A system for the inducible secretion of proteins from Bacillus subtilis during logarithmic growth

Abstract A Bacillus subtilis-Escherichia coli shuttle vector was constructed containing the B. subtilis levansucrase gene promoter and region encoding its signal sequence.
A site for the restriction enzyme Nae I was included to facilitate precise translational fusions to the DNA encoding the levansucrase signal sequence. Fusions of TEM β-lactamase to this construct displayed sucrose-inducible expression and secretion of B. subtilis .     

Anionic polymers of Bacillus subtilis cell wall modulate the folding rate of secreted proteins

In order to characterize the dynamics of the interaction between the emergent membrane translocated exoprotein and the components of Bacillus subtilis cell wall, we examined the kinetics of the in vitro refolding of levansucrase and alpha-amylase, at pH 7 and 37 degrees C, in the presence of polyphosphates (polyP) of various chain lengths (2/=16. In contrast, polyP modulate indirectly the rate of alpha-amylase refolding via their affinity for calcium. These differential effects might explain that the rate of the cell wall translocation of alpha-amylase secretion was found to be half that of levansucrase.     

Production and secretion of pertussis toxin subunits in Bacillus subtilis

Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines. The use of acellular vaccines is predicted to increase sharply in the near future. There is therefore a need to produce PT in a way that makes its purification as easy as possible. Our approach was to express all five PT subunits individually in Bacillus subtilis. We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit. All PT-subunits were secreted and found in the culture supernatant. The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found. The subunits were also present in the membrane fraction of the respective strains.     

枯草芽孢杆菌蛋白质分泌机制研究进展

综述了枯草芽孢杆菌不同蛋白质分泌机制,重点讨论了大多数细菌蛋白分泌的Sec途径,包括Sec途径的信号肽,信号肽酶,SecYEG通道,与分泌有关的各种细胞因子以及Sec途径的限制因素,此外还简要讨论了Tat途径,该途径能够转运折叠迅速或归密的蛋白质。     

Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques

Bacillus subtilis has been developed as a model system for physiological proteomics. However, thus far these studies have mainly been limited to cytoplasmic, extracellular, and cell-wall attached proteins. Although being certainly important for cell physiology, the membrane protein fraction has not been studied in comparable depth due to inaccessibility by traditional 2-DE-based workflows and limitations in reliable quantification. In this study, we now compare the potential of stable isotope labeling with amino acids (SILAC) and (14)N/(15)N-labeling for the analysis of bacterial membrane fractions in physiology-driven proteomic studies. Using adaptation of B. subtilis to amino acid (lysine) and glucose starvation as proof of principle scenarios, we show that both approaches provide similarly valuable data for the quantification of bacterial membrane proteins. Even if labeling with stable amino acids allows a more straightforward analysis of data, the (14)N/(15)N-labeling has some advantages in general such as labeling of all amino acids and thereby increasing the number of peptides for quantification. Both, SILAC as well as (14)N/(15)N-labeling are compatible with 2-DE, 2-D LC-MS/MS, and GeLC-MS/MS and thus will allow comprehensive simultaneous interrogation of cytoplasmic and enriched membrane proteomes.     

过表达TatAdCd转位酶对枯草芽孢杆菌脂肪酶分泌的影响

【目的】研究过表达枯草芽孢杆菌Tat运输途径的Tat Ad Cd转位酶对促进脂肪酶分泌的影响。【方法】用cdd基因的串联启动子和前导区,替换tat AD-CD操纵子的启动子和前导区,并在染色体sac B基因位点整合表达;采用q RT-PCR方法表征tat AD-CD操纵子的表达水平;用脂肪酶表达质粒p HP13L转化Tat Ad Cd转位酶过表达菌株,构建产脂肪酶重组菌。通过测定脂肪酶活性,以及聚丙烯酰胺凝胶电泳,考察Tat Ad Cd转位酶过表达对脂肪酶分泌的影响。【结果】tat AD-CD操纵子被过表达,其胞内m RNA相对水平提高了185倍。Tat Ad Cd转位酶的过表达,使脂肪酶发酵单位提高了40%。【结论】使用cdd基因的串联启动子和前导区,能够有效地过表达目的基因;枯草芽孢杆菌脂肪酶可以同时经由Sec途径和Tat途径分泌;过表达Tat Ad Cd转位酶,能够显著提高脂肪酶的分泌量。     

Coupling of fermentation and microfiltration for α-amylase production from Bacillus amyloliquefaciens

Abstract: A strain of Bacillus amyloliquefaciens secreting α-amylase was cultivated continuously in a fermentor coupled to a filtration unit. Under the tested operating conditions, the maximum flux was 91 m^-2 h^-1 during the first day and 61 m^-2 h^-1 during the 2nd day. The α-amylase retention was around 30%. Compared to a batch process, continuous cultivation with cell recycling led to lower α-amylase concentrations but to a doubling of volumetric productivities.     

Cloning of a thermostable α-amylase gene from Bacillus licheniformis and its expression in Escherichia coli and Bacillus subtilis

Abstract The gene coding for the thermostable α-amylase Bacillus licheniformis has been isolated from a direct shotgun in Escherichia coli using the bacteriophage lambda as a vector. The fragment containing the α-amylase gene has been sub-cloned in pBR322 and its restriction map determined. The α-amylase produced by the E. coli clones retained the thermostability of the B. licheniformis enzyme. Expression and properties of the gene product in E. coli and Bacillus subtilis have been examined.     

高效降解蛋白枯草芽孢杆菌的筛选及促建鲤生长的研究

【目的】从建鲤肠道分离并优选出了一株对蛋白具有强效降解力的菌株,探究其临床生产效果。【方法】使用牛奶平板进行筛选,经常规细菌学鉴定和16S rDNA序列分析确定A7菌株为枯草芽孢杆菌,通过临床饲喂试验探究其对建鲤促生长作用。【结果】A7菌株在牛奶平板上的水解圈直径可达27.5 mm,其最佳固体发酵条件为温度28°C、接种量5%(1.2×109 CFU/mL)、料水比1.0:1.2和发酵时间72 h。临床饲喂试验结果表明,饵料中添加0.5%、1.0%和1.5%的A7株菌粉均能促建鲤生长。其中,添加量为1.0%的试验组,鱼体增重率和蛋白利用率最高,饵料系数最低,与产品对照组和空白对照组相比,均达到了显著性差异(P0.05)。此外,随A7株菌粉添菌量的增加,各试验组肝胰脏和肠道的蛋白酶及淀粉酶活力出现先增大后减小趋势,与产品对照组和空白对照组相比,添加1.0%的试验组增幅最大(P0.05)。研究发现,试验组鱼肌肉中粗蛋白和粗脂肪含量较空白对照组也有明显的增减趋势(P0.05)。【结论】饵料中添加A7株菌粉,可有效促进鱼的生长,降低饵料系数,提高肝胰脏和肠道的蛋白酶及淀粉酶活力,并提高鱼肌肉中的蛋白含量和降低脂肪含量。     

Localisation of the cell wall-associated phosphodiesterase PhoD of Bacillus subtilis

The localisation of phosphate-starvation-induced phosphodiesterase PhoD from Bacillus subtilis was studied by analysing processing, release and immunogold labelling of the sections. Although the processing of the pre-protein was extremely slow, the major fraction of PhoD could be detected at the surface of the cell wall. The results indicate that inefficient processing of the translocated pre-protein keeps PhoD in a cell wall-associated location. The uncleaved signal peptide might function as a membrane anchor.     

Interaction of the Bacillus subtilis chaperone CsaA with the secretory protein YvaY

Bacillus subtilis CsaA was previously characterised as a molecular chaperone with export-related activities. In order to elucidate the functionality of CsaA further, interaction with its postulated substrate YvaY was investigated. Similar binding to carrier immobilised mature and preYvaY revealed that the interaction was not mediated via the signal peptide of preYvaY. Higher affinity to denatured peptides compared to native peptides indicated preferred binding to unfolded proteins. To characterise affinity of CsaA more detailed, binding to preYvaY derived peptides was analysed. CsaA showed affinity to multiple peptides in the scan, mainly correlated to a positive net charge. Affinity of export-specific Escherichia coli chaperone SecB to the carrier immobilised peptides indicated partially overlapping binding characteristics of SecB and CsaA.     

1株枯草芽胞杆菌体外拮抗6种肠道致病菌的研究

目的研究枯草杆菌BS-3株对大肠埃希菌等6种肠道致病菌的拮抗作用。方法通过在体外BS-3菌株分别与大肠埃希菌等6种致病菌混合培养后,观察不同时间内各菌的菌量变化。结果BS-3菌株与6种肠道致病菌混合培养24、48、72和96h,其菌量逐渐增加;6种致病菌的菌量随着培养时间延续逐渐减少,其中产毒性大肠埃希菌、致病性大肠埃希菌和宋内志贺菌与对照组比较差异更明显。结论BS3菌株在培养生长过程中,可抑制大肠埃希菌等6种肠道致病菌的生长。     

3种渗透剂对转基因烟草根系枯草芽孢杆菌纤溶酶(BSFE)分泌的调节

用0.05%～8.00%的甘露醇、山梨醇和聚乙二醇6000等3种渗透调节剂可提高转枯草芽孢杆菌纤溶酶(Bacillus subtilis fibrinolytic enzyme, BSFE)转基因烟草(Nicotiana tabacum L.)根系BSFE的分泌表达水平,其水培液BSFE活性在15 d内基本呈抛物线型变化趋势.经3种渗透剂处理后转BSFE基因烟草水培液的BSFE活性峰值明显高于对照,且出现时间比对照相对延迟1-2d.甘露醇、山梨醇和聚乙二醇6000可作为该转基因烟草根系BSFE分泌表达的有效化学调节剂.     

Protein secretion by the mouse blastocyst: Stimulatory effect on secretion into the blastocoel by transforming growth factor-α

We have previously demonstrated that newly synthesized proteins are secreted into the mouse blastocoel [Dardik and Schultz (1991): Biol Reprod 45:328–333]. In the present study we examine the effect of transforming growth factor-α (TGF-α) on these proteins. We observe that TGF-α stimulates secretion of these newly synthesized proteins into the blastocoel and apical medium, which faces the zona pellucida, by about 65%. Although one-dimensional gel electrophoretic analysis does not reveal any marked differences in the patterns of newly synthesized proteins secreted into the blastocoel in response to TGF-α, zymography reveals a marked stimulation in the secretion of several gelatinases into the blastocoel and apical medium. These results suggest additional functions for TGF-α in mouse preimplantation development. © 1993 Wiley-Liss, Inc.     

Diversity of the growth patterns of Bacillus subtilis colonies on agar plates

Abstract A Bacillus subtilis strain showed a variety of colony growth patterns on agar plates. The bacterium grew to a fractal colony through the diffusion-limited aggregation process, a round colony reminiscent of the Eden model, a colony with a straight and densely branched structure similar to the dence branching, morphology, a colony spreading without any openings, and a colony with concentric rings, on plates with various agar and nutrient concentrations. The microstructures of these colonies were also characteristic and dynamic. The patterns of these bacterial colonies were thought to grow in relation to the diffusion of nutrient in the agar plate.