Abstract:PhoPR is an important two-component regulatory system in Mycobacterium tuberculosis. PhoP is essential for virulence as a response regulator involved in cell wall lipid biosynthesis and regulation of gene expression. In this review, the structure, function and vaccine application of PhoP were summarized, as well as some questions that need to be solved.

Abstract:Understanding the biotransformation mechanism of chlorinated hydrocarbons in contaminated site is of great significance to the in-situ bioremediation. Therefore, we summed up the overlapping composition of chlorinated hydrocarbons and analyzed statistically the concentration variations and degradation rate of chlorinated hydrocarbons in various landfill which were regarded as one of the most typical compound pollution sites. The statistical data indicated that chloralkane and chloroalkene concentration ranged 0.20 to 32.45 and 0.50 to 32.45 μg/m³, respectively, which were the main components. We also found that biodegradation rates of chlorinated hydrocarbons decreased with the number of attached chlorine atoms in landfill cover. Then, we summarized the biodegradation mechanism of chlorinated hydrocarbons under different environmental conditions. The results implied that chlorinated hydrocarbons biodegradation incorporated aerobic co-metabolism, halorespiration and anaerobic reductive dechlorination involved in a wide range of substrates and a variety of functional microbes. Based on of these analyses, we constructed biodegradation models of chlorinated hydrocarbons in landfill cover. Finally, the possible development of chlorinated hydrocarbons biological removal in the future was predicated.

Abstract:[Objective] In addition to Streptococcus suis serotype 2, Streptococcus suis serotype 9 (SS9) is also a currently prevalent serotype and a zoonotic pathogen. In our previous study, SS9 DNA nuclease (SsnA) was considered as a candidate virulence factor. To clarify the impact of SsnA on SS9 virulence, we constructed ssnA mutant (ΔssnA) and studied its biological functions. [Methods] We evaluated the virulence of wild type strain and ΔssnA in a zebrafish infection model and compared the adherence rate to HEp-2 cells, the survival rate in pig blood, and enzymatic activity between wild type stain and ΔssnA. [Results] In a zebrafish infection experiment, the 50% lethal dose value of ΔssnA was 11.2-fold higher than that of wild type strain. The adherence rate of ΔssnA to HEp-2 cells was only 60.61% of the wild strain level. The survival rate of ΔssnA in pig blood was declined to 71.88% of wild strain level. The enzymatic activity assay showed that SsnA can degrade both linear and circular DNA. [Conclusion] SsnA contributes to SS9 virulence in a zebrafish infection model, the adherence to HEp-2 cells, and the survival in pig blood. SsnA is indeed an essential virulence factor for SS9.

Abstract:[Objective] I studied the community structure and diversity of culturable moderate halophilic bacteria isolated from Qrhan Salt Lake.[Methods] I isolated and cultured the moderate halophilic bacteria on different selective media. After the 16S rRNA gene sequences was amplified and measured, I constructed the phylogenic tree, analyzed the community structure and calculated the diversity indexes according to the 16S rRNA gene information. [Results] A total of 421 moderate halophilic bacteria were isolated from water and mud samples in Qrhan Salt Lake. The 16S rRNA gene information showed that 4 potential novel species belonged to the family Bacillaceae. Eighty-three model strains belonged to 3 phylurms 6 families 16 genus. Among them, Bacillus sp., Oceanobacillus sp. and Halomonas sp. were dominant species. Diversity analysis showed that the diversity of strains isolated from water sample was higher than that from mud sample, but the dominance degree of strains isolated from mud sample was higher than that from water sample. [Conclusion] The genetic diversity of moderate halophilic bacteria isolated from Qrhan Salt Lake was abundant. Also, there were dominant and novel species of culturable moderate halophilic bacteria in this lake.

Abstract:[Objective] To study the function and mechanism of STM14_3514 gene that encoded in Salmonella pathogenicity island (SPI)-1 of Salmonella enterica serovar Typhimurium strain ATCC 14028. [Methods] We constructed STM14_3514 mutant strain and a complemented strain of the mutant. Through mice experiment, attachment assays, invasion assays, macrophage replication assays, western blot, and Quantitative real-time PCR analysis (qRT-PCR), we compared the virulence of the mutant strain to that of the wild-type 14028. [Results] STM14_3514 mutant shows increased virulence to mice, and the bacterial number of STM14_3514 mutant in liver, spleen, and ileum was more abundant than that of the wild-type strain. The increased virulence of STM14_3514 mutant is caused by its elevated invasion ability to epithelial cells (>2-fold and P<0.05). qRT-PCR and western blot results show that STM14_3514 reduced the expression of HilA and another SPI-1invasion locus. Moreover, the repression of HilA by STM14_3514 is mediated by HilC.[Conclusion] STM14_3514 is a negative regulator in SPI-1, which can repress HilA and SPI-1invasion locus through HilC, and possibly contribute to the repression on SPI-1 after bacterial invasion.

Abstract:[Objective] To study the effect of amphipathic helix characteristics of FtsZ (236-245) domain on FtsZ assembly and interaction of FtsZ with FtsA in Escherichia coli strains. [Methods] We constructed FtsZ and its mutant's plasmids by molecular clone and site-directed mutagenesis, and purified targeted proteins using affinity chromatography. QN23-QN29 strains were constructed by linear DNA homologous recombination and P1 transduction. We observed cellular localization patterns of FtsZ and its mutants in E. coli by living cell imaging experiments, examined membrane binding properties of FtsZ mutants by membrane proteins isolation and Western blot analysis, and analyzed interaction of FtsZ/FtsZ* with FtsA by Co-immunoprecipitation and far Western blot. Native gel separation and in vitro polymerization experiments were done to check effects of FtsZ point mutation on FtsZ assembly. [Results] Yfp-labeled FtsZ^E237A/K and FtsZ^E241A/K mutant proteins failed to localize in E. coli strains, assemble into functional Z-ring structure, and had decreased function of FtsZ (wt). In vitro experiments showed that E237A/K and E241A/K mutations of FtsZ decreased the polymerization efficiency of FtsZ monomer, weakened FtsZ*-FtsA interaction and changed membrane binding properties of FtsZ. [Conclusion] FtsZ E237 and E241 are critical amino acids that affect the amphipathic helix characteristics of FtsZ (236-245) domain, FtsZ assembly and FtsZ-FtsA interaction in E. coli strains.

Abstract:[Objective] We studied the interactions in a co-culture of two bacteria. [Methods] By pairwise co-culturing of 36 Escherichia coli and 36 Staphylococcus aureus strains, we monitored the growth of each species in an interaction environment. We identified numerous Single Nucleotide Polymorphisms (SNPs) by whole-genome sequencing used as genetic markers to predict variations in phenotypic traits. Genome-wide association study (GWAS) was applied to identify loci that controlled competition between the two species. [Results] In E. coli, 162 significant SNPs affected the change of maximum growth rate by comparing initials strains with those grown in co-culture, and 36 significant SNPs affected the change of maximum growth rate comparing monoculture and co-culture strains. Five of the significant E. coli genes we identified after annotation this time were also reported in other evolutionary studies. We also identified 85 significant SNPs in S. aureus that affected the change of maximum growth rate by comparing initial strains with those grown in monoculture. About the change of bacterial numbers, we found that 706 significant SNPs were associated in E. coli and 129 in S. aureus. Thirteen of the E. coli significant genes in this study were also verified in previous evolutionary reports. [Conclusion] We found several significant genes both in monoculture and co-culture affecting the interaction of E. coli and S. aureus. GWAS has the potential to study interspecific interactions of bacteria.

Abstract:[Objective] The objective of the study was to assess the antimicrobial activity of silver nanoparticles (AgNPs) against multiple drug resistant strains. [Methods] Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AgNPs against three model microbes, namely Escherichia coli, Staphylococcus aureus, Candida albicans were measured by microdilution broth method. Time-kill curve within 24 h was made according to colony count method after three model microbes were treated with a series concentration of AgNPs. Post-antibiotic effect was tested by colony count method. Finally, we determined the antimicrobial efficacy against multiple drug resistant strains in biological safety laboratory grade 2 (BSL-2). [Results] AgNPs with a diameter of 5 nm to 30 nm were synthesized by the biological method. The zeta potential was -19.5 mV. The time-kill curve of the three model microbes showed time-dependent antibacterial activity. The effect of AgNPs on E. coli and C. albicans after "antibiotic effect" increased with time, there was no obvious "post-antibiotic effect" on S. aureus. Both MIC values and MBC values of AgNPs for the three model microbes were between 1 μg/mL and 4 μg/mL. However, the MIC value of AgNPs for the three human multidrug-resistant strains was 6 μg/mL to 26 μg/mL and MBC value of AgNPs was 10 μg/mL to 32 μg/mL. The MIC values of AgNPs for 14 animal multi-drug resistant strains were between 4 μg/mL and 10 μg/mL, and the MBC values were between 8 μg/mL and 16 μg/mL. The MBC/MIC values of all the tested strains were less than 2. [Conclusion] AgNPs is a time-dependent antimicrobial agent with different "post-antibiotic effect", which can inhibit both human and animal-derived multi-drug resistant bacteria.

Abstract:[Objective] To study the potential of using glucose as carbon source to produce microalgae biomass and biochemical components, such as photosynthetic pigments, lipids, carbohydrates and proteins by tropical marine microalgae Chloralla sp. HN08.[Methods] We compared the growth characteristics of Chloralla sp. HN08 cells under photoautotrophic and mixotrophic (10 g/L glucose was added into the medium) conditions. The photosynthesis, specific growth rates, cell densities, and the content of cell's major components including lipids, starch, soluble sugar, and soluble protein were determined and compared.[Results] Glucose (10 g/L in medium) could promote Chlorella growth and increase the final cell density under light condition. However, cells declined gradually under heterotrophic condition. Under mixotrophic condition, the specific growth rate and the final cell density were 6.8 and 1.3 times as that of cells under photoautotrophic condition, respectively. The content of soluble sugar, starch, and lipids in mixotrophic cells was also significantly higher (P<0.05) than that in photoautotrophic cells. However, the content of soluble protein and photosynthetic pigments of mixotrophic cells was significantly lower (P<0.05) than that of autotrophic cells. Algae culture with glucose addition showed a higher light saturation as well as respiration rate. No significant difference in net photosynthesis rate was found between autotrophic and mixotrophic cultures (P>0.05). [Conclusion] Under light condition, glucose as a carbon source can promote lipids and starch accumulation, as well as biomass production.

Abstract:[Objective] To study the phylogenetic and genetic heterogeneity of 23 Acidithiobacillus strains from various geographical locations, as well as the relationship between the DNA fingerprinting classification and geographical origin of Acidithiobacillus. [Methods] Partial 16S-23S rRNA gene intergenic spacer (ITS) was used to construct corresponding phylogenetic trees based on the sequence homology. rus gene amplification and rep-PCR assay with two different primers (BOXAIR and ERIC) were performed to analyze genetic heterogeneity of Acidithiobacillus strains from diverse environment. [Results] Acidithiobacillus revealed a great genetic heterogeneity. The whole isolates were classified into five groups by ITS sequence analysis. This result was similar with that obtained by rep-PCR. Acidithiobacillus ferrooxidans strains were always divided into two groups of phylogenetic and BOXAIR fingerprinting cluster analysis. However, these were clustered one group in the ERIC dendrogram. Genotypic analysis of the rus gene suggested that different iron oxidation pathways have been evolved in these closely related bacteria. Taken together, the iron oxidation pathway of Acidithiobacillus and phylogenetic groups have no obvious correlation. ITS gene has been proven very useful in distinguishing closely related species or subspecies of Acidithiobacillus, to BOXAIR-PCR, which has been recommended as reliable tool for genetic heterogeneity analysis of Acidithiobacillus.

Abstract:[Objective] Pooling of multiple samples is widely used in studying general patterns of microbial communities that are heterogeneously structured in space. Pooling strategies and the number of sequence reads generate biases in the description of diversity and community structure of root-associated fungi. Therefore, we developed a molecular toolbox for fast and accurate identification of the root-associated fungal community of Rododendron species. [Methods] Multiple root samples of R. lutescens and R. bureavii were collected for DNA extraction. Effects of two different pooling strategies, i.e. pooling samples prior to vs. post PCR, on fungal species composition were studied by comparing results within host species. [Results] Species richness and Shannon-Wiener index of fungal communities of clone library constructed by pooling samples after PCR were higher than that of pooling prior to PCR. High frequency fungal species were detected by both pooling strategies, whereas infrequent species detected by the two strategies differed. Notably, the prior to PCR pooling strategy effectively alleviated the unwanted amplification of host plant sequences when fungal specific primer ITS1f and ITS4 were used. Accumulation curves of fungal species suggested that sequencing at least 50 clones can fully reflect species composition of clone library of the two Rhododendron root-associated fungal community. [Conclusion] Clone library constructed by post PCR pooling of samples is better in providing accurate views of fungal diversity and community structure of Rhododendron species.

Abstract:[Objective] Three soil types in different salt contents were taken as the experiment objectives. We evaluated the effect of various saline alkali soil types on diversity of bacterial community structure in spermosphere soil during water absorption and germination of peanut seeds.[Methods] The V3-V4 region of 16S ribosomal RNA genes was amplified using PCR, and the PCR products were then analyzed using Illumina high-throughput sequencing technology. [Results] (1) The diversity of soil bacterial community in saline alkali soil was higher than that in non-saline alkali soil. Especially, the highest diversity was in spermosphere soil from Qingtuo. (2) The microflora structures in different soils were distinct at the class level. Soil bacteria in four samples were classified into six classes, including Proteobacteria, Actinobacteria, Actinobacteria, Bacteroidetes, Acidobacteria and Firmicutes. Proteobacteria and Actinobacteria groups were dominant in colonies. The analysis of whole samples colony structure showed that the difference of type and abundance at phylum and genus level during different adsorption time was most significant (P<0.05). (3) The analysis of beta diversity and phylogenetic distances of constructed phylogenetic trees revealed that the sequenced clones fell into two major groups within the domain bacteria.[Conclusion] The diversity of bacteria community compositions in the high salt content soil was higher. There were obvious differences in microbial community structure of different soil types at class level, primarily in the Proteobacteria and Actinobacteria. The type and abundance of microbial colonies at both phylum and genus levels were affected by the seed germination time. However, there was no influence on the genetic distance between the samples from the same soil type.

Abstract:[Objective] To reflect the importance of nitrite reductase (NIR) in the environment, we studied its distribution. [Methods] The sequences of NIR were searched in the sequenced genome database at NCBI based on previous reported NIR sequences. The sequence similarity was done by multiple sequence alignment, and phylogenetic relationship was evaluated via constructing the phylogenetic tree. Furthermore, their distribution in the marine metagenome was studied by metagenomics. [Results] The homologues of these two enzymes were 397 and 812 strains in sequenced genome, and the proportion was 8 and 15.7 percent, respectively. Almost all of archaea containing type II NIR. They have high identity by multiple sequence alignment analysis. The cofactor, the substrate and the cooper binding sites in type II were high conserved, suggesting that these enzymes had the specific function in denitrification. Phylogenetic analysis showed the two enzymes may have the common ancestor. In marine metagenome analysis, type I and II have 6 and 35 reads per 100000 reads, respectively, the two types of NIRs have the biggest proportion at the tropical south pacific area. [Conclusion] Collectively, we suggested NIR, especially type II, play a key role in bioremediation of nitrogen contamination.

Abstract:[Objective] The physiological function of membrane protein RHOGL009301 in Rhodococcus sp. R04 and the metabolic properties of the mutant strain were studied to determine the relationship between the physiological function of the membrane protein and the transport of benzoate. [Methods] The RHOGL009301 gene and the green fluorescent protein gene were fused for expressing in Rhodococcus erythropolis, and the location of RHOGL009301 was observed by Delta Vision. The RHOGL009301 gene was knocked out by homologous recombination, and the growth of wild strain and deficient strain in different carbon sources were compared. The internal and external metabolites of the wild strain and the deficient strain when grown on biphenyl and benzoate were measured by HPLC, and the changes of metabolite concentration in different growth conditions were analyzed. [Results] A fusion gene that contained RHOGL009301 gene and the green fluorescent protein gene was co-expressed in Rhodococcus erythropolis and localized on the cell membrane. The deficient strain R04ΔMP of RHOGL009301 gene was obtained. The biomass of the deficient strain was significantly reduced in biphenyl and benzoate culture, and its growth rate was slowed down. HPLC analysis showed that the deletion of RHOGL009301 gene inhibited the transport of benzoate.[Conclusion] RHOGL009301 membrane protein is one of the proteins involved in metabolism and transport of benzoate. Based on sequence homology analysis, we can conclude that the membrane protein is a novel benzoate transport protein.