Insights into copper effect on Proteus hauseri through proteomic and metabolic analyses.

This is the first-attempt to use liquid chromatography coupled with tandem mass (LC-MS-MS) in deciphering the effects of copper ion on Proteus hauseri. Total 941 proteins in copper-addition (+Cu) group and 898 proteins in non-copper-addition (-Cu) group were found, which containing 221 and 178 differential proteins in +Cu and -Cu group, respectively. Differential proteins in both groups were defined into 14 groups by their functional classification which transport/membrane function proteins were the major different part between the two groups, which took 19.5% and 7.7%, respectively. The result of BioCyc and KEGG analyses on metabolic pathway indicated that copper could interrupted the pathway of chemotaxis CheY and inhibited the swarming of P. hauseri, which provided a potential in controlling the pathogenicity of this strain.

Simple, effective protein extraction method and proteomics analysis from polyunsaturated fatty acids-producing micro-organisms.

Polyunsaturated fatty acids (PUFAs) are valuable ingredients in the food and pharmaceutical products due to their beneficial influence on human health. Most studies paid attention on the production of PUFAs from oleaginous micro-organisms but seldom on the comparative proteomics of cells. In the study, three methods (i.e., cold shock, acetone precipitation and ethanol precipitation) for lipid removal from crude protein extracts were applied in different PUFAs-producing micro-organisms. Among the selective strains, Schizochytrium was used as an oleaginous strain with high lipid of 60.3 (w/w%) in biomass. The Mortierella alpina and Cunninghamella echinulata were chosen as the low-lipid-content strains with 25.8 (w/w%) and 21.8 (w/w%) of lipid in biomass, respectively. The cold shock resulted as the most effective method for lipid removed, thus obtained higher protein amount for Schizochytrium. Moreover, from the comparative proteomics for the three PUFAs-producing strains, it showed more significant proteins of up or down-regulation were explored under cold shock treatment. Therefore, the essential proteins (i.e., polyunsaturated fatty acid synthase) and regulating proteins were observed. In conclusion, this study provides a valuable and practical approach for analysis of high PUFAs-producing strains at the proteomics level, and would further accelerate the understanding of the metabolic flux in oleaginous micro-organisms.

Enzymatic exploration of catalase from a nanoparticle producing and biodecolorizing algae Shewanella xiamenensis BC01.

Shewanella xiamenensis (SXM) was found to produce nanoparticles (NPs) under aerobic condition. The oxidoreductase enzymatic activities including of catalase, manganese peroxidase, laccase, NADH dehydrogenase, flavin reductase, azoreductase and Fe reductase are first investigated. Catalase showed the greatest enzymatic activity among all oxidoreductases in SXM, which with strong activities in multiple substrates of ABTS, guaiacol and 2,6-DMP. The optimum temperature, pH, concentrations of H2O2 and 2,6-DMP for this enzyme were found to be 65 °C, pH 4.0, 128.7 mM and 10 mM, respectively. Finally, from the kinetic parameters and structure simulation of catalase, implied that SXM would potentially apply in bioremediation, microbe fuel cells (MFCs) and nano-biotechnology based on its distinguished enzymatic system.

Direct proteomic mapping of Streptomyces roseosporus NRRL 11379 with precursor and insights into daptomycin biosynthesis.

This first-attempt study provided liquid chromatography tandem mass (LC-MS/MS) proteomics approach to explore precursor effects on daptomycin synthesis from Streptomyces roseosporus NRRL 11379. Among all, 357 and 691 differential proteins from 601 proteins in precursor group (144 h+) and 935 proteins in non-precursor group (144 h-) were identified, respectively. Through the simulation of the 2D-protein mapping, most proteins were found in isoelectric points ranged of 4.5-10.0 as well as Mws ranged 10-100 kDa. As a result, LC-MS/MS analysis was consistence with the analytical results of two-dimensional electrophoresis (2DE) but provided much intact profiles of proteins by precursor effect on S. roseosporus. To have more insight exploration, differential proteins associated to Streptomyces spp. were defined into 14 groups of their functional classification. The major differential proteins were in transport/membrane functional group with an occupation of 12.4% for 144 h+ and 5.2% for 144 h-, respectively. LC-MS/MS results as a direct proteomic mapping approach reveal more daptomycin synthetic and regulation-related proteins from precursor group in terms of methyltransferase, ATP-binding cassette (ABC) transporters, resistance proteins and regulators.

Daptomycin antibiotic production processes in fed-batch fermentation by Streptomyces roseosporus NRRL11379 with precursor effect and medium optimization.

Sodium decanoate was first found to be an effective precursor for synthesis of daptomycin from Streptomyces roseosporus NRRL11379 which was increased to 71.55-fold, compared with decanoic acid. The optimal flow rate of precursor was at 600 mg/(L day) after 48 h fermentation. From protein analysis via SDS-PAGE and identification of Tandem MS/MS afterwards, it deciphered that guanosine pentaphosphate synthetase, PNPase, tripeptidylamino peptidase primarily dealing with daptomycin synthesis. By applying Taguchi's L16 in culture optimization, the best yield was obtained from the medium with 60 g/L dextrin, 10 g/L dextrose, 1.0 g/L molasses, and 8 g/L yeast extract, respectively. The fed-batch fermentation, applied with feedback control of dextrin, stimulated the production up to 812 mg/L at 288 h. To our best knowledge, the daptomycin production in this study is significantly higher than that in previous studies and can make it more widely used in pharmaceutical industry.

Exploring metal effects and synergistic interactions of ferric stimulation on azo-dye decolorization by new indigenous Acinetobacter guillouiae Ax-9 and Rahnella aquatilis DX2b.

The first-attempt study deciphered metal-interacting effects on dye-decolorizing capabilities of indigenous bioelectricity-generating strains, Acinetobacter guillouiae Ax-9 and Rahnella aquatilis DX2b. Most of the metallic ions were inhibitory to color removal capabilities of these strains. However, with supplementation of 5 mM ferric chloride, specific decolorization rate (SDR) of Ax-9 increased by 55.48% compared to Fe(3+)-free conditions. In contrast, SDR of DX2b decreased 75.35% due to the inhibition of ferric chloride. On the other hand, ferric citrate could stimulate SDR of DX2b for 21.5% at same dosage. Enzymatic assay indicated that Fe reductase activity was consistent with synergistic effects of ferric chloride on Ax-9, and ferric citrate on DX2b. Protein analysis via SDS-PAGE and identification of Tandem MS/MS afterwards showed that outer membrane protein (Omp) primarily deals with decolorization as a channeling regulation. Moreover, molecular modeling and bioinformatics data also provided detailed evidences to confirm the biological significance of Omp.

Copper ion-stimulated McoA-laccase production and enzyme characterization in Proteus hauseri ZMd44.

The novel bioelectricity-generating bacterium of Proteus hauseri ZMd44 has been first identified to produce McoA-laccase (EC 1.10.3.2) induced by copper sulphate. The optimal concentration of copper is 3 mM as supplementation at the beginning of culture or early exponential growth phase, during which laccase is predominantly synthesized. Moreover, the whole cellular and intracellular activities of laccase increase in the degrees of inducible copper concentrations. A possible mechanism for this phenomenon is that copper ions enhance the laccase genetic transcription level during the laccase synthesis thus granting this strain in copper tolerance. McoA-laccase belongs to typical type 1 (T1) Cu site laccase by electron paramagnetic resonance (EPR) analysis of intracellular enzyme. From our results, the optimal temperature and pH are 60°C and pH 2.2, respectively. The kinetic profiles show that this enzyme is stable under 50°C and in the slightly acidic environment, making it a potentially useful enzyme in dye decolorization, paper-pulp bleaching and bioremediation industries.