Shouwen Chen

Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis.

A new capillary zone electrophoresis (CZE) method for the analysis of dipicolinic acid, a specific component found in spores but not in vegetative cells, was used to determine spore concentration in Bacillus thuringiensis according to the relationship between the spore concentration and the content of dipicolinate. The quantitative relationship was established by using purified spores. Electrolyte conditions that affected the separation efficiency of dipicolinate and the reproducibility were investigated. With 10 mM phosphate, 10 mM ethylenediaminetetraacetic acid and 0.25 mM tetradecyltrimethylammonium bromide at pH 6.2 as the carrier electrolyte, dipicolinate can be determined within 8 min at an applied voltage of -25 kV (anode at detector) and a capillary temperature of 25 degrees C. The method has a high separation efficiency with which the number of theoretical plates is above 300,000 plates m(-1). The relative standard deviations for migration time and peak area are less than 0.5% and 2.0%, respectively. The detection limit for dipicolinate was 10 ng ml(-1), which corresponds to 7.2 x 10(5) spores ml(-1). The method was used to determine spores in fermentation broths, and the results obtained agreed well with the values obtained by plate counting.

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.

Co-producing iturin A and poly-γ-glutamic acid from rapeseed meal under solid state fermentation by the newly isolated Bacillus subtilis strain 3-10

The strain 3-10 was isolated from soil and identified as B. subtilis according to morphological and physiological characteristics and nucleotide sequence of 16S rRNA. It co-produced anti-fungal iturin A and fertilizer synergist of poly-γ-glutamic acid (γ-PGA) under solid state fermentation (SSF) with rapeseed meal. The co-production of iturin A and γ-PGA reached 5.3 and 51.3xa0g/kg-dry weight culture, respectively, and the number of viable cells reached 1.9xa0×xa01010xa0CFU/g-dry weight culture. In pot tests, the shoot length and dry weight of watermelon seedlings treated by the SSF culture improved by 48.0 and 30.8%, respectively compared to the control; and its biocontrol effect on watermelon fusarium wilt achieved 89.6%. These results highlight a novel strategy to exploit the low-cost and widely available rapeseed meal as dual-functional bio-organic fertilizer under SSF by B. subtilis.

Identification of three Zwittermicin A biosynthesis-related genes from Bacillus thuringiensis subsp. kurstaki strain YBT-1520.

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6xa0kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.

Fusion of the genes for AHL-lactonase and S-layer protein in Bacillus thuringiensis increases its ability to inhibit soft rot caused by Erwinia carotovora

Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. To synthesize and express the N-acyl-homoserine lactonase (AHL-lactonase) in the extracellular space of B. thuringiensis, the aiiA4Q7 gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. When compared with wild-type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.

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.

Propanol Addition Improves Natamycin Biosynthesis of Streptomyces natalensis

The effects of the addition of short-chain fatty acids and lower alcohols on the production of natamycin from Streptomyces natalensis F4-245 was investigated, and propanol was found to be the most effective additive. Under the optimal condition of propanol addition, the maximal natamycin titer reached 10.38xa0g/l, which was 17xa0% higher than that of the control. Metabolites analysis showed the concentrations of amino acids and acetyl-CoA were enhanced while those of organic acids in tricarboxylic acid (TCA) cycle were reduced. This work demonstrates that the addition of propanol is an effective strategy to increase natamycin yield through regulating metabolite node and pools of precursors.

Supplementations of ornithine and KNO3 enhanced bacitracin production by Bacillus licheniformis LC-11

During the fermentative process of Bacillus licheniformis LC-11, the dissolved oxygen (DO) and ornithine (Orn) content in the medium fell to zero, indicating that they were the potential limiting factors for cell growth and/or bacitracin biosynthesis. In addition, given that the nitrate-reducing system existing in B. licheniformis could favor cell anaerobiosis, the impacts of KNO3 and Orn supplementations were therefore evaluated for improving bacitracin production in both a shaking flask and a 10-L fermentor. Orn supplementation (0.04xa0g/L) at 16xa0h or KNO3 addition (0.5xa0g/L) in the production medium enhanced bacitracin production by 5.8 and 3.7xa0%, respectively. The combined effects of KNO3 and Orn supplementations were then conducted via a two-level central composite design, resulting in a predicted maximum bacitracin yield of 821.81xa0U/mL if KNO3 and Orn were supplemented at 514.39xa0mg/L and 45.35xa0mg/L, respectively. This predicated yield was then verified experimentally in a 10-L fermentor, achieving a 10.8xa0% increased bacitracin production (825xa0U/mL) over that of the control.