Rapid Identification of Legionella Pathogenicity by Surface-Enhanced Raman Spectroscopy.

OBJECTIVE: To establish Surface-enhanced Raman Spectroscopy (SERS) can be used as a rapid and reliable method to distinguish virulent strain and mild strain of L. pneumophila. METHODS: Mortality data were collected from company departments through administrative documents, death certificates, etc. Trend analyses of cancer mortality were performed on the basis of 925 cancer deaths between 2001 and 2010. RESULTS: Our results indicated that the peaks of high virulence strains reached ⋝4000. This criterion was verified by subsequent cell experiments. In addition, we also conducted SERS rapid identification on the virulence of several collected clinical strains and obtained accurate results. CONCLUSION: The present study indicates that the established SERS protocol can be used as a rapid and reliable method to distinguish virulent and mildly virulent strains of L. pneumophila, which can be further used in clinical samples.

The periplasmic PDZ domain-containing protein Prc modulates full virulence, envelops stress responses, and directly interacts with dipeptidyl peptidase of Xanthomonas oryzae pv. oryzae.

PDZ domain-containing proteases, also known as HtrA family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell-envelope stress responses. These proteases have diverse functions through proteolysis- and nonproteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes revealed that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidence demonstrating that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell-envelope stress responses.

Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing.

Bacillus amyloliquefaciens has been a major workhorse for the production of a variety of commercially important enzymes and metabolites for the past decades. Some subspecies of this bacterium are recalcitrant to exogenous DNA, and transformation with plasmid DNA is usually less efficient, thereby limiting the genetic manipulation of the recalcitrant species. In this work, a methodology based on electro-transformation has been developed, in which the cells were grown in a semicomplex hypertonic medium, cell walls were weakened by adding glycine (Gly) and DL-threonine (DL-Thr), and the cell-membrane fluidity was elevated by supplementing Tween 80. After optimization of the cell-loosening recipe by response surface methodology (RSM), the transformation efficiency reached 1.13 ± 0.34 × 10(7) cfu/µg syngeneic pUB110 DNA in a low conductivity electroporation buffer. Moreover, by temporary heat inactivation of the host restriction enzyme, a transformation efficiency of 8.94 ± 0.77 × 10(5) cfu/µg DNA was achieved with xenogeneic shuttle plasmids, a 10(3)-fold increase compared to that reported previously. The optimized protocol was also applicable to other recalcitrant B. amyloliquefaciens strains used in this study. This work could shed light on the functional genomics and subsequent strain improvement of the recalcitrant Bacillus, which are difficult to be transformed using conventional methods.

[Pathway analysis for production of L-leucine by Brevibacterium flavum TK0303].

Brevibacterium flavum is used for the production of a number of amino acids in the biotechnology industry. The yield of producing a metabolite is ultimately limited by the ability of the central metabolism and the desired biosynthesis pathway. Pathway analysis is a very useful tool for metabolic engineering, which can be applied to increase the yield of a metabolite or channeling a metabolite into desired pathways. It does not require any kinetic parameters and only uses the Stoichiometric equations. Pathway analysis for production of L-leucine by Brevibacterium flavum TK0303 at steady state was conducted in this paper. Theoretical yield and flux distribution for optimal pathway were determined. It is also concluded that pyruvate and acetyl-coenzyme A are the key nodes of the L-leucine biosynthesis pathway by analyzing the flux distributions of different modes. According to the pathway analysis, the production of L-leucine is expected to be raised by strengthening the flux of the key nodes (pyruvate and acetyl-coenzyme A) through changing the environmental factors. Because the flux of TCA cycle in Brevibacterium flavum TK0303 is weak, the production of L-leucine must be provided enough amido by adding glutamic acid to the fermentation medium. NH4Ac is both a carbon source and a nitrogen source, which could be helpful to the production of L-leucine. The effects of glutamic acid and NH4Ac on the production of L-leucine were further studied. The production of L-leucine increased 56% by adding glutamic acid. By improving the concentration of NH4Ac, the biosynthesis of L-leucine was greatly strengthened too. The results indicate that the flux of L-leucine can be largely increased by changing the chemical regulatory factors such as NH4Ac and glutamic acid and the modes established by pathway analysis prove to be efficient to describe the metabolic network of L-leucine production by Brevibacterium flavum TK0303.

Isolation and identification of a novel polysaccharide-peptide complex with antioxidant, anti-proliferative and hypoglycaemic activities from the abalone mushroom.

A novel antioxidant polysaccharide-peptide complex LB-1b from the fruiting bodies of the edible abalone mushroom (Pleurotus abalonus) was purified and identified. The structural characteristic of LB-1b was identified by FTIR (Fourier-transform IR), 13C NMR and 1H NMR spectroscopy. LB-1b is a polysaccharide-peptide complex that contains glucose, rhamnose, glucuronic acid and galactose in the molar ratio of 22.4:1:1.7:1.6 and the N-terminal sequence of its peptide moiety has also been determined. The N-terminal amino acid sequence of LB-1b, IPKERKEFQQAQHLK, showed some resemblance to antioxidant enzymes. LB-1b exhibited high antioxidant activity in erythrocyte haemolysis in vitro and the anti-proliferative activity towards hepatoma HepG2 cells and breast cancer MCF7 cells with an IC50 of 24 and 14 µM respectively. LB-1b also demonstrated hypoglycaemic activity in drug-induced diabetic mice and anti-HIV-1 RT (reverse transcriptase) with an IC50 value of 12.5 µM.

Isolation of a polysaccharide with antiproliferative, hypoglycemic, antioxidant and HIV-1 reverse transcriptase inhibitory activities from the fruiting bodies of the abalone mushroom Pleurotus abalonus.

OBJECTIVE: The intent of this study was to purify and characterize a polysaccharide named LA from the fruiting bodies of the edible mushroom Pleurotus abalones. METHODS: The 120-kDa polysaccharide was obtained by extraction with boiling water, ethanol precipitation, ion exchange chromatography on Diethylaminoethyl-cellulose and gel filtration on Sephadex G-200. KEY FINDINGS: The LA was composed of glucose, rhamnose, glucuronic acid, xylose, galactose and arabinose in the molar ratio of 26.3:2.7:1:1.4:1.8:1.2. The FITR and 1H-NMR spectrum of LA disclosed that it was a saccharide with an α-configuration. Its 13C-NMR spectrum revealed that its main chain was [→6)-α-D-Gly(1→]n. The LA exhibited antioxidant activities, especially in scavenging 1,1- diphenyl-2-picryl-hydrazyl radicals and hydroxyl radicals. It manifested antiproliferative activity towards breast cancer MCF7 cells with an IC50 of 3.7 µm, and also exerted some antiproliferative activity against HepG2 cells. It manifested a hypoglycemic action on diabetic mice. It inhibited HIV-1 reverse transcriptase with an IC50 of 8.7 × 10(-2) µm. CONCLUSIONS: The polysaccharide from the abalone mushroom.

Optimization of fermentation conditions for the biosynthesis of L-threonine by Escherichia coli.

In this study, the fed-batch fermentation technique was applied to improve the yield of L-threonine produced by Escherichia coli TRFC. Various fermentation substrates and conditions were investigated to identify the optimal carbon source, its concentration and C/N ratio in the production of L-threonine. Sucrose was found to be the optimal initial carbon source and its optimal concentration was determined to be 70 g/L based on the results of fermentations conducted in a 5-L jar fermentor using a series of fed-batch cultures of E. coli TRFC. The effects of glucose concentration and three different feeding methods on the production of L-threonine were also investigated in this work. Our results showed that the production of L-threonine by E. coli was enhanced when glucose concentration varied between 5 and 20 g/L with DO-control pulse fed-batch method. Furthermore, the C/N ratio was a more predominant factor than nitrogen concentration for L-threonine overproduction and the optimal ratio of ammonium sulfate to sucrose (g/g) was 30. Under the optimal conditions, a final L-threonine concentration of 118 g/L was achieved after 38 h with the productivity of 3.1 g/L/h (46% conversion ratio from glucose to threonine).