Screening for Candidate Genes Associated with Biocontrol Mechanisms of Bacillus pumilus DX01 Using Tn5 Transposon Mutagenesis and a 2-DE-Based Comparative Proteomic Analysis.

A total of 1467 mutants of the biocontrol bacterium Bacillus pumilus DX01 were obtained by Tn5 insertional mutagenesis and subjected to the determination of antagonistic capabilities. Compared with the wild-type strain DX01, the mutant M25 was identified to have the most significant reduction in antagonistic capability against the phytopathogen Bipolaris maydis and extracellular proteinase activity. The integration site of the exogenous T-DNA in the genome of mutant M25 was revealed in the coding region of malony CoA-ACP transacylase (MCAT) gene (mcat), which belongs to a polyketide synthase (PKS) gene cluster, DX01pks of B. pumilus DX01. Furthermore, the whole DX01pks gene cluster was cloned using Illumina Solexa sequencing technology, and it has a modular framework different from the other two gene clusters involved in polyketide synthesis in B. amyloliquefaciens FZB42 (pks1) and B. subtilis 168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol of B. pumilus DX01, the changes in the relative level of expression of total proteins between the original strain DX01 and the mutant M25 were detected by 2-DE-based proteomic analysis. A total of twenty differentially expressed proteins were identified upon the mcat gene transposition mutagenesis. Of these proteins, seven proteins were up-regulated in expression level and the other proteins were down-regulated. Taken together, the results in this study showed that Tn5 transposon mutagenesis of B. pumilus DX01 can lead to a significant change of antiphytopathogen ability, and the DX01pks gene cluster possibly play a potential role in the biocontrol processes of this bacterium.

Isolation, characterization, genomic sequencing, and GFP-marked insertional mutagenesis of a high-performance nitrogen-fixing bacterium, Kosakonia radicincitans GXGL-4A and visualization of bacterial colonization on cucumber roots.

A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology. The analyses of average nucleotide identity based on BLAST+ (ANIb) and correlation indexes of tetra-nucleotide signatures (Tetra) showed that the NF isolate GXGL-4A is closely related to the Kosakonia radicincitans type strain DSM 16656. Therefore, the isolate GXGL-4A was eventually classified into the species of Kosakonia radicincitans and designated K. radicincitans GXGL-4A. A high consistency in composition and gene arrangement of nitrogen-fixing gene cluster I (nif cluster I) was found between K. radicincitans GXGL-4A and other Kosakonia NF strains. The mutants tagged with green fluorescence protein (GFP) were obtained by transposon Tn5 mutagenesis, and then, the colonization of gfp-marked K. radicincitans GXGL-4A cells on cucumber seedling root were observed under fluorescence microscopy. The preferential sites of the labeled GXGL-4A cell population were the lateral root junctions, the differentiation zone, and the elongation zone. All these results should benefit for the deep exploration of nitrogen fixation mechanism of K. radicincitans GXGL-4A and will definitely facilitate the genetic modification process of this NF bacterium in sustainable agriculture.

[Correlation between polymorphisms in the human leukocyte antigen-DQB1 alleles and hepatitis B with primary hepatocellular carcinoma].

OBJECTIVE: To investigate the correlation between polymorphisms in human leukocyte antigen (HLA)-DQB 1 and primary liver cancer (PLC) with hepatitis B virus (HBV) and to search for susceptibility and resistance genes related to PLC with HBV. METHODS: One hundred and eighteen patients with HBV-related liver cancer were enrolled from the First Hospital of Shanxi Medical University. Patients were stratified by family history of hepatitis B (39 with; 79 without) and HBV DNA positivity (60 positive, ≥1*10(3) IU/mL; 58 negative, <1*10(3) IU/mL). The HLA-DQB 1 genotype was determined by PCR and direct nucleotide sequence analysis genotyping. Allele frequencies were calculated by the direct counting method. Betweengroup comparisons were carried out with the Chi-square test or Mann-Whitney U test. RESULTS: The allele frequencies of HLA-DQBl*0202 and HLA-DQBl*0301 were significantly higher in patients with hepatocellular carcinoma (HCC) than the control group (1 1.8% and 29.3% vs. 7.6% and 21.1%; U=2.43 and 3.09, P<0.05, RR=1.581 and 1.477). The allele frequencies of HLA-DQB1*0202 and HLADQB 1*0301 were significantly higher in patients with HCC and familial history of hepatitis B than in the normal population (14.1% and 29.5% vs. 7.6% and 21.1%; U=3.76 and 3.16, P less than 0.05, RR=1.928 and 1.495). The allele frequency of HLA-DQB 1*0301 was significantly higher in the HBV DNA positive group than in the HBV DNA negative group (35.0% vs. 23.3%; x2=5.543, P less than 0.05, RR=1.775), while the frequency of HLA-DQB1*0302 was significantly lower in the HBV DNA positive group than in the HBV DNA negative group (10.9% vs. 14.7%; x2=4.604, P<0.05, RR=0.229). CONCLUSIONS: The HLA-DQB 1 *0202 and HLA-DQB 1*0301 alleles may represent susceptibility for PLC with hepatitis B as well as for familial hepatitis B liver cancer. The HLA-DQB 1*0301 allele may support replication of HBV DNA, facilitating progression to liver cancer. The HLA-DQB1*0302 allele may inhibit replication of HBV DNA and reduce the incidence of liver cancer.

Biosynthesis of Resveratrol in Blastospore of the Macrofungus Tremella fuciformis.

Tremella fuciformis is a known edible macrofungus that has medicinal value. It is widely cultivated in China and its products are distributed worldwide. In this study, a novel bioconversion system was established to produce resveratrol in the blastospore of T. fuciformis (bTf). The expression vector ptro1-4cl-rs that contains 4-coumarate:coenzyme A ligase gene (4cl) and resveratrol synthase gene (rs) was transformed into bTf by LiAc/PEG-mediated transformation. PCR and southern blotting analysis verified the successful integration of the exogenous 4cl and rs genes into the genome of bTf. HPLC analysis confirmed that two transformants can convert p-coumaric acid into resveratrol (0.92 and 0.83 µg/g resveratrol of dry weight within 7 days). This study is the first to report about the transformation and expression of resveratrol biosynthetic genes in bTf. This research is a significant step toward obtaining resveratrol-producing T. fuciformis strains, which not only satisfy the demand of resveratrol market, but also expand the category of functional food.