Niche and ecosystem preference of earliest diverging fungi in soils.

Within the supergroup Rotosphaeromycetes, or "Holomycota"/"Nucletmycea", there are several well-recognised unicellular clades in the earliest diverging fungi (EDF). However, we know little about their occurrence. Here, we investigated EDF in the rhizosphere and bulk soils from cropland, forest, orchard, and wetland ecosystems around the Beijing-Hebei area, China, to illustrate their niche and ecosystem preference. More than 500 new operational taxonomic units (OTUs) of EDF were detected based on the 18S rRNA genes. Microsporida and Aphelida constitute dominant groups, whereas Rozellosporida was quite rare. Although the EDF community was site-specific, the soil chemical characteristics, vegetation, and other eukaryotic microorganisms were the key factors driving the occurrence of EDF. Moreover, the stochastic process consisted the most of the EDF community assembly.

Distinctive microbiota distribution from healthy oral to post-treatment apical periodontitis.

Post-treatment apical periodontitis (PoAP) occurs when root canal treatment has not adequately eliminated bacterial invasion and infection. Yet little is known about the bacterial composition and changes related to the etiology and pathogenesis of PoAP. In this study, clinical samples classified as root apex (HARD) and periapical granulation tissues (SOFT) were separately collected from 10 patients with PoAP. The microbiota of each sample was characterized by 16S rRNA gene sequencing, and the obtained dataset was coanalyzed with 20 NCBI sequence read archive (SRA) datasets of healthy oral (HO) and primary apical periodontitis (PAP). We observed 2522 operational taxonomic units (OTUs) belonging to 29 phyla, and all samples shared 86.5% of the sequence reads. The OTUs affiliated with Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria, were identified as core microbiota, which accounted for nearly 90% of 16S rRNA sequences in all samples. However, the principal coordinates analysis (PCoA) of the beta diversity demonstrated that the three periapical statuses have distinct microbial compositions. Compared with HO and PoAP, Actinomyces has a significantly increased abundance in PAP. The microbial diversities in PoAP were significantly lower than those in the HO and PAP (p<0.05). The relative abundance of most bacterial taxa was decreasing, except that Clostridia and Synergistia were increased. Furthermore, we explored the potential metabolic differences of the microbial communities by KEGG pathway prediction. We revealed that the microbiota of PoAP might have a more active metabolic capacity, including carbohydrate metabolism, energy metabolism, and enzyme cofactor/carrier biosynthesis (p<0.05). Our study revealed that invasion of opportunistic pathogens such as Clostridia and Synergistia might play a significant role in PoAP, thus guiding the further study of complex microbial-host interactions and the development of more effective diagnostic and therapeutic methods.

Phylogeny and Metabolic Potential of the Methanotrophic Lineage MO3 in Beijerinckiaceae from the Paddy Soil through Metagenome-Assembled Genome Reconstruction.

Although the study of aerobic methane-oxidizing bacteria (MOB, methanotrophs) has been carried out for more than a hundred years, there are many uncultivated methanotrophic lineages whose metabolism is largely unknown. Here, we reconstructed a nearly complete genome of a Beijerinckiaceae methanotroph from the enrichment of paddy soil by using nitrogen-free M2 medium. The methanotroph labeled as MO3_YZ.1 had a size of 3.83 Mb, GC content of 65.6%, and 3442 gene-coding regions. Based on phylogeny of pmoA gene and genome and the genomic average nucleotide identity, we confirmed its affiliation to the MO3 lineage and a close relationship to Methylocapsa. MO3_YZ.1 contained mxaF- and xoxF-type methanol dehydrogenase. MO3_YZ.1 used the serine cycle to assimilate carbon and regenerated glyoxylate through the glyoxylate shunt as it contained isocitrate lyase and complete tricarboxylic acid cycle-coding genes. The ethylmalonyl-CoA pathway and Calvin-Benson-Bassham cycle were incomplete in MO3_YZ.1. Three acetate utilization enzyme-coding genes were identified, suggesting its potential ability to utilize acetate. The presence of genes for N2 fixation, sulfur transformation, and poly-ß-hydroxybutyrate synthesis enable its survival in heterogeneous habitats with fluctuating supplies of carbon, nitrogen, and sulfur.

Revealing the community and metabolic potential of active methanotrophs by targeted metagenomics in the Zoige wetland of the Tibetan Plateau.

The Zoige wetland of the Tibetan Plateau is one of the largest alpine wetlands in the world and a major emission source of methane. Methane oxidation by methanotrophs can counteract the global warming effect of methane released in the wetlands. Understanding methanotroph activity, diversity and metabolism at the molecular level can guide the isolation of the uncultured microorganisms and inform strategy-making decisions and policies to counteract global warming in this unique ecosystem. Here we applied DNA stable isotope probing using 13 C-labelled methane to label the genomes of active methanotrophs, examine the methane oxidation potential and recover metagenome-assembled genomes (MAGs) of active methanotrophs. We found that gammaproteobacteria of type I methanotrophs are responsible for methane oxidation in the wetland. We recovered two phylogenetically novel methanotroph MAGs distantly related to extant Methylobacter and Methylovulum. They belong to type I methanotrophs of gammaproteobacteria, contain both mxaF and xoxF types of methanol dehydrogenase coding genes, and participate in methane oxidation via H4 MPT and RuMP pathways. Overall, the community structure of active methanotrophs and their methanotrophic pathways revealed by DNA-SIP metagenomics and retrieved methanotroph MAGs highlight the importance of methanotrophs in suppressing methane emission in the wetland under the scenario of global warming.

Two Metagenome-Assembled Genomes of Hydrogen-Dependent Methanomassiliicoccales Methanogens from the Zoige Wetland of the Tibetan Plateau.

Wetlands in the Tibetan Plateau play a crucial role in global carbon cycling. Here, we report the metagenome-assembled genomes (MAGs) of two hydrogen-dependent methanogens from the Zoige wetland of the Tibetan Plateau. The novel species belong to Methanomassiliicoccales, the seventh euryarchaeal methanogenic order.

High-performance detection of Mycobacterium bovis in milk using digital LAMP.

This paper described a high-performance molecular test for the detection of Mycobacterium bovis (M. bovis) based on digital loop-mediated isothermal amplification (dLAMP). M. bovis is a persistent pathogen that causes zoonotic tuberculosis and can infect both animals and human beings. The detection of M. bovis in milk samples is critical for effective control and prevention of zoonotic diseases but there lacks effective and sensitive methods. Here, we developed a convenient and low-cost system for M. bovis detection in milk, which incorporated automated DNA extraction and dLAMP by interfacial emulsification technique. Versus real-time PCR, dLAMP provides higher accuracy and sensitivity for direct M. bovis detection in milk, offering a limit of detection of 14 CFU/mL within 2 h. The dLAMP system can become a powerful platform for the detection of pathogens in complex samples and provide more reliable guidance for food safety testing, epidemiological research and clinical diagnosis.

High-throughput single-cell cultivation reveals the underexplored rare biosphere in deep-sea sediments along the Southwest Indian Ridge.

Microorganisms in the deep sea play vital roles in marine ecosystems. However, despite great advances brought by high throughput sequencing and metagenomics, only a small portion of microorganisms living in the environment can be cultivated in the laboratory and systematically studied. In this study, an improved high-throughput microfluidic streak plate (MSP) platform was developed to speed up the isolation of microorganisms from deep-sea sediments and evaluated with deep-sea sediments collected from the Southwest Indian Ridge (SWIR). Based on our previously reported MSP method, we improved its isolation efficiency with a semi-automated droplet picker and improved humidity control to enable long-term cultivation with a low-nutrient medium for up to five months according to the slow-growing nature of most deep-sea species. The improved MSP method allows the isolation of microbes by selection and investigation of microbial diversity by high throughput sequencing of the pooled sample cultures. By picking individual droplets and scale-up cultivation, a total of 772 strains that were taxonomically assigned to 70 species were isolated from the deep-sea sediments in the SWIR, including 15 potential novel species. On the other hand, based on 16S rRNA gene amplicon sequencing analysis, the microbial diversity of the SWIR was studied and documented with culture-dependent and independent methods in this study. The superiority of the MSP platform in revealing the rare biosphere was also evaluated based on amplicon sequencing. The results show that droplet-based single-cell cultivation of the MSP has a much higher ability than traditional agar plate cultivation in obtaining microbial species and more than 90% of operational taxonomic units (OTUs) detected in the MSP pool belong to the rare biosphere. Our results indicate the high robustness and efficiency of the improved MSP platform in revealing the environmentally rare biosphere, especially for slow-growing species. Overall, the MSP platform has a superior ability to recover microbial diversity than conventional agar plates and it was found to hold great potential for recovering rare microbial resources from various environments.

Interfacial Nanoinjection-Based Nanoliter Single-Cell Analysis.

Single-cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here, described is a simple method named interfacial nanoinjection (INJ), which can miniaturize various single-cell assays to be performed in nanoliter water-in-oil droplets on standard microwell plates. The INJ droplet handler can adjust droplet volumes for multistep reactions on demand with high precision and excellent monodispersity, and consequently enables a wide range of single-cell assays. Importantly, INJ can be coupled with fluorescence-activated cell sorting (FACS), which is currently the most effective and accurate single-cell sorting and isolation method. FACS-INJ pipelines for high-throughput plate well-based single-cell analyses, including single-cell proliferation, drug-resistance testing, polymerase chain reaction (PCR), reverse-transcription PCR, and whole-genome sequencing are introduced. This FACS-INJ pipeline is compatible with a wide range of samples and can be extended to various single-cell analysis applications in microbiology, cell biology, and biomedical diagnostics.

Full-scale anaerobic reactor samples would be more suitable than lab-scale anaerobic reactor and natural samples to inoculate the wheat straw batch anaerobic digesters.

This study evaluated the effects of the inocula from natural wetland, lab-scale and full-scale anaerobic reactors on wheat straw anaerobic digestion. Three replicate batch reactors were constructed for each inoculum to investigate the reactor performances and microbial communities. Reactors seeded with full-scale reactor samples were started up most rapidly, achieved the highest methane production, and were recognized as the higher efficient reactors. The dominance of acetoclastic methanogens, including Methanosaeta and Methanoscrina, was crucial for the higher efficient reactors, whereas hydrogenotrophic methanogens were dominant in other reactors. Genus Treponema, which could enhance the cellulose degradation and conduct homoacetogenesis, was first reported to be dominant in the bacterial communities of high efficient reactors. Inoculum sources and process conditions were suggested to be the deterministic factors in shaping the microbial communities in the higher efficient reactors. These findings contribute to the startup of new anaerobic reactors.

Upland Soil Cluster Gamma dominates methanotrophic communities in upland grassland soils.

Aerobic methanotrophs in upland soils consume atmospheric methane, serving as a critical counterbalance to global warming; however, the biogeographic distribution patterns of their abundance and community composition are poorly understood, especial at a large scale. In this study, soils were sampled from 30 grasslands across >2000 km on the Qinghai-Tibetan Plateau to determine the distribution patterns of methanotrophs and their driving factors at a regional scale. Methanotroph abundance and community composition were analyzed using quantitative PCR and Illumina Miseq sequencing of pmoA genes, respectively. The pmoA gene copies ranged from 8.2 × 105 to 1.1 × 108 per gram dry soil. Among the 30 grassland soil samples, Upland Soil Cluster Gamma (USCγ) dominated the methanotroph communities in 26 samples. Jasper Ridge Cluster (JR3) was the most dominant methanotrophic cluster in two samples; while Methylocystis, cluster FWs, and Methylobacter were abundant in other two wet soil samples. Interestingly, reanalyzing the pmoA genes sequencing data from existing publications suggested that USCγ was also the main methanotrophic cluster in grassland soils in other regions, especially when their mean annual precipitation was <500 mm. Canonical Analysis of Principal Coordinates including all soil samples indicated that the methanotrophic community composition was significantly correlated with local environmental factors, among which mean annual precipitation and pH showed the strongest correlations. Variance partitioning analysis showed that environmental factors and spatial distance were significant factors affecting the community structure of methanotrophs, and environmental properties were more important factors. Collectively, these findings indicate that atmospheric methane may be mainly oxidized by USCγ in upland soils. They also highlight the key role of water availability and pH in determining the abundance and community profiles of grassland soil methanotrophs.

Methylococcaceae are the dominant active aerobic methanotrophs in a Chinese tidal marsh.

Although coastal marshes are net carbon sinks, they are net methane sources. Aerobic methanotrophs in coastal marsh soils are important methane consumers, but their activity and populations are poorly characterized. DNA stable-isotope probing followed by sequencing was used to determine how active methanotrophic populations differed in the main habitats of a Chinese coastal marsh. These habitats included mudflat, native plant-dominated, and alien plant-dominated habitats. Methylococcaceae was the most active methanotroph family across four habitats. Abundant methylotroph sequences, including methanotrophs and non-methane-oxidizing methylotrophs (Methylotenera and Methylophaga), constituted 50-70% of the 16S rRNA genes detected in the labeled native plant-dominated and mudflat soils. Methylotrophs were less abundant (~ 20%) in labeled alien plant-dominated soil, suggesting less methane assimilation into the target community or a different extent of carbon cross-feeding. Canonical correspondence analysis indicated a significant correlation between the active bacterial communities and soil properties (salinity, organic carbon, total nitrogen, pH, and available phosphorus). Importantly, these results highlight how changing vegetation or soil features in coastal marshes may change their resident active methanotrophic populations, which will further influence methane cycling.

Dynamic Sessile-Droplet Habitats for Controllable Cultivation of Bacterial Biofilm.

Bacterial biofilms play essential roles in biogeochemical cycling, degradation of environmental pollutants, infection diseases, and maintenance of host health. The lack of quantitative methods for growing and characterizing biofilms remains a major challenge in understanding biofilm development. In this study, a dynamic sessile-droplet habitat is introduced, a simple method which cultivates biofilms on micropatterns with diameters of tens to hundreds of micrometers in a microfluidic channel. Nanoliter plugs are utilized, spaced by immiscible carrier oil to initiate and support the growth of an array of biofilms, anchored on and spatially confined to the micropatterns arranged on the bottom surface of the microchannel, while planktonic or dispersal cells are flushed away by shear force of aqueous plugs. The performance of the aforementioned method of cultivating biofilms is demonstrated by Pseudomonas aeruginosa PAO1 and its derived mutants, and quantitative antimicrobial susceptibility testing of PAO1 biofilms. This method could significantly eliminate corner effects, avoid microchannel clogging, and constrain the growth of biofilms for long-term observations. The controllable sessile droplet-based biofilm cultivation presented in this study should shed light on more quantitative and long-term studies of biofilms, and open new avenues for investigation of biofilm attachment, growth, expansion, and eradication.

Anthropogenic protection alters the microbiome in intertidal mangrove wetlands in Hainan Island.

Intertidal mangrove wetlands are of great economic and ecological importance. The regular influence of tides has led to the microbial communities in these wetlands differing significantly from those in other habitats. In this study, we investigated the microbiomes of the two largest mangrove wetlands in Hainan Island, China, which have different levels of anthropogenic protection. Soil samples were collected from the root zone of 13 mangrove species. The microbial composition, including key functional groups, was assessed using Illumina sequencing. Bioinformatics analysis showed that there was a significant difference in the microbiomes between the protected Bamen Bay and the unprotected Dongzhai Bay. The overall microbiome was assigned into 78 phyla and Proteobacteria was the most abundant phylum at both sites. In the protected wetland, there were fewer marine-related microbial communities, such as sulfate-reducing bacteria, and more terrestrial-related communities, such as Verrucomicrobia methanotrophs. We also observed distinct microbial compositions among the different mangrove species at the protected site. Our data suggest that the different microbiomes of the two mangrove wetlands are the result of a complex interaction of the different environmental variables at the two sites.

Aerobic methanotroph diversity in Sanjiang wetland, Northeast China.

Aerobic methanotrophs present in wetlands can serve as a methane filter and thereby significantly reduce methane emissions. Sanjiang wetland is a major methane source and the second largest wetland in China, yet little is known about the characteristics of aerobic methanotrophs in this region. In the present study, we investigated the diversity and abundance of methanotrophs in marsh soils from Sanjiang wetland with three different types of vegetation by 16S ribosomal RNA (rRNA) and pmoA gene analysis. Quantitative polymerase chain reaction analysis revealed the highest number of pmoA gene copies in marsh soils vegetated with Carex lasiocarpa (10(9) g(-1) dry soil), followed by Carex meyeriana, and the least with Deyeuxia angustifolia (10(8) g(-1) dry soil). Consistent results were obtained using Sanger sequencing and pyrosequencing techniques, both indicating the codominance of Methylobacter and Methylocystis species in Sanjiang wetland. Other less abundant methanotrophy, including cultivated Methylomonas and Methylosinus genus, and uncultured clusters such as LP20 and JR-1, were also detected in the wetland. Methanotroph diversity was almost the same in three different vegetation covered soils, suggesting that vegetation types had very little influence on the methanotroph diversity. Our study gives an in-depth insight into the community composition of aerobic methanotrophs in the Sanjiang wetland.

Bacterial community structure in two permafrost wetlands on the Tibetan Plateau and Sanjiang Plain, China.

Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10(10) 16S rRNA gene copies per gram of wet soil in both wetlands, with 10(8) pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.

The diversity and abundance of bacteria and oxygenic phototrophs in saline biological desert crusts in Xinjiang, northwest China.

Although microorganisms, particularly oxygenic phototrophs, are known as the major players in the biogeochemical cycles of elements in desert soil ecosystems and have received extensive attention, still little is known about the effects of salinity on the composition and abundances of microbial community in desert soils. In this study, the diversity and abundance of bacteria and oxygenic phototrophs in biological desert crusts from Xinjiang province, which were under different salinity conditions, were investigated by using clone library and quantitative PCR (qPCR). The 16S rRNA gene phylogenetic analysis showed that cyanobacteria, mainly Microcoleus vagnitus of the order Oscillatoriales, were predominant in the low saline crusts, while other phototrophs, such as diatom, were the main microorganism group responsible for the oxygenic photosynthesis in the high saline crusts. Furthermore, the higher salt content in crusts may stimulate the growth of other bacteria, including Deinococcus-Thermus, Bacteroidetes, and some subdivisions of Proteobacteria (ß-, γ-, and δ-Proteobacteria). The cpcBA-IGS gene analysis revealed the existence of novel M. vagnitus strains in this area. The qPCR results showed that the abundance of oxygenic phototrophs was significantly higher under lower saline condition than that in the higher saline crusts, suggesting that the higher salinity in desert crusts could suppress the numbers of total bacteria and phototrophic bacteria but did highly improve the diversity of salt-tolerant bacteria.

Community structure, abundance, and activity of methanotrophs in the Zoige wetland of the Tibetan Plateau.

The Zoige wetland of the Tibetan Plateau is a high-altitude tundra wetland and one of the biggest methane emission centers in China. In this study, methanotrophs with respect to community structure, abundance, and activity were investigated in peat soils collected in the vicinity of different marshland plants that dominate different regions of the wetland, including Polygonum amphibium, Carex muliensis, and Eleocharis valleculosa (EV). 16S rRNA gene and particulate methane monooxygenase gene (pmoA) clone library sequence data indicated the presence of methanotrophs with two genera, Methylobacter and Methylocystis. Methylococcus, like pmoA gene sequences, were also retrieved and showed low similarity to those from Methylococcus spp. and thus indicates the existence of novel methanotrophs in the Zoige wetland. Quantitative polymerase chain reaction (qPCR) assays were used to measure the abundance of methantrophs and detected 10(7) to 10(8) of total pmoA gene copies per gram dry weight of soil in the three marshes. Group-specific qPCR and reverse transcriptase qPCR results found that the Methylobacter genus dominates the wetland, and Methylocystis methanotrophs were less abundant, although this group of methanotrophs was estimated to be more active according to mRNA/DNA ratio. Furthermore, EV marsh demonstrated the highest methanotrophs abundance and activity among the three marshes investigated. Our study suggests that both type I and type II methanotrophs contribute to the methane oxidation in the Zoige wetland.

Diversity of methanotrophs in Zoige wetland soils under both anaerobic and aerobic conditions.

Zoige wetland is one of the most important methane emission centers in China. The oxidation of methane in the wetland affects global warming, soil ecology and atmospheric chemistry. Despite their global significance, microorganisms that consume methane in Zoige wetland remain poorly characterized. In this study, we investigated methanotrophs diversity in soil samples from both anaerobic site and aerobic site in Zoige wetland using pmoA gene as a molecular marker. The cloning library was constructed according to the pmoA sequences detected. Four clusters of methanotrophs were detected. The phylogenetic tree showed that all four clusters detected were affiliated to type I methanotrophs. Two novel clusters (cluster 1, cluster 2) were found to relate to none of the recognized genera of methanotrophs. These clusters have no cultured representatives and reveal an ecological adaptation of particular uncultured methanotrophs in Zoige wetland. Two clusters were belonging to Methylobacter and Methylococcus separately. Denaturing gradient gel electrophoresis gel bands pattern retrieved from these two samples revealed that the community compositions of anaerobic soil and aerobic soil were different from each other while anaerobic soil showed a higher metanotrophs diversity. Real-time PCR assays of the two samples demonstrated that aerobic soil sample in Zoige wetland was 1.5 times as much copy numbers as anaerobic soil. These data illustrated that methanotrophs are a group of microorganisms influence the methane consumption in Zoige wetland.