Zhixia Ji

Genome Sequence of Bacillus licheniformis WX-02

Bacillus licheniformis is an important bacterium that has been used extensively for large-scale industrial production of exoenzymes and peptide antibiotics. B. licheniformis WX-02 produces poly-gamma-glutamate increasingly when fermented under stress conditions. Here its genome sequence (4,270,104 bp, with G+C content of 46.06%), which comprises a circular chromosome, is announced.

Expression of glr gene encoding glutamate racemase in Bacillus licheniformis WX-02 and its regulatory effects on synthesis of poly-γ-glutamic acid

The glr gene, which encodes glutamate racemase involved in the conversion of l-glutamic acid to its D-isomer, was cloned and expressed in Bacillus licheniformis WX-02. Overexpression of the glr gene not only increased the production of poly-γ-glutamic acid (γ-PGA) by 22.5% but also increased the proportion of d-glutamate in γ-PGA from 77 to 85%. The activity of glutamate racemase was higher than in the original strain throughout cultivation. This is the first report that overexpression of the glr gene could enhance the l- and d-glutamate conversion in B. licheniformis WX-02 and increase the proportion of d-glutamate in γ-PGA and the yield of γ-PGA.

Sample preparation for the metabolomics investigation of poly-gamma-glutamate-producing Bacillus licheniformis by GC–MS

Metabolomics aims to analyze global intracellular metabolites of organisms. To study the intracellular metabolites of poly-γ-glutamate-producing Bacillus licheniformis, the quenching and extraction conditions were assessed and optimized. It indicated that perchloric acid was an appropriate quenching solution for B. licheniformis by measuring livability of cells, leakage of ATP, energy charge and intracellular metabolites. 0.85% NaCl was an appropriate washing solvent for a sample because it did not lead to serious leakage and would not affect operation of GC-MS. Among the four different extraction methods (cold pure methanol, PM; hot ethanol/water (75:25 v/v), HE; cold methanol/water (50:50 v/v), MW; and cold chloroform/methanol/water (50:25:25 v/v/v), CMW), HE was superior to others on the basis of the energy charge and intracellular metabolites, which could effectively inhibit enzymatic activities and extract more intracellular metabolites. The method could obtain some metabolites which were involved in the most important synthesis pathways of poly-γ-glutamate, including glycolysis, citric acid cycle and glutamate metabolism. It is the first evaluation of a sample preparation of poly-γ-glutamate-producing bacteria, which might be used as one model for the preparation of polymer-producing samples for metabolomics analysis.

Decreased Tobacco-Specific Nitrosamines by Microbial Treatment with Bacillus amyloliquefaciens DA9 during the Air-Curing Process of Burley Tobacco

Tobacco specific nitrosamines (TSNA) mainly consisting of N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are a group of toxic components threatening human health. To inhibit TSNA formation in tobacco leaves, a high nitrite reductive strain with low nitrate reduction ability was isolated and applied to tobacco leaves in an attempt to lower the nitrite precursor of TSNA. By morphology, physiology, biochemistry, and 16S rDNA sequence analysis, the strain DA9 was identified as Bacillus amyloliquefaciens. Under the optimized fermentation parameters (glucose 40 g/L, NH4Cl 4 g/L, corn steep liquor 8 g/L, MnSO4 0.01 g/L, KH2PO4 1.0 g/L, MgSO4 0.3 g/L, initial pH 7.0, inoculum age 6 h, inoculum size 3%, temperature 37 °C), the maximum cell dentisity of 1.2 × 10(9) CFU/mL was obtained at 36 h. The DA9 cell suspensions were applied in the air-curing process of the Burley tobacco (Eyan 6) leaves. The treatment by DA9 cells lowered 32% of the nitrite content and 47% of total TSNA content in the tobacco leaves, and the concentrations of the NNN, NNK, and NAT were decreased by 48%, 12%, and 35%, respectively. Collectively, this study provides a promising strain and a novel strategy for decreasing TSNA during the air-curing process.