In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3'→5' exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.
DNA-Directed DNA Polymerase/metabolism* ; Bacillus Phages/genetics* ; Metagenome

Small urban wetlands are widely distributed and susceptible to human activities, serving as important sources and sinks of carbon. Microorganisms play a crucial role in carbon cycle, while limited studies have been conducted on the microbial diversity in small urban wetlands and the functions of microbiome in carbon fixation and metabolism. To probe into the microbiome-driven carbon cycling in small urban wetlands and dissect the composition and functional groups of microbiome, we analyzed the relationships between the microbiome structure, element metabolism pathways, and habitat physicochemical properties in sediment samples across three small wetlands in Huzhou City, and compared them with natural wetlands in the Zoige wetland. High-throughput sequencing of 16S rRNA gene amplicons and metagenomics was employed to determine the species and functional groups. Sixty medium to high-quality metagenome-assembled genomes (MAGs) were constructed, including 55 bacterial and 5 archaeal taxa, and their potential in driving elemental cycles were analyzed, with a focus on carbon fixation. Several bacterial species were found to encode a nearly complete carbon fixation pathway, including the Calvin cycle, the reductive tricarboxylic acid cycle, the Wood-Ljungdahl pathway, and the reductive glycine pathway. There were several potential novel carbon-fixing bacterial members, such as those belonging to Syntrophorhabdus (Desulfobacterota) and UBA4417 (Bacteroidetes), which had high relative abundance in the wetland microbiome. Unveiling the genetic potential of these functional groups to facilitate element cycling is of great scientific importance for enhancing the carbon sequestration capacity of small urban wetlands.
Wetlands ; Microbiota/genetics* ; Carbon Cycle/genetics* ; Bacteria/classification* ; RNA, Ribosomal, 16S/genetics* ; China ; Cities ; Geologic Sediments/microbiology* ; Archaea/classification* ; Metagenomics ; Metagenome

Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (T_m) values of mutants V80C and D226C/S281C were increased by 5.2 ℃ and 6.9 ℃, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.
Plastics/metabolism* ; Polyethylene Terephthalates/metabolism* ; Biodegradation, Environmental ; Metagenome

The development of high-throughput sequencing techniques enabled a deeper and more comprehensive understanding of environmental microbiology. Specifically, the third-generation sequencing techniques represented by nanopore sequencing have greatly promoted the development of environmental microbiology research due to its advantages such as long sequencing reads, fast sequencing speed, real-time monitoring of sequencing data, and convenient machine carrying, as well as no GC bias and no PCR amplification requirement. This review briefly summarized the technical principle and characteristics of nanopore sequencing, followed by discussing the application of nanopore sequencing techniques in the amplicon sequencing, metagenome sequencing and whole genome sequencing of environmental microorganisms. The advantages and challenges of nanopore sequencing in the application of environmental microbiology research were also analyzed.
Environmental Microbiology ; High-Throughput Nucleotide Sequencing ; Metagenome ; Nanopore Sequencing ; Nanopores

The research on the relationship between gut microbiota and human health continues to be a hot topic in the field of life science. Culture independent 16S rRNA gene high-throughput sequencing is the current main research method. However, with the reduction of sequencing cost and the maturity of data analysis methods, shotgun metagenome sequencing is gradually becoming an important method for the study of gut microbiome due to its advantages of obtaining more information. With the support from the human microbiome project, 30 805 metagenome samples were sequenced in the United States. By searching NCBI PubMed and SRA databases, it was found that 72 studies collecting about 10 000 Chinese intestinal samples were used for metagenome sequencing. To date, only 56 studies were published, including 16 related to metabolic diseases, 16 related to infectious and immune diseases, and 12 related to cardiovascular and cerebrovascular diseases. The samples were mainly collected in Beijing, Guangzhou, Shanghai and other cosmopolitan cities, where great differences exist in sequencing platforms and methods. The outcome of most studies are based on correlation analysis, which has little practical value in guiding the diagnosis and treatment of clinical diseases. Standardizing sampling methods, sequencing platform and data analysis process, and carrying out multi center parallel research will contribute to data integration and comparative analysis. Moreover, insights into the functional verification and molecular mechanism by using the combination of transcriptomics, proteomics and culturomics will enable the gut microbiota research to better serve the clinical diagnosis and treatment.
China ; Gastrointestinal Microbiome/genetics* ; Humans ; Metagenome ; Microbiota ; RNA, Ribosomal, 16S/genetics* ; United States

PURPOSE: The roles of gut microbiota on the natural course of atopic dermatitis (AD) are not yet fully understood. We investigated whether the composition and function of gut microbiota and short-chain fatty acids (SCFAs) at 6 months of age could affect the natural course of AD up to 24 months in early childhood.METHODS: Fecal samples from 132 infants were analyzed using pyrosequencing, including 84 healthy controls, 22 transient AD and 26 persistent AD subjects from the Cohort for Childhood Origin of Asthma and Allergic Diseases (COCOA) birth cohort. The functional profile of the gut microbiome was analyzed by whole-metagenome sequencing. SCFAs were measured using gas chromatography-mass spectrometry.RESULTS: Low levels of Streptococcus and high amounts of Akkermansia were evident in transient AD cases, and low Clostridium, Akkermansia and high Streptococcus were found in children with persistent AD. The relative abundance of Streptococcus positively correlated with scoring of AD (SCORAD) score, whereas that of Clostridium negatively correlated with SCORAD score. The persistent AD group showed decreased gut microbial functional genes related to oxidative phosphorylation compared with healthy controls. Butyrate and valerate levels were lower in transient AD infants compared with healthy and persistent AD infants.CONCLUSIONS: Compositions, functions and metabolites of the early gut microbiome are related to natural courses of AD in infants.
Asthma ; Butyrates ; Child ; Clostridium ; Cohort Studies ; Dermatitis, Atopic ; Fatty Acids, Volatile ; Gas Chromatography-Mass Spectrometry ; Gastrointestinal Microbiome ; Humans ; Infant ; Metabolomics ; Metagenome ; Oxidative Phosphorylation ; Parturition ; Streptococcus

Microbes are the most important commensal organisms in humans, animals and plants, and are the major habitants in soil, sediment, water, air and other habitats. The analysis of microbiome in these habitats has become a basic research technique. As a fast developing technology in recent years, microbiome sequencing and analysis have been widely used in human health, environmental pollution control, food industry, agriculture and animal husbandry and other fields. In order to sort out and summarize the current status, development and application prospects of microbiome sequencing and analysis technologies, this special issue has prepared a collection of 16 papers in this field, that comprise sample preservation and processing, single microbe genome sequencing and analysis, and microbiome feature analysis in special habitats, microbiome related databases and algorithms, and microbiome sequencing and analysis expert consensus. It also introduced in detail the development trend of the microbiome sequencing and analysis, in order to promote the rapid development of the microbiome sequencing and analysis industry and scientific research in China, and provide necessary reference for the healthy development of related industries.
Animals ; Bacteria/genetics* ; China ; High-Throughput Nucleotide Sequencing ; Humans ; Metagenome ; Microbiota/genetics* ; RNA, Ribosomal, 16S

Virome is the collective term for the viral collection or viral metagenomes that are distributed in various environments. Viruses can be found in bodies of water, glaciers, plants, animals, and even some viruses, which are classified as eukaryotes, prokaryotes and subviruses. Viruses play very important role in maintaining environmental homeostasis and ecosystem balance, and are especially closely related to human health. In recent years, with the advancement of sequencing technology and data analysis, we are able to gain more insights into the virome and explore its potential role in the ecological niche by metagenomic sequencing. A large amount of viral data have been obtained from glaciers, oceans, and various plants and animals, and numerous unknown viruses have been discovered. Virome has been studied mainly through metagenomic data mining, as well as virus-like particles separation and enrichment. To date, several different methods for viral isolation and enrichment exist, and numerous bioinformatic analyses of the virome have been performed. However, there is a lack of specific and complete reviews on the enrichment and data analysis methods for the virome. Thus, our review will summarize viral isolation and enrichment methods and data analysis, and present some of the landmark research conducted by the enrichment method, to provide a reference for researchers of interest and further advance the field of virome research.
Animals ; Humans ; Metagenome ; Metagenomics ; Microbiota/genetics* ; Virome ; Viruses/genetics*

Metagenomic next-generation sequencing (mNGS) could be used for pathogen detection from nearly all types of clinical samples. Especially, the unique diagnostic capability of pathogen mNGS detecting unknown causative agent of infectious diseases makes this method become an importation complement and irreplaceable component for conventional routine laboratory test. However, the complexity of the testing process, the rapid product update, and the insufficiency in quality control and evaluation methods that all make clinical transformation, industry development, and regulation of this technology full of challenge and uncertainty. This review briefly introduces the technical advantages and challenges, and describes the general workflow and quality control steps in details. Finally, it focuses on current considerations regarding quality evaluation methods and standards for pathogen mNGS.
Communicable Diseases ; High-Throughput Nucleotide Sequencing ; Humans ; Metagenome ; Metagenomics ; Quality Control

Strain is the fundamental unit in microbial taxonomy. The functional diversity among strains has great influence on host phenotypes. With the development of microbiome research, knowing the composition and functional capacities of complex microbial communities at the strain level has become increasingly valuable in scientific research and clinical applications. This review introduces the principles of bioinformatics algorithms for strain analysis based on metagenomic data, the applications in microbiome research and directions of future development.
Algorithms ; Computational Biology ; Metagenome ; Metagenomics ; Microbiota/genetics*