Global transcription pattern of virulent S. pneumoniae strain D39 (NCTC) compared to that of strains R6 and D39 (Lilly). Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. D39 is an historically important serotype 2 strain that was used in experiments by Avery and coworkers to demonstrate that DNA is the genetic material. Although isolated nearly a century ago, D39 remains extremely virulent in murine infection models and is perhaps the strain used most frequently in current studies of pneumococcal pathogenicity. To date, the complete genome sequences have been reported for only two S. pneumoniae strains; TIGR4, a recent serotype 4 clinical isolate, and laboratory strain R6, an avirulent, unencapsulated derivative of strain D39. We report herein the genome sequences of two different isolates of strain D39 and the corrected sequence and updated annotation of strain R6. Comparisons of these three related sequences allowed deduction of the likely sequence of the D39 progenitor and mutations that arose in each isolate. Despite its numerous repeated sequences and IS elements, the serotype 2 genome has remained remarkably stable during cultivation, and one of the D39 isolates contains only 5 relatively minor mutations compared to the deduced D39 progenitor. In contrast, laboratory strain R6 contains 71 single base pair changes, 6 deletions, 4 insertions, and has lost the cryptic pDP1 plasmid compared to the D39 progenitor strain. Many of these mutations are in or affect the expression of genes that play important roles in regulation, metabolism, and virulence. The nature of the mutations that arose spontaneously in these three strains, relative global transcription patterns determined by microarray analyses, and the implications of the D39 genome sequences to studies of pneumococcal physiology and pathogenesis are presented and discussed. Keywords: bacterial strain comparison, bacterial isolate comparison Overall design: Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction. Data were also normalized with background subtraction (not shown); expression ratios did not differ significantly with or without background subtraction. Tables of average fold changes are included as supplementary material for the accompanying manuscript (Genome Sequence of Avery's Virulent Serotype 2 Strain D39 of Streptococcus pneumoniae and Comparison With That of Unencapsulated Laboratory Strain R6, Lanie J.A. et al, J. Bact, accepted).

肺炎链球菌（Streptococcus pneumoniae，又称肺炎球菌）是引发多种严重黏膜及侵袭性疾病的主要人类呼吸道致病菌。D39是一株具有重要历史意义的2型血清型菌株，曾被埃弗里（Avery）及其同事用于实验，证明DNA是遗传物质。尽管已分离近一个世纪，D39在小鼠感染模型中仍具有极强的毒力，或许是当前肺炎球菌致病性研究中使用最频繁的菌株。截至目前，仅两株肺炎链球菌菌株完成了完整基因组序列报道：一株是近年分离的4型血清型临床分离株TIGR4，另一株是实验室菌株R6——它是D39的无荚膜无毒衍生物。本文报道了两株不同D39分离株的基因组序列，以及R6菌株的校正序列与更新注释。通过对这三株相关菌株的序列比对，我们推导了D39祖先菌株的可能序列，并确定了各分离株中出现的突变。尽管2型血清型基因组存在大量重复序列与插入序列元件（IS elements），但其在传代培养过程中仍保持了极高的稳定性；其中一株D39分离株与推导的D39祖先菌株相比，仅存在5处相对轻微的突变。相比之下，实验室菌株R6与D39祖先菌株相比，存在71处单碱基替换、6处缺失、4处插入，且丢失了隐性pDP1质粒。这些突变中有许多位于或影响调控、代谢与毒力相关基因的表达。本文还介绍并讨论了这三株菌株自发突变的特征、通过微阵列分析（microarray analyses）确定的整体转录谱，以及D39基因组序列对肺炎球菌生理学与致病机制研究的意义。关键词：细菌菌株比较、细菌分离株比较。实验整体设计：将细菌菌株于37℃、5% CO₂环境下在富营养培养基（脑心浸液培养基，Brain Heart Infusion, BHI）中进行指数生长期培养，按照相关样本记录中的方法进行处理。样本来自三次独立生物学重复，并包含一次染料互换实验。数据采用Lowess（亚栅格）法进行归一化，未进行背景扣除；同时也采用了带背景扣除的归一化方法（数据未展示），但两种方法得到的表达比值无显著差异。平均倍数变化表格已作为补充材料附于随刊手稿《Genome Sequence of Avery's Virulent Serotype 2 Strain D39 of Streptococcus pneumoniae and Comparison With That of Unencapsulated Laboratory Strain R6, Lanie J.A. et al, J. Bact, accepted》中。