Abstract:Potential relationships between toilet flushing or toilet plume-related microorganism aerosol and pathogenic microbe transmission have been widely concerned. Continuing studies or reports have been published from time to time. This review aimed to summarize toilet bio-aerosol related research and progress, to discuss the potential effect of toilet flushing, flush with lid open or closed and toilet surface on aerosol-related pathogenic microbe transmission, including virus, bacteria and parasites. Furthermore, this review also discussed the potential health risks that toilet-related microbial aerosol may have on human and made suggestions about public health protection and future research prospection.

Abstract:All amino acids (AAs) other than glycine are chiral, existing in the forms of D-amino acids (DAAs) or L-amino acids (LAAs). Compared to widely studied LAAs, our knowledge about DAAs is still very limited. Herein, we review the advances in studying the physiological roles and functions of DAAs in bacteria. Though DAAs are not involved in protein synthesis, they are found in a wide range of organisms, especially bacteria. It is known that DAAs, especially non-canonical D-amino acids (NCDAAs), have a wide variety of special functions in bacterial physiology. As building blocks, DAAs are important components of peptidoglycan in bacterial cell wall, non-ribosomal peptides and poly-γ-glutamate. At the individual cell level, DAAs regulate bacterial surface charges and autolysin activity, inhibit the germination of bacterial spores, regulate cell wall remodeling during stationary phases and virulence of pathogenic bacteria. While at the population level, DAAs play important roles in biofilm development and bacterial ecology. Additionally, certain DAAs can support the growth of certain bacteria directly as nutrients, though some others act as inhibitors of bacterial growth.

Abstract:[Objective] To analyze the microbial community of fermented grains during Yanghe strong-aroma liquor fermentation process, and to establish the correlation between the microorganisms and the biosynthesis of main organic acids.[Methods] The microbial community of fermented grains was analyzed by metagenome sequencing. Principal component analysis and partial least-squares regression analysis were used to find the key microorganisms correlated with the biosynthesis of main organic acids.[Results] The fermentation process can be divided into two periods (0-14 d and 15-60 d), according to the change of microbial composition and the biosynthesis of organic acids. The number of microbes significantly correlated with the biosynthesis of main organic acids from 0 to 15 d was significantly higher than that from 15 d to 60 d. The core microorganisms involved in the biosynthesis of major organic acids were characterized belonging to seven genera:Lactobacillus, Staphylococcus, Saccharomyces, Naumovozyma, Issatchenkia, Psychrobacillus and Rhizopus.[Conclusion] The core and key microorganisms involved in the biosynthesis of major organic acids during Chinese strong-aroma liquor fermentation process were identified. The results may laid the practical foundation and theoretical basis for elucidating the mechanism of acid production in Chinese strong-aroma liquor fermentation process and guarantee of the quality control of liquor.

Abstract:[Objective] The objective of this study was to analyze the function of the HMG-box transcription factor LELCRP1 (Lentinula edodes lignocellulase genes regulation protein 1) in the expression of lignocellulase genes.[Methods] The RNAi vector of lelcrp1 was constructed by double-joint and homologous recombination, then transformed into the mycelia of Lentinula edodes heterotypic strain W1 by Agrobacterium tumefaciens-mediated transformation (ATMT). The copy number of the inserted fragment in the genome of strain W1 was detected by Southern blotting. Fluorescent quantitative PCR was used to detect the expression level of lignocellulase genes in RNAi transformants, and the mycelial growth rate was measured on MYG plates.[Results] We got four RNAi transformants which were significantly down-regulated by 6 to 7-fold compared with the original strain W1. The results of Southern blotting showed that the lelcrp1 gene fragment had been successfully integrated into the genome of Lentinula edodes in a single copy. Analysis of expression profiles of 26 lignocellulase genes in two RNAi transformants revealed that 9 cellulase genes, 1 hemicellulase gene, 2 auxiliary enzymes AA9 gene and 1 manganese peroxide gene were down-regulated significantly, compared to the original strain. The mycelial growth rate of RNAi transformants were significantly slower than the original strain.[Conclusion] The expression of lelcrp1 was silenced successfully by RNAi, resulting in down-regulation of the expression level of some cellulose and ligninase genes. The HMG-box domain transcription factor could regulate the expression of lignocellulase genes.

Abstract:[Objective] This study was designed to screen the functional microbial strains suitable for the Pleurotus eryngii substrate fermentation, so as to improve its feed quality.[Methods] First, to choose suitable strains, which may have strong cellulase, ligninase and antibacterial activities. We used sifting mediums combined with the cellulase, ligninase and antibacterial activities tests. After that, we identified the species of selected strains according to their 16S rRNA (bacteria) and 18S rDNA (fungi) gene sequence analyses. Then, compound microbial inoculants were generated by mixing isovolumetric microbial solutions of each strain and used for the solid fermentation of Pleurotus eryngii substrate.[Results] We successfully acquired and identified 6 strains. Bacillus tequilensis strain P11, Pichia fermentans strain R8 and Kluyveromyces marxianus strain MU5 showed higher cellulase activity. Bacillus amyloliquefaciens subsp. and Plantarum strain MU7 showed higher ligninase activity, and Weissella paramesenteroides strain R4 and Pediococcus acidilactici strain R9 showed higher antibacterial activity. Once they were mixed and used to ferment Pleurotus eryngii substrate for 7 days, all the main indexes tested met the optimal criteria and remained stable. Compared with the EM solution fermented Pleurotus eryngii substrate, the neutral detergent fiber and acidic detergent fiber of the compound agent fermented Pleurotus eryngii substrate were significantly decreased by 19.6% and 21.44% respectively (P<0.05). The content of crude protein, crude ash and ether extract increased by 10.44%, 5.26% and 123.53% respectively (P<0.05).[Conclusion] We successfully obtained six strains, the mixture of them used to ferment the Pleurotus eryngii substrate can significantly enhance its feed quality.

Abstract:[Objective] Microbial resources remain a major focus of biotechnology development, because of their crucial value for fundamental life science research and for development of the bio-economy. China, as one of the two largest producers of scientific publications in the world, will play a global role in research and exploitation of microbial resources. The objective of this study was to analyze progress in microbial resource research and patenting in China to provide strategic directions for research and bio-industry.[Methods] Data were analyzed from culture collections, publications and patents to gain a picture of the status and trends in microbial resources research and exploitation in China. The data were benchmarked against those of other countries. Based on these analyses, we provide here suggestions for planning and policy to research, manage and exploit microbial resources.[Conclusion] Establishment of various microbial resource platforms in China reflects the increasing attention paid to this scientific area. The high number of publications annually and patents licensed indicate impressive progress in research and bio-industrial applications. Comprehensive resource preservation and development of research and application systems will provide a solid support for the bio-economy in China and other countries.

Abstract:[Objective] The objective of this study was to evaluate the influence of environment factors on fungal communities during succession periods along the chrono-sequence in forefront of Tianshan No. 1 glacier.[Methods] Fungal community composition in oligotrophic environment on the forefront of a receding glacier was analyzed by sequencing of 18S rRNA gene fragment of DNA samples from cryoconite and sub-and proglacial sediment near the glacier terminus.[Results] Fungal sequences obtained from zone represented 5 fungal phyla:Ascomycota (52.7%), Basidiomycota (16.9%), Chytridiomycota (15.1%), Zygomycota (2.4%) and Glomeromycota (1.2%). Although the proportion of Ascomycota outclass Basidiomycota-related sequences was identified in younger substrate, Basidiomycota sequences related to Microbotryomycetes, Tremellomycetes and Agaricomycetes increased gradually along the chrono-sequence. Representative sequences related to parasitic, pathogenic and airborne fungal lineages showed deposition of ecdemic fungi and anthropogenic activities directly or indirectly influenced fungal composition and spatial distribution in glacier ecosystem.[Conclusion] Our results suggest the forefront of Tianshan No. 1 glacier provide a favorable platforms for diversity and community succession of fungi adapted to oligotrophic, no vegetated and cold conditions. The different community structure was observed during succession periods along the chrono-sequence in forefront of Tianshan No. 1 glacier.

Abstract:[Objective] Application of antagonistic endophyte is a potential strategy for biological control of plant diseases. In this study, the antagonistic endophytic bacteria were isolated and screened from healthy mulberry, to provide candidate strains for the biological control of mulberry fruit sclerotiniose.[Methods] Tissue isolation method was used to isolate the endophytic bacteria from healthy mulberry tissues. The endophytic bacterium with strong and stable antagonistic activity on the pathogens of mulberry fruit sclerotiniose was screened by inhibition zone method. The antagonistic bacterium was identified through morphological features, cultural, physiological and biochemical characteristics and 16S rDNA phylogenetic analysis. Antimicrobial spectrum and thermal stability of cell-free fermentation supernatant were assayed by mycelial growth rate. The fermentation conditions and medium composition were optimized through single factor and orthogonal experiment.[Results] In total 55 endophytic bacteria were isolated from healthy mulberry. Among them, strain XP-27 was found to have strong and stable antagonistic activity against Sclerotinia sclerotiorum PZ-2 (the pathogen of mulberry fruit sclerotiniose). Morphological features, cultural, physiological and biochemical characteristics indicated that XP-27 belongs to genus of Bacillus. Based on 16S rDNA phylogenetic analysis, XP-27 was close to the Bacillus methylotrophicus and within the same clade with several strains of Bacillus methylotrophicus. Therefore, strain XP-27 was identified as Bacillus methylotrophicus, and named to be B. methylotrophicus XP-27. Antimicrobial spectrum assayed results showed that strain XP-27 had variously antagonistic activity on 10 plant pathogens and had excellent heat stability. The results of fermentation optimization showed that the optimal medium was composed of 1.00% beef extract, 1.50% starch, 0.05% K₂HPO₄, 0.10% MgSO₄·7H₂O and the optimal culture conditions were inoculation size of 7% for 120 h at 30℃ with initial pH 8.0.[Conclusion] The above results indicated that mulberry endophytic bacterium B. methylotrophicus XP-27 has strong antagonistic activity on S. sclerotiorum PZ-2, being a candidate strain for developing the biological control agent against mulberry fruit sclerotiniose.

Abstract:[Objective] To mine the genetic resource of novel cold-active α-amylases and reveal their cold-adaptation mechanism are of importance to deepen our understanding of cold-active enzyme and provide key information for the molecular improvement of α-amylase.[Methods] Based on the genome sequence of Thermoascus crustaceus JCM12803, we cloned an α-amylase-encoding gene (Tcamy), and inserted it into the expression vector pPIC9. The gene product was heterologously expressed in Pichia pastoris GS115 and characterized. By using amino acid sequence analysis and homologous modeling, we studied the cold-adaptation mechanism of TcAmy in viewpoint of sequence-structure-function relationship.[Results] TcAmy is a typical cold-active α-amylase, showing optimal activity at 35℃ and remaining 27% maximal activity even at 0℃. Sequence and structure analysis indicated that in comparison to thermostable counterparts, TcAmy has low N-glycosylation degree, decreased Pro and Arg contents and increased Gly content, and less disulfide bridges and ionic bonds.[Conclusion] We obtained a novel cold-active α-amylase, with low N-glycosylation degree, specific amino acid composition and intermolecular interactions, all contributing to its cold-adapted property.

Abstract:[Objective] By comparing the bacterial and fungal communities in alpine grassland soil and their degraded soil in Northwest Yunnan, we investigated the effect of vegetation degradation on microbial communities in alpine grassland soils.[Methods] Bacterial 16S rRNA genes and fungal ITS genes were chosen as the target genes. Quantitative PCR was performed to measure the gene copies to assess the abundance of microbial communities. Illumina Hiseq sequencing and bioinformatics analysis were carried out to determine microbial community composition and community structure.[Results] After grassland degradation, soil pH value was greatly increased by 0.65 units, and soil moisture, total organic carbon, dissolved nitrogen and C/N ratio were significantly decreased by 18.4%, 67.5%, 47.2% and 1.2%, respectively. Grassland degradation significantly reduced soil bacterial and fungal community abundances by 92.4% and 94.9%, respectively. Grassland degradation significantly altered the β-diversity of soil bacterial and fungal community, but had no effect on that of α-diversity. In addition, grassland degradation changed the species composition of soil bacterial and fungal communities at the OTU level, and the fungal OTUs changed largely. Grassland degradation had no effect on bacterial community composition at the phylum level, but changed the composition at the class level (such as Acidimicrobiia, Betaproteobacteria, Chloroplast etc.). No significant difference was detected in fungal community composition between grassland soils and degraded soils.[Conclusion] These findings suggested that the vegetation degradation in alpine grassland lead to a decline in soil quality and microbial abundance, and changes in microbial community structure.

Abstract:[Objective] The effects of antibiotics on bacteria are complex, and bacterial response to antibiotics is just beginning to be understood using systems biology. We previously showed that a hemerythrin-like protein, MSMEG_3312, is involved in erythromycin susceptibility. In this study, we explore the mechanisms of collective antibiotic tolerance to erythromycin in mycobacteria through the hemerythrin-like protein MSMEG_3312.[Methods] We analyzed MSMEG_3312 secondary structure using spectrophotometric and circular dichroism (CD) methods. Tandem mass tag(TMT)-labeled quantitative proteomics was used to compare protein level changes between the wild type strain mc² 155 and the knockout strain Δmsmeg_3312, following bioinformatics analysis. Differentially expressed proteins were also verified by qPCR. To confirm our analyses' conclusions that transporters are involved in MSMEG_3312-related erythromycin susceptibility, we also measured the concentration of mycobacterial erythromycin in vivo in the wild type strain mc² 155 and Δmsmeg_3312 using an erythromycin ELISA kit.[Results] Initially, we confirmed that MSMEG_3312 is a redox-related hemerythrin-like protein using spectrophotometric and CD analysis. Quantitative proteomic analysis revealed that Δmsmeg_3312 has eight up-regulated proteins, including three transporters, and 14 down-regulated proteins, compared with the wild type strain mc² 155, while growing in 7H9 medium. In contrast, 448 proteins were identified as being differentially expressed between mc² 155 and Δmsmeg_3312, when treated with erythromycin, of which 11 were identified as up-regulated transporter proteins, and 26 were associated with amino acid synthetic pathways. The intracellular erythromycin concentration in Δmsmeg_3312 was also lower than in mc² 155.[Conclusion] We show that MSMEG_3312 mediates erythromycin resistance due to collective antibiotic tolerance arising from antibiotic titration and high-density populations.

Abstract:[Objective] The aim was to analyze the sequence polymorphism, gene transcriptional pattern and functions of the RXLR effector PcAvh2 from Phytophthora capsici.[Methods] We cloned PcAvh2 gene by high-fidelity PCR from 31 P. capsici isolates, 2 P. parasitica isolates and 1 P. cactorum isolate. Gene expression changes during the developmental stages (mycelia, zoosporangia, zoospores and germinated cysts) and infection period (1.5, 3, 6, 12, 24, 36, 72 h post-inoculation) of P. capsici were monitored by quantitative RT-PCR. PVX-based agroinfiltration assay was performed to examine if PcAvh2 could suppress plant immunity triggered by 6 effectors (BAX, INF1, PsojNIP, PsCRN63, PsAvh241 and R3a/Avr3a). The CaCl₂/PEG-mediated protoplast transformation of P. capsici was conducted to silence PcAvh2 and determine if its silencing affected the pathogen's virulence.[Results] PcAvh2 is a typical RXLR effector and possesses 10 alleles in the population. Furthermore, it also exists in P. parasitica and P. cactorum. The expression of PcAvh2 was up-regulated during the host infection by P. capsici. It can suppress the plant immunity induced by all 6 effectors. Intriguingly, silencing of PcAvh2 in P. capsici significantly compromised the pathogen's virulence on host plants.[Conclusion] RXLR effector PcAvh2 is one of important pathogenicity factors in P. capsici.

Abstract:[Objective] Our study aims to understand the microbial diversity of endophytic bacterial community of different rice tissues.[Methods] We adopted high-throughput sequencing technology to study the microbial community structure and composition of bacterial endophytes in rice plant. We extracted DNA of the microbial community of bacterial endophytes in the rice var. No. 43 of Ningjing. The bacterial 16S rRNA gene hypervariable region V5-V7 was detected by high-through sequencing technologies.[Results] In total 610 OTUs were obtained from leaf bacterial endophytes, 411 OTUs from rice stem, 174 OTUs from rice root. Based on the results of species classification, leaf endophytic bacteria were represented by 22 phyla that mainly comprised 40 classes, 103 orders, 198 families, and 399 genera. Among them, the predominant genera were Rhodococcus and Lactobacillus, and their relative abundances were 21.00% and 9.19%, respectively. Stem endophytic bacteria were represented by 19 phyla that mainly comprised 31 classes, 85 orders, 169 families, and 306 genera. Among them, the predominant genera were Rhodococcus and Ralstonia, and their relative abundances were 19.25% and 13.52%, respectively. Root endophytic bacteria were represented by 9 phyla that mainly comprised 19 classes, 44 orders, 82 families, and 140 genera. Among them, the predominant genera were Enterobacter and Escherichia, and their relative abundances were 81.13% and 10.89%, respectively. Seventy-eight of the OTUs were represented in all the rice endophytic samples. Actinobacteria were strongly associated with other bacteria. The higher species abundance of root bacterial endophytic community could regulate various metabolic networks compared to the stem and leaf.[Conclusion] The diversity of bacterial endophytic community associated to rice plants has important ecological significance.

Abstract:[Objective] We aimed to reveal a novel function of bagZH in Streptomyces sp. Tü4128, which encoded a tyrosinase-like copper enzyme.[Methods] The bagZH gene was deleted through homologous recombination, and the secondary metabolites were detected and analyzed by HPLC and LC-ESI-MS. The activity of the BagH enzyme expressed by E. coli BL21(DE3) was measured by biochemical assays. The catalytic products of the enzyme were analyzed by LC-ESI-MS, in which o-aminophenol and 3,4-AHBA were used as substrates.[Results] HPLC analysis showed that the production of bagremycin significantly decreased when bagZH was deleted. Complementation of bagZH gene expression cassettes in the mutant increased the accumulation of bagremycin. LC-ESI-MS results showed that the molecular weight of the new product with a retention time of 3.18 min was 286.32 g/mol, which was consistent with the predicted molecular weight of the product synthesized by esterification of 3,4-AHBA in vivo. In vitro biochemical analysis demonstrated that BagH can catalyze the oxidation of o-aminophenol (protecting group).[Conclusion] Our findings revealed for the first time that bagZH participated in the biosynthesis of bagremycin by encoding a tyrosinase-like copper enzyme. BagH protected the biosynthetic intermediates by catalyzing the oxidation of 3,4-AHBA to a nitroso derivative (protecting group). After condensation with p-coumanic acid, the nitroso-group is reduced by a reductase in vivo to generate bagremycin A and B. The results obtained in this study provide a basis and reference for in-depth study of the mechanism of bagremycin and rational design and transformation of high-yielding strains.

Abstract:[Objective] Our research focuses on the detection and the distribution of some important virulence genes in Pasteurella multocida associated with bovine respiratory disease (BRD).[Methods] In total 237 nasal swabs were collected from August 2017 to April 2018 in 8 areas of China. The bacteria were isolated to record the colony morphology and Gram staining, and phylogenetic tree based on the 16S rRNA gene sequences of the 31 isolates was drawn. Serotype classification was performed, and total 23 virulence factors were determined by PCR.[Results] In total 31 strains of P. multocida were identified from 237 samples. Serum typing found all the isolates were type A strains and phylogenetic tree showed the 31 strains related to HB01 strain (China), USDA-ARS-USMARC-60712, USDA-ARS-USMARC-60214, ATCC 43137 and 36950. PCR was used for detecting 23 virulence-associated genes occurred in those isolates, demonstrating that each P. multocida carried 17 to 19 virulence-associated genes with high concentration ratio.[Conclusion] Type A P. multocida were dominant strains in which 7 strains from different areas of China belong to one cluster, virulent genes tadD and hgbA were lost in this cluster as well as virulent hsf-1 was carried. The analysis result reminds the virulence genotyping distribution has relevant to serum typing.