Diplonemea (diplonemids) is one of the most abundant and species-rich protist groups in marine environments; however, their community structures among local and seasonal samples have not yet been compared. In the present study, we analyzed four diplonemid community structures around the Izu Peninsula, Japan using barcode sequences amplified from environmental DNA. These sequences and the results of statistical analyses indicated that communities at the same site were more similar to each other than those in the same season. Environmental variables were also measured, and their influence on diplonemid community structures was examined. Salinity, electrical conductivity, and temperature, and their correlated variables, appeared to influence the structures of diplonemid communities, which was consistent with previous findings; however, since the results obtained did not reach statistical significance, further studies are required. A comparison of each diplonemid community indicated that some lineages were unique to specific samples, while others were consistently detected in all samples. Members of the latter type are cosmopolitan candidates and may be better adapted to the environments of the studied area. Future studies that focus on the more adaptive members will provide a more detailed understanding of the mechanisms by which diplonemids are widely distributed in marine environments and will facilitate their utilization as indicator organisms to monitor environmental changes.
Fermentative nitrogen-fixing bacteria have not yet been examined in detail in thermal environments. In the present study, we isolated the thermophilic fermentative bacterium, strain YA01 from a hot spring. This strain grew at temperatures up to 78°C. A phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain YA01 belonged to the genus Caldicellulosiruptor, which are fermentative bacteria in the phylum Firmicutes, with 97.7–98.0% sequence identity to its closest relatives. Strain YA01 clearly exhibited N2-dependent growth at 70°C. We also confirmed N2-dependent growth in the relatives of strain YA01, Caldicellulosiruptor hydrothermalis 108 and Caldicellulosiruptor kronotskyensis 2002. The nitrogenase activities of these three strains were examined using the acetylene reduction assay. Similar activities were detected for all tested strains, and were slightly suppressed by the addition of ammonium. A genome analysis revealed that strain YA01, as well as other Caldicellulosiruptor, possessed a gene set for nitrogen fixation, but lacked the nifN gene, which encodes a nitrogenase iron-molybdenum cofactor biosynthesis protein that is commonly detected in nitrogen-fixing bacteria. The amino acid sequences of nitrogenase encoded by nifH, nifD, and nifK shared 92–98% similarity in Caldicellulosiruptor. A phylogenetic tree of concatenated NifHDK sequences showed that NifHDK of Caldicellulosiruptor was in the deepest clade. To the best of our knowledge, this is the first study to demonstrate the nitrogen-fixing ability of fermentative bacteria at 70°C. Caldicellulosiruptor may have retained an ancient nitrogen-fixing enzyme system.
Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120 min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens.
Phaseolus vulgaris is a grain cultivated in vast areas of different countries. It is an excellent alternative to the other legumes in the Venezuelan diet and is of great agronomic interest due to its resistance to soil acidity, drought, and high temperatures. Phaseolus establishes symbiosis primarily with Rhizobium and Ensifer species in most countries, and this rhizobia-legume interaction has been studied in Asia, Africa, and the Americas. However, there is currently no evidence to show that rhizobia nodulate the endemic cultivars of P. vulgaris in Venezuela. Therefore, we herein investigated the phylogenetic diversity of plant growth-promoting and N2-fixing nodulating bacteria isolated from the root nodules of P. vulgaris cultivars in a different agroecosystem in Venezuela. In comparisons with other countries, higher diversity was found in isolates from P. vulgaris nodules, ranging from α- and β-proteobacteria. Some isolates belonging to several new phylogenetic lineages within Bradyrhizobium, Ensifer, and Mesorhizobium species were also specifically isolated at some topographical regions. Additionally, some isolates exhibited tolerance to high temperature, acidity, alkaline pH, salinity stress, and high Al levels; some of these characteristics may be related to the origin of the isolates. Some isolates showed high tolerance to Al toxicity as well as strong plant growth-promoting and antifungal activities, thereby providing a promising agricultural resource for inoculating crops.
IgA nephropathy is one of the leading causes of chronic kidney disease in Japan. Since the origin and mechanisms by which IgA nephropathy develops currently remain unclear, a confirmed disease diagnosis is currently only possible by highly invasive renal biopsy. With the background of the salivary microbiome as a rich source of biomarkers for systemic diseases, we herein primarily aimed to investigate the salivary microbiome as a tool for the non-invasive diagnosis of IgA nephropathy. In a comparison of salivary microbiome profiles using 16S rRNA amplicon sequencing, significant differences were observed in microbial diversity and richness between IgA nephropathy patients and healthy controls. Furthermore, recent studies reported that patients with IgA nephropathy are more likely to develop inflammatory bowel diseases and that chronic inflammation of the tonsils triggered the recurrence of IgA nephropathy. Therefore, we compared the salivary microbiome of IgA nephropathy patients with chronic tonsillitis and ulcerative colitis patients. By combining the genera selected by the random forest algorithm, we were able to distinguish IgA nephropathy from healthy controls with an area under the curve (AUC) of 0.90, from the ulcerative colitis group with AUC of 0.88, and from the chronic tonsillitis group with AUC of 0.70. Additionally, the genus Neisseria was common among the selected genera that facilitated the separation of the IgA nephropathy group from healthy controls and the chronic tonsillitis group. The present results indicate the potential of the salivary microbiome as a biomarker for the non-invasive diagnosis of IgA nephropathy.
We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains—Synechocystis sp. PCC6803 and Synechococcus sp. WH7803—were grown in conventional media, and media with low ammonium, low sulfate and a high CO2/low O2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.
Carbonyl sulfide (COS) is the most abundant and long-lived sulfur-containing gas in the atmosphere. Soil is the main sink of COS in the atmosphere and uptake is dominated by soil microorganisms; however, biochemical research has not yet been conducted on fungal COS degradation. COS hydrolase (COSase) was purified from Trichoderma harzianum strain THIF08, which degrades COS at concentrations higher than 10,000 parts per million by volume from atmospheric concentrations, and its gene cos (492 bp) was cloned. The recombinant protein purified from Escherichia coli expressing the cos gene converted COS to H2S. The deduced amino acid sequence of COSase (163 amino acids) was assigned to clade D in the phylogenetic tree of the β-carbonic anhydrase (β-CA) family, to which prokaryotic COSase and its structurally related enzymes belong. However, the COSase of strain THIF08 differed from the previously known prokaryotic COSase and its related enzymes due to its low reactivity to CO2 and inability to hydrolyze CS2. Sequence comparisons of the active site amino acids of clade D β-CA family enzymes suggested that various Ascomycota, particularly Sordariomycetes and Eurotiomycetes, possess similar enzymes to the COSase of strain THIF08 with >80% identity. These fungal COSase were phylogenetically distant to prokaryotic clade D β-CA family enzymes. These results suggest that various ascomycetes containing COSase contribute to the uptake of COS by soil.
Cyanobacteria thrive in diverse environments. However, questions remain about possible growth limitations in ancient environmental conditions. As a single genus, the Thermosynechococcus are cosmopolitan and live in chemically diverse habitats. To understand the genetic basis for this, we compared the protein coding component of Thermosynechococcus genomes. Supplementing the known genetic diversity of Thermosynechococcus, we report draft metagenome-assembled genomes of two Thermosynechococcus recovered from ferrous carbonate hot springs in Japan. We find that as a genus, Thermosynechococcus is genomically conserved, having a small pan-genome with few accessory genes per individual strain as well as few genes that are unique to the genus. Furthermore, by comparing orthologous protein groups, including an analysis of genes encoding proteins with an iron related function (uptake, storage or utilization), no clear differences in genetic content, or adaptive mechanisms could be detected between genus members, despite the range of environments they inhabit. Overall, our results highlight a seemingly innate ability for Thermosynechococcus to inhabit diverse habitats without having undergone substantial genomic adaptation to accommodate this. The finding of Thermosynechococcus in both hot and high iron environments without adaptation recognizable from the perspective of the proteome has implications for understanding the basis of thermophily within this clade, and also for understanding the possible genetic basis for high iron tolerance in cyanobacteria on early Earth. The conserved core genome may be indicative of an allopatric lifestyle—or reduced genetic complexity of hot spring habitats relative to other environments.
Fragmented and primer ligated dsRNA sequencing (FLDS) is a sequencing method applicable to long double-stranded RNA (dsRNA) that enables the complete genome sequencing of both double- and single-stranded RNA viruses. However, the application of this method on a low amount of dsRNA has been hindered by adaptor dimer formation during cDNA amplification and sequence library preparation. We herein developed FLDS ver. 3 by optimizing the terminal modification of an oligonucleotide adaptor and the conditions of adaptor ligation. We also examined the concentration of Mg2+ in the PCR reaction for cDNA amplification and the purification method of amplified cDNA. Fine sequence reads were successfully obtained from metagenomic shotgun sequencing libraries constructed from 10 and 100 pg dsRNA, and these libraries exhibited weaker detection sensitivity for low-abundance dsRNAs (viral genomes and genome segments) than that constructed from 1 ng of dsRNA. We also report the utility of capillary electrophoresis for dsRNA quantification. The FLDS ver. 3 package expands the frontiers of our knowledge in RNA virus diversity and evolution.
Malodorous emissions are a crucial and inevitable issue during the decomposition of biological waste and contain a high concentration of ammonia. Biofiltration technology is a feasible, low-cost, energy-saving method that reduces and eliminates malodors without environmental impact. In the present study, we evaluated the effectiveness of compost from cattle manure and food waste as deodorizing media based on their removal of ammonia and the expression of ammonia-oxidizing genes, and identified the bacterial and archaeal communities in these media. Ammonia was removed by cattle manure compost, but not by food waste compost. The next-generation sequencing of 16S ribosomal RNA obtained from cattle manure compost revealed the presence of ammonia-oxidizing bacteria (AOB), including Cytophagia, Alphaproteobacteria, and Gammaproteobacteria, and ammonia-oxidizing archaea (AOA), such as Thaumarchaeota. In cattle manure compost, the bacterial and archaeal ammonia monooxygenase A (amoA) genes were both up-regulated after exposure to ammonia (fold ratio of 14.2±11.8 after/before), and the bacterial and archaeal communities were more homologous after than before exposure to ammonia, which indicates the adaptation of these communities to ammonia. These results suggest the potential of cattle manure compost as an efficient biological deodorization medium due to the activation of ammonia-oxidizing microbes, such as AOB and AOA, and the up-regulation of their amoA genes.
Clone libraries of bacterial 16S rRNA genes (a total of 1,980 clones) were constructed from the leaf blades, petioles, taproots, and lateral roots of sugar beet (Beta vulgaris L.) grown under different fertilization conditions. A principal coordinate analysis revealed that the structures of bacterial communities in above- and underground tissues were largely separated by PC1 (44.5%). The bacterial communities of above-ground tissues (leaf blades and petioles) were more tightly clustered regardless of differences in the tissue types and fertilization conditions than those of below-ground tissues (taproots and lateral roots). The bacterial communities of below-ground tissues were largely separated by PC2 (26.0%). To survey plant growth-promoting bacteria (PGPBs), isolate collections (a total of 665 isolates) were constructed from the lateral roots. As candidate PGPBs, 44 isolates were selected via clustering analyses with the combined 16S rRNA gene sequence data of clone libraries and isolate collections. The results of inoculation tests using sugar beet seedlings showed that eight isolates exhibited growth-promoting effects on the seedlings. Among them, seven isolates belonging to seven genera (Asticcacaulis, Mesorhizobium, Nocardioides, Sphingobium, Sphingomonas, Sphingopyxis, and Polaromonas) were newly identified as PGPBs for sugar beet at the genus level, and two isolates belonging to two genera (Asticcacaulis and Polaromonas) were revealed to exert growth-promoting effects on the plant at the genus level for the first time. These results suggest that a community analysis-based selection strategy will facilitate the isolation of novel PGPBs and extend the potential for the development of novel biofertilizers.