Abstract:Salmonella is a Gram-negative facultative intracellular pathogen that can infect vast array of hosts and cause a series of diseases, sometimes even life-threatening systemic diseases. As an indispensable virulence determinant associated with the systemic infections, Salmonella pathogenicity island 2 (SPI2) encodes type III secretion system 2 (T3SS2) which is induced after invasion, and the T3SS2 secreted effectors are essential for Salmonella to survive and replicate inside various cell types. In recent years, this issue remains the focus of pathogenic research. This review focuses on the aspects of gene characterization of SPI2, regulation of SPI2 gene expression, the structure and assembly of T3SS2, the T3SS2 effectors and some vaccine candidates associated with T3SS2 to present the current understanding of Salmonella T3SS2.

Abstract:Arthrobacter species are found ecologically diverse and can survive in various environments. Many strains of these species have metabolic versatility and can degrade many environmental pollutants. Arthrobacter species are thought to play important roles in catabolism of environmental pollutants in nature. In recent years, the genomes of many Arthrobacter strains have been sequenced, which provides comprehensive information to clarify the molecular mechanisms related to environmental adaptability of Arthrobacter species. These genomics findings revealed several features that are commonly observed in Arthrobacter strains allowing for survival under stressful conditions. These include an array of genes associated with sigma factors and responses to oxidative, osmotic, starvation and temperature stresses. The genomics basis of their environmental adaptability are reviewed, which is expected to provide useful information for applying Arthrobacter strains in pollution remediation and shed some light on other bacterial environmental adaptability researches.

Abstract:[Objective] To reveal the relationship between Deuteromycetes community and the environmental in Kiaochow Bay of the Yellow Sea. [Methods] Using recorded pollution survey, we used molecular methods to study seasonal and spatial variation of Deuteromycetes community diversity in different polluted waters of Kiaochow Bay of the Yellow Sea, China. [Results] Denaturing gradient gel electrophoresis fingerprints varied obviously among different sites of similar level of pollution. Moreover, sequence analysis of recovered dominant bands exhibited the existence of plenty of uncultivable fungi, among which Penicillium was the dominant genus. Furthermore, in heavily polluted estuary, there were abundant animal pathogens such as amoeba and Pythium as well as Deuteromycetes. These discoveries demonstrate that the Deuteromycetes community structure is closely related to marine environment, and are indicative of different level of marine contamination. [Conclusion] The relationship between Deuteromycetes community and different level of pollution and seasons varied were closely related.

Abstract:[Objective] We studied the influences of water pressure and temperature on denitrification, and detected its nitrogen removal characteristics for providing evidence to remediate the micro-polluted reservoir source water. [Methods] Mixed oligotrophic aerobic denitrification bacteria was obtained through enrichment, domestication, and screening processes, which was isolated from sediment in the source water reservoir; and the nitrogen removal characteristics was detected by an in-situ biological inoculation experiment (DO at 3-8 mg/L). [Results] Nitrate of the hard flask system (with water pressure influence) was removed completely, however, at 0.5, 5 m water layer, the nitrate removal rate of the soft flask reached 90.66%, 100%, other layers reached 99.61%, 80.55%, 67.01%, 64.73%. No nitrite accumulated. Because of bacteria death, ammonia had a slight increase. At the end of the experiment, in the 0.5, 5.0, 7.5, 10.0, 12.5 and 15.0 m water layer, the total nitrogen removal rates of hard flask reached 50.11%, 61.49%, 56.24%, 44.50%, 36.80% and 38.73%, however, that of soft system reached 33.47%, 60.61%, 43.98%, 36.28%, 27.52% and 28.57%. OD₆₀₀ and pH first rose and then dropped. The mixed bacteria had prominent nitrogen removal ability between 11℃ and 30℃. [Conclusion] The mixed bacteria have a strong adaptability to temperature and the water pressure has a disadvantage to the nitrogen removal.

Abstract:[Objective] To investigate yeasts diversity in Qilu Lake and analyze the influence of environmental factors on yeast diversity. [Methods] Yeasts were isolated by in-situ cultivation and analyzed for the D1/D2 domain of large subunit (26S) ribosomal DNA and morphological characterization. We analyzed yeast species richness and species distribution in the Qilu Lake. [Results] In total 321 isolated yeasts were identified to 14 genera and 27 species. Rhodosporidium kratochvilovae and Aureobasidium pullulans were the dominating species in the lake, representing 29.6% and 16.8% of the total strains, respectively. Principal component analysis (PCA) showed that the total phosphorus was an important environmental factor affecting the distribution of Rhodosporidium and the pH affected Cryptococcus yeasts distribution. [Conclusion] There was a high diversity of yeasts community in the Qilu Lake.

Abstract:[Objective]To explore the diversity of species and biological activities of the endophytes from Huperzia serrata that is a wild medicinal plant under state protection (category ii), and to discover and collect endophytic bacteria from medicinal plants. [Methods]Huperzia serrata samples were collected from Sichuan and Fujian Provinces. Culture-dependent method was used to obtain endophytes from the surface-sterilized plant samples. The diversity of the isolates was analyzed according to the 16S rRNA gene sequences information. Jaccard index, Shannon-wiener Index, Simpson Index and Pielou Index were calculated. Then six screening models were followed to study the physiological activities of the isolates, based on which we evaluated the diversity of biological activities of the endophytes from Huperzia serrata and their potential medicinal value. [Results] A total of 356 endophytic bacteria were purified from Huperzia serrata, and the analysis results of their 16S rRNA gene sequences showed that they affiliated to 41 genera of 26 families in the phyla Actinobacteria, Firmicutes and Proteobacteria. The numbers and biodiversity indexes of endophytes from the aboveground part and belowground part of Huperzia serrata were approximately equivalent. Among them 11 potential novel species belonged to the genera Amycolatopsis, Angustibacter, Arthrobacter, Curtobacterium, Frondihabitans, Glaciihabitans, Jatrophihabitans, Luteimicrobium, Massilia, Naumannella and Tardiphaga, and 1 novel genus of the family Dermacoccaceae was discovered. The screening for anti-microbial results from these 356 isolates were as follows: the activity rates of against Enterococcus faecalis, Klebsiella pneumonia, Mycobacterium smegmatis and Xanthomonas campestris were 9.0%, 1.4%, 2.2% and 0.8% respectively. Of them 4.5% exhibited activities on the screening model of statins-like antihyperlipidemics showing inhibition of Sporobolomyces salmonicolor SS04; 8.6% of them had the activities of against HIV-1. In total, the fermentation broths from 74 strains exhibited activities on at least one screening model, the positive rate among the isolates was 20.8%. [Conclusion] This study demonstrated that the endophytic bacteria from Huperzia serrata were of great significant bio-diversity and antibiotic diversity, therefore, they could be an ideal microbial resource for further discovery of new natural products.

Abstract:[Objective] In order to investigate effects of leucyl aminopeptidase on mycelia morphology, growth rate, spinosad yield and protein expression in Saccharopolyspora spinosa by disrupting its encoding gene pepA and analyzing the characteristics of engineered S. spinosa. [Methods] The pepA gene of S. spinosa was amplified based on the conserved sequence and cloned into Escherichia coli-Streptomyces shuttle vector pOJ260 to generate pOJ260-pepA, which was transformed into S. spinosa by conjugation. Mycelium observation, SDS-PAGE and HPLC were used to analyze the engineered strain. [Results] Mycelia in S. sp-ΔpepA displayed a much higher degree of fragmentation and fewer branches compared to that of parental strain. Meanwhile, the growth rate of S. sp-ΔpepA was retarded and its biomass was reduced. Shake-flask fermentation demonstrated that spinosad yield increased by 122% in S. sp-ΔpepA strain compared to that of parental strain. SDS-PAGE analysis showed that protein expression profile of the engineered strain significantly changed. [Conclusion] The pepA gene negatively regulates the biosynthesis of spinosad and disruption of pepA gene could affect the mycelial morphology and growth of S. spinosa.

Abstract:[Objective] We studied the effects of nalidixic acid, norfloxacin and kanamycin on the transfer frequency of SXT/R391 element ICEValA056-1 in Vibrio alginolyticus. [Methods] The circular ICEValA056-1 in V. alginolyticus A056 was detected by PCR. Conjugation experiments were conducted between V. alginolyticus A056 and Escherichia coli VB111 to explore the frequency variation of the integrating conjugative elements transfer after donor strain A056 was cultured in Luria Broth containing nalidixic acid or norfloxacin or kanamycin in different concentrations for 15 min or 30 min. [Results] Circular ICEValA056-1 was detected in V. alginolyticus A056, indicating that ICEValA056-1 had the potential to transfer. Treatment with 40 μg/mL nalidixic acid for 30 min increased the transfer frequency of ICEValA056-1 to19.59 folds. Treatment with 50 μg/mL norfloxacin for 15 min increased the transfer frequency of ICEValA056-1 to 31.25 folds. The transfer frequency of ICEValA056-1 had no significant changes under treatment with different concentrations of kanamycin for 30 min. [Conclusion] This study indicates that some antibiotics can obviously increase the transfer frequency of ICEValA056-1, and that antibiotics abuse and arbitrarily discharge might intensify dissemination of integrating conjugative elements from V. alginolyticus to other bacteria.

Abstract:[Objective] To identify the function of glucose dehydrogenase (GDH) and gene expression level in the process of solubilizing phosphorus. [Methods] Phosphate solubilizing bacteria (PSB) were isolated and purified by soluble phosphorus circle method, and identified by Vitek 2 system and 16S rRNA sequence. The phosphate solubilization capacity and GDH activity of PSB were determined. GDH genes were cloned by PCR and the relative expression level of both genes under different conditions were determined by real-time quantitative PCR. [Results] Two PSB were identified as Pseudomonas sp. and Enterobacter sp. and the highest phosphorus solubilizing capability was 558 μg/mL for the former and 478 μg/mL for the latter. GDH genes of the two bacteria were cloned and the fragments were 2007 bp and 2066 bp. Different GDH activity and GDH gene expression were cultivated under the condition of different phosphorus sources and pH value. GDH gene expression of strain wj1 was higher than the other under high phosphorus, and the result was opposite under phosphorus stress. However, GDH gene expression of strain wj3 was lower in all phosphorus levels. The expression of GDH gene and the change of the enzyme activity were not obviously related with phosphorus solubilizing capability for strain wj3. [Conclusion] There were different characteristics of GDH activity and GDH gene expression in two isolated strains that have different phosphate solubilizing mechanisms.

Abstract:[Objective] The purpose of the study was to reveal geographic region-related Acidithiobacillus spp. distribution and allopatric speciation. Phylogenetic and diversity analysis was done to expand our knowledge on microbial phylogeography, diversity-maintaining mechanisms and molecular biogeography. [Methods] We amplified 16S rRNA gene and RubisCO genes to construct corresponding phylogenetic trees based on the sequence homology and analyzed genetic diversity of Acidithiobacillus spp.. [Results] Thirty-five strains were isolated from three different regions in China (Yunnan, Hubei, Xinjiang). The whole isolates were classified into five groups. Four strains were identified as A. ferrivorans, six as A. ferridurans, YNTR4-15 Leptspirillum ferrooxidans and HBDY3-31 as Leptospirillum ferrodiazotrophum. The remaining strains were identified as A. ferrooxidans. Analysis of cbbL and cbbM genes sequences of representative 26 strains indicated that cbbL gene of 19 were two copies (cbbL1 and cbbL2) and 7 possessed only cbbL1. cbbM gene was single copy. In nucleotide-based trees, cbbL1 gene sequences of strains were separated into three sequence types, and the cbbL2 was similar to cbbL1 with three types. Codon bias of RubisCO genes was not obvious in Acidithiobacillus spp.. [Conclusion] Strains isolated from three different regions in China indicated a great genetic diversity in Acidithiobacillus spp. and their 16S rRNA/RubisCO genes sequence was of significant difference. Phylogenetic tree based on 16S rRNA genes and RubisCO genes was different in Acidithiobacillus spp..

Abstract:[Objective] We aimed to detect the CRSPR-Cas system of six Streptococcus thermophilus. [Methods] Bioinformatics method was used to predict CRSPR-Cas system of nine S. thermophilus that published in National Center for Biotechnology Information. Four primers were designed according to the flanking sequences of standard strains and the CRISPR-Cas system of six S. thermophilus have been detected by PCR method. [Results] S. thermophilus S4 had a Cas9 gene, others all had Cas9 gene, Cas10 gene and Cas9^* gene. In addition, 79 and KLDS3.0207 still had Cas3 gene. [Conclusion] Signature genes amplification of CRSPR-Cas system could predict the type of CRSPR-Cas system in unsequenced strains, these findings will help establish the foundation for the study of CRSPR-Cas system in lactic acid bacteria.

Abstract:[Objective] The purpose of this study was to study the distribution, diversity and cytotoxic activity of endophytic bacteria of Sonneratia apetala collected from Maowei Sea, Qinzhou city. [Methods] The 16S rRNA gene sequencing and MTT were used to explore the diversity and cytotoxic activity of endophytic bacteria isolated from different organs and tissues of Sonneratia apetala. [Results] Total of 38 isolates were obtained. The result of diversity analysis showed that these isolates could be phylogenetically classified into 21 genera and 12 families, based on their 16S rRNA gene sequencing. Of them 5 were potential new genera or new genus. Five strains (R74, R71, S92, S85 and S84) had cytotoxic activity against human liver carcinoma Hep G2 cell line. [Conclusion] Endophytic bacteria of Sonneratia apetala are genetically diverse and most of them have abundant new bioactivities.

Abstract:[Objective] Huperzine A (HupA) was approved as a drug for the treatment of Alzheimer's disease. The HupA biosynthetic pathway was started from lysine decarboxylase (LDC), which catalyzes lysine to cadaverine. In this study, we cloned and expressed an LDC gene from a HupA-producing endophytic fungus, and tested LDC activities. [Methods] An endophytic fungus Shiraia sp. Slf14 from Huperzia serrata was used. LDC gene was obtained by RT-PCR, and cloned into pET-22b(+) and pET-32a(+) vectors to construct recombinant plasmids pET-22b-LDC and pET-32a-LDC. These two recombinant plasmids were transformed into E. coli BL21, cultured for 8 h at 24℃, 200 r/min with 1×10^-3 mol/L IPTG into medium to express the LDC proteins, respectively. LDC proteins were purified by Ni²⁺ affinity chromatography. Catalytic activities were measured by Thin Layer Chromatography. At last, the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software. [Results] LDC and Trx-LDC were expressed in E. coli BL21 successfully. SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 kDa and 42.7 kDa respectively, which are consistent with bioinformatics analysis. In addition, TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities. [Conclusion] This work can provide fundamental data for enriching LDC molecular information and reveal the HupA biosynthetic pathway in endophytic fungi.

Abstract:[Objective] We studied the difference of bacterial community composition among glacial snow, moraine deposits and glacial soil on Chongce Ice Cap of West Kunlun Mountains. [Methods] Based on traditional culture-dependent and 16S rRNA sequence analysis, we analyzed the community structure of bacteria on the level of genus and phylum. [Results] Results show that glacial bacteria were composed of Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes on the phylum level. Glacial snow was dominated by Proteobacteria, whereas glacial soil and moraine deposits were dominated by Actinobacteria. On the genus level, glacial soil was dominated by Arthrobacter, while glacial snow was dominated by Methylobacterium, Modestobacter, Hymenobacter, Brevundimonas and Bacillus. Bacterial composition was similar between glacial soil and moraine deposits, but different from glacial snow. Skermanella may be unique on Chongce Ice Cap. [Conclusion] Our study indicated the vulnerability of bacterial diversity in glacial snow with glacial retreat, and the importance of bacterial resources preservation on glacial snow environments.

Abstract:[Objective] We isolated and screened endophytic and rhizosphere bacteria with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase from halophyte Limonium sinense (Girard) Kuntze collected from Jiangsu coastal area and investigated their diversity and plant growth promoting potential. [Methods] Strains were obtained from inner tissues and rhizosphere soils using pure culture cultivation method and identified by 16S rRNA gene sequencing and phylogenetic analysis. Their potential plant growth promoting index of nitrogen fixation, phosphate solubilization, indoleacetic acid (IAA) production and NaCl tolerance ability were evaluated. [Results] Eighteen strains with ACC deaminase were obtained and 13 of them exhibited more than 20 nmol α-KA/(mg Pr·h) ACC deaminase activity. Nine isolates produced IAA, 11 had nitrogen fixation ability and 7 of them had phosphate solubilization ability. Most of the isolates could grow under 0%-13% NaCl. The results of 16S rRNA sequencing showed that these strains belonged to seven genera, with Arthrobacter as the most predominant genus. Among them, strain KLBMP 5180 was found to be a potential novel species of the genus Arthrobacter. [Conclusion] The halophyte plants Limonium sinense (Girard) Kuntze located in the area of coastal shoal contain a variety of symbiotic bacteria with ACC deaminase as well as the source of novel species. Some of them had good research prospect in the future.