Abstract:

Abstract:Microbiology data centers has become an essential part of strategic resources of a country. The National Microbiology Data Center (NMDC, https://nmdc.cn/) allows a huge amount of microbiological data to be organized and integrated in an effective way, and shared in an open manner, which is crucial for the research, utilization, and sustainable development of microbiological resources. This paper summarizes the progress of the recently founded NMDC platform, with respect to core resources, services, and functions, in order to foster the applications and practices of microbiological knowledge in academic and industrial users.

Abstract:Biopart databases are the key platforms to collect, sort and share data and physical bioparts, which may promote the research and application of synthetic biology. The research progress of building of biopart database is summarized in this review. There are several databases or registries of bioparts appeared abroad, such as Registry of Standard Biological Parts. In these databases, OpenMTA has been applied to share and distribute bioparts freely; besides, "Synthetic Biology Open Language, SBOL" was developed to exchange the biopart data among different databases; recently a project named as ‘bionet’ has been put on progress to manage, metadata and physical objects of bioparts by decentralized way. We have recently established a biopart database called "Registry and database of bioparts for synthetic biology (RDBSB)" (RDBSB, https://www.biosino.org/rdbsb/or https://www.biosino.org/npbiosys/) by working together with other research institutes. This biopart dabase has been established based on a "data standard for catalytic bioparts" system compatible with SBOL language, and a series of developed standard functional test methods for different sorts of bioparts as well as a set up safe and efficient submission system for bioparts. Until now, RDBSB has collected more than 300000 catalytic bioparts, of which more than 70000 ones have been described with their functional characterization supported by Scientific literatures preserved more than 10000 physical bioparts and 5000 chassis strains, and set up an efficient sharing system to distribute the data and physical bioparts through its website. In the past two years, RDBSB has attracted one million visits per year, and provided more than 100 Services per year to build customized bioparts or chassis strains to researchers or the customized bio-technologic services of build bioparts or chassis for companies and scientific research units per year, which demonstrated its facilitation in the research and application of synthetic biology. However, more breakthroughs are still required in the collection and investigation of regulatory bioparts and innovation of mechanisms for sharing data, metadata physical bioparts and related tools. We are looking forward to building a database with convergence of data, metadata physical objects and design tools to better serve the synthetic biology research basing on a basic regulation including biopart data sourcing, multi-layer reviewing, resource sharing, biopart data disclosure, secured biopart data and authorized access.

Abstract:Enzymes catalyze most biochemical reactions in living organisms, and the high efficiency, chemo-, regio- and stereoselectivity make enzymes appealing catalysts in many fields. The digitalization of enzyme-related information is of great importance to both academic research and industrial applications. Microbial enzyme resource databases establish foundations for the mining, engineering, and utilization of new biocatalysts. This mini-review will briefly introduce the current development of enzyme databases and discuss the enzyme resources applications facilitated by databases.

Abstract:Amplicon sequencing is the most widely used sequencing method to evaluate microbial diversity in virtually all environments. Thus, appropriate and specific primers are needed to amplify amplicon regions in amplicon sequencing. For this purpose, the community currently uses probeBase, which curates rRNA-targeted probes and primers. However we found that 63.58% of the primers in probeBase have problematic issues in the short name, full name, and/or position. Furthermore, the current convention for short names causes ambiguity. We here introduce our new Database of Primer Scientific Names (DPSN), which is a manually curated database for the 173 primers from probeBase and 42 new added primers complete with a new short name convention. Building on the work of probeBase, we provide a more user-friendly and standardized system. The new short primer naming convention has three basic components:5' position on the sense strand, version, and direction. An additional character for the name of the taxonomic group is also added in front of the name for convenient use. Furthermore, DPSN contains primers for large subunit as well. In order to separate them from the primers for small subunit, a header character is also recommended. All 173 primers in probeBase were corrected according to this new rule, and are stored in DPSN, which is expected to facilitate accurate primer selection and better standardized communication in this field.

Abstract:In recent years, quarantine pests, especially phytopathogenic fungi, posed serious threats to the biological security of China. Thus, the reinforcement of defense systems for those invasive fungal species is essential to prevent alien biological threats. Due to the lack of reference materials and basic databases related to those phytopathogenic fungi, the identifications of these quarantine fungi remain ineffective, posing high risks of biological invasion that may lead to significant economic losses and ecological damages. In view of the shortcomings in the inspection and quarantine of important invasive fungal species, we established a reference database, which includes reference information from the voucher specimens, including the standard molecules, morphological characteristics, and multi-locus reference sequences. An integrative online identification and inspection platform was established by integrating multiple computer packages, including platform for initial multi-objective inspection of fungal species from quarantine samples, and the "one-stop shop" identification package of fungal species based on multi-locus sequences. The database can be freely accessed at website:www.casbrc.org/pqfungi. Construction of the reference databases and inspection platforms may greatly enhance the inspection efficiency of alien fungal pathogens, and have been playing increasingly important role in the construction of "intelligent customs" of China.

Abstract:[Objective] Developing a website that collects environmental microbial information and can quickly synthesize environmental control microbial populations.[Methods] With the support of the "Chinese Academy of Sciences Strategic Biological Resources Service Network Plan", relying on the resource-rich environmental microbial entity collection library, we improved the information on function, environmental adaptation and physiological and biochemical characteristics of the deposited strains, and established environmental microbial information repository (website:http://www.envimicrobe.com/em.cib.cn). [Results] The information repository mainly includes two modules:"resource library" and "group synthesis system". The resource database collects and integrates information on the functional characteristics, environmental adaptability, physiological and biochemical characteristics of the strains. The population synthesis system matches the feature information of these functional bacteria with the pollutant information and environmental condition parameter information of the target contaminants, and then quickly synthesizes targeted environmental management microbial populations, and develops environmental microbial compound products. [Conclusion] The application of this information database not only helps researchers quickly obtain relevant microbial resource information and carry out environmental microbial research efficiently and quickly, but also promotes the industrialization and application of environmental microbial resources.

Abstract:[Objective] Isolation and screening of cultivated microorganisms in Baijiu is an important research focus in Baijiu industry. The aim of this paper is to construct the database of cultivated microorganisms from the ecosystem of Chinese Baijiu (CMBaijiu). [Methods] The data of CMBaijiu is collected from the published literatures on the Baijiu microorganisms and microbial information from Baijiu ecosystem by strain preservation center. The database contains three main functions:(1) Retrieval of cultivated microorganisms in Baijiu:the information of cultivated microorganisms in Baijiu can be retrieved by the strain name, isolated locations, the medium and the metabolites, so as to obtain the detailed physiological, biochemical and taxonomic information of the microorganisms. (2) Retrieval of media information:medium information including culture composition and preparation methods can be retrieved by specific components, culture media numbers and names. (3) Data update:online uploading information of new cultivated microorganisms and media.[Results] At present, a total of 1221 kinds of culturable microorganisms and 295 kinds of medium information are collected in the CMBaijiu. The website of CMBaijiu is http://cmbaijiu.i-sanger.com/. [Conclusion] CMBaijiu is the first database of cultivated microbial information in Chinese Baijiu industry and could be help for the study of cultivated microorganisms in Baijiu.

Abstract:This article is based on the statistical analysis of the data of the "Microorganisms Deposits and Samples (2001-2019)" published by the World Intellectual Property Organization (WIPO). The results show that the global patent microbial deposits have shown a steady growth trend, China ranks first in the world in terms of the number of patent strains, with most of the world's patent strains preserved (31386, accounting for 39.86%). After 2018, the global sample of strains increased sharply, Among which the United States was still the country with the largest number of patent strains (24153 strains, accounting for 96.63% of the total sample), and the sample rate of China's patent strains is at a low level (1034 strains, accounting for 3.29%). The phenomenon of "high preservation, low distribution" of patent strains in China is serious, and the level of industrial development and utilization should be appropriately promoted.

Abstract:Calcineurin is a serine/threonine (Ser/Thr) protein phosphatase and generally conserved in fungal genus. Its upstream signaling pathway were composed of Ca²⁺ channel (CCH1), transporter (MID1), calcium ion sensing protein (CaM), calmodulin-dependent phosphatase and etc. Calcineurin is the only phosphatase in fungi that is regulated by calcium ions and calmodulin, and plays a central role in the calcium signal cascade that regulates fungal Ca²⁺ homeostasis. Through calcium signaling cascade pathway, it participates in biological processes that regulates the growth, development and virulence formation of fungi for response to changes in external environmental factors, as well as fungi can adapt to various environment and maintain normal life activities. This review summarizes the signal composition of fungal calcineurin and the upstream and downstream signal transduction pathways, as well as the regulation of cell growth and metabolism, the formation of virulence, and tolerance resistance. With the advantage of the regulation of fungal metabolite synthesis, the mining of calcium regulating phosphatase signal as a potential synthetic biological element and regulatory switch was also proposed.

Abstract:The CRISPR/Cas system, an adaptive immune system widely found in most bacteria and archaea, is composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas). The CRISPR/Cas system can recognize and bind foreign invading nucleic acid. Then the cleavage activity of Cas protein is activated that can cleave and degrade the invasive nucleic acid. The specific sequence recognition and cleavage activity of the CRISPR/Cas system is applied to nucleic acid detection that provides a new research idea to improve detection performance such as detection sensitivity and specificity. This study introduces the development of the CRISPR/Cas system, its mechanism of action, etc. In addition, we also summarize the representative applications of diversified Cas proteins in nucleic acid detection. The advantages and disadvantages of CRISPR/Cas technology in nucleic acid detection are discussed, and future research is proposed. In sum, this review aims to provide references for the detection of pathogenic microorganisms by using a nucleic acid detection method based on CRISPR/Cas technology.

Abstract:Streptomyces is a genus of high GC content Actinomycetes with a complex morphological differentiation cycle and strong secondary metabolism ability. It can use the precursor compounds and energy produced by its primary metabolism to synthesize a variety of complex structure, diverse function and biologically active secondary metabolites, which has important value in the fields of agriculture, food, animal husbandry, industry, and medical research. The late morphologic differentiation of Streptomyces is often accompanied by the biosynthesis of secondary metabolites, both of which are regulated by complex networks, and the morphology of Streptomyces has a great impact on the yield of secondary metabolites. A comprehensive understanding of the growth cycle of Streptomyces will deepen the knowledge of the relationship between Streptomyces morphological differentiation and secondary metabolite synthesis. In this paper, we review the process of morphological differentiation, the common regulatory factors involving in morphological differentiation and antibiotic synthesis, and the relationship between Streptomyces morphology and yield, which will help us to better understand the process of antibiotic synthesis. It will also inspire the insights about shortening the fermentation cycle, the construction of high-yield engineered strains, the research and development of new fungicides and the synthesis of new antibiotics.

Abstract:Stability in microorganisms of urban green space (UGS) is a key factor to maintain its functioning of ecosystems. Rapid urbanization in the Anthropocene era would change soil physical and chemical properties of UGS, input emerging contaminants, aggravate the potential risk of microbial ecosystem and alter the diversities of microbial community and ecosystem function. These will further influence the service functions of UGS ecosystem. This article reviews the characteristics of UGS microorganisms, and the effects of urbanization on the UGS microbiome including soil, phyllosphere and air; antibiotic resistance genes (ARGs); pathogenic microorganisms; and rare species. Compared with the nature-derived microbes, UGS microorganisms generally have a higher heterogeneity, and are greatly affected by human activities. In the meantime, the level of ARGs and the number of human pathogenic bacteria have increased significantly, reflecting the disturbance of urbanization to the health and function of UGS ecosystems. We suggest that more attention to the impact of urbanization on microorganisms of UGS should be paid in future research, and to provide reliable theoretical supports for the risk assessment on human health.

Abstract:African swine fever (ASF) is a hemorrhagic and fatal infectious disease caused by African swine fever virus (ASFV). ASF is endemic or epidemic in Africa, Asia, and Europe and causes huge economic losses to the pig industry. ASFV has a large DNA genome encoding more than 150 proteins, including many nonessential genes-encoded proteins associating with ASFV virulence, viral replication, immunoescape and unknown functions. Currently, a number of ASF live attenuated vaccines have been developed by deleting virulence-related nonessential genes. Generally, these vaccines have safety concerns, although they are able to provide partial to full protection. Systematic identification of more nonessential genes, especially virulence-related genes, will not only contribute to the development of safer gene-deleted ASF vaccines, but also benefit the understanding of the ASF pathogenesis. This review systematically summarizes the functions of known nonessential genes of ASFV, with focus on those involved in virulence, regulation of viral replication and escape of host antiviral immunity, and puts forward suggestions for the identification and functional study of unknown nonessential genes of ASFV.

Abstract:Salmonella are a group of zoonotic pathogens threatening the public health. The invasion of Salmonella into humans and livestock causes typhoid and paratyphoid fever, gastroenteritis, septicaemia, and extraintestinal focal infections. Antimicrobials are an effective treatment for serious Salmonella infections. However, the extensive use of antimicrobials in clinical practice and animal husbandry has led to increasing antimicrobial resistance among Salmonella. Integrons, mobile genetic elements ubiquitous in bacteria, can efficiently capture exogenous genes and ensure their expression. Moreover, these integrons could be complexed with transposons, plasmids, etc., consequently enabling the intra- and interspecies dissemination of multiple antimicrobial resistance genes in bacteria. Over the past two decades, the emergence of new gene arrangements and complex integrons in gene cassettes has led to the rapid evolution of integron systems. Integrons play a significant role in the spread of antimicrobial resistance in Salmonella. In this paper, we summarized the molecular structure, classification, and action mechanism of integron systems, and reviewed the progress of research on class I, II, and III integron-mediated antimicrobial resistance presented in Salmonella, together with the available detection methods, aiming to provide a reference for the research on antimicrobial resistance of Salmonella.

Abstract:Streptococcus suis is an important bacterial pathogen for pigs. It can cause meningitis, sepsis and arthritis in pigs, leading to severe economic losses to the pig industry. In addition, S. suis can also infect humans and is considered a zoonotic pathogen. In order to better prevent and control the disease caused by S. suis, it is necessary to use molecular epidemiological methods to elucidate the epidemiological characteristics of S. suis, including virulence typing, temporal and spatial distribution, transmission route, source of infection, and genetic determinants of transmission. At present, there are several commonly used methods including multilocus sequence typing, pulsed-field gel electrophoresis, whole genome sequencing, and PCR-based methods. This article introduces the principles of the above-mentioned methods and the application examples in the epidemiology of S. suis, and analyzes their advantages and disadvantages, which contributes to better revealing the epidemiological characteristics of S. suis, and providing a reference for formulating prevention and control strategies for the disease caused by S. suis.

Abstract:The type Ⅵ secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Vibrio parahaemolyticus also harbours T6SS gene clusters and possesses two T6SSs, T6SS1 and T6SS2. [Objective] In previous work, we identified several potential T6SS effectors by using comparative proteomics. In this study, VPA1500 was chosen to explore the roles on biological characteristics and pathogenicity of Vibrio parahaemolyticus.[Methods] The deletion mutant ΔVPA1500 and complementary strain CΔVPA1500 were constructed by using homologous recombination technology. Growth characteristics, anti-bacterial activity in vivo, motility, the transcription level of flagella-related genes, and biofilm formation ability were analyzed in the wild-type strain (WT), ΔVPA1500 and CΔVPA1500. Furthermore, cytotoxicity to host cell, lethality rate in mice, bacterial colonization, and histopathological changes were also analyzed between WT and ΔVPA1500. [Results] Compared with WT, there were no significant changes in growth characteristics, swarming ability, and biofilm formation of ΔVPA1500 (P>0.05), while swimming ability was significantly decreased (P<0.05). Transmission electron micrographs showed that VPA1500 deletion affected the formation of bacterial flagella in V. parahaemolyticus. qPCR results also showed that the VPA1500 gene significantly inhibited the transcription level of some flagella-related genes in WT. Bacterial competition experiments showed that the deletion of VPA1500 reduced the anti-bacterial activity of Vibrio parahaemolyticus against E. coli in vitro. However, ΔVPA1500 showed significantly weaker cytotoxicity to Hela cells than WT. In addition, ΔVPA1500 exhibited attenuated virulence in mice that showed lower a lethality rate than that of the wild-type strain. Moreover, VPA1500 deletion affected the colonization in heart, liver, spleen, and kidney of mice, whereas the complementation strain restored the virulence to resemble that of WT. Histopathological analyses further demonstrated that detection of VPA1500 could reduce the damage of Vibrio parahaemolyticus to tissues in mice. [Conclusion] The VPA1500 plays an important role in swimming motility, pathogenicity, and anti-bacterial activity in V. parahaemolyticus.

Abstract:[Objective] To analyze the prophages carried by 82 strains of Acinetobacter baumannii with completely sequenced genomes, then identify antibiotic resistance genes and virulence factors encoded by prophages. [Methods] Phage Search Tool Enhanced Release (PHASTER) software was used to predict the prophages carried by A. baumannii. The Comprehensive Antibiotic Research Database (CARD) and Virulence Factors Database (VFDB) online analysis software were used to predict the antibiotic resistance genes and virulence factors encoded by the prophages, respectively. [Results] A total 472 prophages were found, of which 201 were "intact", 91 were "questionable", and 180 were "incomplete". On average, at least two intact prophages were carried per A. baumannii genome. The percentage of the prophage genome ranged between 4% and 6% in each A. baumannii genome. Twenty-nine out of the 472 prophages carried 77 resistance genes, which belong to 14 different antimicrobial types, could be grouped into 15 different drug families, and could be classified into six antibiotic resistance mechanisms. One hundred and thirty-two out of the 472 prophages encode putative virulence genes, grouped into 38 virulence gene and 34 virulence factor classes. Most of these prophages encode one or two virulence factors, and a few of them encode three or more virulence factors. Analysis of component ratios of possible virulence factor host sources indicated Neisseria meningitidis, Shigella dysenteriae, and Legionella pneumophila, as well as A. baumannii, as host sources.[Conclusion] A. baumannii generally carries prophages, but the proportion of prophage genes in the genome of A. baumannii is not high. Some prophages carry antibiotic resistance genes, mainly aminoglycosides, sulfonamides, and beta-lactams. Nearly 30% of the prophages carry virulence genes. Prophages may play an important role in the acquisition and spread of antibiotic resistance, and evolution of pathogenicity in A. baumannii.

Abstract:[Objective] By studying the diversity and community structure of fungi in the rhizosphere soil of different halophytes in the Ebinur Lake wetland in Xinjiang, we provide theoretical support for the degradation and restoration of the Ebinur Lake wetland and the in-depth study of fungi. [Methods] We used high-throughput sequencing technology to determine the fungal amplicon ITS1 region to analyze the fungal community diversity in the rhizosphere soil of six halophytes in the Ebinur Lake Wetland, and combined with the relevant soil physical and chemical factors to analyze the relationship between the environment and the diversity and richness of fungal communities. [Results] The diversity and richness of fungal communities in the rhizosphere soil of the six halophytes in Ebinur Lake wetland were different. The fungi in the rhizosphere of Suaeda glauca had the highest diversity and the fungi in the rhizosphere of Phragmites australis had the highest richness. The analysis of fungal community composition showed that the fungal communities in the soil samples mainly belonged to Ascomycota and Basidiomycota, and Ascomycota was the dominant phylum. Alternaria was the dominant genus shared by all the six plants, but its abundance varies among different plants, with the highest abundance in Cynanchum sibiricum and the lowest in Euphorbia soongarica. There was a significant negative correlation between pH and fungal diversity, and a significant positive correlation between total phosphorus and fungal richness. The influence of pH, electrical conductivity and organic matter on dominant fungi was the most significant. [Conclusion] The rhizosphere soil fungal community composition and diversity of the six halophytes were significantly different in Ebinur Lake Wetland. The diversity and abundance of fungi in the rhizosphere soil of Suaeda glauca and Phragmites australis were higher than those of other plants, and Ascomycetes and Alternaria were the main soil fungal genera in Ebinur Lake Wetland. The research results could provide theoretical guidance for the ecological restoration of Ebi Lake wetland.

Abstract:[Objective] We analyzed the metabolic pathways and key genes of A82846B synthesis of Kibdelosporangium aridum CCTCC M2020063.[Methods] We used the second-generation sequencing technology Illumina and the third-generation sequencing technology PacBio to perform whole-genome sequencing of Kibdelosporangium aridum CCTCC M2020063. We also used Glimmer to predict the coding sequence. To identify the secondary metabolite, high performance liquid chromatography and liquid chromatograph mass spectrometer was applied. To predict the secondary metabolite synthesis gene cluster, AntiSMASH 5.0 software was applied as well. Besides, focusing on the analysis of its mbtH-like gene, geneious software was adopted in this paper to analyze A82846B synthesis-related genes. [Results] Sequence analysis showed that the strain belonged to Kibdelosporangium aridum, containing a plasmid-free linear genome chromosome with the full-length gene sequence of 12475688 bp and the GC content of 66.27%. This linear genome chromosome had 11900 open reading frames and a total of 47 gene clusters. This strain was capable to synthesize A82846B, and the biosynthesis-related genes were located in Cluster32, which contained 33 genes. Overexpression of the mbtH-like gene gene07864 promoted the synthesis of A82846 by 26.42%. The halogenase gene was gene07859, being highly similar to the halogenase of vancomycin and balimycin. [Conclusion] In this study, we carried out the genome sequence analysis of Kibdelosporangium aridum CCTCC M2020063, and we also obtained the information of A82846B biosynthesis-related functional genes, which provided a strong basis for the metabolic pathway and engineering modification of A82846B.

Abstract:[Objective] To identify the macrolide antibiotic bafilomycins from the strain SCSIO 40020 isolated from the Pearl River Estuary sediment. To clone and heterologously express the biosynthetic gene cluster (BGC) of bafilomycins.[Methods] The strain SCSIO 40020 was identified based on phylogenetic analysis of its 16S rRNA gene sequence. The generated compounds from SCSIO 40020 were purified via normal phase-column chromatography and semi-preparative chromatography. The chemical structures of the isolated compounds were subsequently elucidated by comprehensive spectroscopic analyses. The bafilomycins BGC was identified by using bioinformatics approach. Through screening the bacterial artificial chromosome (BAC) library of this strain, the BAC containing the bafilomycins BGC was introduced into three Streptomyces hosts by conjugation for expression. Afterwards, the fermentation culture of the recombinant strains were analyzed on high-performance liquid chromatography (HPLC). [Results] The strain SCSIO 40020 was identified as Streptomyces genus and two macrolide compounds bafilomycins A₁and D were isolated from the fermentation crude extract. The bafilomycins BGC was cloned and successfully expressed in three heterologous Streptomyces hosts. In addition, the biosynthetic pathway of bafilomycins was proposed. [Conclusion] The bafilomycin-producing strain Streptomyces sp. SCSIO 40020 was obtained from the Pearl River Estuary. The BAC heterologous expression system of this strain was successfully established. Moreover, the bafilomycins BGC was successfully expressed in Streptomyces lividans SBT18, Streptomyces coelicolor M1154 and Streptomyces albus J1074 for the first time, which laid solid foundation for the production of bafilomycin analogues and mining other interesting BGCs from Streptomyces sp. SCSIO 40020 in the further study.

Abstract:[Objective] To explore the potential microbial resources and functions, this study analyzed the diversity of endophytic bacteria in the roots and stems of Dendrobium officinale cultivated on trunks and in greenhouses. [Methods] The composition and distribution of the endophytic bacterial community were analyzed by Illumina MiSeq High-throughput sequencing. Meanwhile, the endophytic bacteria were isolated using the traditional culture method and identified by 16S rRNA gene sequence analysis. The Kirby-Bauer test was used for detecting antimicrobial activity. [Results] The results showed that the culture-independent bacteria mainly belonged to Pseudomonas (30.52%), followed by Mycobacterium (10.22%) and Brachybacterium (8.32%). The abundance and diversity of endophytic bacteria of Dendrobium officinale cultivated on trunks were higher than that cultivated in greenhouse. The endophytic bacteria of Dendrobium officinale in root showed more abundance and diversity than in the stems. Bacillus (18.37%) accounted for the highest proportion of the culturable endophytic bacteria, followed by Curtobacterium (8.16%). Meanwhile, 8 strains showed antimicrobial activity against at least one pathogen in this study. [Conclusion] The result demonstrated that there was a huge resource of endophytic bacteria in the Dendrobium officinale. The distribution of the endophytic bacterial community was influenced by the difference of cultivation patterns and tissues. In addition, the strains with antimicrobial activity could be used as biological control of pathogenic bacteria. This study provides a diverse endophytic microbial resource of Dendrobium for further exploring and utilizing.

Abstract:[Objective] Expressing the bacterial siderophore synthetase PchE under the ADH2 promoter in Saccharomyces cerevisiae system with the PPTase Sfp from Bacillus subtilis, to explore the heterologous expression of activated bacterial protein in eukaryotic system. [Methods] The sfp gene was amplified from Escherichia coli BAP 1. Both pchE gene and pchE tandem with sfp were cloned to the yeast-E. coli shuttle vector pXW55, and transformed into the Saccharomyces cerevisiae BJ5464-npgA strain. After the purification steps including affinity chromatography and ion-exchange chromatography, HPLC was used to detect whether the PchE from E. coli and Saccharomyces cerevisiae maintain catalytic activity in the biochemical reaction in vitro.[Results] In the Saccharomyces cerevisiae expression system, prokaryotic protein PchE was obtained with high purity. And no matter assisted by the bacterial or fungal PPTase, PchE can be modified and synthesize intermediate product HPT-Cys. [Conclusion] It was firstly demonstrated that both fungal gene npgA and bacterial gene sfp can modify bacterial nonribosomal peptide synthase. With the comparison of protein expression between yeast and bacterial host, the giant protein PchE expressed by yeast has higher purity and fewer nonspecific bands. This suggested that yeast host might be more suitable for expressing and purifying functional giant protein.

Abstract:[Objectives] To evaluate the application of whole genome sequencing technology on the detection of Salmonella serotype and antimicrobial resistance phenotype. [Methods] Two hundred and ninty strains isolated from chicken from 1950 to 2015 in China were serotyped and tested for antimicrobial sensitivity. The whole genomes were extracted and sequenced, and the serotype and antimicrobial resistance of each Salmonella strain were analyzed by submitting the genomic sequences to SeqSero and ResFinder databases, respectively. Results of serotypes and antimicrobial resistances obtained by conventional detection methods and whole genome sequencing analysis were compared, and the compliance status of two detection methods were analyzed. [Results] The results showed that the major serotypes were S. enteritidis and S. gallinarum (≥ 84.5%). The coincidence rate of Salmonella serotype between the results of conventional detection method and whole genome sequencing analysis was 97.6%. The results of minial inhibitory concentration to 11 drugs indicated that the Salmonella isolates had the higher resistance rate to sulfisoxazole (39.3%), tetracycline (39.0%) and colistin (39.0%), but showed lower resistance rates to other antimicrobial agents. The predicted resistance of meropenem, florfenicol, azithromycin and amoxicillin/clavulanic acid was 100% by whole genome sequencing, and the coincidence rate of enrofloxacin, tetracycline, trimethoprim-sulfamethoxazole, ampicillin, ceftiofur, and sulfisoxazole exceeded 95.0%. [Conclusions] High accuracy and sensitivity of whole genome sequencing technology in Salmonella serotype and antimicrobial resistance was seen in this study. As an effective tool, whole genome sequencing technology has a good application prospects on serotyping and antimicrobial resistance analysis of Salmonella.

Abstract:[Objective] MotA, an important flagellar motor protein regulating flagellar motility, is one of components of transmembrane proton channel. In this study, we explored the role of motA in Azorhizobium caulinodans ORS571 in the free-living and symbiotic states. [Methods] ∆motA mutant was constructed by homologous recombination and tri-parental conjugation. We tested the characterizations of the growth state, swimming motility, nitrogen fixation, exopolysaccharides (EPS) production, biofilm formation, and host colonization between wild-type and ∆motA mutant. [Results] ∆motA mutant showed defective cell motility ability but the growth property was not impaired, and its nitrogen fixation, EPS production, biofilm formation, and host colonization were lower than that of the wild-type. [Conclusion] In A. caulinodans ORS571, MotA plays role in motility, nitrogen fixation, EPS production, biofilm formation, and root colonization.

Abstract:[Objective] NtrC is a DNA-binding transcriptional regulator which plays an important role in activating the transcription of nitrogen assimilation genes and maintaining nitrogen supply. The objectives of this study were to explore its physiological function and mechanism in Aeromonas hydrophila.[Methods] In this study, the homologous recombination method was used to construct the ntrC deletion strain of Aeromonas hydrophila, and the physiological phenotype of the deletion strain was measured and analyzed with the wild strain as the control. A data independent acquisition-based quantitative proteomics method was performed to compare the protein expression differences between the wild-type and ntrC deletion strains.[Results] After the ntrC gene was knocked out, the tolerance of Aeromonas hydrophila to nitrogen deficiency, osmotic pressure, heavy metal ions, oxidation and different antibiotic stress was significantly changed, and these phenotypes could be recovered in its rescued strains. Quantitative proteomics was used to compare the protein expression between the WT and the ntrC deletion strain. Results showed that ntrC may be involved in the regulation of biosynthesis of amino acids, ascorbate and aldose metabolism pathways. [Conclusion] This study elucidated the important role of NtrC in Aeromonas hydrophila and its impact on the biological function of the bacteria. Moreover, the relationship between the proteins directly or indirectly regulated by NtrC and the physiological phenotype was discussed, the results provide valuable information for the prevention and control of aquatic pathogens in the future.

Abstract:[Objective] To express and purify the endonuclease V (Saci_0544) from Sulfolobus acidocaldarius, identify its endonuclease activity and enzymatic characterization. [Methods] The endonuclease V (SacEndoV) from Sulfolobus acidocaldarius was expressed in E. coli and purified by affinity chromatography; Oligonucleotides with different types of damage were used as substrates to identify the cleavage activity of SacEndoV. [Results] SacEndoV specifically cleaves damaged DNA substrates containing deoxyinosine. Compared with double-stranded DNA substrates, the enzyme has a clear preference for single-stranded DNA substrates in vitro. The enzyme activity of SacEndoV is excellent in the temperature range of 70-95℃. And its enzyme activity depends on the divalent metal ion, Mg²⁺ is the best cofactor. The optimal reaction pH of SacEndoV is 7.5-8.0, and NaCl with a concentration higher than 200 mmol/L will significantly inhibit its cleavage activity. The structural integrity of deoxyribonucleotide adjacent to the 3' end of deoxyinosine in the damaged DNA is of great significance for SacEndoV to recognize and cleave the corresponding substrates. The presence of AP sites at the 3' end of deoxyinosine prevents SacEndoV from cleaving damaged DNA. In addition, it has been determined that SacEndoV has cleavage activity on damaged RNA substrates containing inosine.[Conclusion] This study confirmed that SacEndoV is a typical endonuclease V with substrate specificity for damaged DNA containing deoxyinosine, and participates in the repair of deoxyinosine in Sulfolobus acidocaldarius.

Abstract:[Objective] Continuous cropping obstacle reduced the production and quality of agricultural products,and application of rhizosphere beneficial microbes with the function of degrading autotoxic substances, inhibiting the growth of plant pathogen and promoting plant growth was an important strategy for suppression of continuous cropping obstacle. Screening and revealing the function of peanut plant-growth promoting rhizobacteria with the potential to alleviate soil degeneration caused by continuous cropping obstacle, would provide scientific evidence for the application in agricultural produce. [Methods] Screening high-efficiency plant growth-promoting rhizobacteria with function of degrading autotoxic substances, inhibiting the growth of plant pathogen and promoting plant growth from 12-year peanut filed with continuous cropping obstacle, and the phylogenetic analysis was performed by 16S rRNA gene sequencing to determine the taxonomic status. [Results] 7 plant growth-promoting rhizobacteria with high-efficiency in degrading autotoxic substances were isolated from the rhizosphere from 12-year continuous cropping peanut soil, and according to phylogenetic analysis, identified as Klebsiellasp. B02, Klebsiella sp. B07, Klebsiella sp. B15, Bacillus sp. B28, Acinetobacter sp. P09, Brucella sp. VA05 and Bacillus sp. CA04. The result of growth-promoting experiments indicated that 7 strains showed the ability of IAA-producing, 3 strains showed the ability of nitrogen-fixing, 4 strains showed the ability of phosphate solubilization, 2 strains showed the activity of potassium solubilizing. The result of antagonistic activity showed that 2 strains could against to plant pathogen, and belong to the specie of Bacillus genus. Bacillus sp. B28 was selected for peanut pot experiment, the results showed that strain Bacillus sp. B28 can significantly alleviate the inhibition of phenolic acid on peanut seed germination and promote the growth of peanut seedling. [Conclusion] This research screened plant growth-promoting rhizobacteria with high-efficiency in alleviate continuous cropping obstacles, provided strains and scientific evidence for the application in agricultural produce.

Abstract:[Objective] To characterize novel acquired trimethoprim resistance gene dfrB7, and to determine the biochemical basis of acquired trimethoprim resistance for DfrB7 coded by novel dfrB7 and previously known representative Family B DHFRs.[Methods] Phylogenetic analysis of previously reported DfrB proteins and the novel DfrB7 was performed. PCR-amplified dfr genes were cloned into pACYC184 and pET15b(+) vectors, followed by transformation into Escherichia coli. The dfr-pACYC184 plasmid containing E. coli strains were tested for trimethoprim susceptibility by microdilution broth method. Enzymatic catalysis parameters were determined by analyzing the NADPH:dihydrofolate oxidoreductase activities. Isothermal titration calorimetry was performed to measure the dissociation constants between TMP and DHFRs. [Results] Novel dfrB7 gene conferred trimethoprim resistance (MIC ≥ 1024 mg/L) when it was cloned into E. coli. Phylogenetic analysis showed that dfrB7 encodes a Family B DHFR. Novel dfrB7 and previously known representative dfr genes were overexpressed and purified for the analysis of enzymatic parameters and TMP affinity. Comparing with chromosomal DHFR, both DfrB1 and DfrB7 showed significantly lower activities, and Family B DHFRs have drastic lower affinities to trimethoprim.[Conclusion] DfrB7 encoded by a novel dfrB7 gene has common characteristics of Family B DHFRs. The acquired resistance to trimethoprim is caused by the low affinities of Family B DHFRs to trimethoprim.

Abstract:[Objective] Secreted proteins play an important role in the pathogenic process of phytopathogenic fungi. Predecessors mostly used a single strain to predict and analyze secreted proteins. There are no reports on the prediction and comparative studies of secreted proteins in multiple types of fungi. [Methods] This study is based on the whole genome sequence of fungi with a variety of different nutrition methods. According to the characteristics of the secreted protein, online analysis programs such as SignalP v5.0 and ProtComp v9.0 were used to analyze the secreted proteins of 19 fungi, which including model organisms, hemibiotrophic, necrotrophic and biotrophic. [Results] The results showed that among the about 130000 protein sequences of the above-mentioned fungi, the proportion of secreted proteins was 0.74% to 4.83%. Among them, the secreted proteins of biotrophic accounted for the highest proportion, with an average of 3.51%. The average ratio of hemibiotrophic and model organisms was the lowest, with an average of 1.78% and1.36%. At the same time, biotrophic have the most types of functions, 433 species, followed by necrotrophic, with 266 species, and model organisms have the least functional types, at 100, among which functions are hypothetical protein and non-characteristic. [Conclusion] This research has laid a solid theoretical foundation for the in-depth analysis of secreted proteins in realizing the pathogenic mechanism of fungi with different nutritional methods.

Abstract:Caves are extreme environments with permanent darkness and limited nutrients, which serve as natural laboratories to study the subsurface deep biosphere. Although microbial communities have demonstrated strong niche specificity in different caves, the environmental driving mechanism of microbial communities and the ecological processes responsible for community assembly in different niches particularly those in different substrates e. g. solid versus liquid samples were poorly understood.[Objective] Here we aim to explore the environmental driving mechanisms and community assembly process of bacterial communities in different substrates e. g. solid versus liquid samples combined with physicochemical properties.[Methods] To this end, a karst cave, the Luohandu Cave in Guilin city, Guangxi province, locating in the typical karst region in southwestern China, was selected for a systematic investigation of microbial communities. Solid samples (weathered rocks and sediments) and liquid samples (dripping water, pool water and ground river water samples) were collected along the cave and subjected to high-throughput sequencing of 16S rRNA gene. Meanwhile, physicochemical properties of these samples were analyzed.[Results] Results showed strong niche specificity of microbial communities in the Luohandu cave. Actinobacteria and Acidobacteria dominated bacterial communities of weathered rocks and sediments, respectively. In contrast, water samples were dominated by γ-Proteobacteria. Redundancy analysis (RDA) between microbial communities and environmental variables demonstrated that temperature, weathering index and SO₄^2- concentration significantly affect microbial communities in solid samples. USCγ and Pseudomonas positively associated with temperature, while Pseudonocardia, Solirubrobacter and Gemmatimonas negatively correlated with temperature. However, microbial communities in water samples were significantly controlled by electrical conductivity (EC) and dissolved oxygen (DO) as indicated by RDA. The co-occurrence network of bacterial communities was characterized by a good modularity with nodes from liquid samples locating in the same module, indicating a niche preference. Positive links dominated in the network, suggesting a corporative strategy among different microbial groups to survive in caves. Different ecological processes were found to be responsible for bacterial community assembly in different substrates. Deterministic process (48.75%) and stochastic process (51.25%) contributed almost equally to bacterial community assembly in solid samples, whereas stochastic process (64.76%) dominated the microbial community assembly in liquid samples. As for the individual ecological processes, homogenizing dispersal, undominated and dispersal limit in stochastic process contributed 40.42%, 10.46% and 3.13% to community assembly respectively; whereas homogenous selection and heterogeneous selection within deterministic process contributed 26.75% and 21.99%, respectively, in solid samples. In liquid samples, individual processes contributed to community assembly in order from high to low were undominated (28.57%), homogenous selection (25.71%), homogenizing dispersal (24.76%), dispersal limitation (10.48%), and heterogeneous selection (9.52%).[Conclusion] Our results for the first time revealed the different environmental driving mechanisms and different community assembly processes underlining microbial communities living in different substrates (solid versus liquid). This study offers a new window to understand the interactions among microbes and their environments, and interactions among different microbial groups in the subsurface biosphere in caves.

Abstract:[Objective] Nicotinamide adenine dinucleotide (NAD⁺) is an important cofactor in organisms, and its intracellular concentration plays an important role in NAD⁺-dependent redox reaction and related biochemical synthesis. In order to strengthen the synthesis of cofactors, we optimized the induction conditions and increased the copies of key enzyme genes to elevate the intracellular NAD⁺ concentration.[Methods] First, the induction temperature, inducer concentration, and induction time were optimized for the starting strain NA016 of Escherichia coli. Meanwhile, the transcriptional levels of overexpressed genes in metabolic modification were determined by real-time fluorescence quantitative PCR. Subsequently, the correlations between NAD⁺ content and the transcription levels of these overexpressed genes were explored. Finally, the copies of the genes positively regulating NAD⁺ production were up-regulated to improve the intracellular NAD⁺ concentration.[Results] At the optimized induction conditions, the NAD⁺ concentration in NA016 increased by 35.37%. The genes nadE and pncB positively regulated the production of NAD⁺, and up-regulating the copies of these two genes in NA016 increased the NAD⁺ concentration by 22.46% to 41.66 μmol/g DCW.[Conclusion] Optimizing the induction conditions and up-regulating the copy number of key enzyme genes can increase the NAD⁺ concentration. These research findings provide reference for the study of NAD⁺synthesis.