枯草芽孢杆菌核黄素操纵子与ribC基因的修饰与遗传效应

[目的]研究核黄素操纵子(rib)组成型高表达,以及ribC基因低水平表达对枯草芽孢杆菌过量合成核黄素的影响.[方法]在染色体原位修饰启动子,用mRNA稳定子替换mRNA前导区,使rib操纵子组成型高表达;修饰ribC基因的启动子,降低ribC基因的表达水平.采用qRT-PCR方法,表征基因的相对表达水平;通过摇瓶发酵,测定重组菌的生物量和核黄素产量,表征相关基因修饰所表现的遗传效应.[结果]用gsiB mRNA稳定子替换核黄素操纵子的mRNA前导区,使其相对表达水平提高了约1 500倍.ribC基因启动子-35区的首个碱基由“T”突变为“C”,使ribC基因的表达水平下降了97％以上.得到的重组菌株LX34在补加蔗糖20 g/L的LB培养基上摇瓶发酵36 h,可积累核黄素2.1 g/L,同时生物量没有明显下降.[结论]使用gsiB mRNA稳定子,能够有效地提高目标基因或操纵子的表达水平;启动子-35区首个碱基的点突变,能够有效降低ribC基因的表达水平;rib操纵子过量表达和ribC基因低水平表达,使细胞能够过量合成并积累核黄素.     

短小芽孢杆菌碱性蛋白酶基因启动子的克隆、鉴定及其应用

利用TAIL-PCR(Thermal asymmetric interlaced PCR)从短小芽孢杆菌基因组中扩增到碱性蛋白酶基因编码区上游的启动子片段。对该片段的序列测定和分析表明, 此片段长797 bp, 但与基因表达有关的序列长约390 bp。对启动子片段进行不同长度的缺失突变, 以获得最小的基因启动子片段, 结果表明, 该基因起始密码子上游约160 bp的DNA片段就可以启动基因的表达。将含有该片段的碱性蛋白酶基因WApQ3插入大肠杆菌-芽孢杆菌穿梭质粒载体pSUGV4中, 构建了碱性蛋白酶基因表达质粒pSUBpWApQ3。将该质粒分别转入枯草芽孢杆菌和短小芽孢杆菌中表达, 可在胞外检测到碱性蛋白酶活性, 最高酶活分别为466.5 U/mL和3060 U/mL。     

枯草芽孢杆菌核黄素操纵子rib operon的克隆与表达

目的:构建产核黄素的枯草芽孢杆菌基因工程菌.方法:以穿梭载体pEB03构建核黄素操纵子的表达质粒载体pGJB13和pGJB14,与质粒pMX45分别转化产核黄素的枯草芽孢杆菌GJ07,并通过发酵摇瓶实验检测核黄素的产量.结果:得到产核黄素的工程菌GJ13 、GJ14和GJ08,在以蔗糖为碳源的发酵条件下,GJ08可产核黄素820mg/L,提高了约55%.结论:得到了产核黄素的高产菌种G J08.     

苏云金芽孢杆菌芽孢萌发相关基因gerM的克隆及功能研究

依照蜡状芽孢杆菌gerM基因的保守序列设计引物,从苏云金芽孢杆菌中扩增出640bp的DNA片段。以此为探针,从苏云金芽孢杆菌部分基因组酶切文库中成功地克隆到了一个4·5kb的DNA片段。序列分析表明,该片段包含一个完整的开放阅读框,其预测的编码产物与枯草芽孢杆菌GerM蛋白具有很高的同源性,将该基因命名为gerM。RT-PCR分析表明,gerM基因仅在芽孢形成的过程中表达。通过同源重组的策略构建了gerM基因的阻断突变株。研究表明,gerM基因的破坏影响苏云金芽孢杆菌芽孢萌发的速率和比例。     

地衣芽孢杆菌YP1A耐有机溶剂蛋白酶基因的克隆与功能表达

根据B．licheniformis YP1A来源的碱性蛋白酶具有的高强度耐有机溶剂性能及相关数据库分析,采用PCR克隆B．licheniformis YP1A耐有机溶剂碱性蛋白酶基因,序列分析显示该基因（1264bp）包含启动子与编码380个氨基酸的开放阅读框（ORF）,ORF包括信号肽、前肽及编码254个氨基酸的成熟肽序列。相关基因分析表明,YP1A耐有机溶剂碱性蛋白酶基因与地衣芽孢杆菌ATCC14580的碱性蛋白酶基因仅有6个氨基酸残基差异：构建2种含YP1A碱性蛋白酶CDS的组成型穿梭表达载体pHY／aprYP与pHY／aprP43,前者采用YP1A蛋白酶自带的启动子,后者则采用来自于质粒pP43NMK的P43强启动子。利用这2种表达载体在枯草芽孢杆菌WB800中成功进行蛋白酶的功能表达．其中P43强启动子的表达能力明显优于碱性蛋白酶自带的启动子,表达的蛋白酶比酶活为395U／ml。重组菌表达的碱性蛋白酶在体积分数50％的亲水及疏水有机溶剂中表现出了很好的耐受性,验证了克隆基因为地衣芽孢杆菌YP1A的高强度耐有机溶剂碱性蛋白酶基因.     

枯草芽孢杆菌基因启动子的分离与鉴定

利用启动子探针型载体pSUPV4直接在大肠杆菌 (Escherichiacoli)中分离枯草芽孢杆菌 (Bacillussubtilis)WB6 0 0的基因启动子片段 ,获得 5 5个具有卡那霉素抗性的重组子。对 3个抗性最高的重组子pSU -Bs2 ,pSU -Bs4 ,pSU -Bs8进行序列测定和同源性分析发现 ,所克隆到的基因启动子片段均来自于枯草芽孢杆菌的基因组 ,并且具有枯草杆菌基因启动子的保守序列。对抗性最高的Bs2片段进一步研究表明 ,它可以在大肠杆菌中高效地启动来自于短小芽孢杆菌的碱性蛋白酶基因的表达 ,也能在枯草芽孢杆菌中启动卡那霉素抗性基因的表达。     

地衣芽孢杆菌α—淀粉酶基因在枯草芽孢杆菌中的诱导表达

采用PCR技术扩增了sacB基因的启动子-信号序列,并将扩增的序列重组进含地衣芽孢杆菌α-淀粉酶基因的质粒载体上构建了含α-淀粉酶基因的分泌型表达载体pSA60。将pSA60转化枯草芽孢杆菌QB1098后,α-淀粉酶基因在sacB基因启动子-信号序列的调控和蔗糖的诱导下获得表达,表达产物分泌至胞外。     

少根根霉脂肪酶基因在枯草芽孢杆菌中的诱导表达

利用PCR技术从少根根霉基因组中扩增出脂肪酶成熟肽基因ral,并从枯草芽孢杆菌基因组中扩增出sacB基因的启动子-信号序列(SacB);通过搭桥PCR将SacB序列与ral基因融合,并将该基因表达盒连接到枯草杆菌分泌表达载体pGJ103中构建了脂肪酶基因的诱导表达载体pGJ103-SacB-ral。将重组载体转化至枯草芽孢杆菌后,少根根霉脂肪酶成熟肽基因在SacB启动子-信号序列的调控和蔗糖的诱导下获得表达,产物分泌至胞外。     

肌苷生产菌枯草芽孢杆菌ATCC 13952的全基因组测序及序列分析

摘要:【目的】枯草芽孢杆菌ATCC 13952是一株肌苷工业生产菌株。为深入研究ATCC 13952菌株积累肌苷的分子机制以及为进一步分子育种研究提供序列背景信息,有必要解析ATCC 13952菌株的基因组序列信息。【方法】本研究采用高通量测序和Sanger测序相结合对ATCC 13952菌株进行全基因组测序,然后使用相关软件对测序数据进行基因组组装、基因预测与功能注释、GO/COG 聚类分析、共线性分析等。【结果】枯草芽孢杆菌ATCC 13952整个基因组大小为3876276 bp,GC含量为45.8%,序列已提交至GenBank 数据库,登录号为CP009748。比较基因组及嘌呤代谢相关基因分析结果显示:枯草芽孢杆菌ATCC 13952与其他几株芽孢杆菌具有较好的基因组共线性关系,嘌呤代谢相关基因编码的蛋白与标准菌株比较发生了一些缺失和突变。【结论】本研究首次报道了一株肌苷生产菌枯草芽孢杆菌ATCC 13952的全基因组序列,分析了基因组基本特征,初步探讨了该菌株积累肌苷的分子机制,为后续的进一步分子育种提供了理论基础。     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9Kb的基因片段.核酸序列分析结果表明,该片段全长1984bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes.

In this paper, we report the identification, cloning, and complete nucleotide sequence of four genes from Actinobacillus pleuropneumoniae that are involved in riboflavin biosynthesis. The cloned genes can specify production of large amounts of riboflavin in Escherichia coli, can complement several defined genetic mutations in riboflavin biosynthesis in E. coli, and are homologous to riboflavin biosynthetic genes from E. coli, Haemophilus influenzae, and Bacillus subtilis. The genes have been designated A. pleuropneumoniae ribGBAH because of their similarity in both sequence and arrangement to the B. subtilis ribGBAH operon.     

Characterization of the chemotaxis fli Y and che A genes in Bacillus cereus

This paper describes the first identification of chemotaxis genes in Bacillus cereus. We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B. cereus strains, ATCC 14579 and ATCC 10987. While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B. subtilis, is one of the three genes encoding proteins of the flagellar switch complex. Although the sequences and relative position of cheA and fliY were found to be identical in the two B. cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B. cereus ATCC 14579 and ATCC 10987. Evidence is shown that the genomic organization and the expression of fliY and cheA in B. cereus differ significantly from that described for B. subtilis, which is considered a model microorganism for chemotaxis in gram-positive bacteria.     

Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium

The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B. cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of B. anthracis or B. cereus ATCC 14579, and the precise corresponding sequence from B. cereus ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B. subtilis, but most do not have homologues in B. subtilis. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B. anthracis. Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.     

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.     

Physical maps of the genomes of three Bacillus cereus strains.

NotI restriction maps of the chromosomes from Bacillus cereus ATCC 10876, ATCC 11778, and the B. cereus type strain ATCC 14579 have been established and compared with the previously established map of B. cereus ATCC 10987. Between 10 and 14 NotI fragments were observed, ranging from 15 to 1,300 kb, in digests of DNA from the various strains. The sizes of the genomes varied between 5.4 and 6.3 Mb. The maps were constructed by hybridization of 42 random probes, prepared from B. cereus ATCC 10987 libraries, to fragments from partial and complete NotI digests, separated by pulsed-field gel electrophoresis. Nine probes were specific for ATCC 10987 only. Probes for five B. subtilis and five B. cereus genes were also used. The NotI restriction fragment patterns of the four strains were strikingly different.     

Assay and characterization of a strong promoter element from B. subtilis

A new strong promoter fragment isolated from Bacillus subtilis was identified and characterized. Using the heat stable beta-galactosidase as reporter, the promoter fragment exhibited high expression strength both in Escherichia coli and B. subtilis. The typical prokaryotic promoter conservation regions were found in the promoter fragment and the putative promoter was identified as the control element of yxiE gene via sequencing assay and predication of promoter. To further verify and characterize the cloned strong promoter, the putative promoter was sub-cloned and the beta-Gal directed by the promoters was high-level expressed both in E. coli and B. subtilis. By means of the isolated promoter, an efficient expression system was developed in B. subtilis and the benefit and usefulness was demonstrated through expression of three heterologous and homogenous proteins. Thus, we identified a newly strong promoter of B. subtilis and provided a robust expression system for genetic engineering of B. subtilis.