus Brevibacillus, including Brevibacillus levickii LMG 22481(T), were below 59%. From the chemotaxonomic and physiological data and the levels of DNA-DNA relatedness, these two strains should be classified as representing a novel species of the genus Brevibacillus, for which the name Brevibacillus fulvus sp. nov. (type strain K2814(T) = JCM 18162(T) = ATCC BAA-2417(T) = DSM 25523(T)) is proposed.