Comprehensive analysis of metabolites produced by co-cultivation of Bifidobacterium breve MCC1274 with human iPS-derived intestinal epithelial cells.

Examining how host cells affect metabolic behaviors of probiotics is pivotal to better understand the mechanisms underlying the probiotic efficacy in vivo. However, studies to elucidate the interaction between probiotics and host cells, such as intestinal epithelial cells, remain limited. Therefore, in this study, we performed a comprehensive metabolome analysis of a co-culture containing Bifidobacterium breve MCC1274 and induced pluripotent stem cells (iPS)-derived small intestinal-like cells. In the co-culture, we observed a significant increase in several amino acid metabolites, including indole-3-lactic acid (ILA) and phenyllactic acid (PLA). In accordance with the metabolic shift, the expression of genes involved in ILA synthesis, such as transaminase and tryptophan synthesis-related genes, was also elevated in B. breve MCC1274 cells. ILA production was enhanced in the presence of purines, which were possibly produced by intestinal epithelial cells (IECs). These findings suggest a synergistic action of probiotics and IECs, which may represent a molecular basis of host-probiotic interaction in vivo.

Isolation of Aquatic Plant Growth-Promoting Bacteria for the Floating Plant Duckweed (Lemna minor).

Plant growth-promoting bacteria (PGPB) can exert beneficial growth effects on their host plants. Little is known about the phylogeny and growth-promoting mechanisms of PGPB associated with aquatic plants, although those of terrestrial PGPB have been well-studied. Here, we report four novel aquatic PGPB strains, MRB1-4 (NITE P-01645-P-01648), for duckweed Lemna minor from our rhizobacterial collection isolated from Lythrum anceps. The number of L. minor fronds during 14 days co-culture with the strains MRB1-4 increased by 2.1-3.8-fold, compared with an uninoculated control; the plant biomass and chlorophyll content in co-cultures also increased. Moreover, all strains possessed an indole-3-acetic acid production trait in common with a plant growth-promoting trait of terrestrial PGPB. Phylogenetic analysis showed that three strains, MRB-1, -3, and -4, were affiliated with known proteobacterial genera (Bradyrhizobium and Pelomonas); this report is the first to describe a plant-growth promoting activity of Pelomonas members. The gammaproteobacterial strain MRB2 was suggested to be phylogenetically novel at the genus level. Under microscopic observation, the Pelomonas strain MRB3 was epiphytic and adhered to both the root surfaces and fronds of duckweed. The duckweed PGPB obtained here could serve as a new model for understanding unforeseen mechanisms behind aquatic plant-microbe interactions.

Complete Genome Sequence of Luteitalea sp. Strain TBR-22.

We report a complete genome sequence of a novel bacterial isolate, strain TBR-22, belonging to the class Vicinamibacteria of the phylum Acidobacteria, which was isolated from duckweed fronds. The genome expands our knowledge of the lifestyle of this abundant but rarely characterized phylum.

Draft Genome Sequence of Bryobacteraceae Strain F-183.

Here, we report a draft genome sequence of a bacterial strain, F-183, isolated from a duckweed frond. Strain F-183 belongs to the family Bryobacteraceae of the phylum Acidobacteria, and its genomic information would contribute to understanding the ecophysiology of this abundant but rarely characterized phylum.

Novel Plant-Associated Acidobacteria Promotes Growth of Common Floating Aquatic Plants, Duckweeds.

Duckweeds are small, fast growing, and starch- and protein-rich aquatic plants expected to be a next generation energy crop and an excellent biomaterial for phytoremediation. Despite such an importance, very little is known about duckweed-microbe interactions that would be a key biological factor for efficient industrial utilization of duckweeds. Here we first report the duckweed growth promoting ability of bacterial strains belonging to the phylum Acidobacteria, the members of which are known to inhabit soils and terrestrial plants, but their ecological roles and plant-microbe interactions remain largely unclear. Two novel Acidobacteria strains, F-183 and TBR-22, were successfully isolated from wild duckweeds and phylogenetically affiliated with subdivision 3 and 6 of the phylum, respectively, based on 16S rRNA gene sequence analysis. In the co-culture experiments with aseptic host plants, the F-183 and TBR-22 strains visibly enhanced growth (frond number) of six duckweed species (subfamily Lemnoideae) up to 1.8-5.1 times and 1.6-3.9 times, respectively, compared with uninoculated controls. Intriguingly, both strains also increased the chlorophyll content of the duckweed (Lemna aequinoctialis) up to 2.4-2.5 times. Under SEM observation, the F-183 and TBR-22 strains were epiphytic and attached to the surface of duckweed. Taken together, our findings suggest that indigenous plant associated Acidobacteria contribute to a healthy growth of their host aquatic plants.

Comparative Analysis of Microbial Communities in Fronds and Roots of Three Duckweed Species: Spirodela polyrhiza, Lemna minor, and Lemna aequinoctialis.

The microbial communities inhabiting the fronds of duckweeds have not been investigated in as much detail as those on the roots. We herein examined the microbial communities in three duckweed species using 16S rRNA amplicon sequencing and compared them to those on the roots. The microbial compositions of the fronds were distinct from those of the roots in the three species. Various types of taxonomic bacteria, including rarely cultivated phyla, Acidobacteria, Armatimonadetes, and Verrucomicrobia, were also isolated from the fronds, but at a slightly lower abundance than those from the roots. These results suggest that duckweed fronds are an alternative source for isolating rare and novel microbes, which may otherwise be recalcitrant to cultivation using conventional strategies.

Insight into Energy Conservation via Alternative Carbon Monoxide Metabolism in Carboxydothermus pertinax Revealed by Comparative Genome Analysis.

Carboxydothermus species are some of the most studied thermophilic carboxydotrophs. Their varied carboxydotrophic growth properties suggest distinct strategies for energy conservation via carbon monoxide (CO) metabolism. In this study, we used comparative genome analysis of the genus Carboxydothermus to show variations in the CO dehydrogenase-energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H2 production (hydrogenogenic CO metabolism). Indeed, the ability or inability to produce H2 with CO oxidation is explained by the presence or absence of this gene cluster in Carboxydothermus hydrogenoformans, Carboxydothermus islandicus, and Carboxydothermus ferrireducens Interestingly, despite its hydrogenogenic CO metabolism, Carboxydothermus pertinax lacks the Ni-CO dehydrogenase catalytic subunit (CooS-I) and its transcriptional regulator-encoding genes in this gene cluster, probably due to inversion. Transcriptional analysis in C. pertinax showed that the Ni-CO dehydrogenase gene (cooS-II) and distantly encoded energy-converting-hydrogenase-related genes were remarkably upregulated with 100% CO. In addition, when thiosulfate was available as a terminal electron acceptor in 100% CO, the maximum cell density and maximum specific growth rate of C. pertinax were 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H2 produced was only 62% of the amount of CO consumed, less than expected according to hydrogenogenic CO oxidation (CO + H2O → CO2 + H2). Accordingly, C. pertinax would couple CO oxidation by Ni-CO dehydrogenase II with simultaneous reduction of not only H2O but also thiosulfate when grown in 100% CO.IMPORTANCE Anaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche by scavenging potentially toxic CO and producing H2 as an available energy source for thermophilic microbes. This hydrogenogenic carboxydotrophy relies on a Ni-CO dehydrogenase-energy-converting hydrogenase gene cluster. This feature is thought to be common to these organisms. However, the hydrogenogenic carboxydotroph Carboxydothermus pertinax lacks the gene for the Ni-CO dehydrogenase catalytic subunit encoded in the gene cluster. Here, we performed a comparative genome analysis of the genus Carboxydothermus, a transcriptional analysis, and a cultivation study in 100% CO to prove the hydrogenogenic CO metabolism. Results revealed that C. pertinax could couple Ni-CO dehydrogenase II alternatively to the distal energy-converting hydrogenase. Furthermore, C. pertinax represents an example of the functioning of Ni-CO dehydrogenase that does not always correspond to its genomic context, owing to the versatility of CO metabolism and the low redox potential of CO.

Genomic Analysis of Calderihabitans maritimus KKC1, a Thermophilic, Hydrogenogenic, Carboxydotrophic Bacterium Isolated from Marine Sediment.

Calderihabitans maritimus KKC1 is a thermophilic, hydrogenogenic carboxydotroph isolated from a submerged marine caldera. Here, we describe the de novo sequencing and feature analysis of the C. maritimus KKC1 genome. Genome-based phylogenetic analysis confirmed that C. maritimus KKC1 was most closely related to the genus Moorella, which includes well-studied acetogenic members. Comparative genomic analysis revealed that, like Moorella, C. maritimus KKC1 retained both the CO2-reducing Wood-Ljungdahl pathway and energy-converting hydrogenase-based module activated by reduced ferredoxin, but it lacked the HydABC and NfnAB electron-bifurcating enzymes and pyruvate:ferredoxin oxidoreductase required for ferredoxin reduction for acetogenic growth. Furthermore, C. maritimus KKC1 harbored six genes encoding CooS, a catalytic subunit of the anaerobic CO dehydrogenase that can reduce ferredoxin via CO oxidation, whereas Moorella possessed only two CooS genes. Our analysis revealed that three cooS genes formed known gene clusters in other microorganisms, i.e., cooS-acetyl coenzyme A (acetyl-CoA) synthase (which contained a frameshift mutation), cooS-energy-converting hydrogenase, and cooF-cooS-FAD-NAD oxidoreductase, while the other three had novel genomic contexts. Sequence composition analysis indicated that these cooS genes likely evolved from a common ancestor. Collectively, these data suggest that C. maritimus KKC1 may be highly dependent on CO as a low-potential electron donor to directly reduce ferredoxin and may be more suited to carboxydotrophic growth compared to the acetogenic growth observed in Moorella, which show adaptation at a thermodynamic limit.IMPORTANCECalderihabitans maritimus KKC1 and members of the genus Moorella are phylogenetically related but physiologically distinct. The former is a hydrogenogenic carboxydotroph that can grow on carbon monoxide (CO) with H2 production, whereas the latter include acetogenic bacteria that grow on H2 plus CO2 with acetate production. Both species may require reduced ferredoxin as an actual "energy equivalent," but ferredoxin is a low-potential electron carrier and requires a high-energy substrate as an electron donor for reduction. Comparative genomic analysis revealed that C. maritimus KKC1 lacked specific electron-bifurcating enzymes and possessed six CO dehydrogenases, unlike Moorella species. This suggests that C. maritimus KKC1 may be more dependent on CO, a strong electron donor that can directly reduce ferredoxin via CO dehydrogenase, and may exhibit a survival strategy different from that of acetogenic Moorella, which solves the energetic barrier associated with endergonic reduction of ferredoxin with hydrogen.

Draft Genome Sequences of Two Hydrogenogenic Carboxydotrophic Bacteria, Carboxydocella sp. Strains JDF658 and ULO1, Isolated from Two Distinct Volcanic Fronts in Japan.

Hydrogenogenic carboxydotrophs may provide hydrogen as primary energy for the microbial community via carbon monoxide oxidation. To investigate the genetics of carbon monoxide metabolism, we report here the draft genome sequences of the hydrogenogenic carboxydotrophs Carboxydocella sp. strains JDF658 (2.60 Mbp; G+C content, 49.2%) and ULO1 (2.70 Mbp; G+C content, 48.8%).

Draft Genome Sequences of Carboxydothermus pertinax and C. islandicus, Hydrogenogenic Carboxydotrophic Bacteria.

Carboxydothermus spp. are some of the most studied carbon monoxide-oxidizing anaerobic thermophiles. For further investigation into the carbon monoxide metabolism of Carboxydothermus spp., we report here the draft genome sequences of the hydrogenogenic carboxydotrophs Carboxydothermus pertinax (2.47 Mb; G+C content, 40.7%) and C. islandicus (2.39 Mb; G+C content, 42.0%).

Draft Genome Sequences of Methanoculleus horonobensis Strain JCM 15517, Methanoculleus thermophilus Strain DSM 2373, and Methanofollis ethanolicus Strain JCM 15103, Hydrogenotrophic Methanogens Belonging to the Family Methanomicrobiaceae.

The familyMethanomicrobiaceaecomprises hydrogen- and formate-utilizing methanogens. Genome sequencing of nine species ofMethanomicrobiaceaehas been conducted so far. Here, we report three additional draft genome sequences ofMethanomicrobiaceae, those ofMethanoculleus horonobensisJCM 15517 (=T10(T)),Methanoculleus thermophilusDSM 2373 (=CR-1(T)), andMethanofollis ethanolicusJCM 15103 (=HASU(T)).

Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal environments.

Carboxydotrophic anaerobic thermophiles have been isolated from various hydrothermal environments and are considered to be important carbon monoxide (CO) scavengers or primary producers. However, the ecological factors that influence the distribution, abundance and CO-oxidizing activities of these bacteria are poorly understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus spp. in a hot spring sample and found that they constituted up to 10% of the total bacterial cells. In this study, we investigated environmental features, potential microbial CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot springs to determine environmental factors that affect CO oxidizers and to see whether Carboxydothermus spp. are common in these environments. We detected potential microbial CO-oxidation activities in samples that showed relatively high values of total organic carbon, total nitrogen, oxidation-reduction potential and soil-water content. The abundance of Carboxydothermus spp. did not correlate with the presence of potential microbial CO-oxidation activities; however, Carboxydothermus spp. were detected in a wide range of environments, suggesting that these bacteria are widely distributed in spite of the relatively low population size. This study implies that thermophilic CO oxidizers occur in a wide range of environments and oxidize CO in somewhat oxidative environments rich in organic matter.

Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster.

A unique [Ni-Fe-S] cluster (C-cluster) constitutes the active center of Ni-containing carbon monoxide dehydrogenases (CODHs). His(261), which coordinates one of the Fe atoms with Cys(295), is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys(295), we constructed CODH-II variants. Ala substitution for the Cys(295) substitution resulted in the decrease of Ni content and didn't result in major change of Fe content. In addition, the substitution had no effect on the ability to assemble a full complement of [Fe-S] clusters. This strongly suggests Cys(295) indirectly and His(261) together affect Ni-coordination in the C-cluster.

A thermophilic, hydrogenogenic and carboxydotrophic bacterium, Calderihabitans maritimus gen. nov., sp. nov., from a marine sediment core of an undersea caldera.

A hydrogenogenic, carboxydotrophic marine bacterium, strain KKC1(T), was isolated from a sediment core sample taken from a submerged marine caldera. Cells were non-motile, Gram-stain-negative, 1.0-3.0 µm straight rods, often observed with round endospores. Strain KKC1(T) grew at 55-68 °C, pH 5.2-9.2 and 0.8-14 % (w/v) salinity. Optimum growth occurred at 65 °C, pH 7.0-7.5 and 2.46 % salinity with a doubling time of 3.7 h. The isolate grew chemolithotrophically, producing H2 from carbon monoxide (CO) oxidation with reduction of various electron acceptors, e.g. sulfite, thiosulfate, fumarate, ferric iron and AQDS (9,10-anthraquinone 2,6-disulfonate). KKC1(T) grew heterotrophically on pyruvate, lactate, fumarate, glucose, fructose and mannose with thiosulfate as an electron acceptor. When grown mixotrophically on CO and pyruvate, C16 : 0 constituted almost half of the total cellular fatty acids. The DNA G+C content was 50.6 mol%. The 16S rRNA gene sequence of KKC1(T) was most closely related to those of members of the genus Moorella with similarity ranging from 91 to 89 %. Based on physiological and phylogenetic novelty, we propose the isolate as a representative of a new genus and novel species with the name Calderihabitans maritimus gen. nov., sp. nov.; the type strain of the type species is KKC1(T) ( = DSM 26464(T) = NBRC 109353(T)).

Carboxydothermus pertinax sp. nov., a thermophilic, hydrogenogenic, Fe(III)-reducing, sulfur-reducing carboxydotrophic bacterium from an acidic hot spring.

A novel anaerobic, Fe(III)-reducing, hydrogenogenic, carboxydotrophic bacterium, designated strain Ug1(T), was isolated from a volcanic acidic hot spring in southern Kyushu Island, Japan. Cells of the isolate were rod-shaped (1.0-3.0 µm long) and motile due to peritrichous flagella. Strain Ug1(T) grew chemolithoautotrophically on CO (100% in the gas phase) with reduction of ferric citrate, amorphous iron (III) oxide, 9,10-anthraquinone 2,6-disulfonate, thiosulfate or elemental sulfur. No carboxydotrophic growth occurred with sulfate, sulfite, nitrate or fumarate as electron acceptor. During growth on CO, H(2) and CO(2) were produced. Growth occurred on molecular hydrogen as an energy source and carbon dioxide as a sole carbon source. Growth was observed on various organic compounds under an N(2) atmosphere with the reduction of ferric iron. The temperature range for carboxydotrophic growth was 50-70 °C, with an optimum at 65 °C. The pH(25 °C) range for growth was 4.6-8.6, with an optimum between 6.0 and 6.5. The doubling time under optimum conditions using CO with ferric citrate was 1.5 h. The DNA G+C content was 42.2 mol%. Analysis of 16S rRNA gene sequences demonstrated that this strain belongs to the thermophilic carboxydotrophic bacterial genus Carboxydothermus, with sequence similarities of 94.1-96.6% to members of this genus. The isolate can be distinguished from other members of the genus Carboxydothermus by its ability to grow with elemental sulfur or thiosulfate coupled to CO oxidation. On the basis of phylogenetic analysis and unique physiological features, the isolate represents a novel species of the genus Carboxydothermus for which the name Carboxydothermus pertinax sp. nov. is proposed; the type strain of the novel species is Ug1(T) (=DSM 23698(T)=NBRC 107576(T)).