The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. To assess the function of PshX, we produced a ∆pshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ∆pshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer toward the P₈₀₀ special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.

缺氧光养细菌Heliobacterium modeticaldum含有一个光化学反应中心蛋白复合物（称为 HbRC），由 PshA 多肽的同源二聚体和新发现的称为 PshX 的多肽的两个拷贝组成，PshX 是结合两个细菌叶绿素g分子的单个跨膜螺旋。为了评估 PshX 的功能，我们制作了Hbt的ΔpshX菌株。通过利用内源性Hbt。谦虚IA 型 CRISPR-Cas 系统有助于突变体选择。我们通过将同源重组和基于 CRISPR 的选择步骤分成两个质粒转化来优化该系统，从而实现无标记基因替换。野生型和ΔpshX菌株纯化的 HbRC 的荧光和低温吸光度表明，与 PshX 结合的细菌叶绿素在整个 HbRC 中具有最低的位点能量。这表明 PshX 作为一个低能天线亚基，参与熵辅助向上的能量向 P ₈₀₀特殊细菌叶绿素g的转移一对。我们进一步讨论了 PshX 可能在 HbRC 稳定性中发挥的作用，它在其他 heliobacterial 物种中的保护，以及在其他反应中心的类似位置产生和维持单 TMH 亚基的进化压力。