A risk estimation method for depression based on the dysbiosis of intestinal microbiota in Japanese patients.

Introduction: Early detection of depression is important for preventing depression-related suicides and reducing the risk of recurrence. This study explored the association between depression and intestinal microbiota and developed a depression risk-estimation method based on this. Methods: The intestinal microbiota of Japanese patients with depression (33 males and 35 females) and disease-free controls (246 males and 384 females) in their 20's to 60's were compared by sex using 16S rRNA gene amplicon sequencing. A depression-risk estimation method was developed using structural equation modeling. Results: Intestinal bacteria taxa that differed between depression and control groups were identified based on effect size (absolute value greater than 0.2). Neglecta was more abundant, while Coprobacter, Butyricimonas, Clostridium_XlVb, and Romboutsia were less abundant in the male depression group compared to the male control group. In the female depression group, Massilimicrobiota, Merdimonas, and Sellimonas were more abundant, whereas Dorea and Agathobacter were less abundant compared to the female control group. Several of the intestinal bacterial taxa that were less abundant in depression were associated with butyrate or hydrogen production. Using these depression-associated intestinal bacteria as indicators, risk-estimation models using structural equation modeling for depression were developed. In the risk-estimation models for males and females, the areas under the receiver operating characteristic curve were 0.72 and 0.70, respectively, indicating that these models can distinguish between individuals with and without depression. Conclusions: This study provides insights into depression etiology and aids in its early detection and treatment.

Characteristics of Intestinal Microbiota in Japanese Patients with Mild Cognitive Impairment and a Risk-Estimating Method for the Disorder.

Intestinal microbiota may play a significant role in the development and progression of mild cognitive impairment (MCI). In addition, sex differences in the prevalence of MCI and intestinal microbiota are likely to exist. Therefore, this study investigated the association between MCI and intestinal microbiota by comparing Japanese patients in their 70s with MCI (11 males and 18 females) and disease-free controls (17 males and 23 females), taking sex into account. In both sexes, Clostridium_XVIII, Eggerthella, Erysipelatoclostridium, Flavonifractor, and Ruminococcus 2 were the more abundant taxa in the MCI group, whereas Megasphaera, Oscillibacter, Prevotella, Roseburia, and Victivallis were less abundant. Based on these characteristics, it was hypothesized that the composition of the intestinal microbiota in the MCI group leads to dysregulation of the intestinal microbiota, increased intestinal and blood-brain barrier permeability, and increased chronic neuroinflammation, with the long-term persistence of these abnormalities ultimately leading to cognitive decline. Furthermore, risk estimation models for MCI based on intestinal microbiota data were developed using structural equation modeling. These tests discriminated between the MCI and control groups. Incorporating these factors into intestinal microbiota testing using stool samples may be an efficient method to screen individuals with MCI.

Sex Differences in Intestinal Microbiota and Their Association with Some Diseases in a Japanese Population Observed by Analysis Using a Large Dataset.

In recent years, many studies have focused on the relationship between intestinal microbiota and human health, but the impact of sex has not yet been sufficiently investigated. In this study, sex differences in the intestinal microbiota of a Japanese population were investigated by age group, using a large dataset constructed for a cross-sectional study. α-diversity analysis indicated that the impact of sex differences varied among the 20s-50s age groups but tended to be smaller among the 60s-70s age groups. Fusobacterium, Megamonas, Megasphaera, Prevotella, and Sutterella were more common among males, whereas Alistipes, Bacteroides, Bifidobacterium, Odoribacter, and Ruthenibacterium were common among females. Next, intestinal bacteria potentially associated with 12 diseases were investigated for each sex. The results indicate that many of these differ between males and females, and among age groups. Thus, sex and age should be considered for studies on intestinal microbiota and disease association, prevention, and treatment approaches that target them.

Calcite formation induced by Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens and Rheinheimera texasensis.

A wide range of bacterial species are able to induce calcium carbonate precipitation. Using our own laboratory-preserved strains, we have newly discovered that Ensifer sp. MY11e, Microbacterium sp. TMd9a1, Paeniglutamicibacter sp. MSa1a, Pseudomonas sp. GTc3, and Rheinheimera sp. ATWe6 can induce the formation of calcite crystals on an agar medium. Type strains of their closely related species (Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens, and Rheinheimera texasensis) could also induce calcite formation. Although the initial pH value of the agar medium was 6.1, the pH of the agar media containing calcite, induced by cultivation of the 10 bacterial strains, increased to 8.0-8.4. The ammonification (oxidative deamination) of amino acids may been responsible for this increase in pH. The crystals formed both on and around the bacterial colonies. Furthermore, when these strains (excepting two Microbacterium strains) were cultivated on a cellulose acetate membrane filter (0.20 µm pore size) resting on the surface of the agar medium (i.e., in the membrane filter culture method), the crystals formed on the agar medium separate from the bacterial cells. These results indicate that the bacterial cells did not necessarily become nucleation sites for these crystals. We also investigated whether the studied strains could be applied to the biocementation of sand, and found that only two Ensifer strains were able to form large sand lumps.

Bacterial community structures in air conditioners installed in Japanese residential buildings.

The bacterial community structures in four Japanese split-type air conditioners were analyzed using a next-generation sequencer. A variety of bacteria were detected in the air filter of an air conditioner installed on the first floor. In the evaporator of this air conditioner, bacteria belonging to the genus Methylobacterium, or the family of Sphingomonadaceae, were predominantly detected. On the other hand, the majority of bacteria detected in the air filters and evaporators of air conditioners installed on the fifth and twelfth floors belonged to the family Enterobacteriaceae. The source of bacteria belonging to the family Enterobacteriaceae may have been aerosols generated by toilet flushing in the buildings. Our results suggested the possibility that the bacterial contamination in the air conditioners was affected by the floor level on which they were installed. The air conditioner installed on the lower floor, near the ground, may have been contaminated by a variety of outdoor bacteria, whereas the air conditioners installed on floors more distant from the ground may have been less contaminated by outdoor bacteria. However, these suppositions may apply only to the specific split-type air conditioners that we analyzed, because our sample size was small.

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.

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.

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).

Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI.

Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcγRI and human Fc, at 1.80 Šresolution, revealing an unique hydrophobic pocket at the surface of hFcγRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcγRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcγRI.

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.

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.

The binding of soluble recombinant human Fcγ receptor I for human immunoglobulin G is conferred by its first and second extracellular domains.

Human FcγRI is a high affinity receptor for the Fc portion of human immunoglobulin G (IgG), and has extracellular, transmembrane and cytoplasmic regions. The extracellular region of human FcγRI, which is the part that interacts with human IgG, is comprised of three immunoglobulin-like domains. Unlike low affinity Fcγ receptors (FcγRII and FcγRIII), FcγRI has a unique third extracellular domain (D3). This study investigated the contribution of D3 to the binding between recombinant human FcγRI (rhFcγRI) and human IgG. The three extracellular domains and the first and second extracellular domains of human FcγRI were expressed by Escherichia coli as rhFcγRI and rhFcγRI-D1D2, respectively. The binding specificity of rhFcγRI-D1D2 to human IgG subclasses was the same as that of rhFcγRI. From surface plasmon resonance analysis, the binding affinity of rhFcγRI-D1D2 for human IgG1/κ was high (the equilibrium dissociation constant: KD=8.04 × 10(-10)M), but slightly lower than that of rhFcγRI (KD=2.59 × 10(-10)M). While the association of rhFcγRI-D1D2 with human IgG1/κ was same as that of rhFcγRI, the dissociation of rhFcγRI-D1D2 was faster than that of rhFcγRI. From these results, D3 of rhFcγRI would not contribute directly to the binding specificity and association of rhFcγRI, but to the holding bound human IgG.

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 recombinant human Fcγ receptor I by directed evolution.

Human FcγRI is a high-affinity receptor for human IgG. On the basis of its binding activity, recombinant human FcγRI (rhFcγRI) has several possible applications, including as a therapeutic reagent to treat immune complex-mediated disease and as a ligand in affinity chromatography for purification of human IgG. As the stability and production rate of rhFcγRI are low, it would need to be engineered for use in such applications. In this study, we demonstrated engineering of rhFcγRI by directed evolution through random mutagenesis and integration of mutations. Engineered rhFcγRI was expressed by Escherichia coli. Screening identified 19 amino acid mutations contributing to the thermal stability and production rate of rhFcγRI. By integration of these mutations, engineered rhFcγRI containing all 19 amino acid mutations (enFcRd) was constructed and showed markedly enhanced thermal stability (transition midpoint temperature [Tm] = 65.6°C) and production rate (3.27 mg L-medium(-1) OD(600)(-1)) compared with wild-type rhFcγRI (Tm = 48.5°C; production rate, 0.07 mg L-medium(-1) OD(600)(-1)) without a change in the specificities of binding to human IgG subclasses. Moreover, the binding affinity of enFcRd for human IgG1/к (equilibrium dissociation constant [K(D)] = 0.80 × 10(-10) M) was higher than that of wild-type rhFcγRI (K(D) = 1.23 × 10(-10) M). Our study showed that substantial engineering of rhFcγRI is possible.

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli.

Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K D =[1.7±0.2]×10−10 M) and IgG3 (K D=[1.1±0.2]×10−10 M) were similar to those of glycosylated rhFcγRI.

Regulation of glycerol metabolism in Enterobacter aerogenes NBRC12010 under electrochemical conditions.

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD(+)/NADH. At a NAD(+)/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD(+)/NADH. Exogenous NADH was oxidized to NAD(+) by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.

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 F(2) 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.

Tuberibacillus calidus gen. nov., sp. nov., isolated from a compost pile and reclassification of Bacillus naganoensis Tomimura et al. 1990 as Pullulanibacillus naganoensis gen. nov., comb. nov. and Bacillus laevolacticus Andersch et al. 1994 as Sporolactobacillus laevolacticus comb. nov.

Two thermophilic strains, designated 607T and 606b, were isolated from a compost pile in Japan. The novel strains were Gram-positive, aerobic, spore-forming rods. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strains 607T and 606b were closely related to Bacillus naganoensis (94.0-94.1% similarity) and separated from clusters of the related genera Bacillus (<91.9%) and Sporolactobacillus (91.0-92.5%). In addition, some chemotaxonomic and physiological characteristics of strains 607T and 606b differed from those of B. naganoensis and the two related genera. Several differences in physiological characteristics and 16S-23S rRNA gene internal transcribed spacer region nucleotide sequences were observed between strains 607T and 606b; however, DNA-DNA hybridization indicated that these two strains belonged to the same species. From these results, it is proposed that strains 607T and 606b represent the type species of a new genus, Tuberibacillus calidus gen. nov., sp. nov., with strain 607T (=JCM 13397T=DSM 17572T) as the type strain. In addition, the results of phylogenetic analyses, as well as chemotaxonomic and physiological characterization, indicated that B. naganoensis and Bacillus laevolacticus did not belong to the genus Bacillus. Based on these results, it is proposed that B. naganoensis and B. laevolacticus should be transferred to Pullulanibacillus naganoensis gen. nov., comb. nov. and Sporolactobacillus laevolacticus comb. nov., respectively.

Planifilum fimeticola gen. nov., sp. nov. and Planifilum fulgidum sp. nov., novel members of the family 'Thermoactinomycetaceae' isolated from compost.

Four thermophilic, Gram-positive strains, designated H0165(T), 500275(T), C0170 and 700375, were isolated from a composting process in Japan. The isolates grew aerobically at about 65 degrees C on a solid medium with formation of substrate mycelia; spores were produced singly along the mycelia. These morphological characters resembled those of some type strains of species belonging to the family 'Thermoactinomycetaceae', except that aerial mycelia were not formed. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the closest related species to the isolates were members of the family 'Thermoactinomycetaceae', but that the isolates formed an independent phylogenetic lineage. Some chemotaxonomic characters of the isolates, such as DNA G+C contents of 58.7-60.3 mol%, MK-7 as the major menaquinone and cellular fatty acid profiles, differed from those of members of the family 'Thermoactinomycetaceae'. DNA-DNA hybridization showed that the isolates could be divided into two genomic groups, strain H0165(T) and the other three strains. These results indicated that the four isolates should be classified into two species of a novel genus in the family 'Thermoactinomycetaceae', for which the names Planifilum fimeticola gen. nov., sp. nov. (type strain H0165(T)=ATCC BAA-969(T)=JCM 12507(T)) and Planifilum fulgidum sp. nov. (type strain 500275(T)=ATCC BAA-970(T)=JCM 12508(T)) are proposed.