Tandem genes of Chlamydia psittaci that encode proteins localized to the inclusion membrane
Tandem genes of Chlamydia psittaci that encode proteins localized to the inclusion membrane
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DOI：
10.1046/j.1365-2958.1998.00867.x
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发表时间：
1998-06-01
影响因子：
3.6
通讯作者：
Hackstadt, T
中科院分区：
文献类型：
--
作者：
Bannantine, JP;Rockey, DD;Hackstadt, T
衣原体是专性细胞内细菌，在非酸化液泡内复制，称为包涵体。为了鉴定细胞内生命周期所特有的衣原体蛋白，用感染豚鼠的恢复期抗血清和针对福尔马林固定的纯化衣原体初级体（EBs）的抗血清对裸热衣原体DNA的lambda表达文库进行了差异筛选。一个文库克隆被鉴定出含有两个开放阅读框（orf），具有编码类似大小的蛋白质的潜力，大约为20 kDa。这些蛋白随后被命名为IncB和IncC。测序结果显示，克隆的插入物在incS上游有一个强大肠杆菌样启动子序列，orf之间有一个36nt的基因间区。对incB和incC上游区域的序列分析显示，两个orf与氨基酸转运体和钠依赖性转运体具有很强的同源性。针对IncB或IncC的抗血清免疫印迹表明，这些蛋白存在于C. psiffaci感染的HeLa细胞中，但在纯化的EBs中不存在或低于检测水平。逆转录聚合酶链反应证明incB和incC转录在一个操纵子中，免疫荧光显微镜显示incB和incC都定位于感染细胞的包膜上。在IncA、IncB和IncC之间没有明显的一级序列相似性，但每个都包含与IncA相似的大小和特征的大型疏水结构域。对新近完成的沙眼衣原体血清D基因组数据库的分析发现，沙眼衣原体ORFs编码的是与incS和incC同源的基因，表明这些基因在衣原体中是保守的。
Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole, termed an inclusion. To identify chlamydial proteins that are unique to the intracellular phase of the life cycle, a lambda expression library of Chlamydia psittaci DNA was differentially screened with convalescent antisera from infected guinea pigs and antisera directed at formalin-fixed purified chlamydial elementary bodies (EBs). One library clone was identified that harboured two open reading frames (ORFs) with coding potential for similar-sized proteins of approximate to 20 kDa. These proteins were subsequently termed IncB and IncC. Sequencing of the cloned insert revealed a strong Escherichia coli-like promoter sequence immediately upstream of incS and a 36nt intergenic region between the ORFs. Sequence analysis of the region upstream of incB and incC revealed two ORFs that had strong homologies to an amino acid transporter and a sodium-dependent transporter. Immunoblotting with antisera directed at IncB or IncC demonstrated that these proteins are present in C. psiffaci-infected HeLa cells but are absent or below the level of detection in purified EBs. Reverse transcriptase-polymerase chain reactions provided evidence that incB and incC are transcribed in an operon, Immunofluorescence microscopy demonstrated that IncB and IncC are each localized to the inclusion membrane of infected cells. No primary sequence similarity is evident between IncA, IncB or IncC, but each contains a large hydrophobic domain of similar size and character as in IncA. Analysis of the recently completed C. trachomatis serovar D genome database has revealed C. trachomatis ORFs encoding homologues to incS and incC, indicating that these genes are conserved among the chlamydiae.