EXPRESSION AND PHASE VARIATION OF GONOCOCCAL OPA GENES

淋球菌 OPA 基因的表达和相变

• 批准号: 3803279
• 负责人: R J BELLAND
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3803279
• 关键词: Escherichia coli; Neisseria gonorrhoeae; bacterial DNA; bacterial proteins; chimeric proteins; conformation; gene expression; genetic transcription; genetic translation; membrane proteins; molecular cloning; nucleic acid repetitive sequence; recombinant DNA; transcription termination

The major surface components of the human pathogen Neisseria gonorrhoeae change dramatically during the course of gonorrheal disease in male volunteers. Changes involve the expression states of particular genes in conjunction with the genetic content of the genes being expressed. The Opa family of outer-membrane proteins change expression states as a result of a unique translational switching mechanism involving the insertion or deletion of variable numbers of pentameric coding repeat elements (CR) within that segment of the gene coding for the signal-peptide of the Opa pre-protein. The direct, tandem repeat elements have the capacity to form an intramolecular triplex (H-DNA) in response to negative supercoiling torsion. The opa CR region is the first naturally occurring prokaryotic DNA sequence shown to adopt this non B-DNA conformation and is also unique in that the sequence occurs within the coding sequence of the gene. The phase variation frequencies of repeated genetic elements which form H-DNA are significantly lower than elements with the same repetitive nature which are unable to form H-DNA. Transcription of both types of repeat elements results in increased phase variation frequencies, but this effect is less pronounced in H-DNA forming repetitive sequences. Transcription of opa genes is constitutive for the 5' end of the gene but shows a strong termination when the genes are in the "off" configuration. The termination may be linked to the formation of the H-DNA structure and if so, represents a novel termination mechanism which could allow the cell to conserve nucleotide triphosphates. The enzymes responsible for generation of negative supercoiling (gyrA/B) were cloned and are being purified to determine their effect on the transcription of opa and the relationship between transcription and H-DNA formation. A number of Opa proteins have been constitutively expressed as beta-lactamase-Opa fusions in E. coli and have been shown to segregate to the outer-membrane in a conformation resembling that seen in N. gonorrhoeae. Recombinant Opa+ bacteria mimic the interactions seen between human neutrophils and gonococcal strains expressing the analogous Opa. Recombinant strains also show an ability to adhere to certain eukaryotic cell lines in in vitro binding assays.

人类病原体淋病奈瑟菌的主要表面成分 男性淋病病程中的剧烈变化 志愿者。变化涉及到特定基因的表达状态 与正在表达的基因的遗传含量相联系。奥帕 外膜蛋白家族改变表达状态的结果 一种独特的平移切换机构，涉及插入或 删除数量可变的五聚体编码重复序列(CR) 在编码OpA信号肽的基因片段内 前蛋白质。直接串联重复元件具有形成 对负超螺旋反应的分子内三链(H-DNA) 扭力。OPA CR区是第一个自然存在的原核生物 DNA序列显示采用这种非B-DNA构象，并且也是唯一的 因为该序列出现在该基因的编码序列内。这个 构成H-DNA的重复遗传元件的相变频率 显著低于具有相同重复性质的元素 不能形成H-DNA。两种类型重复元件的转录 导致相变频率增加，但这种影响较小 在H-DNA中发音，形成重复序列。OPA转录 基因是基因5‘端的组成部分，但表现出很强的 当基因处于“关闭”状态时终止。终结者 可能与H-DNA结构的形成有关，如果是这样，代表 一种新的终止机制，可以允许细胞保存 三磷酸核苷酸。这些酶负责产生 克隆了负超卷曲(gyra/B)，并正在进行纯化以 确定它们对OPA转录的影响及其相互关系 转录和H-DNA形成之间的关系。许多OpA蛋白都有 在大肠杆菌中结构性表达为β-内酰胺酶-OpA融合蛋白 已经被证明以构象的形式分离到外膜 与淋病奈瑟菌相似。重组OPA+细菌模拟 人中性粒细胞与淋球菌株之间的相互作用 表达与之类似的Opa。重组菌株也显示出一种能力 在体外结合试验中与某些真核细胞系结合。