Key message

Homologous genes for the peptidoglycan precursor flippase MurJ, and peptidoglycan hydrolases: lytic transglycosylase MltB, and dd-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens.

Abstract

The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and ld-carboxypeptidase ldcA in addition to a putative dd-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens.

中文翻译：

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苔藓植物叶绿体分裂需要编码脂质 II 翻转酶 MurJ 和肽聚糖水解酶的基因

关键信息

肽聚糖前体翻转酶 MurJ 和肽聚糖水解酶的同源基因：裂解转糖基酶 MltB 和dd -羧肽酶 VanY 是苔藓立球藻中叶绿体分裂所必需的。

抽象的

苔藓Physcomitrella patens被用作模型植物来研究质体肽聚糖的生物合成。在细菌中，MurJ 翻转酶将肽聚糖前体从细胞质转运到周质。在这项研究中，我们在P. patens基因组中鉴定了一个MurJ同源物 ( PpMurJ ) 。细菌利用肽聚糖降解和循环途径进行细胞分裂。我们还在P. patens基因组中搜索了与细菌肽聚糖水解酶同源的基​​因，并鉴定了与裂解转糖基酶mltB、N-乙酰氨基葡萄糖苷酶nagZ和ld-羧肽酶ldcA同源的基因。除了先前报道的推定的dd -羧肽酶vanY。此外，我们还发现了一个β-内酰胺酶样基因（Pplactamase）。在叶绿体中检测到具有 PpMltB 或 PpVanY 的 GFP 融合蛋白，而具有 PpNagZ、PpLdcA 或 Pplactamase 的融合蛋白位于细胞质中。寻找 PpMurJ-GFP 融合蛋白的实验失败了。P. patens中的PpMurJ基因破坏导致原核细胞中出现大叶绿体。与野生型植物的叶绿体数量（38.9±4.9）相比，PpMltB敲除和PpVanY敲除的叶绿体数量较少（分别为 14.3 ± 6.7 和 28.1 ± 5.9）。在PpNagZ、PpLdcA或Pplactamase单敲除后，没有观察到叶绿体数量的差异。PpMltB/PpVanY双敲除细胞中的叶绿体数量与PpMltB单敲除细胞中的叶绿体数量相似。重组 PpMltB 蛋白的酶谱分析揭示了其肽聚糖水解酶活性。我们的结果表明 PpMurJ、PpMltB 和 PpVanY 在青苔P. patens的叶绿体分裂中起关键作用。