基因串联策略强化枯草芽孢杆菌终止子的功能及其在基因表达中的应用

在底盘微生物中使用高性能终止子能够显著增强基因终止效率,维持新合成mRNA的稳定性,提升外源基因表达性能.而目前缺乏专一用于枯草芽孢杆菌的终止子元件,限制了复杂功能基因电路的设计.[目的]在枯草芽孢杆菌中挖掘新的高性能终止子,并进一步重新设计,丰富适用于这一底盘的高性能人工终止子.[方法]将枯草芽孢杆菌终止子和枯草芽孢...     

枯草芽孢杆菌168新型转录终止子的构建与表征（英文）

转录终止子作为一种位于终止密码子后的调控信号,负责终止DNA的转录和RNA的释放。文中首次改造并分析了来源于噬菌体的λt_o终止子的发卡结构与富含尿嘧啶的序列对枯草芽孢杆菌168中基因转录终止效率以及mRNA稳定性的影响。结果表明,相对于野生型的λt_0终止子,突变体M3、M11和M12表现出了更高的转录终止效率,突变体M3、M4和M11更有利于上游绿色荧光蛋白mRNA的稳定。另外,我们发现插入RNase作用位点同样提高了mRNA的稳定性。研究结果表明终止子中的发卡环对转录终止不是必需的,同时,结果也证明了转录终止子可以作为一种潜在的工具用于提高枯草芽孢杆菌中mRNA的稳定性以及相应酶的表达。     

枯草芽孢杆菌转录调控网络的连接结构及分解

对枯草芽孢杆菌（Bacillus subtilis）转录调控网络的全局连接性质进行分析之后,基于一种自上而下的思路,将重点放在最大弱连通体,提出了基于距离的分解方法,并对得出的模块进行了明确的生物学功能定义。研究结果表明基于距离的分解方法对于标识转录调控网络中的生物学功能模块十分有效.     

枯草芽孢杆菌噬菌体载体的构建

以φ0105DI：It为原始株构建的重组噬菌体φ105S35和φ10 5S36具有自主侵染能力和溶源化特征。其基因组内插入的lkb片段上的cat,基因赋予二者所在宿主以氯霉素抗性,在两株噬菌体中插入位点相同,即原φ105DI ：It的smal酶切片段D、E之间,但插入片段在二者中的定向相反。与cat基因同时引入的单一BamHI和Xbal位点提供了外源DNA的插入位置。重组噬菌体DNA可高效转染枯草芽孢杆菌原生质体。因此φ105S35和币φ105S36可作为枯草芽孢杆随系统的载体而被利用。     

杀虫防病基因工程枯草芽孢杆菌的构建

分别以枯草芽孢杆菌大肠杆菌穿梭质粒pHB201和pRP22为载体,通过感受态转化方法,将Bt-HD-1杀虫蛋白基因cry1Ac导入了水稻纹枯病生防菌株枯草芽孢杆菌B916。工程菌株质粒酶切电泳分析、Southern印迹分析和杀虫生物活性测定结果证实了cry1Ac基因的导入及其在B916中的有效表达。抑菌测定证明工程菌株保持了原野生型菌株良好的抑菌活性。质粒稳定性分析表明以载体pRP22构建的工程菌株Bs2249具有良好的稳定性,而以载体pBH201构建的工程菌株Bs2014则不稳定。此外,实验还证实Bt基因的导入与表达对B916的生长没有不良影响。     

枯草芽孢杆菌感受态研究新进展

在枯草芽孢杆菌中,感受态的形成受到一种二元信号转导系统的调节,这种系统对胞外的感受态信息素浓度作出感应而激活晚期感受态基因的表达。各种晚期感受态蛋白分别负责外源DNA的吸附、吸收和内源化,它们共同构成了DNA的运输系统。初步探讨了枯草芽孢杆菌感受态调节在细胞生长和进化中的意义。     

Efficiency of transcriptional terminators in Bacillus subtilis

To promote more efficient synthesis of heterologous gene products in a Bacillus subtilis host, we have developed a system for rapidly testing the effect of a putative terminator on in vivo gene expression. Terminator structures from the Bacillus amyloliquefaciens amyE gene, the Bacillus licheniformis penP gene, the B. subtilis bglS gene, and the Bacillus thuringiensis cry gene were subcloned and inserted into a vector in such a way as to disrupt expression of the cat-86 gene. Comparisons are made between gene expression levels and the stabilities of the respective stem-loop structures.     

Identification and characterization of new DNA replication terminators in Bacillus subtilis

A functional DNA replication terminator of Bacillus subtilis contains two overlapping binding sites, A and B, for the replication terminator protein (RTP). A degenerate 17-mer oligonucleotide corresponding to the consensus B site has been used to detect four new terminators in the B. subtilis chromosome, in addition to the previously identified and closely spaced IRI and IRII. All the new terminators lie in the terminus region of the chromosome, on both sides of IRI and IRII, with their positions spanning <1O% of its length. Their DNA sequences are characterized by clearly identifiable A- and B-binding sites. They bind RTP in a manner indistinguishable from IRI, although precise affinities have not been compared. Each new terminator is functional in causing fork arrest when present in a plasmid replicating in B. subtilis . Three of the four were tested for polarity in fork-arrest activity and exhibited the polarity expected. The total of six terminators now identified in B. subtilis have been named TerI-TerVI . TerI and TerII correspond to the previously identified IRI and IRII, respectively. The chromosomal orientations of all but one of the terminators ( TerIV ) have been established and they conform to an arrangement similar to that in Escherichia coli in which two opposed groups of polar terminators provide a replication-fork trap ensuring that the approaching forks meet within a restricted region of the chromosome. The development of a strikingly similar arrangement of terminators in the two organisms, despite the lack of any detectable similarity in their respective DNA terminators and terminator proteins, emphasizes the importance of the replication-fork trap in each case.     

Search for additional replication terminators in the Bacillus subtilis 168 chromosome.

The Bacillus subtilis 168 chromosome is known to contain at least six DNA replication terminators in the terminus region of the chromosome. By using a degenerate DNA probe for the consensus terminator sequence and low-stringency hybridization conditions, several additional minor hybridizing bands were identified. DNA corresponding to the most intense of these bands was cloned and characterized. Although localized in the terminus region, it could not bind RTP and possibly represents a degenerate terminator. A search of the SubtiList database identified an additional terminator sequence in the terminus region, near glnA. It was shown to bind RTP and to function in blocking replication fork movement in a polar manner. Its orientation conformed to the replication fork trap arrangement of the other terminators. The low-stringency hybridization experiments failed to identify any terminus region-type terminators in the region of the chromosome where postinitiation control sequences (STer sites) are known to reside. The two most likely terminators in STer site regions, in terms of sequence similarity to terminus region terminators, were identified through sequence searching. They were synthesized and were found not to bind RTP under conditions that allowed binding to terminus region terminators. Neither did they elicit fork arrest, when present in a plasmid, under stringent conditions. It is concluded that the STer site terminators, at least the first two to the left of oriC, do not have the typical consensus A+B site makeup of terminus region terminators.     

Suppressor system in Bacillus subtilis 168

Multiple auxotrophic strains of Bacillus subtilis 168 were tested for joint one-step reversion of two or more auxotrophic markers to the wild-type phenotype. Mu8u5u5, a strain requiring leucine, methionine, and threonine, yielded revertants that grew without added methionine or threonine and proved to have a suppressor gene. When transferred by transformation with deoxyribonucleic acid, this suppressor gene also suppressed the adenine mutation in another strain, Mu8u5u6. The one-step double revertants fell into two distinct classes: strains of class su(+) (I) grow well in broth; strains of class su(+) (II) grow poorly. Strains su(+) (II) tend to revert frequently to the su(+) (I) or su(-) state. Conditional lethal mutants of phage phie were isolated which can grow on the su(+) and not on the su(-) strains.     

Bacteriophage Interference in Bacillus subtilis 168

Strains of Bacillus subtilis lysogenic for temperate bacteriophage SPO2 inhibit the development of bacteriophage phi1. After infection by bacteriophage phi1, DNA and RNA synthesis in the lysogenic host terminates, culminating in cell death. Bacteriophage SPO2 also prevents the production of bacteriophage phi105. Mechanisms for these two types of bacteriophage interference are discussed.     

Utilization of subsidiary chromosomal replication terminators in Bacillus subtilis

The Bacillus subtilis merodiploid strain GSY1127 contains a large nontandem duplication of a portion of its chromosome within its left (anticlockwise) replication segment. This causes displacement of the replication terminus region to a noticeably asymmetric location relative to oriC. The utilization of the subsidiary replication terminators, TerIII and TerV, in the merodiploid strain has been compared with that in B. subtilis 168. It is shown that TerIII is utilized to a significant extent in GSY1127 and that TerV is used only marginally at the most. Neither of these terminators is used to a measurable extent in the 168 strain. It is concluded that TerIII and TerV do indeed function as backups to the major terminator TerI, as has been generally thought. It is further concluded that, in the 168 strain, the vast majority of clockwise forks are arrested at the highly efficient TerI terminator, with fork fusion between the approaching forks occurring frequently while the clockwise fork is stationary at TerI.