Abstract:There are large number of complex and diverse microbiota in gastrointestinal tract, and the gut microbes play an important role in maintaining gut environment homeostasis, not only affecting nutrient absorption and energy metabolism, but also regulating host physiological functions. Intestinal microorganisms can use nutrients of the host and then produce microbial metabolites, finally form host-microbe metabolic axis between host and gut microbes. The axis plays an important role in animal nutrition metabolism and immune homeostasis, and eventually affects the overall metabolism of host. We reviewed the concept of host-microbe metabolic axis, gut-liver axis, gut-brain axis, the interaction between gut microbiota and the host intestinal metabolism axis, and its impact on host health, with the aim to deepen our understanding about the contribution of intestinal microbes to host metabolism.

Abstract:H₂S is the third gaseous signaling molecule next to nitric oxide and carbon monoxide, but studies on its physiological functions in bacteria are just emerging. In this paper, we review recent findings regarding endogenous production and physiological functions of H₂S in facultative anaerobic bacteria, partly based on our own research on Shewanella oneidensis. There are two principal H₂S producing pathways in S. oneidensis:one is through cysteine degradation, and the other is via inorganic sulfur respiration. Endogenous H₂S could either benefit mutual growing bacteria by supplying energy and inorganic, or inhibit competing bacteria. Our review attaches particular importance to the role of H₂S in bacterial oxidative stress response. On one hand, H₂S is able to directly inhibit heme-containing catalase, enhancing killing by H₂O₂. On the other hand, H₂S could activate oxidative response as a signaling molecule, leading to cell protection from the oxidative stress due to elevated expression of H₂O₂ scavenging and repairing systems. Intriguingly, the dominance of either role is determined by H₂S-treating time, that is, inhibition is the immediate response whereas activation of oxidative stress response needs extended treatment. The elucidation of endogenous production and its physiological function of H₂S in facultative anaerobic bacteria would improve understanding of biogeochemical sulfur recycling, and facilitate control of infectious bacterial pathogens.

Abstract:Members of the genus Microlunatus exhibit many potential advantages in managing the environmental pollution caused by phosphorus. The genus was proposed by Nakamura and co-workers with the name Microlunatus phosphovorus as the type species in 1995. Up to date, the genus Microlunatus encompasses seven validly described species, which were isolated from various environments. Members of the genus Microlunatus share the following genus-specific characteristics, possessing LL-2, 6-diaminopimelic acid in the cell wall peptidoglycan, MK-9(H₄) as the predominant menaquinone and diphosphatidylglycerol and phosphatidylglycerol as the phospholipid pattern. Based on the taxonomic results of two newly isolated strains of the genus Microlunatus and the related reference reports, this review summarizes the research advances of the genus Microlunatus, including the genus establishment, taxonomic characteristics, their distribution in the environments, as well as the application prospect in chemical and medical industry.

Abstract:Aedes (a genus of mosquitoes) transfers Zika virus (ZIKV) to humans. About two billion people worldwide live in ZIKV-affected areas. The outbreak of ZIKV in Central and South America threats public health worldwide, especially for pregnant women and their fetuses. ZIKV infection has become one of the major causes of neonatal congenital microcephaly and adult Guillain-Barre syndrome. No effective vaccine and treatment are available now against ZIKV infection. The first Zika vaccine is a DNA vaccine which affords complete protection against ZIKV and induces a high level of specific antibody titers. Advantages of DNA vaccines are simple to design and produce, safe for adults and fetuses, and without virulence recovery induced by reproducible vaccines. Some positive vaccines including traditional and emerging ones are in research and some progresses have been achieved. This article reviews the current situation and progress of Zika vaccines.

Abstract:[Objective] To identify and clone the polymerase Ⅲ U6 promoter from Cryptococcus neoformans (CnU6 promoter), and verify if CnU6 promoter can effectively transcribe shRNA and gRNA of CRISPR/Cas9 system.[Methods] Combining the C. neoformans genome information published in GenBank database and RNA-seq library data from our laboratory, we obtained the U6 RNA sequence with high transcriptional level by bioinformatics analysis. The putative CnU6 promoter was ligated upstream of shRNA and gRNA by EasyGeno and overlapping PCR respectively. Based on shRNA-mediated target gene silence phenotype by RNAi and gene mutation by gRNA-guided Cas9 nuclease mediated target sites editing by CRISPR/Cas9 system, we could identify if CnU6 promoter could drive the transcription of short RNA.[Results] CnU6 promoter could drive the transcribtion of shRNA, which could silence the target gene, and gRNA, which could guide Cas9 nuclease to cut the target site.[Conclusion] The CnU6 promoter from C. neoformans was successfully identified and cloned, which could drive the transcription of shRNA and gRNA efficiently.

Abstract:[Objective] Ageratina adenophora, a most harmful invasive plant in China, contains many toxicants to animals, plants and microbes. To understand the toxicity of its decomposed materials by microbial inoculants to soil microbes and plants is beneficial to produce organic fertilizer (OF).[Methods] A field experiment, including the control (CK), sole chemical fertilizer (CF), pure OF, and combination of CF and OF (CF+OF), was conducted to study the effect of OF on soil bacteria, and yield and quality of pepper.[Results] Providing soil with OF resulted in higher microbial biomass than CF. Both OF and CF+OF increased bacterial community indexes in contrast to CF that increased microbial dominant index in soils. Actinobacteria and Proteobacteria accounted for more than 50% of the total bacteria in each soil examined. Among top 20 predominant bacteria, 7 were ubiquitous in all studied soils and 6 to 8 were unique in each soil. Furthermore, there were higher nutrient concentrations in the soil supplied with CF in early fruit stage, whereas that with CF+OF contained higher available phosphorus and rapidly available potassium than CF, and alkaline hydrolyzed nitrogen was similar in late fruit period. Pepper plants fertilized with CF+OF absorbed more nitrogen, phosphorus, and potassium, produced 14.4% more fruits than CF and improved pepper fruit quality.[Conclusion] OF application enriched soil bacterial groups, increased soil microbial biomass, and pepper fruit yield and quality. Organic fertilizer made from A. adenophora improves soils for pepper plants.

Abstract:[Objective] To identify non-coding RNAs in Haloferax mediterranei through high-throughput RNA sequencing, bioinformatics analysis and molecular techniques.[Methods] After H. mediterranei cells under log phase of growth were treated with different salt concentrations for 30 minutes, total RNA was extracted for the following strand-specific RNA sequencing and differential RNA sequencing. These RNA-seq data were used to identify the genome-wide ncRNAs and to predict the 5' and 3'-ends of the transcripts by bioinformatics analysis. A few selected ncRNAs were further confirmed by Northern blotting and Circularized RNA reverse transcription-PCR analysis.[Results] We identified 105 highly credible ncRNAs. Expression of four ncRNAs showed difference in different salt concentrations. We confirmed the expression, length of transcripts, transcription start and termination sites of incRNA1436 and incRNA1903 by Northern blotting and CR-RT-PCR.[Conclusion] We identified the ncRNAs of H. mediterranei in a genome-wide scale, including identification of a few ncRNAs involved in the responses of H. mediterranei to different salt concentrations. Our results have provided fundamental data and novel insights for future study of the function of ncRNA in haloarchaea.

Abstract:Pyocyanin, an important virulence factor, is synthesized and secreted by Pseudomonas aeruginosa PAO1and plays a critical role in pathogen-host interaction during infection. Sigma³⁸ (σ³⁸, σS) is a central regulator for many virulence production in pathogens.[Objective] Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma³⁸ factor in Pseudomonas aeruginosa PAO1.[Methods] We first cloned the flanking fragments of rpoS from the chromosomal DNA of P. aeruginosa PAO1 and constructed the deletion mutant ΔrpoS with the insertion of gentamycin resistance cassette (aacC1). Complementation of rpoS was then carried out after construction and introduction of pME10S (containing the whole rpoS region). Finally, we created the mutant ΔrpoSphz1 and ΔrpoSphz2, and measured pyocyanin production by these mutants in GA medium, using the parental strain Δphz1 and Δphz2 as controls.[Results] In GA medium, pyocyanin production by mutant ΔrpoS increased dramatically in comparison with the wild-type strain PAO1. Production of pyocyanin, however, was decreased to the level of the wild-type strain with complementation of the derivative ΔrpoS harboring pME10S. Mutant ΔrpoSphz2 produced much more pyocyanin than mutant Δphz2. Mutant ΔrpoSphz1, however, produced much less pyocyanin than mutant Δphz1.[Conclusion] By positively regulating the expression of phz2 and negatively regulating the phz1, sigma³⁸ factor exerts negative modulation on pyocyanin biosynthesis in P. aeruginosa PAO1.

Abstract:[Objective] To study the exopolysaccharide (EPS) of endophytic fungal Fusarium redolens 6WBY3 isolated from Fritillaria unibracteata var. wabuensis (FUW) including its antioxidant activities.[Methods] We isolated the EPS from the culture medium of strain 6WBY3 using the methods of degreasing with organic solvent, precipitation with ethanol, decoloration with macroporous resins, deproteinization with protease in combination with sevag reagent, desalination with dialysis and separation with DEAE-cellulose ion exchange chromatography. Then, we analyzed the EPS fractions using high-performance gel-permeation chromatography with evaporative light scattering detector (HPGPC-ELSD), ultra violet (UV) spectra, scanning electron microscope (SEM), PMP precolumn derivatization with high performance liquid chromatography (PMP-HPLC), fourier transform infrared (FT-IR) spectroscopy and ¹H-nuclear magnetic resonance (¹H NMR) methods. We evaluated the antioxidant activity of the EPS using DPPH and ABTS radical scavenging activities and iron ion chelating ability methods.[Results] We obtained two homogeneous EPSs, namely 6WBY3EPS-3 and 6WBY3EPS-4, with the molecular weight (Mw) of 17.41×10⁶ and 8.84×10⁵ Da, respectively. 6WBY3EPS-3 was composed of mannose, glucose and galactose in a molar ratio of 8.16:4.96:10.00, while 6WBY3EPS-4 was composed of mannose, rhamnose, glucose and galactose in a molar ratio of 8.08:1.71:6.32:10.00. The results of SEM showed that 6WBY3EPS-3 was the irregular multilateral body, and 6WBY3EPS-4 was rules of quadrilateral body. The results of FT-IR and ¹H NMR analysis exhibited that 6WBY3EPS-3 was acidic polysaccharose with abundant galactofuranose, mannofuranose and a few α-D-glucopyranose, while 6WBY3EPS-4 possessed pyranose ring mainly. Those two EPSs had anomeric hydrogen with α-(main) and β-glycosidic configuration. Furthermore, the results of antioxidant activity suggested that both 6WBY3EPS-3 and 6WBY3EPS-4 had a weak DPPH radical scavenging ability, a moderate ABTS radical scavenging activity and a moderate iron ion chelating effect.[Conclusion] The two EPSs from the endophytic fungus 6WBY3 were firstly obtained and investigated. Our investigation indicated that the EPS from 6WBY3 had the application potential as an antioxidant in medicine and food industries. In addition, this work can also provide theory reference for improving development of EPS from other endophytic fungi.

Abstract:[Objective] To establish a pipeline for unknown transcriptional start site (TSS) identification without radioactivity, we used genetic fragment analysis system and replenished two steps regarding prediction and evaluation.[Methods] We used unknown TSSs of GroEL genes from M. xanthus as a case. Firstly, we predicted the potential TSSs through bioinformatics databases. According to the prediction, we designed and synthesized fluorescence labeled primers to carry out the reverse transcription reactions. Further, we took advantage of the genetic fragment analysis system to identify TSSs with internal standards. Finally, we applied the normal distribution theory to evaluate the data.[Results] We determined the numbers, abundances and accurate sites of the TSSs:GroEL1 has one promoter and the site is TSS₂₈₆, whereas GroEL2 has two promoters, and the sites are TSS₅₄₈ and TSS₅₀₂. TSS₂₈₆ is 14.3 times more abundant than TSS₅₄₈ and TSS₅₄₈ is 13.8 times more than TSS₅₀₂.[Conclusion] The bioinformatics analyzing indicates the range for the experimental design. TSS determination through genetic fragment analysis system is safer, more automatic and accurate. Normal distribution theory further refines the reliability of results. Combination of the three techniques establishes a more complete pipeline of primer extension for unknown TSS determination.

Abstract:[Objective] We constructed Bordetella bronchiseptica QH0814 hcp mutant to characterize its pathogenicity.[Methods] Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QH0814Δhcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose (LD₅₀) and the infection capacity.[Results] There was no significant variation of the growth rate between the mutant and the parental strain. Compared with the parental strain, the adherence ability of the mutant did not change remarkably. However, the invasion ability decreased significantly. Mice lethal test showed that the LD₅₀ of the mutant was higher than that of the parental strain. Correspondingly, the bacterial colonization of the mutant in mice blood, lung and liver was much less than that of the parental strain.[Conclusion] The knocking-out of the hcp gene had no influence on bacterial growth, but it could attenuate significantly the invasion and colonization of the bacterium. Therefore, the gene may play a role in the pathogenesis of Bordetella bronchiseptica.

Abstract:[Objective] In order to provide scientific data for studying the ecology of phage infecting Sinorhizobium meliloti, we examined morphological characteristics of rhizobiophages and their phylogenetic status of the major captain protein g23.[Methods] Rhizobiophages were isolated by the double-layer plate method with host Sinorhizobium meliloti USDA1002^T. The morphological characteristic of rhizobiophages were studied by transmission electron microscope. Meanwhile, rhizobiophage DNA was extracted, and the g23 that encodes the major capsid protein of bacteriophages was chosen as objective gene in PCR amplification.[Results] Three rhizobiophages were isolated, all had an icosahedral head with approximately 81 to 86 nm in diameter and a long contractile tail with 54 to 70 nm in length. Basic local alignment search tool searches in website of national center for biotechnology information (NCBI) revealed that the g23 amino acid sequences obtained in this study had high identity with each other, but had very lower identity with those from T-evens, PseudoT-evens, SchizoT-evens and ExoT-evens. Phylogenetic analysis showed that the isolated g23 sequences formed a unique clade with those clones obtained from different ecosystem.[Conclusion] All results indicated that the isolated rhizobiophages belong to family Myoviridae, a new group of T4 phages, which had lower identity with the g23 clones obtained in different environment.

Abstract:[Objective] In order to reveal key metabolites and metabolic pathways of avermectin, comparative metabolomics approach was used to analyze the difference of key intracellular metabolites of Streptomyces avermitilis in different fermentation media. Then the rational feeding approach was used to enhance avermectin production.[Methods] Mycelial samples in M1 and M2 media were analyzed by GC-MS based comparative intracellular metabolomics. Based on the deficiency of relative metabolic pathways, single precursor supplement and combined precursors supplement were exerted, and the optimized medium M3 was obtained to enhance avermectin production.[Results] In total 232 intracellular metabolites were identified and 70 of them were accurately matched, and 21 metabolites influencing avermectin biosynthesis were finally verified by PCA and PLS analyses. Among them, lactic acid, pyruvic acid, succinic acid, threonine, isoleucine, valine and lipids determined the avermectin titer more obviously. When supplying with these precursors together to M2 at different time points, the titer increased by 10.4% from 5.36 g/L to 5.92 g/L.[Conclusion] The production of avermectin increased apparently with the help of comparative metabolomics analysis and rational feeding optimization. It also provides a new approach to enhance the yield of bioproducts.

Abstract:[Objective] The aim of this study was to isolate Lactobacillus plantarum acid-sensitive mutants with lower H⁺-ATPase activity, and to study the mechanism of H⁺-ATPase regulation in Lactobacillus plantarum.[Methods] We used neomycin to isolate acid-sensitive mutants of L. plantarum, and measured H⁺-ATPase activity and lactic acid production of wild-type and mutants. Genomic DNA was extracted from the wild-type ZUST and two mutants (ZUST-1, ZUST-2), and used as PCR templates. H⁺-ATPase genes of the strain were amplified, and the PCR products were sequenced. Sequence similarity of H⁺-ATPase was analyzed. Real-time RT-PCR was used to evaluate the relative quantification of the H⁺-ATPase genes expression.[Results] The growth of the mutants was characterized in MRS broth, which revealed that their cell biomass and acid production were lower than that of the wild-type. H⁺-ATPase activity of the mutants ZUST-1 and ZUST-2 was 10.1% and 28.8% lower than that of the wild-type. Results showed that atpA gene of the mutants ZUST-1 and ZUST-2 existed 22 mutations by alignment of the wild-type sequence, and atpC gene of ZUST-2 existed 6 mutations. Mutants ZUST-1 and ZUST-2 atpA gene expression were 41.1% and 35.7% lower than that of the wild-type in exponential phase, 43.6% and 14.2% in stationary phase, respectively. The atpC gene expression of ZUST-1 was similar to that of the wild-type in exponential phase, and was 30% higher than that of the wild-type in stationary phase, and ZUST-2 atpC gene was not expressed.[Conclusion] The mutants with lower H⁺-ATPase activity were found to up-regulate the expression of H⁺-ATPase genes in stationary phase, except ZUST-2 atpC gene was not expressed. H⁺-ATPase activity has an important connection with the difference in gene expression of atpA and atpC. The results of this study will pave the way for gaining further insights into the mechanism of the H⁺-ATPase-defective mutants.

Abstract:[Objective] In order to get excellent strains for aquiculture water purification, we screened lactic acid bacteria from the aquaculture environment and intestinal tract of shrimp.[Methods] The potential water purification ability of lactic acid bacteria at normal and low temperature was evaluated in the simulated wastewater. Morphological physio-biochemical characteristics, 16S rRNA gene sequence analysis were used to identify strain r13. Single factor test and orthogonal-design experiment were applied to optimize fermentation for r13.[Results] In total 136 lactic acid bacteria strains were isolated from 3 samples. The results of water purification test suggested r13 had higher removal ability of nitrite and ammonia from water. After 72 h treatment by r13, nitrite with 11.5 mg/L in the water was completely removed and ammonia degradation rate was 29.1% with 13.0 mg/L original concentration. According to morphological, physio-biochemical characteristics and 16S rRNA gene sequence analysis, r13 was identified as Lactobacillus plantarum. The optimal fermentation condition for r13 was 6.0 g/L yeast extract, 20.0 g/L glucose, 4.0 g/L sodium acetate, 2.0 g/L diammonium hydrogen citrate, 2.0 g/L monopotassium phosphate, 50 mL tomato juice, with inoculation rate 5% (V/V), at pH 6.0 and 34℃. Under this condition cultured for 72 h, the bacterial biomass reached 28.4 g/L wet weight and cell counting reached 4.4×10⁹ CFU/mL.[Conclusion] Considering high nitrite removal ability, we suggested that r13 would be promising microorganism for water purification in aquiculture.