肺炎链球菌是寄生在人类上呼吸道上主要的病原菌之一。肺炎链球菌可以引起急性肺炎、脑膜炎、中耳炎和败血症等，每年在全球范围内至少导致1百多万人死亡。多糖荚膜是肺炎链球菌最重要的致病因子，主要通过其多糖分子的负电荷来排斥白细胞的吞噬，帮助细菌逃逸宿主免疫系统的检测和杀菌功能。肺炎链球菌能够产生至少97种不同的多糖荚膜（血清型），每种荚膜都是由单个（37型）或多个（其他型）基因（基因簇）所编码的多糖聚合酶来合成的。 目前对肺炎链球菌荚膜合成基因在多糖合成和转运方面的功能已经有了大量的研究，但是在国际范围内对这些基因的转录调控机制仍然了解甚少。本课题的主要研究目标是阐述肺炎链球菌荚膜合成基因的转录机制。首先，我们研究分析了血清型2 D39菌株荚膜基因簇的转录特点。确定荚膜基因的转录起始位点位于cps2A基因（荚膜基因簇的第一个基因）上游第25th的鸟嘌呤G。通过染色体突变和荧光报告系统，揭示荚膜基因的转录不但依赖于cps2A基因上游核心启动子区域，而且还需要其上游的三个增强子元件（转座子插入序列IE、RUP和一个链接序列SS）。这些序列在D39菌株染色体上的敲除和置换实验显示核心启动子和每个增强子元件在荚膜合成和致病性方面都具有重要的贡献。其次，我们测定了225株肺炎链球菌临床菌株荚膜基因启动子和增强子DNA序列，结果发现这个区域在增强子元件的排布和DNA序列上具有高度的多样性。主要表现为：1）序列本身和长短的变化；2）核酸序列组合的多样化；3）保守序列的选择性组合；4）与荚膜基因非一一对应的关系。启动子-增强子置换实验明确地揭示：荚膜基因增强子元件序列的变异会导致其下游荚膜基因转录水平的显著差异和荚膜厚度的相应变化。最后，增强子元件的变异可以导致肺炎链球菌菌株在致病性方面的显著性差异。综上所述，本课题在国际上第一次揭示了肺炎链球菌通过多个不同的增强子元件来调控荚膜基因的转录水平，从而调控其荚膜的厚度。肺炎链球菌还可以通过自然转化所介导的DNA序列平行转移来相互交换荚膜基因，改变荚膜基因增强子元件在染色体上的相对排布位置和DNA序列，由此调控荚膜厚度及其相关的生物学特性。本课题为充分理解肺炎链球菌荚膜合成的调控机理奠定了可靠的理论基础，为进一步阐明肺炎链球菌在致病性方面的分子机制提供了重要的信息。

Streptococcus pneumoniae (pneumococcus) is a commensal in the upper respiratory tract and also a major human pathogen of acute pneumonia, meningitis, otitis media, and septicemia. Pneumococcal infections are globally responsible for at least one million of deaths every year. The capsular polysaccharide (CPS) is the major virulence factor of S. pneumoniae, enabling the bacteria to evade phagocytic killing by the intrinsic negative charge of CPS. S. pneumoniae produces at least 97 antigenically different types of capsule, each of which is synthesized by the capsule gene locus. The genetic and biochemical principles of the CPS synthesis have been extensively documented. While the type-37 capsule is synthesized by a single polysaccharide polymerase gene (tts), the production of all the other known CPSs is realized by different sets of multiple cps genes in the same locus of the pneumococcal chromosome. Except for the type-3 cps genes, the gene clusters of the other 95 CPS types consist of the common and type-specific cps genes. It is known that these cps genes are transcribed from the promoter upstream of cpsA, the first gene in the cps gene clusters. It has also been obserbed that the production level of the pneumococcal capsule is altered by multiple conditions. However, the transcription of the cps locus is poorly understood at the present time. The goal of this dissertation project is to determine how the cps genes of S. pneumoniae are transcribed and transcriptionally regulated. We first characterized the transcriptional features of the cps locus in the type-2 virulent strain D39. The initial analysis revealed that the cps genes are cotranscribed from a major transcription start site at the 25th (G) upstream of cps2A (the first gene of cps locus). The primier extension, for the first time, revealed that the 17 genes in the cps locus are co-transcribed as an operon. Using unmarked chromosomal truncations and a luciferase-based transcriptional reporter, we showed that the promoter of the cps operon consists of four functional modules: core promoter and three enhancer sequences (putative transposable insertion element - IE, repeat unit of the pneumococcus - RUP, and a spacing sequence - SS). The importance of these promoter modules in the transcription of the cps operon was functionally confirmed by significant reduction of the knockout mutants in capsule production and virulence in a mouse systemic infection model. We subsequently assessed the sequence feature of the cps promoter region in 225 clinical pneumococcal isolates. The result revealed entensive modular and sequence variations in the promoter region of these isolates, which are characterized by postional shuffling of the IE and RUP module, as well as mosaic combinations of the enhancer modules with nucleotide polymorphisms. These sequence variations appear to be a result of horizontal DNA exchange mediated by the natural competence. The promoter replacement experiments showed remarkable functional impact of the strain-to-strain variations in the cps promoter on the levels of the cps gene mRNA, capsule production, and virulence. These results strongly suggest that sequence varionations in the cps promoter represent a novel adaptation mechanism of S. pneumoniae by diversifying the capsule thickness among the strains, in addition to the well-known capsule type switch. In summary, this study, for the first time, discovered four functional modules in the cps promoter of S. pneumoniae. All of these promoter modules are required for the full transcription of the cps operon and capsule production. Sequence and modular variations in the cps promoter among the clinical isolates can significantly influence the transcription of the cps genes and capsule production. The insightful information derived from this project has substantially enhaced our understanding of the molecular mechanisms governing the formation of the pneumococcal capsule and thereby the pathogenesis of this important human pathogen.