REGULATION OF STREPTOCOCCUS GORDONII GLUCOSYLTRANSFERASE

戈登链球菌只有一种葡萄糖转移酶(GTF) 从蔗糖中产生葡聚糖。GTF活性的差异，这 都受到环境和生长条件的影响，影响能力 戈登葡萄球菌在体外定植表面，因此可能会影响 它们在体内建立生态环境的能力。自S。 戈登尼细胞及其产物存在于早期牙菌斑中， 它们充当附着其他口腔生物的基质， 包括潜在的病原体，因此可能在 确定成熟斑块的组成。与变形链球菌不同的是， 有多个GTF基因，但基因调控尚不清楚 机制上，戈登氏链霉菌只有一个GTF基因。GTF的表达 结构基因gtfG受上游基因rgG正向调控， 唯一被描述的GTF基因的调控决定因素。戈登氏链霉菌 在高(SPP+)和高(SPP+)之间经历自发的可逆位相变化 低水平(SPP-)的GTF活性。RGG可通过反式作用增加GTF 在SPP+和SPP-菌株中均有活性。变化的分子基础 在gtfG中的表达未知，但有初步证据表明 涉及的机制不止一种。这些可能包括框架移位或 RGG或GTFG中的重排；或者，位于远端的调控 S基因可能影响RGG和/或GTFG的表达。拟议的研究将 试图确定与gtfg监管相关的因素，并 有助于在DNA水平上阐明相位变化的潜在机制。 将比较SPP+和SPP-相变异体的核苷酸序列 RgG和gtfG基因的结构差异及其侧翼 区域，这可能会影响它们的表达。戈登氏链霉菌的远侧区 影响RGG和/或gtfG表达的DNA将在 粪肠球菌系统，其允许有效地筛选许多质粒 克隆人。含有rgG和/或gtfG LacZ转录的质粒DNA 融合将被引入粪肠球菌和由此产生的菌株 将与携带随机克隆的亲和质粒转化 Gordonii DNA。任何影响rgg和/或gtfG表达的克隆，如 通过菌落颜色的变化来监测，反映了β- 半乳糖苷酶活性，将进一步鉴定。因为Lacz 转录融合只在另一种类型的S.gordonii中微弱表达 记者制度是研究已确定的监管要素所必需的 戈登氏链霉菌的遗传背景。因此，氯霉素 乙酰转移酶(CAT)转录融合将整合到 监测rgG和/或gtfG表达的戈登链霉菌染色体 通过CAT活性。携带特征性远端调控基因的质粒 DNA片段将被转化到这些戈登链球菌融合菌株中 进行额外的基因研究。最后，戈登葡萄球菌的菌株具有 染色体猫的融合将被用于研究环境影响 GtfG及其调控基因(S)在DNA水平上的表达它 希望通过这些研究，旨在更好地了解监管 GtfG的表达，将提供对口腔微生物生态的洞察 和牙齿健康。

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.