Abstract:[Objective] The genA inactive mutant was constructed on the basis of Micromonospora purpurea G1008, and then the function of genA was studied by analyzing the secondary metabolites. [Methods] Plasmid pAB103 used for the genA in-frame deletion was constructed and transformed into Micromonospora purpurea G1008 by conjugation. Apramycin resistance and PCR amplification were used to confirm Micromonospora purpurea GA1048. [Results] Gentamicin A2, instead of gentamicin C complex, was accumulated in Micromonospora purpurea GA1048, comparing with Micromonospora purpurea G1008. [Conclusion] The inactivation of genA led to the changes of gentamicin biosynthesis flow, and genA might be responsible for the methylation at C-3″ of garosamine.

Abstract:[Objective] To obtain an engineering strain producing G418 single component by directional reconstruction of Micromonospora purpurea G1008 producing gentamicins. [Methods] Using the temperature-sensitive plasmid pKC1139 as vector, the recombinant plasmid pQB303 was constructed and introduced into the M. purpurea G1008 by conjugation. The genQ*-disruption mutant was screened out by replica plating and PCR analysis. Use MS and TLC for analysis of metabolites. [Results] A genQ*-disruption strain, named as M. purpurea GQ175, was obtained to produce G418 single component. The level of antibiotic production was 828 mg/L, similar to the level of the parent strain. [Conclusion] The engineering strain GQ175 produced G418 single component, which had great value for industrial development. Collectively, these results demonstrated that the C-6′ dehydrogenase gene for gentamicin biosynthesis is genQ* and there is no other isoenzyme genes in M. purpurea G1008.

Abstract:[Objective] The aim of this study was to describe carbon metabolic characteristics and regulation mechanisms of Lactobacillus casei strain at the genome-level. [Methods] Pathway Tools, based on BioCyc and MetaCyc database, was used for genome-wide level of carbon metabolism comparative analysis of 12A and other 7 L. casei strains published complete genome sequences. [Results] Nine sugars could be transferred and metabolized by L. casei 12A inside cells. Various oligosaccharides and polysaccharides could be hydrolyzed into galactose and glucose by strain 12A outside cells. In particular, strain 12A could generate ethanol and its by-products through heterolactic fermentation and mixed acid fermentation pathways. [Conclusion] L. casei 12A can metabolize multiple types of carbohydrate, thus the choice of its fermentation substrates is broad. And strain 12A can be used as a specific strain for industrial ethanol production. In addition, it is reliable that comparative genomics is used for analyzing and establishing relationship between genetic structure and bacterial metabolic capability.

Abstract:

Abstract:[Objective] We detected and analyzed N-acyl-homoserine lactones (AHLs) of Acinetobacter spp. (M1) that was isolated from spoilage Litopenaeus vannamei. We also investigated the effects of bacteria growth stages and environmental factors on activities of signaling molecules. [Methods] The reporter strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136) and TLC (Thin-Layer Chromatography) method were used to detect and analyze AHLs activities of M1. [Results] AHLs secreted by M1 were N-3-oxo-C6-HSL and N-3-x-oxo-C8-HSL. The activities of AHLs increased first and then decreased with the prolongation of the culture time under appropriate culture conditions, and reached the maximum in late logarithmic stage. The ability of M1 producing AHLs was reduced by weak acid and alkalescent envirornment, the optimum pH of M1 secreting AHLs was pH 7.0. The concentration of NaCl had no significant effect on the whole ability of M1 secreting AHLs, while high concentration promoted the ability of individual to product AHLs. The optimal temperature for strain M1 to produce AHLs was 30 °C, and the relative higher or lower temperature inhibited the AHLs secretion. [Conclusion] The AHLs of strain M1 were N-3-oxo-C6-HSL and N-3-oxo-C8-HSL. Quorum sensing systems of M1 was regulated by both cell density and environmental factors.

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Abstract:[Objective] In order to provide a scientific basis for further research of marine siderophores, detection and separation methods of the siderophores from Synechococcus sp. PCC 7002 were investigated. [Methods] Synechococcus sp. PCC 7002 was cultivated in iron-limited Medium A. We used two-layer plate, mixture plate and traditional chromazurol S plate to detect the siderophores qualitatively. The relative amount of siderophores was detected by the chromazurol S shuttle assay. Amberlite XAD-2 resin and immobilized metal-chelating affinity chromatography were used for siderophores separation. Two different elution methods (pH reduced elution and competitive elution) were utilized in immobilized metal-chelating affinity chromatography. [Results] Mixture plate was more rapid, efficient and convenient among the three qualitative detection methods. The relative amount of siderophores from the iron-limited culture was up to 93.50%. Siderophores adsorbed sufficiently into Amberlite XAD-2 resin and the separating efficiency was relatively high when the sample pH was adjusted to 2. Strong fluorescence of siderophores from Synechococcus sp. PCC 7002 was observed at 254 nanometer ultraviolet light. [Conclusion] In present study, we got the effective detection and separation methods of siderophores from Synechococcus sp. PCC 7002.

Abstract:[Objective] to investigate the diversity of marine fungi producing extracellular protease isolated from the intertidal zone of Naozhou Island in the South China Sea. [Methods] The samples of both seawater and sediment were collected from the intertidal zone of Naozhou Island in South China Sea, and used to investigate the diversity of the fungi producing extracellular proteases by culture-dependent method, protease producer plate assay, and a phylogenetic analysis based on the sequences containing internal transcribed spacer 1, 5.8S rRNA gene and internal transcribed spacer 2 (ITS1-5.8S-ITS2). [Results] A total of 198 isolates of fungi were isolated, purified and collected from the samples on the plates of Potato dextrose agar (PDA) medium prepared with 50% seawater. Amongst these isolates, 178 strains were identified successfully by PCR amplification, sequencing, BLAST and phylogenetic analysis of the ITS1-5.8S-ITS2 sequences. All these sequences showed ≥98% identity with the existing relative ITS1-5.8S-ITS2 sequences in the GenBank database, except 10 strains showed the identity <97% with their closest match suggesting their possibility of being novel species. These 178 strains represented 66 species, belonging to 45 genera of 27 families in 16 orders, 6 classes, spreading in two phyla, Ascomycota and Basidiomycota. The predominant genus was Penicillium, which takes a great proportion of 28.70%, followed by Aspergillus, which takes a proportion of 11.24%. There were 83 identified strains showed clear zone around their colonies on the PDA plates supplemented with skim milk indicating they could produce extracellular proteases. [Conclusion] A total of 178 strains of fungi were isolated and identified from the intertidal zone of Naozhou Island in the South China Sea. Amongst these strains, 10 strains would be novel specie Candidates, and 83 strains could produce extracellular proteases.

Abstract:[Objective] The purpose of this paper is to isolate and screen lead-zinc resistant strains from lead-zinc tailings areas. Its influence factors and mechanism of Pb(II) and Zn(II) adsorption also were studied. This work is expected to provide a reference for bioremediation of heavy metal pollution. [Methods] The fungus HA which was isolated and screened from lead-zinc tailings areas was used. The main influence factors (i.e., initial concentration of Pb(II) and Zn(II), pH, inoculation amounts) on the removal of Pb(II) and Zn(II) were investigated. And the related adsorption mechanism was also discussed by isothermal adsorption model, adsorption kinetics analysis and IR analysis. [Results] The resistant strain HA screened from tailings areas was identified as Aspergillus oryzae. In the experimental range of initial concentration of Pb(II) and Zn(II) (100–800 mg/L), the removal of Pb(II) and Zn(II) decreased with the increase of the initial concentration of Pb(II) and Zn(II). The growth of HA turned into stable period after 25 h, and the removal of Pb(II) and Zn(II) was relatively stable. The maximum biosorption was found at initial Pb(II) and Zn(II) of 100 mg/L and pH 5.0. Under such conditions, the adsorption rates of Pb(II) and Zn(II) was 97.8% and 54.1%, respectively. When the inoculation quantity of HA was 1 mL, the Pb(II) and Zn(II) adsorption rate of increment was the fastest. The adsorption process of Pb(II) and Zn(II) by HA was in accordance with Langmuir adsorption model, and its adsorption was given priority with monolayer adsorption. During the dynamic adsorption process of Pb(II) and Zn(II) by HA, a pseudo-second-order adsorption kinetics model was more suitable for describing the adsorption performance of HA, and the effect of HA on adsorption of Pb(II) was significantly higher than on Zn(II). IR analysis showed that the functional groups of HA such as hydroxyl, alkyl, amide, carbonyl and phosphate group were involved in the adsorption process of Pb(II) and Zn(II) by HA. [Conclusion] HA is a fungus having stronger adsorption ability for Pb(II) and Zn(II), and this research results of influence factors and mechanism of Pb(II) and Zn(II) adsorption would provide a guidance for bioremediation of heavy metal pollution.

Abstract:[Objective] To investigate the diversity of endophytic actinomycetes isolated from Chinese medicinal plant Melia toosendan from different regions of Sichuan province. [Methods] Seven kinds of selective isolation media were used to isolate endophytic actinomycetes from the roots, stems, leaves, bark, and fruits of Melia toosendan. 16S rRNA gene-RFLP was used to analysis the diversity and community structure of endophytic actinomycetes. [Results] A total of 403 strains of endophytic actinomycetes were isolated and purified. The number of isolates was varied with different sampling sites, media and plant tissues. Eighty-six strains were obtained from the samples in Guangyuan, which was the highest. The least was the samples from Mianyang, only 12 strains. One hundred and forty-eight strains of actinomycetes were isolated from the bark, accounting for 36.7% of the total strains. Only 31 strains were isolated from the fruits, accounting for 7.6% of the total strains. Although the quantity of isolates from the roots was very low, the isolation frequency was the highest. The most ideal isolation media were numbers 5 and 3, indicating that this two media were most suitable for the growth of endophytic actinomycetes. The results of 16S rRNA gene PCR-RFLP analysis indicated that all of the strains were clustered together at the 68% similarity level and divided into 10 genetic types at the 84% similarity level. Thirty-seven representative strains were chosen for 16S rRNA sequencing. The results of 16S rRNA sequencing and phylogenetic tree ananlysis of 16S rRNA of endophytic actinomycetes showed that the tested strains belonged to Streptomyces, Kitasatospora, Arthrobacter, and Kribbella, among which Streptomyces was dominant, accounting for 91% of the representative strains. [Conclusion] The results presented above indicate that the main endophytic actinomycetes belong to Streptomyces, Kitasatospora, Arthrobacter, and Kribbella in Chinese medicinal plant Melia toosendan in Sichuan province.

Abstract:[Objective] To understand all-level structures of Aeromonas veronii MreB protein, and study the conservation of MreB protein and its reliability as a molecular marker. [Methods] Online software such as ProtParam, PredictProtein and SWISS-MODEL were used to analyze the primary structure and physicochemical properties of the protein, and predict its secondary and tertiary structures. Amino acid sequence comparative analysis and phylogenetic study were done on MreB proteins in several types of bacteria through software like ClustalX 2.0, ESpript 3.0 and MEGA 6. [Results] MreB protein of Aeromonas veronii is an acidic protein composed of 346 amino acids, which is mainly located in the cell membrane. Secondary structure of the protein mainly consists of α-helices, extended long chains and random coil structures, of which random coils share the highest proportion. The results of homology modeling reveal that the tertiary structure of the protein contains 12 α-helical structures, 5 β-folded structures, 8 β-hairpin structures, 22 β-turns and 4 γ-turns. [Conclusion] Detailed physicochemical information on Aeromonas veronii MreB protein is obtained. The model built by homology modeling presents a reasonable spatial structure, and receives a relatively high PROCHECK score, which provides some structural basis for experimental research. Moreover, with conserved sites and structural conservation, MreB protein belongs to a highly conserved protein with appropriate variability. Therefore, it can be used as a molecular marker tool in the classification and identification of bacteria.

Abstract:[Objective] To obtain endophytes which has the plant-growth promote function from alfalfa. [Methods] Two strains were isolated and purified from fresh tissues of alfalfa, and then analyzed by 16s rRNA gene sequence, identified by the physiological and biochemical methods. The plant-growth promote properties to alfalfa of two strains were detected. [Results] Two alfalfa endophytes, ASR16 (Pantoea vagans) and ALR33 (Bacillus pumilus) were obtained. Both of the endophytes can produce IAA and siderophore, have phosphate solubilization activity, and can promote the growth of alfalfa effectively. [Conclusion] Two plant endophytes, ASR16 and ALR33, with plant-growth promotion to alfalfa could be used as the basis of bacterial manure research.

Abstract:[Objective] To identify microbes from rotted root of Pinellia ternata, so as to provide clues for efficient prevention of this plant disease. [Methods] Pathogens were isolated and their pathogenicity was confirmed by Koch’s rule. These pathogens were further studied through etiological, morphological and bacteriological techniques. In addition, 16S rRNA sequence analysis was done for bacterial pathogens, ITS and TEF sequence were used analysis for fungal pathogens. [Results] Twelve strains of bacterial pathogens and five strains of fungal pathogens were found in rotted tuber of P. ternata from three different cultivating places in Guizhou. The twelve strains of bacterial pathogens belonged to Pectobacterium carotovorum subsp. carotovorum. Among the fungal pathogens, ZJ and Z3 belonged to Fusarium oxysporum, and D3, D5 and H1 belonged to F. solani. [Conclusion] The pathogens that cause root-rot of Pinellia ternata in Guizhou include bacterial pathogens and fungal pathogens.

Abstract:[Objective] To understand the influence of Qi-Wei-Bai-Zhu-San on blood of diarrheal mice with dysbacteriosis. [Methods] Diarrheal mice model with dysbacteriosis was made by feeding antibiotics, then treated with intragastric administration of Qi-Wei-Bai-Zhu-San and 1/2 amount of ultra-micro Qi-Wei-Bai-Zhu-San. The blood was collected by the eyeball method and analyzed with the CA-500 blood automatic instrument. [Results] The values of PLT (platelet), MCV (mean corpuscular volume) and WBC (white blood cell) in the model group mice were lowed than that in control group mice (P<0.01 or P<0.05). After treatment by gavage, the values of PLT, MCV and WBC of the traditional decoction group and 1/2 amount of ultra-micro Qi-Wei-Bai-Zhu-San increased, and the MCV of traditional decoction group returned to normal and had no statistical difference comparing with control group (P>0.05). There were no statistics differences in the values of PLT and MCV between ultra-micro 1/2 amount group and control group (P>0.05). It implied that the blood recovered to normal. Qi-Wei-Bai-Zhu-San could promote the production of PLT and WBC, and improve the MCV. The WBC of 1/2 amount of ultra-micro Qi-Wei-Bai-Zhu-San group increased more than that of traditional decoction group. [Conclusion] Qi-Wei-Bai-Zhu-San could control immune function of diarrheal mice with dysbacteriosis. The functions of 1/2 amount of ultra-micro Qi-Wei-Bai-Zhu-San were better than that of traditional Qi-Wei-Bai-Zhu-San.

Abstract:[Objective] This study wanted to isolate and evaluate the probiotic properties of a broad-spectrum antibacterial lactic acid bacterium from Chinese traditional fermented soybean paste which was sampled from Jianhe country, Guizhou Province. [Methods] The strain DJ-04 was screened by antibacterial activity test, which was identified by physiological and biochemical characteristics and classification of 16S rRNA. The strain was examined for a series of assays including tolerance to artificial gastric intestinal juice, bile salt, and osmotic pressure. [Results] The strain DJ-04 was identified as Lactobacillus plantarum. It had an antibacterial activity against five pathogenic bacterium, including Escherichia coli, Salmonella sp., Staphylococcus aureus, Shigella sp., and Pseudomonas aeruginosa. DJ-04 had a strong tolerance to acid. The number of viable cells after 3 hours of incubation in pH 2.5 reached more than 107 CFU/mL. DJ-04 had a strong tolerance to artificial gastric intestinal juice since no significant changes were observed after 3 hours of incubation. The number of viable cells after 24 hours of incubation in 0.2 g/100 mL bile salt was above 108 CFU/mL. In addition, the number of viable cells after 24 hours of incubation in MRS containing 10 g/100 mL NaCl was more than 107 CFU/mL. [Conclusion] The isolated Lactobacillus plantarum DJ-04 had good tolerance to artificial gastric intestinal juice, bile salt, and osmotic pressure, suggesting that it could be a potential probiotics candidate for beneficial use.

Abstract:[Objective] To select and identify an endophytic fungus isolated from wheat head collected from a Kashin-Beck disease area of Qinghai province, and study its influences of culture conditions on the production of T-2 toxin. [Methods] Primary selection of toxin-producing strain was using embryonic bud inhibition and yeast fungistatic tests on all isolated strains. Strain re-selection was carried out by TLC and HPLC assay to check the extract of the strain. Strain 5-5m-1 was identified based on its colonial and microscopic morphological characters and ITS sequencing. The optimization of culture condition for T-2 toxin production was investigated by single factor experiments and orthogonal design method. [Results] The morphological characters of the strain 5-5m-1 were similar to that of Fusarium poae. The ITS sequencing showed that the sequence of strain 5-5m-1 had high similarity to that of the strain F. poae. The optimal culture conditions of the strain to produce T-2 toxin were corn solid medium, alternative illumination with 25 °C during the day and 15 °C at night. [Conclusion] Strain 5-5m-1 was identified as Fusarium poae. Culture conditions have great influence on its ability to produce T-2 toxin. The results will provide important references for further studies on the mechanism of T-2 toxin production and the measure to prevent mycotoxin contamination.

Abstract:During the last 30 years, plant endophytes deserve more attention around the world although its definition still remains confused and controversy. In this review, major historic events in endophyte researches and their scientific significances were discussed. Scientists firstly focused on special roles of endophytic fungi within woody plants and Gramineae plants. After anticancer natural product taxol was found to be produced by a plant endophytic fungus in 1993, biologically active metabolites were widely screened from plant endophytic microbes in many countries. Though comparing and contrasting the endophytes from different tissues of annual grasses, perennial grasses, woody plants and vine plants, common and specific properties of these endophytes were reviewed. The tendency of researches on endophytes were revealed. Plant endophytes contributed a lot to the total amount of microbe species on the earth. This review pointed that “the real situation of plants in the nature were microbes-plants symbiota”. Therefore, plant breeding were actually improvement of microbes-plants symbiota. Finally, the influences of endophytes on the ecology, biology, microbial evolution, species formation, plant-endophyte co-metabolism, plant protection, animal husbandry, veterinary science, forestry and some other fields were also discussed. Endophyte researches exhibit another view of microbial resources, significant impacts should be observed in many areas. The significance of endophytes not only rely on their niche, the ecological characterics, but also the synergism between endophytes and their hosts, resulting some new functions and new metabolites due to the co-metabolism and interactions.

Abstract:Nitrate/nitrite-dependent anaerobic methane oxidation (Nx-damo) is a newly demonstrated microbiological pathway for C and N cycle. This finding is significant for not only the understanding of the mechanisms of microorganisms driven cycling of biogenic elements, but also the development of sustainable wastewater treatment technology focusing on nitrogen removal and mitigation of green-house gas release. In this paper, the current advances in Nx-damo, including the identification and natural distribution of responsible microorganisms, their enrichment and the effect factors, as well as the physiological property and metabolic pathways, were reviewed. Advantages and potentials of Nx-damo based wastewater treatment technology were evaluated. Finally, perspectives on expected research and development of Nx-damo were suggested from microbiological and engineering aspects. Purpose of this review is to provide valuable information to promote more research on this field.

Abstract:To determine the structure, classification and functional aspects of G protein-coupled receptor (GPCR) in filamentous fungi, and to determine the relationship of GPCR between filamentous fungi and other model organisms. Based on the reported GPCR in the model organisms and filamentous fungi, the conserved domain was analyzed by SMART web service, and the genetic relationship between filamentous fungi and other model organisms was identified by Clustal X and MEGA software. There were seven transmembrane domains of typical GPCR and novel GPCR with PIPK, RGS and other conserved domains in filamentous fungi. The classification of GPCR from different scholars was identificated, as well as novel GPCR has special function. The model organisms GPCR and filamentous fungi GPCR can be grouped into each category. The number of GPCR of filamentous fungi was less than that of the model organism, and the number of GPCR was not the same among fungal taxa. GPCR in filamentous fungi contains a number of other conserved domains in addition to a typical seven transmembrane, which provide important theoretical basis for further study on its function.

Abstract:Manufactured nanomaterials have been widely used in life because of their unique physical and chemical properties. The environmental behaviors and biological safety of manufactured nanomaterials in water had become a hotspot in research of environmental science. The effects of manufactured nanomaterials in water-biofilm system and its influencing mechanisms are the key scientific problems, which are urgent to be solved. This review discusses the toxicity effects of nanomaterials on aquatic organisms, which cause bacterial death, inhibiting the growth of algae. The main influences of manufactured nanomaterials on biofilms are bacterial activity, microbial community structure, net pollution activity, and related key enzyme activity. The toxicity mechanism of nanomaterials can be investigated adsorption on biofilms or penetrate into the cell membrane, lead to the DNA damage, lipid peroxidation, protein modifications and oxidative stress of the bacteria biofilms by producing excess reactive oxygen species. Also, the adsorption mechanism of biofilm for nanomaterials was discussed, and illustrates the adsorption of biofilm on nanomaterials is the most important ways of its removal from water. The results of this review provide an important theoretical basis on further study the mechanism of interacation between nanomaterials and biofilms.

Abstract:Francisella is a gram-negative fastidious bacteria. F. tularensis, the type species of Francisella genus, causes a zoonotic disease tularemia. Francisella was isolated from soil, grass, moor and infected animals. It can spread with aerosols, infected arthropod, contaminated water and food. In recent years, several species of Francisella were identified, including F. guangzhouensis and other potential pathogenic species, isolated from air-conditioning cooling water in Guangzhou. However, the identification and epidemic risks of these genus were unclear yet. This review summarized the recent advances in the classification, identification and epidemic of Francisella.

Abstract:Human gastrointestinal is a diversified and live microecological system, and a body site of scientific interest. Intestinal tract comprises 100 trillion and nearly 1 000 to 1 150 bacterial phylotypes that interact and influence the health of the human host. The influences could be positive or with potential threats. In this paper, the relationship between gastrointestinal microbiota and diseases, the effect of probiotics on human health, and the new approach of researching on the gastrointestinal microbiota are reviewed to provide references for domestic researchers, and to raise the awareness of general public regarding gastrointestinal health

Abstract:Endophytes are widely distributed in roots, stems and seeds of tobacco plants. It has been well documented that tobacco plants infected by endophytes are generally more competitive compared with uninfected plants, as indicated by better growth, stronger resistance to diseases and pests, and lower content of nitrosamines. This paper reviewed recent research progresses in biodiversity of endophytes in tobacco plants and their important role in tobacco cultivation, and summarized the functioning mechanisms of endophytes. Finally, the weaknesses in current research were discussed, and research perspectives were also proposed.

Abstract:Lycopene is a member of important carotenoids with antioxidation, anti-aging, immunity enhancement, and other physiological functions. Although commercial production of lycopene has been realized in some enterprise, poor lycopene production remains the key restricting factor for lycopene biosynthesis by Blakeslea trispora. Combining with the latest achievements of lycopene production by B. trispora, this mini-review summarized the genetic breeding of strain, the optimization of fermentation process, chemical regulation, and the prospects of future research.

Abstract:[Objective] Bartonella bacilliformis is a fastidious haemotropic gram-negative bacterium, an etiological agent of a life-threatening illness, termed Carrion’s disease or bartonellosis. We developed a rapid, highly sensitive and specific assay, based on real-time fluorescence quantitative PCR for identifying B. bacilliformis. [Methods] A primer pair and probe set for B. bacilliformis were designed based on a species-specific gene of B. bacilliformis, based on bioinformatics method by scanning and comparing of all bacteria on GenBank data. The annealing temperature and the final concentration of the TaqMan probe and primers were optimized. The amplification product of the target gene was cloned into pEASY-T vector for preparing the standard and making a standard curve. The standard curve was made using 10×dilution series of the reference plasmid and the efficiency and linearity of PCR amplification were analyzed. Specificity, sensitivity and reproducibility of the PCR system were assessed. [Results] The optimized annealing temperature is 60 °C and the probe and primer concentration are 200 nmol/L for 20 μL reaction system. The TaqMan probe-based fluorescence quantitative PCR did not show cross reactivity with other Bartonella species, non-Bartonella bacteria and the other animals. The correlation coefficient R2 and E-value of the standard curve were 1.0 and 98.18%, respectively. [Conclusion] The real-time PCR with TaqMan-MGB probe assay is highly specific and sensitive for the detection of B. bacilliformis.