The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, approximately 1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.

译文

:分布式基因组假设(DGH)指出,细菌物种中的每个菌株都从基于种群的超基因组中获得独特的基因分布,其比任何给定菌株的基因组都要大很多倍。自然感染人群通常是多克隆的,大多数慢性细菌病原体具有高度发展的水平基因转移机制的观察结果提示了DGH,并提供了解释哺乳动物宿主面对适应性防御机制时慢性感染如何持续存在的手段和机制。先前已确定DGH对专性病原体的有效性,我们希望评估其对机会细菌病原体的适用性。这是通过构建和分析包含约216,000个功能性克隆的高度冗余的集合基因组文库完成的,该文库由铜绿假单胞菌的12种低通道临床分离株,6种耳泻分离株和6个其他身体部位构建而成。对来自该文库的3,214个随机挑选的克隆(平均插入片段大小,约1.4 kb)进行序列分析,结果表明,对于铜绿假单胞菌原型菌株PAO1的所有基因组序列,其中348个(10.8%)克隆是唯一的。在这些独特序列内的开放阅读框的假想翻译证明了与许多细菌毒力因子和以前在铜绿假单胞菌中未鉴定出的其他蛋白质的蛋白质同源性。进行了基于PCR和逆转录PCR的分析,以分析这些序列的70个开放阅读框子集在11种临床菌株中的分布和表达模式。这些序列在临床分离株中分布不均,只有一半或34个新序列出现在一个或两个单独的菌株中。表达谱分析表明这些序列中的绝大多数都被表达,强烈暗示它们编码功能蛋白。

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