Chlamydia trachomatis is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the elementary body (EB) and reticulate body (RB), each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiations require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and an AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, ClpX is encoded within an operon 3′ to clpP2. We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro. Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2. Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation.

中文翻译：

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沙眼衣原体的ClpX和ClpP2直系同源基因在生物的生长和发育中具有离散的基本功能。

沙眼衣原体是专性的细胞内细菌，经历复杂的发育周期，其中细菌在功能和形态上不同的两种形式（基本体（EB）和网状体（RB））之间进行区分，每种形式都表达自己的专门蛋白质组。初级（EB到RB）分化和次级（RB到EB）分化都需要蛋白质更新，我们假设蛋白酶对于介导分化至关重要。Clp蛋白酶系统在细菌中非常保守，对蛋白质更新非常重要。最低限度，该系统依赖于丝氨酸蛋白酶亚基ClpP和AAA + ATPase（例如ClpX），该酶识别并展开ClpP降解的底物。在衣原体中，ClpX在操纵子3'中编码为clpP2。我们目前的证据表明衣原体ClpX和ClpP2直系同源物对于生物的生存能力和发育至关重要。我们在这里证明衣原体ClpX是一种功能性ATP酶，并在体外形成预期的同六聚体。缺乏ATPase活性的ClpX突变体的过表达对DNA复制或继发分化的影响有限，但尽管如此，仍注意到EB活力降低，并观察到EB形态上的缺陷。相反，催化性失活的ClpP2突变体的过表达通过减少生物总数来显着影响发育周期进程。使用CRISPR干扰阻止clpP2X转录导致细菌生长减少，这种作用在反式中得到了补充由clpP2的质粒复制而成。两者合计，我们的数据表明ClpX和相关的ClpP2在衣原体发育周期的进展和分化中发挥不同的功能。