Shen Kai, Antalis Patricia, Gladitz John, Sayeed Sameera, Ahmed Azad, Yu Shujun, Hayes Jay, Johnson Sandra, Dice Bethany, Dopico Richard, Keefe Randy, Janto Benjamin, Chong William, Goodwin Joseph, Wadowsky Robert M, Erdos Geza, Post J Christopher, Ehrlich Garth D, Hu Fen Z
Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, 320 East North Ave., 11th Floor South Tower, Pittsburgh, PA 15212, USA.
Infect Immun. 2005 Jun;73(6):3479-91. doi: 10.1128/IAI.73.6.3479-3491.2005.
We hypothesize that Haemophilus influenzae, as a species, possesses a much greater number of genes than that found in any single H. influenzae genome. This supragenome is distributed throughout naturally occurring infectious populations, and new strains arise through autocompetence and autotransformation systems. The effect is that H. influenzae populations can readily adapt to environmental stressors. The supragenome hypothesis predicts that significant differences exist between and among the genomes of individual infectious strains of nontypeable H. influenzae (NTHi). To test this prediction, we obtained 10 low-passage NTHi clinical isolates from the middle ear effusions of patients with chronic otitis media. DNA sequencing was performed with 771 clones chosen at random from a pooled genomic library. Homology searching demonstrated that approximately 10% of these clones were novel compared to the H. influenzae Rd KW20 genome, and most of them did not match any DNA sequence in GenBank. Amino acid homology searches using hypothetical translations of the open reading frames revealed homologies to a variety of proteins, including bacterial virulence factors not previously identified in the NTHi isolates. The distribution and expression of 53 of these genes among the 10 strains were determined by PCR- and reverse transcription PCR-based analyses. These unique genes were nonuniformly distributed among the 10 isolates, and transcription of these genes in planktonic cultures was detected in 50% (177 of 352) of the occurrences. All of the novel sequences were transcribed in one or more of the NTHi isolates. Seventeen percent (9 of 53) of the novel genes were identified in all 10 NTHi strains, with each of the remaining 44 being present in only a subset of the strains. These genic distribution analyses were more effective as a strain discrimination tool than either multilocus sequence typing or 23S ribosomal gene typing methods.
我们推测，作为一个物种，流感嗜血杆菌拥有的基因数量比任何单个流感嗜血杆菌基因组中的基因数量要多得多。这个超基因组分布于自然发生的感染群体中，新菌株通过自身感受态和自身转化系统产生。其结果是流感嗜血杆菌群体能够很容易地适应环境压力源。超基因组假说预测，不可分型流感嗜血杆菌（NTHi）个体感染菌株的基因组之间存在显著差异。为了验证这一预测，我们从慢性中耳炎患者的中耳积液中获得了10株低传代NTHi临床分离株。从混合基因组文库中随机选择771个克隆进行DNA测序。同源性搜索表明，与流感嗜血杆菌Rd KW20基因组相比，这些克隆中约10%是新的，并且它们中的大多数与GenBank中的任何DNA序列都不匹配。使用开放阅读框的假设翻译进行氨基酸同源性搜索，发现与多种蛋白质具有同源性，包括先前在NTHi分离株中未鉴定出的细菌毒力因子。通过基于PCR和逆转录PCR的分析确定了其中53个基因在10株菌株中的分布和表达。这些独特的基因在10个分离株中分布不均匀，在浮游培养物中这些基因的转录在50%（352次中有177次）的情况下被检测到。所有新序列都在一个或多个NTHi分离株中被转录。17%（53个中的9个）的新基因在所有10株NTHi菌株中都被鉴定出来，其余44个基因中的每一个仅存在于部分菌株中。这些基因分布分析作为一种菌株鉴别工具比多位点序列分型或23S核糖体基因分型方法更有效。
