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The pink-pigmented facultative methylotrophs (PPFMs), a major bacterial group found in the plant phyllosphere, comprise two genera: Methylobacterium and Methylorubrum. They have been separated into three major clades: A, B (Methylorubrum), and C. Within these genera, however, some species lack either pigmentation or methylotrophy, which raises the question of what actually defines the PPFMs. The present study employed a comprehensive comparative genomics approach to reveal the phylogenetic relationship among the PPFMs and to explain the genotypic differences that confer their different phenotypes. We newly sequenced the genomes of 29 relevant-type strains to complete a dataset for almost all validly published species in the genera. Through comparative analysis, we revealed that methylotrophy, nitrate utilization, and anoxygenic photosynthesis are hallmarks differentiating the PPFMs from the other Methylobacteriaceae. The Methylobacterium species in clade A, including the type species Methylobacterium organophilum, were phylogenetically classified into six subclades, each possessing relatively high genomic homology and shared phenotypic characteristics. One of these subclades is phylogenetically close to Methylorubrum species; this finding led us to reunite the two genera into a single genus Methylobacterium. Clade C, meanwhile, is composed of phylogenetically distinct species that share relatively higher percent G+C content and larger genome sizes, including larger numbers of secondary metabolite clusters. Most species of clade C and some of clade A have the glutathione-dependent pathway for formaldehyde oxidation in addition to the H4MPT pathway. Some species cannot utilize methanol due to their lack of MxaF-type methanol dehydrogenase (MDH), but most harbor an XoxF-type MDH that enables growth on methanol in the presence of lanthanum. The genomes of PPFMs encode between two and seven (average 3.7) genes for pyrroloquinoline quinone-dependent alcohol dehydrogenases, and their phylogeny is distinctly correlated with their genomic phylogeny. All PPFMs were capable of synthesizing auxin and did not induce any immune response in rice cells. Other phenotypes including sugar utilization, antibiotic resistance, and antifungal activity correlated with their phylogenetic relationship. This study provides the first inclusive genotypic insight into the phylogeny and phenotypes of PPFMs.

Motile bacteria often exhibit chemotaxis toward favorable compounds. However, the diversity of bacteria that are attracted to a given substance is largely unknown. This study aimed to reveal the diversity of bacteria with natural chemotaxis towards methanol. We tried to enrich environmental chemotactic bacteria using a glass capillary that is half-filled with methanol solidified with agarose as a trap (“chemotaxis fishing”). The pilot experiment using methanol-chemotactic Methylobacterium aquaticum strain 22A enriched the cells by 46-fold. The method was then applied to bacterial suspensions from paddy water and plants. Depending on the isolation sources and the methods of motility induction, methylotrophic bacteria were enriched 1.2–330-fold. The fished isolates belong to 32 species in 18 genera, mainly containing Acinetobacter, Methylobacterium and Pseudomonas species. Our chemotaxis fishing unveiled a part of diversity of the bacteria with natural chemotaxis towards methanol.

Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 μg EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 μg EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains.

Methylobacterium species are methylotrophic bacteria that widely inhabit plant surfaces. In addition to studies on methylotrophs as model organisms, research has also been conducted on their mechanism of plant growth promotion as well as the species-species specificity of plant-microbe interaction. We employed whole-cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (WC-MS) analysis, which enables the rapid and accurate identification of bacteria at the species level, to identify Methylobacterium isolates collected from the rice seeds of different cultivars harvested in Japan, Thailand, and Kenya. Rice seeds obtained from diverse geographical locations showed different communities of Methylobacterium species. We found that M. fujisawaense, M. aquaticum, M. platani, and M. radiotolerans are the most frequently isolated species, but none were isolated as common species from 18 seed samples due to the highly biased communities in some samples. These findings will contribute to the development of formulations containing selected species that promote rice growth, though it may be necessary to customize the formulations depending on the cultivars and farm conditions.

Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca 2+ -dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF . This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La 3+ . We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La 3+ ; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place not only for MDH genes but also the subsequent formaldehyde oxidation pathways and respiratory chain, which might be due to the direct oxidation of methanol to formate by XoxF. In addition, a considerable number of genes of unknown function, including AT-rich genes, were found to be upregulated in the presence of lanthanides. This study provides first insights into the specific reaction of methylotrophic bacteria to the presence of lanthanides, emphasizing the biological relevance of this trace metal.

Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place not only for MDH genes but also the subsequent formaldehyde oxidation pathways and respiratory chain, which might be due to the direct oxidation of methanol to formate by XoxF. In addition, a considerable number of genes of unknown function, including AT-rich genes, were found to be upregulated in the presence of lanthanides. This study provides first insights into the specific reaction of methylotrophic bacteria to the presence of lanthanides, emphasizing the biological relevance of this trace metal., Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca2+-dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF. This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La3+. We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La3+; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place not only for MDH genes but also the subsequent formaldehyde oxidation pathways and respiratory chain, which might be due to the direct oxidation of methanol to formate by XoxF. In addition, a considerable number of genes of unknown function, including AT-rich genes, were found to be upregulated in the presence of lanthanides. This study provides first insights into the specific reaction of methylotrophic bacteria to the presence of lanthanides, emphasizing the biological relevance of this trace metal.

WOS: 000399232900008 PubMed ID: 27902265 A novel facultatively methanol-utilizing bacterial strain, SM30(T), was isolated from rice rhizosphere. Strain SM30(T) was Gram-stain-negative, aerobic, motile, short rods, and grew optimally at pH 7 and at 28 degrees C. It could tolerate 0 to 2%(w/v) NaCl. Based on 16S rRNA gene sequence comparisons, strain SM30(T) was most closely related to Pleomorphomonas oryzae DSM 16300(T), with a low similarity of 94.17 %. One of the lanthanide metals, lanthanum, could enhance its growth slightly on methanol. Phylogenetic trees, based on the mxaF, xoxF and cpn60 genes of SM30(T) showed its distinct phylogenetic position with respect to species with validly published names. Polymerase chain reaction (PCR) amplification of the nifH and growth on nitrogen-free medium indicated that strain SM30(T) is a diazotroph. The major cellular fatty acids were summed feature 8 (containing 18:1 omega 7c and 18:1 omega 6c) and cyclo 19:0 omega 8c. The major quinone was ubiquinone 10. The DNA G+C content was 74.6 mol%. Based on the genotypic and phenotypic characteristics, strain SM30(T) represents a novel genus and species, for which the name Oharaeibacter diazotrophicus gen. nov., sp. nov. is proposed with the type strain SM30(T) (= NBRC 111955(T) = DSM 102969(T)). KAKENHIMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of ScienceGrants-in-Aid for Scientific Research (KAKENHI) [15H04476]; Advanced Low Carbon Technology Research and Development Program (ALCA), JST; China Scholarship CouncilChina Scholarship Council; Grants-in-Aid for Scientific ResearchMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of ScienceGrants-in-Aid for Scientific Research (KAKENHI) [15H04476] Funding Source: KAKEN This work was partially supported by KAKENHI (15H04476) to AT, Advanced Low Carbon Technology Research and Development Program (ALCA), JST to AT, and financial support from the China Scholarship Council to HL.

Algal bloom is typically caused by aberrant propagation of a single species, resulting in its predomination in the local population. While environmental factors including temperature and eutrophication are linked to bloom, the precise mechanism of its formation process is still obscure. Here, we isolated a bacterial strain that promotes growth of Heterosigma akashiwo, a Raphidophyceae that causes harmful algal blooms. Based on 16S rRNA gene sequence, the strain was identified as Altererythrobacter ishigakiensis, a member of the class Alphaproteobacteria. When added to culture, this strain facilitated growth of H. akashiwo and increased its cell culture yield significantly. Importantly, this strain did not affect the growth of other raphidophytes, Chattonella ovate and C. antiqua, indicating that it promotes growth of H. akashiwo in a species-specific manner. We also found that, in co-culture, H. akashiwo suppressed the growth of C. ovate. When A. ishigakiensis was added to the mixed culture, H. akashiwo growth was facilitated while C. ovate propagation was markedly suppressed, indicating that the presence of the bacterium enhances the dominance of H. akashiwo over C. ovate. This is the first example of selective growth promotion of H. akashiwo by a marine bacterium, and may exemplify importance of symbiotic bacterium on algal bloom forming process in general.

WOS: 000355955300133 PubMed ID: 26053875 Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant-microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of Japan Science and Technology AgencyJapan Science & Technology Agency (JST) [12-024]; Ryobi Teien Memorial FoundationRyobi Teien Memory Foundation; Japan Society for the Promotion of ScienceMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of Science [23688012]; Grants-in-Aid for Scientific ResearchMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of ScienceGrants-in-Aid for Scientific Research (KAKENHI) [15H04476] Funding Source: KAKEN This work was supported by the Japan Science and Technology Agency, Innovative Seeds Grant, grant number 12-024 (http://www.jst.go.jp/chiiki/seeds/); Research Award from the Ryobi Teien Memorial Foundation (http://www.ryobi.gr.jp/teienzaidan/) (no grant number is available); and Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science, grant number 23688012 (http://www.jsps.go.jp/j-grantsinaid/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A comparative study between the somatosensory-evoked cerebral potential (SEP) and motor cortex-evoked muscle response (MEMR), and between the sensory nerve action potential (SNAP) and peripheral nerve-evoked muscle response (M wave) was performed with respect to their maturational changes in the peak latencies, using 24 rabbits of ages of 10 to 180 days. The peak latencies of SEP, MEMR, SNAP and the M wave all decreased with an increase in age according to a hyperbolic curve of y = axb + c (b less than 0). The adult values of the peak latencies were reached simultaneously at 80 days of age for SEP and MEMR, and at 60 days of age for SNAP and the M wave. These findings indicate that the conduction of the somatosensory and motor pathways becomes fully maturated at the same time. On analysis of the differential coefficients on the above equation, some but substantial differences in the decrease rate of the peak latency were also found between SEP and MEMR, and between SNAP and the M wave. This suggests that there are also some differences in the maturational rate between the two pathways.

ODA, H. and FUJITANI, Y. Concentric Electrodes for Produci. Acupuncture-Like Anesthetic Effects. Tohoku J. Exp. Med., 1990, 160 (3), 169-175-We designed concentric electrodes composed of a center electrode and an outer ring electrode. Electrical stimulation with two sets of such electrodes for 15min as conditioning stimuli was given to the left hand of 35 adult subjects to induce acupuncture-like anesthetic effects. The effects immediately after the conditioning were compared between stimulation through a pair of center electrodes alone at 3Hz (conditioning 1) and simultaneous stimulation of 3Hz through a pair of center electrodes and 100Hz through a pair of outer ring electrodes (conditioning 2). In conditioning 2, modulating effects of 100Hz stimuli through a pair of outer ring electrodes made it possible to increase the voltage strength of 3Hz stimuli through a pair of center electrodes with maintaining the minimum perception of pricking sensation. In both conditioning procedures, muscle twitching was not accompanied. It was found that the respective stimulating current thresholds for faint touch sensation and also for pricking sensation at the right forearm could be elevated significantly more by conditioning 2 (1.54 and 1.40times) than by conditioning 1 (1.20 and 1.14times).

The effect of 25 mg of chlorpromazine (CPZ) on the contingent negative variation (CNV) at Cz and Oz was studied in nine male subjects. Two kinds of warning stimulus (S1)--imperative stimulus--and motor response (MR) were used. A click as S1 was common to both, while either flashes or electrical stimulation was used as S2. Simultaneously with CNV recording, the reaction time of the finger movement as MR to S2 was measured in five of nine subjects. The results obtained were as follows. 1) CNV amplitudes after CPZ administration were decreased significantly both at Cz and Oz when S2 was flashes and only at Cz when S2 was electrical stimulation. 2) Significant positive correlations of CNV amplitudes between Cz and Oz were found both before and after the drug administration. 3) Reaction times to flashes and electrical stimulation as S2 were not affected by CPZ administration. 4) The reaction time to flashes was significantly shorter than that to electrical stimulation irrespective of pre- and post-administration of CPZ.

YAMAUCHI, N., FUJITANI, Y, and OIKAWA, T. Somatosensory Evoked Potentials Elicited by Mechanical and Electrical Stimulation of Each Single Pain or Tactile Spot of the Skin. Tohoku J. exp. Med., 1981, 133 (1), 81-92 Single pain and tactile spots on the dorsum of the right hand in man were stimulated by electric pulses and mechanical taps using a needle to a pain spot and a horse tail bristle to a tactile one. Somatosensory evoked potentials (SEPs) and far field potentials (FFPs) were observed in three volunteers by averaging of 200 or 400 samples of responses recorded from scalp points corresponding to the left and right somatosensory areas (LSA and RSA). For SEP measurements, the difference between responses at LSA and RSA was obtained in order to specify the optimum response at LSA. The patterns of SEPs elicited by mechanical or electrical stimulation to a pain spot were similar to those to a tactile one. The typical SEP to mechanical stimulation consisted of N23, P31, N40, P49, N64, P87, N114 and P147, while thit to electrical stimulation did of N24, P33, N42, P52, N66, P95, N122 and P156. Consequently, mean peak latencies of the later components in SEPs elicited by mechanical stimulation were earlier than those to electrical one. The SEP amplitudes for mechanical stimulation were 1-3μV and were larger than those to electrical one. By mechanical stimulation FFPs could not be obtained, while by electrical stimulation FFPs in peak latency of approximately 14 msec (P14) were seen. No specific components to sensory modality of pain or tactile sensation were observed either in SEPs or in FFPs.

Somatosensory and visual evoked potentials were studied in fourteen hyperthyroid patients and the results were averaged with the use of a digital computor. The amplitude and the peak latency of each component of the potentials were measured. Forty-five subjects served as controls. The following results were obtained. Amplitudes of each component of the somatosensory and visual evoked potentials were greater in hyperthyroid patients than in normal subjects. No significant changes were observed in the peak latencies of each component of the somatosensory evoked potentials, except a slight prolongation in the latency of the initial negative wave of the visual evoked potential in hyperthyroidism. The threshold of the somatosensory evoked potentials in hyperthyroidism showed no significant changes, but on an increase in the stimulus strength, the amplitudes of the potentials increased much more abruptly in the hyperthyroid than in the normal subjects and reached a maximum at the motor threshold. The topographical distribution of the primary response of the somatosensory evoked potential in hyperthyroid patients was similar to that in normal subjects. No significant correlations were observed between the amplitude of the primary response in the somatosensory evoked potential and each value of BMR, 131I-uptake and resin sponge uptake.

Polysomnographic recordings were done over continuous 3 days together with those of electrical and mechanical activities of the stomach using chronically implanted electrodes and force-transducer in unanesthetized and unrestrained state of 4 adult cats. Circadian rhythm of the basic electric rhythm (BER) and mechanical activity in the stomach accustomed to feeding schedule of once a day was divided into the two; the fed state from the end of feeding to 8 to 11 hr later and the succeeding fasting state. However, differentiation of the fasting state into the contractile and noncontractile periods, which is known in dogs, was not evident in cats. The mean BER interval was longer and the standard deviation was smaller in the fed state than in the fasting state. Among stages of wakefulness (W), drowsiness (D), light slow wave sleep (LS), deep slow wave sleep (DS) and REM sleep, the mean BER intervals in every total time during the fed state were compared with one another. A definite unidirectional finding obtained in all 4 cats was only that the mean BER intervals in the stage D were longer significantly than those in the stage W.