探討根瘤菌 Azorhizobium caulinodans ORS571 的染色體分配蛋白 ParA 與 ParB 在類菌體形成的角色

Functional characterization of Azorhizobium caulinodans ORS571 chromosome partition proteins, ParA ParB, on bacteroid formation

簡曉琳(Hsiao-Lin Chien)

指導教授 ： 劉啟德

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2022年)

https://doi.org/10.6342/NTU202203092

摘要

Azorhizobium caulinodans ORS571 根瘤菌的染色體分配系統 (ParABS) 不僅與染色體分離有關也已證實會參與共生結瘤時類菌體 (bacteroid) 的形成。當染色體分配基因 parA 缺失會導致細胞週期缺陷使得染色體套數增加，細胞形態異常，以及在宿主植物上形成未成熟的莖瘤。在本研究中(第二章)建構了parA 過度表現株 PnptII-parA以進一步探討parA基因在共生及非共生時期所扮演的角色。研究結果發現與 parA基因缺失相比，ParA的過度生產並不影響菌體在非共生時的生長、細胞形態、染色體套數及固氮能力。 然而，在與長喙田莖Sesbania rostrata共生時卻觀察到異常的莖瘤表型，包括產生的莖瘤較小、固氮能力明顯下降且受感染的植物細胞數目也較少。此外，也觀察到具有類似末期分化特徵的類菌體，即形態明顯增長且具有相對狹窄的類菌體外膜空間等。然而，目前文獻上並未報導 S. rostrata可生成驅使類菌體進行末期分化的 NCR 胜肽 (nodule-specific cysteine-rich peptides)。儘管 PnptII-parA 在非共生時期的外觀與野生型幾乎相同，但它們的共生相關基因的表現型態卻顯著不同，推測發生在共生時期的異常表型是來自於結瘤過程中過量的 ParA 蛋白質造成細胞週期中斷所引起。
Azorhizobium 根瘤菌的ParB蛋白質具有 helix-turn-helix (HTH) 結構域，推測可以與特定的 DNA 片段結合。論文第三章中利用生物薄膜干涉技術 (BioLayer Interferometry) 來探討ParB蛋白質與各種 DNA片段結合的特性。在檢測該蛋白質與染色體分配相關的功能時，研究結果發現不同濃度的 ParB 均可與高度保守的 parS 序列具專一性結合; 而在檢測其與類菌體形成相關的功能時，當有其他蛋白質 (BSA) 同時存在下，ParB會與類菌體分化相關基因bacA的啟動子區域中具有迴文特徵的序列產生專一性結合。此外，在2 M的ParB，即接近其細胞中濃度的條件下進行結合動力學試驗，發現ParB 對 bacA 啟動子迴文區序列的結合能力較ParB 對parS 快。因此，推測ParB蛋白可能在結瘤過程中擔任轉錄因子的角色用以調節結瘤相關基因的表現。根據上述結果，Azorhizobium 根瘤菌是否藉由 ParA 與 ParB 的交互作用以決定菌體是在非共生態進行正常的染色體分配，或是在結瘤共生時啟動類菌體的分化，仍有待進一步驗證。

關鍵字

莖瘤發育 ； 染色體分配系統-ParA 及ParB ； 固氮作用 ； 類菌體形成蛋白質BacA ； 生物膜干涉技術

並列摘要

The chromosome partitioning system (ParABS) of Azorhizobium caulinodans ORS 571 has been proved to involve in either chromosome segregation during free-living or bacteroid formation during symbiotic nodulation. Deletion of the chromosome partitioning gene parA of ORS571 results in cell cycle defects, pleiomorphic cell shape, and formation of immature stem-nodules on its host plant Sesbania rostrata. In Chapter 2, a parA overexpression mutant (PnptII-parA) was constructed to further investigate the roles of parA gene under free-living and symbiotic states. By contrast to the deletion of parA gene, overproduction of ParA did not affect the cell growth, chromosome segregation, or free-living nitrogen fixation ability of the mutant. On the other hand, distinctive features were observed under symbiosis, such as smaller nodules, reduced nitrogen-fixing capacity, fewer infected plant cells, and swollen polyploid bacteroids with relatively narrow symbiosome space. Several of the morphotype features are reminiscent of the terminally differentiated bacteroides in some inverted repeat-lacking clade (IRLC) indeterminate nodules. However, S. rostrata is not thought to produce the nodule-specific cysteine-rich (NCR) peptides that induce terminal differentiation in rhizobia. Although the appearance of this mutant is almost the same as the wild type under the free-living state, many symbiotic-related genes of them expressed so differently. Accordingly, it suggests that the abnormal symbiosome formation in the stem-nodules is due to cell cycle disruption caused by excess ParA protein in the symbiotic cells during nodulation.
Azorhizobial ParB contains a helix-turn-helix (HTH) domain, which suggests being able to bind with particular sequences of DNA fragments. In Chapter 3, I conducted Bio-layer interferometry (BLI) assay to elucidate the in vitro DNA-binding traits of this protein. For chromosome partitioning, the dose-dependent binding signal responses of ParB and the highly conserved parS locus were detected. For bacteroid formation, azorhizobial ParB could specifically bind to the three palindromic sites in the bacteroid differentiation-related gene bacA while conducting the trials in the presence of an interfering molecule BSA protein. According to the binding kinetics for interactions at the estimated cellular ParB concentration (2 M) in A. caulinodans, I assumed that azorhizobial ParB binds more quickly to the palindromic sequences in the bacA promoter region than to the conserved parS. Accordingly, azorhizobial ParB was deduced to act as a transcription factor for regulating the bacteroid-related genes during nodulation. Taken together, interactions between azorhizobial ParA and ParB proteins may function as a checkpoint, which couples the chromosome partitioning to the onset of bacteroid formation. This assumption remains to be elucidated.

並列關鍵字

stem nodule development ； chromosome partitioning system-ParA and ParB ； nitrogen fixation ； bacteroid forming protein BacA ； BioLayer Interferometry

參考文獻

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