REGULATION OF STREPTOCOCCUS GORDONII GLUCOSYLTRANSFERASE

戈登链球菌葡萄糖基转移酶的调控

• 批准号: 2749339
• 负责人: M Margaret VICKERMAN
• 金额: $ 6.85万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749339
• 关键词: Enterococcus; Streptococcus; Streptococcus sanguis; bacterial genetics; beta galactosidase; chloramphenicol acetyltransferase; fusion gene; gene expression; genetic operator element; genetic promoter element; genetic regulation; genetic strain; hexosyltransferase; oral bacteria; plasmids; regulatory gene; reporter genes; structural genes

Streptococcus gordonii have a single glucosyltransferase (GTF) enzyme that produces glucans from sucrose. Differences in GTF activity, which are affected by environmental and growth conditions, affect the ability of S. gordonii to colonize surfaces in vitro and therefore, may affect their ability to establish in ecological niches in vivo. Since S. gordonii cells and their products are present in early dental plaque, they act as a substratum for the attachment of other oral organisms, including potential pathogens, and therefore may play important roles in determining mature plaque composition. Unlike mutans streptococci, which have multiple gtf genes with as yet undescribed genetic regulatory mechanisms, S. gordonii has a single gtf gene. The expression of the GTF structural gene, gtfG, is positively regulated by the upstream gene rgg, the only described regulatory determinant for a gtf gene. S. gordonii undergo a spontaneous, reversible phase variation between high (Spp+) and low (Spp-) levels of GTF activity. rgg can act in trans to increase GTF activity in both Spp+ and Spp- strains. The molecular basis for changes in gtfG expression is unknown but there is preliminary evidence that more than one mechanism is involved. These could include frameshifts or rearrangements in rgg or gtfG; alternatively, distally located regulatory gene(s) may affect rgg and/or gtfG expression. The proposed studies will attempt to determine factors associated with regulation of gtfG and to help elucidate mechanisms underlying phase variation at the DNA level. Nucleotide sequences of Spp+ and Spp- phase variants will be compared for structural differences in the rgg and gtfG genes and their flanking regions, which may affect their expression. Distal regions of S. gordonii DNA that affect expression of rgg and/or gtfG will be identified in an E. faecalis system that allows efficient screening of many plasmid clones. Plasmid DNA containing rgg and/or gtfG lacZ transcriptional fusions will be introduced into E. faecalis and the resulting strains will be transformed with compatible plasmids carrying randomly cloned & gordonii DNA. Any clones affecting expression of rgg and/or gtfG, as monitored by changes in colony color reflecting changes in beta- galactosidase activity, will be characterized further. Because lacZ transcriptional fusions are only weakly expressed in S. gordonii, another reporter system is necessary to study the identified regulatory elements in the S. gordonii genetic background. Therefore, chloramphenicol acetyltransferase (CAT) transcriptional fusions will be integrated into the chromosome of S. gordonii to monitor expression of rgg and/or gtfG by CAT activity. Plasmids carrying the characterized distal regulatory segments of DNA will be transformed into these S. gordonii fusion strains for additional genetic studies. Finally, S. gordonii strains with chromosomal CAT fusions will be used to study environmental influences on expression of gtfG and its regulatory gene(s) at the DNA level. It hoped that these studies, aimed at better understanding the regulation of gtfG expression, will provide insights into the oral microbial ecology and dental health.

格氏链球菌具有单一的葡糖基转移酶（GTF） 从蔗糖中产生葡聚糖。GTF活性的差异， 受环境和生长条件的影响， 色葡萄戈登氏菌在体外定殖表面，因此，可能影响 它们在体内建立生态位的能力。自S. 戈登氏细胞及其产物存在于早期牙菌斑中， 它们作为附着其它口腔生物体的基质， 包括潜在的病原体，因此可能在 确定成熟牙菌斑的组成。与变形链球菌不同， 具有多个GTF基因，其具有尚未描述的遗传调节 机制，S.戈登氏菌只有一个GTF基因。GTF的表达 结构基因gtfG受上游基因rgg正调控， 唯一描述的GTF基因的调节决定子。S.戈登链球菌 在高（Spp+）和低（Spp+）之间经历自发的、可逆的相位变化。 低水平（Spp-）GTF活性。rgg可以反式作用以增加GTF 在Spp+和Spp-菌株中的活性。变化的分子基础 在gtfG表达是未知的，但有初步证据表明，更多的 不止一种机制。这些可能包括移码或 rgg或gtfG的重排;或者，位于远端的调节 基因可影响rgg和/或gtfG表达。拟议的研究将 尝试确定与gtfG调节相关的因素，并 有助于阐明DNA水平上相位变化的机制。 将比较Spp+和Spp-期变体的核苷酸序列， rgg和gtfG基因及其侧翼的结构差异 这可能会影响它们的表达。S.戈登链球菌 影响rgg和/或gtfG表达的DNA将在本发明的实施方案中鉴定。 E.粪菌系统，允许有效筛选许多质粒 克隆含有rgg和/或gtfG lacZ转录的质粒DNA 将融合基因导入E.粪菌和由此产生的菌株 将用携带随机克隆的& 戈登氏菌DNA任何影响rgg和/或gtfG表达的克隆，如 通过反映β- 半乳糖苷酶活性将进一步表征。因为lacZ 转录融合体在S. gordonii另一个 报告系统是必要的，以研究确定的监管要素 在S.戈登氏菌的遗传背景因此，氯霉素 乙酰转移酶（CAT）转录融合体将被整合到 S. gordonii监测rgg和/或gtfG的表达 CAT活动。质粒携带特征性的远端调节 DNA片段将转化为这些S。戈登融合菌 进行进一步的基因研究最后，S。戈登氏菌 染色体CAT融合将用于研究环境影响 在DNA水平上对gtfG及其调控基因表达的影响。它 希望这些研究，旨在更好地了解监管， gtfG的表达，将提供深入了解口腔微生物生态学 和牙齿健康。