Mechanism and application of molecular self-assembly in Sup35 prion domain of Saccharomyces cerevisiae / 生物工程学报

Sup35 in its native state is a translation termination factor in Saccharomyces cerevisiae. The prion domain of Sup35p can form amyloid-like proteinaceous fibrils in vitro and in vivo. Furthermore, the in-register cross beta-sheet structure of Sup35p amyloid fibrils is similar to those formed in other species. Therefore, studies on mechanism of Sup35p self-assembly can be an appropriate model to study protein misfolding-related diseases and prion biology. Because of its ability to self-assemble into nanowires, the prion domain of Sup35p has been widely used in biotechnology and nanotechnology.

[Identification and characterization of novel antimicrobial protein APn5 against Erwinia carotovora].

OBJECTIVE: Amorphophallus soft rot disease, caused by Erwinia carotovora, affects Amorphophallus industry development. We identified and characterized protein APn5 against Erwinia carotovora, isolated from Bacillus subtilis strain BSn5. METHODS: Protein APn5 was purified from BSn5 culture by ammonium sulfate precipitation with 30% relative saturation and ultrafiltration. Inhibition activity was tested by agar well diffusion assay. Protein APn5 was treated at different temperatures, pH conditions and proteinase. The growth curve of BSn5 was examined; meanwhile, the inhibition activity of supernatant of BSn5 culture in different growth phase was tested. Amino acid sequence of protein APn5 was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and quadrupole-time-of-flight (Q-TOF). RESULTS: Protein APn5 showed a narrow inhibition spectrum, mainly strongly inhibiting the growth of a few plant pathogenic bacteria. Protein APn5 was sensitive to high temperature and alkaline pH, and partial sensitive to trypsin, proteinase K and pronase E. During strain BSn5 growth phase, the inhibition activity was unstable, which would gradually lose on stationary phase and disappeared finally. CONCLUSION: On the basis of the difference on inhibition spectrum and characteristics, protein APn5 was suggested as a novel antimicrobial protein.

[Prediction, overexpression and activity confirmation of adenylation domain in Zwittermicin A biosynthesis gene cluster].

OBJECTIVE: The adenylation domain is required for the substrate activation of non-ribosomal peptide synthesis. The objective of this research was to prove that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis. METHODS: We cloned the adenylation domain in the Zwittermicin A synthesis cluster of Bacillus thuringiensis strain YBT-1520 with PCR amplification. After a series of enzyme digestions and subclonings, new expression vectors pBMB1312 was obtained. In order to detect the proper condition for overexpression, we tried different Isopropyl beta-D-1-thiogalactopyranoside (IPTG) concentration and temperature during overepression. RESULTS: The overexpression protein of this domain could be purified under 20 degrees C, 0.1 mmol/L Isopropyl beta-D-1-thiogalactopyranoside (IPTG), BL21 codon plus RP (DE3) as the host strain. Then, PPi release assay indicated that 2, 3-diaminopropionate, the presume precursor of Zwittermicin A, could be adenylated by the adenylation domain. CONCLUSION: This research confirmed that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis.

Production and characterization of monoclonal antibodies to nucleoprotein of Marburg virus.

Monoclonal antibodies (MAbs) against the nucleoprotein of Marburg virus (MARV-NP) with high specificity and activity would be useful for the diagnosis of MARV. In this report, a recombinant MARV-NP was successfully expressed by an Escherichia coli expression system. After immunization and cell fusion, three mouse hybridomas (1H4, 2G1, and 3B5) producing MAbs to MARV-NP were established. The MAbs obtained were fully characterized using indirect ELISA and Western blot analysis. The ELISA results showed that the MAbs' titers were in the range of 1:6.400 x 10(3) - 1:1.280 x 10(4) in culture fluids, and 1:1.024 x 10(6) - 1:8.192 x 106 in ascitic fluids. The isotypes of the three MAbs were tested to be IgG1(kappa) and all the MAbs recognized the same antigenic epitope. Western blot analyses demonstrated that all the MAbs were directed against MARV-NP with the affinity constants (Kaff) of about 1.100 x 10(9) M(-1), 1.235 x 10(9) M(-1), and 1.408 x 10(9) M(-1) for the MAbs 1H4, 2G1, and 3B5, respectively.

A fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032.

In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed. The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability.

Complete nucleotide sequence of pBMB67, a 67-kb plasmid from Bacillus thuringiensis strain YBT-1520.

The complete nucleotide sequence of a large (67kb) cryptic plasmid pBMB67 from Bacillus thuringiensis strain YBT-1520 was determined. Of the 74 predicted open reading frames (ORFs), 25 (34%) were assigned putative functions, 18 (24%) encoded conserved hypothetical proteins, and 31 (42%) had no homology to any genes present in the current open databases. The ORFs with similar functions were organized in a modular structure; thus, the DNA sequence of pBMB67 could be functionally divided into three modules, including a 39kb transfer region encoding homologs of the Agrobacterium tumefaciens VirB/D4 system components VirB1, VirB4, VirB11, and VirD4, as well as homologs of Gram-positive conjugation proteins. We also found a potential operon that was analogous to the Rap-Phr cassettes from Bacillus subtilis, which are involved in cell-cell communication and transcriptional regulation. Thus, we suggest that pBMB67 is likely to be implicated in cell-cell signaling and plays a role in the regulation of several cellular processes, with the production of exoprotease being one of the candidates.

Expression of Vitreoscilla hemoglobin in Bacillus thuringiensis improve the cell density and insecticidal crystal proteins yield.

The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb+ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb- strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb+ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb- strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition.

Medium optimization by response surface methodology for poly-gamma-glutamic acid production using dairy manure as the basis of a solid substrate.

Dairy manure, supplemented with agro-industrial materials, was used as the solid substrate for high yield of poly-gamma-glutamic acid (gamma-PGA) by Bacillus subtilis CCTCC202048. The solid-state fermentation medium was optimized by response surface methodology. In the first optimization step, a Plackett-Burman design was used to evaluate the influence of related factors. Wheat bran, soybean cake and glutamic acid were found to be more compatible supplement with dairy manure and positively influenced on gamma-PGA production. In the second step, the concentrations of the three supplemental nutrients above were further optimized using a Box-Behnken design. The average gamma-PGA yield (4.70%) in triplicate under optimal conditions was obtained on the laboratory scale, whereas it was 3.58% at compost experiment. These would lay a foundation for lessening the pollution of dairy manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.

Microcalorimetric investigation on the growth model and the protein yield of Bacillus thuringiensis.

A novel microcalorimetric technique based on the bacterial heat output was applied to evaluate the special growth model, the protein expression and the generation time of Bacillus thuringiensis for the first time. The thermogenic curves of the aerobic metabolism of B. thuringiensis strains YBT-833, YBT-1520 and YBT-833-2-1 were determined by using an LKB-2277 BioActivity Monitor. The analysis of the thermogenic curves indicated both the mutant strain and the wild-type strains followed the same linear growth model during sporulation. The metabolism heat output revealed heat output was correlated to the yield of the insecticidal crystal proteins (ICPs) very well, the more protein product, and the less heat output. Based on the data acquired, we proposed that this method could be a useful tool in monitoring the fermentation of B. thuringiensis.

Effect of Sm(3+) ion on growth of Bacillus thuringiensis by microcalorimetry.

By using an LKB-2277 Bioactivity Monitor, cycle-flow method, the thermogenic curves of aerobic growth for Bacillus thuringiensis cry II strain at 28 degrees C have been obtained. The metabolic thermogenic curves of B. thuringiensis cry II contained two distinct patterns: the first reflects the changes during the bacterial growth phase and the second corresponds to the sporulation phase. From these thermogenic curves in the absence and presence of Sm(3+) ions, the thermokinetic parameters such as the growth rate constants k, the interval time tau(I), the maximum power P (max 1) and heat-output Q(log) for log phase, the maximum power P (max 2) and heatoutput Q(stat) for stationary phase, the heat-output Q(spor) for sporulation phase and total heat effects QT are calculated. Sm(3+) ion has promoting action on the growth of B. thuringiensis cry II in its lower concentration range; on the other hand, this ion has inhibitory action on the sporulation of B. thuringiensis in its higher concentration range. We also found that the effects of Sm(3+) ion on B. thuringiensis during the sporulation phase were far greater than that during the bacterial phase. It is concluded that the application of B. thuringiensis for controlling insecticides is not affected by the presence of the rare-earth elements in the environmental ecosystem.

Study of the thermokinetic properties of copper(II) on Escherichia coli growth.

By using an LKB2277 Bioactivity Monitor, stop-flow mode, the power-time curves of Escherichia coli at 37 degrees C affected by Cu(II) were determined. Some parameters, such as growth rate constants k, inhibitory ratio I, the heat output Qlog in the log phase, and the generation times G were obtained. According to these parameters, we found that a low concentration of Cu(II) (0-20 microg/mL) had an promoting action on the growth of E. coli, but a high concentration of Cu(II) (40-100 microg/mL) had an inhibitory action. The toxicity of Cu(II) can also be expressed as the half-inhibitory concentration IC50; the value is 69.7 microg/mL. The assay is quantitative, inexpensive, and versatile.

Microcalorimetric investigation of the effect of manganese(II) on the growth of Tetrahymena shanghaiensis S199.

The heat output of and the effect of manganese (II) on Tetrahymena shanghaiensis S199 growth metabolism has been determined by means of a LKB-2277 BioActivity monitor. Different concentrations of manganese(II) ions have different effects on the growth of T. shanghaiensis. At low concentrations (0-40 microg/mL) culture growth is promoted, whereas high concentrations (60-800 microg/mL) slow growth. Furthermore, concentrations of 1200 microg/mL or greater stop the growth of this protozooa completely.

The action of Cu2+ on Bacillus thuringiensis growth investigated by microcalorimetry.

By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism has been determined at 28 degrees C and effect of Cu2+ on B. thuringiensis growth was studied. Copper has been regarded as an essential trace element for life. Its deficiency may be the cause of diseases. Cu2+ of different concentration have different effects on B. thuringiensis growth metabolism, Cu2+ of low concentration (0-30 micrograms/ml) can promote the growth of B. thuringiensis, and Cu2+ of high concentration (40-120 micrograms/ml) is able to inhibit its growth and B. thuringiensis can't grow at all when the concentration of Cu2+ is up to 130 micrograms/ml.

TRANSFORMATION OF BACILLUS THURINGIENSIS RECIPIENT BMB171 BY ELECTROPORATION / 微生物学通报

This paper reports the optimized electro-transformation parameters of Bacillus thuringiensis plasmid-free mutant strain BMB171 by electroporation, and expressing effect of several cry genes introduced in this recipient. It showed that a highest electro-transformation frequency could be obtained, when SG solution was used as the buffer, and a 10.0V/cm of field strength, one time of pulse as well as a growth phase of recipient cells at the exponential phase (OD650nm value was 0.2~0.3) were selected. The highest of electro-transformation frequency with pHT3101 could reach at 8 ?107 hansformants/?g DNA. The transformation frequencies increased at linear velocity as the concentration increase of pHT3101 from 54.69pg/ml to 3.50?g/mL, then reached saturation afterwards. All plasmids introduced in BMB171 could produce characteristic insecticidal crystal proteins through expression of relevent cry genes carried by them. Meanwhile, these insecticidal crystal proteins could form parasporal crystals, which have characteristic geometric shapes.