长双歧杆菌NCC2705葡萄糖与乳糖代谢的比较蛋白质组学

[目的]以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础,研究长双歧杆菌发酵乳糖和葡萄糖的比较蛋白质组学.[方法]采用ImageMaster 2D Elite Platnum Version 5.0比较分析3倍以上蛋白差异点;利用MALDI-TOF进行差异蛋白鉴定,每个蛋白质点的肽指纹图谱在长双歧杆菌NCC2705菌株的蛋白质数据库用Mascot进行检索;采用Pro-Q磷酸化试剂进行磷酸化蛋白的染色.[结果]鉴定到31个蛋白表达发生显著变化,在乳糖发酵中14个蛋白上调17个蛋白下调.这些蛋白为亲水性酸性蛋白,它们基因的CAI值均在0.5以上,主要包括糖代谢相关蛋白、应激蛋白、转录和翻译相关蛋白,还有一些未知功能的蛋白.此外,有两个蛋白:转醛缩酶(BL0715,transaldolase,tal)L3蛋白点和丙酮酸激酶(BL0988,pyruvate kinase,pyk)G9蛋白点发生了磷酸化作用.[结论]长双歧杆菌NCC2705在乳糖中生长快于葡萄糖,它们的降解途径是相同的;转醛缩酶和丙酮酸激酶发生了翻译后修饰作用,推测转醛缩酶在43T和47S发生了磷酸化,而丙酮酸激酶在65S发生了磷酸化.     

长双歧杆菌NCC2705葡萄糖与果糖代谢的比较蛋白质组学

为揭示长双歧杆菌NCC2705 (Bifidobacterium longum NCC2705)果糖代谢途径, 建立其果糖发酵模型。以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础, 进行了果糖和葡萄糖生长的菌体比较蛋白质组学研究, 利用MALDI-TOF和ESI-MS/MS鉴定差异蛋白, 进一步通过半定量RT-PCR验证二者显著差异表达蛋白。果糖生长的菌体蛋白中鉴定到了所有葡萄糖降解途径中的酶和蛋白质, 另外鉴定到3倍以上差异蛋白点9个, 其对应的5个蛋白在果糖发酵中上调。半定量RT-PCR验证显著差异蛋白, 显示在果糖发酵中具有高水平表达是ABC 转运系统的果糖特异性-结合蛋白BL0033和ATP结合蛋白BL0034。果糖的发酵时间和浓度梯度试验显示诱导时间越长、浓度越高, BL0033的表达量越高。第一, 比较蛋白谱证明果糖和葡萄糖以相同途径降解。第二, BL0033的表达是受果糖诱导的, 果糖的吸收可能是通过一个特殊的转运系统, 即ABC转运系统将果糖从胞外转运到胞内, 其中BL0033和BL0034共同作为系统元件扮演了重要角色。     

长双歧杆菌NCC2705群体感应系统信号分子AI-2的检测

目的:用生物学方法检测长双歧杆菌NCC2705是否产生群体感应系统信号分子AI-2。方法:将长双歧杆菌NCC2705不同时间点的培养上清分别加至AI-2特异报告系统哈氏弧菌BB170中,以空白培养基上清为对照,用荧光光度计对哈氏弧菌发光强度进行计量,推测出长双歧杆菌NCC2705上清中是否含有分泌的AI-2,并由此推断AI-2的活性。结果:通过微孔板检测系统对加入长双歧杆菌NCC2705培养上清的哈氏弧菌BB170进行检测,发现双歧杆菌上清的加入增强了哈氏弧菌BB170发出的荧光强度。结论:长双歧杆菌NCC2705中存在依赖于luxS/AI-2的群体感应系统,并能够分泌有活性的AI-2,为进一步研究长双歧杆菌NCC2705AI-2及luxS基因的功能打下基础。     

长双歧杆菌NCC2705株果糖结合蛋白BL0033基因的克隆及其在大肠杆菌中的表达

目的：克隆长双歧杆菌NCC2705株果糖结合蛋白BL0033的基因,利用大肠杆菌表达GST-BL0033融合蛋白并纯化。方法：以长双歧杆菌NCC2705株基因组为模板,PCR扩增BL0033基因,并将其插入pGEX-4T-1表达载体,转化至大肠杆菌DH5α;提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21,并对表达条件进行摸索;用谷胱甘肽-Sepharose4B树脂对可溶性GST-BL0033融合蛋白进行纯化。结果：PCR扩增的BL0033基因长度接近1000bp,与预期值一致;重组菌在IPTG浓度为0.05mmoL/L的条件下,于16℃诱导过夜后,SDS-PAGE分析可见可溶性表达条带,相对分子质量约60×103,与预期值一致;亲和纯化后,SDS-PAGE结果显示单一的表达条带。结论：克隆了BL0033蛋白的基因,并表达纯化了融合蛋白GST-BL0033,为进一步研究长双歧杆菌NCC2705株BL0033蛋白功能奠定了基础。     

长双歧杆菌BBMN68 可溶性蛋白质图谱的建立

为了建立长双歧杆菌BBMN68蛋白质图谱,采用双向电泳的方法建立了2-D参考图谱,通过MALDI-TOF/MS质谱鉴定和数据库搜索,鉴定到206个蛋白质(占长双歧杆菌BBMN68基因预测总蛋白的11.4%)。通过2-D胶分析,共有800±15(对数期)和800±20(稳定期)个蛋白质,其中282个蛋白点成功鉴定,代表206个不同的蛋白质。另外,分析了实验鉴定蛋白质的等电点和分子量,蛋白功能,密码子偏好性,蛋白质疏水性以及蛋白质细胞定位的分析。研究结果为长双歧杆菌的比较蛋白质组学研究提供了参考图谱和蛋白质基础信息数据。     

长双歧杆菌NCC2705高效转化系统的建立及GFP表达

目的:建立长双歧杆菌NCC2705高效稳定的电转化系统,并以其为宿主表达绿色荧光蛋白(GFP),构建一个稳定的带报告基因的表达载体。方法:从双歧杆菌克隆载体pDG7中切取其在双歧杆菌中复制所必需的pMB1复制子插入质粒pUC19的多克隆位点区,并将gfp基因在双歧杆菌中进行表达;设计双歧杆菌电击转化体系,研究在不同电击电压条件下的电转效率。结果:首先构建了大肠杆菌-长双歧杆菌穿梭质粒pUB1和带有gfp基因的表达质粒pUB2,用电击法将之转化至双歧杆菌,当细菌生长到D600nm约为0.5时制备感受态细胞,在电容25μF、电阻200Ω、电压12.5 kV/cm的电击条件下进行完整质粒转化,可得到较高的转化率。结论:构建了以长双歧杆菌NCC2705为宿主的高效稳定的表达系统,为进一步研究益生菌的分子生物学和功能特性提供了基础。     

长双歧杆菌分泌型表达载体的构建及Tum-5基因的表达

摘要：目的 构建能在双歧杆菌内稳定存在并高效分泌表达外源基因的长双歧杆菌质粒表达载体。方法 以质粒pBAD/glll为基础,以双歧杆菌内源性阿拉伯糖苷酶的分泌性信号肽取代质粒原有的分泌性信号肽,并将双歧杆菌天然质粒的聚合酶基因克隆入载体中,构建表达载体pBBADs。将人Tum-5基因克隆入载体中,构建质粒pBBADs-Tum-5,电转化长双歧杆菌NCC2705,L-阿拉伯糖诱导表达后,Western Blot鉴定基因表达。结果 成功的构建了长双歧杆菌分泌型质粒表达载体,Tum-5基因在双歧杆菌内可以表达。结论 构建的表达载体为双歧杆菌用于肿瘤靶向基因治疗奠定了基础。     

具高抗氧化能力乳酸菌菌株的筛选与鉴定

目的筛选抗氧化能力强的乳酸菌菌株并进行菌种鉴定。方法通过清除DPPH自由基、清除超氧阴离子自由基、还原能力等实验,筛选出高抗氧化能力的乳酸菌菌株。运用16SrDNA序列分析方法,对抗氧化能力强的菌株进行鉴定。结果本研究筛选得到的抗氧化能力较强的菌株有长双歧杆菌NQ1501、La8、La5和保加利亚乳杆菌NQ2508,经16SrDNA鉴定和系统进化分析,La5鉴定为发酵乳杆菌(Lactobacillus fermentum),La8鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。结论本研究筛选出四株具有高抗氧化活性的乳酸菌,分别为长双歧杆菌NQ1501、鼠李糖乳杆菌La8、发酵乳杆菌La5和保加利亚乳杆菌NQ2508。     

双向电泳结合质谱分析长双歧杆菌XY01分泌蛋白质图谱

菌体的分泌蛋白质在宿主和菌体的相互作用之间起着重要的作用. 本研究采用双向凝胶电泳的方法建立了长双歧杆菌XY01分泌蛋白质图谱,通过MALDI-TOF/TOF质 谱鉴定和数据库搜索,对鉴定到的分泌蛋白进行了分析. 共检测到21个蛋白质点, 成功鉴定18个蛋白质点,分别代表14个不同的蛋白质,等电点分布在4.5～7.0之间 ,分子质量分布在20 ～65 kD之间;通过COGs分类和功能分析,信号肽和细胞定位及KEGG代谢通路分析. 结果表明,这些蛋白质对菌体细胞壁/膜的形成、生物信号传导和物质代谢等起着重要作用. 研究结果为长双歧杆菌蛋白质组学和基因组学的研究提供了参考.     

短双歧杆菌A04菌株体外清除自由基的研究

采用化学比色法研究了短双歧杆菌A04菌株及其菌体破碎物对DPPH自由基、脂自由基的清除能力,采用化学发光法检测了其对DNA损伤的保护作用,应用电子自旋共振(ESR)法测定了其清除氧自由基的能力。结果显示:短双歧杆菌A04菌株及其菌体破碎物均具有明显的抗脂质过氧化能力,能有效地清除DPPH和活性氧自由基,同时还能很好地保护DNA免受自由基损伤。     

Analysis of host-inducing proteome changes in bifidobacterium longum NCC2705 grown in Vivo

To investigate the molecular mechanisms underlying the adaptation of Bifidobacterium longum to the intestinal tract, we utilized a new model for rabbit intestinal culture of B. longum and reported the changes in proteomic profiles after incubation in the in vivo environment. By 2D-PAGE coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses, proteomic profiles of B. longum strain NCC2705 grown in the in vivo and in vitro environments were compared. Confirmed by semiquantitative RT-PCR, which exhibited at least a 3-fold change or greater, 19 up-regulated proteins, 14 down-regulated proteins, and 4 proteins with mobility changes were identified during intestinal growth. These identified proteins include key stress proteins, metabolism-related proteins, and proteins related to translation. Our results indicate that some useful proteins are expressed at higher levels in cells during intestinal growth. These proteins reflected the adaptation of B. longum NCC2705 to the intestine, such as EF-Tu which contributes to the retention or attachment as a Bifidobacterium adhesin-like factor, bile salt hydrolase (BSH) which might play an important role in the molecular mechanisms for the initial interaction of probiotic with the intestinal environment, and stress proteins which defend B. longum against the action of bile salts and other harmful ingredients of the gastrointestinal tract (GIT). The most striking fact of our observation was that four proteins GlnA1, PurC, LuxS, and Pgk exhibit clear post-translational modification. Western blot (WB) analysis and Pro-Q Diamond staining revealed that substances of the GIT trigger Pgk and LuxS phosphorylation at Ser/Thr residues for bacteria grown in vivo. These proteins were identified for the first time as bifidobacterial phosphoproteins. Our data suggest that the phosphorylated autoinducer-2 production protein LuxS of B. longum NCC2705 (LuxS-P) is the active form of LuxS and that LuxS-P may play a key role in the regulation of quorum sensing.     

A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.     

Proteomics analysis of Bifidobacterium longum NCC2705 growing on glucose, fructose, mannose, xylose, ribose, and galactose

To investigate the molecular mechanisms underlying carbohydrate uptake and connected metabolic pathways of Bifidobacterium longum NCC2705, the proteomic profiles of bacteria grown on different carbon sources including glucose, fructose, mannose, xylose, ribose, and galactose were analyzed. Our results show that all sugars tested were catabolized via the bifid shunt. Sixty-eight proteins that exhibited changes in abundance of threefold or greater were identified by MS. A striking observation was the differential expression of proteins related to the pyruvate metabolism. Further analysis of acetic acid and lactic acid in the culture supernatants by HPLC at the end of fermentation showed that more lactic acid was produced during growth on fructose, ribose, xylose, galactose and more acetic acid was produced during the fermentation of glucose and mannose. Growth experiments revealed that B. longum NCC2705 preferentially used fructose, ribose, xylose, and galactose with higher growth rates over glucose and mannose. Furthermore, five proteins (GroEL, Eno, Tal, Pgm, and BL0033) exhibited clear phosphorylation modifications at serine and/or tyrosine residues. BL0033, a component of an ATP-binding cassette (ABC) transporter, was significantly more abundant in bacteria grown on fructose and, to a lesser extent, ribose and xylose. RT-PCR analysis revealed that all genes of the ABC transporter are induced in the presence of these sugars suggesting that BL0033, BL0034, BL0035, and BL0036 constitute an ABC transporter with fructose as preferred substrate.     

Fructose uptake in Bifidobacterium longum NCC2705 is mediated by an ATP-binding cassette transporter

Recently, a putative ATP-binding cassette (ABC) transport system was identified in Bifidobacterium longum NCC2705 that is highly up-regulated during growth on fructose as the sole carbon source. Cloning and expression of the corresponding ORFs (bl0033-0036) result in efficient fructose uptake by bacteria. Sequence analysis reveals high similarity to typical ABC transport systems and suggests that these genes are organized as an operon. Expression of FruE is induced by fructose, ribose, or xylose and is able to bind these sugars with fructose as the preferred substrate. Our data suggest that BL0033-0036 constitute a high affinity fructose-specific ABC transporter of B. longum NCC2705. We thus suggest to rename the coding genes to fruEKFG and the corresponding proteins to FruE (sugar-binding protein), FruK (ATPase subunit), FruF, and FruG (membrane permeases). Furthermore, protein-protein interactions between the components of the transporter complex were determined by GST pulldown and Western blot analysis. This revealed interactions between the membrane subunits FruF and FruG with FruE, which in vivo is located on the external side of the membrane, and with the cytoplasmatic ATPase FruK. This is in line with the proposed model for bacterial ABC sugar transporters.     

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.