华癸中生根瘤菌类菌体发育基因bacA的研究
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摘要
华癸中生根瘤菌(Mesorhizobium huakuii)能与宿主植物紫云英共生,并将空气中的氮气转化为植物可利用的化合态氮。迄今为止,对于根瘤菌从侵入线释放到根瘤含菌细胞后至固氮作用开始之前的共生中期,我们依然所知甚少。
本研究从华癸中生根瘤菌克隆到了bacA基因。氨基酸序列比对和跨膜区域的拓扑预测显示:华癸中生根瘤菌的BacA蛋白与苜蓿中华根瘤菌(Sinorhizobiummeliloti)的BacA蛋白具有很高的同源性。系统发育学分析也表明:尽管苜蓿中华根瘤菌的BacA蛋白与大肠杆菌的SbmA蛋白在功能上可以互换,但与大肠杆菌的SbmA蛋白相比,苜蓿中华根瘤菌的BacA蛋白显然与华癸中生根瘤菌的BacA蛋白具有更近的亲缘关系。BacA蛋白失活使华癸中生根瘤菌丧失了与宿主植物建立有效共生固氮的能力。
与华癸中生根瘤菌野生型菌株相比,bacA基因突变株M1对细胞膜完整性检测试剂(酸性pH,乙醇,SDS和结晶紫)更加敏感,但对一种糖肽类抗生素博来霉素的抗性却略有上升。质谱分析表明:BacA蛋白决定了华癸中生根瘤菌LPS结构类脂A组分中超长链脂肪酸(如27-OHC-28:0和29-OHC-30:0等)的特异性修饰。
本研究结果表明:在华癸中生根瘤菌bacA基因突变株中,细胞膜结构已发生了改变。细胞膜性质的改变能降低根瘤菌对释放到宿主细胞内后环境变化的适应性,但目前尚无证据表明这种细胞膜缺陷与类脂A的特异性VLCFA修饰的改变相关。
Mesorhizobium huakuii is capable of fixing atmospheric nitrogen in symbiosis with the legume Astragalus sinicus,which is an economically important forage and green manure growing in winter throughout Eastern Asia.So far,little is known about the molecular basis of events after the bacteria are released from infection threads into the nodule cells and before nitrogen fixation begins.
In this study,a bacA gene homologue of S.meliloti was isolated from M.huakuii. Alignment of BacA amino acid sequences and topological prediction for cytoplasmic membrane-spanning region both demonstrated that the BacA protein of M.huakuii showed high homology to S.meliloti.Furthermore,phylogenetic analysis illustrated that BacA of M.huakuii was much closer to BacA of S.meliloti rather than SbmA of E.coli, which is functionally interchangeable with BacA of S.meliloti.Inactivation of the bacA gene abolished the ability of M.huakuii to establish a successful symbiosis with its host.
Compared with wild-type M.huakuii,the bacA mutant M1 was more sensitive to cell envelope-disrupting agents(acid pH,ethanol,SDS,and crystal violet) and showed increased resistance to bleomycin,a glycolpeptide antibiotic.Mass spectrometry analysis revealed that BacA affected the very-long-chain fatty acids(27-OHC-28:0 and 29-OHC-30:0) contents of lipid A in M.huakuii.
The evidences from this study suggest that the cell envelope was altered in the bacA mutant of M.huakuii.This alteration of cell envelope may also frustrate bacterial attempts to survive the environmental chang encountered within host cells,while there is no evidence whether this change of cell envelope is related to the unusual lipid A modification.
本研究从华癸中生根瘤菌克隆到了bacA基因。氨基酸序列比对和跨膜区域的拓扑预测显示:华癸中生根瘤菌的BacA蛋白与苜蓿中华根瘤菌(Sinorhizobiummeliloti)的BacA蛋白具有很高的同源性。系统发育学分析也表明:尽管苜蓿中华根瘤菌的BacA蛋白与大肠杆菌的SbmA蛋白在功能上可以互换,但与大肠杆菌的SbmA蛋白相比,苜蓿中华根瘤菌的BacA蛋白显然与华癸中生根瘤菌的BacA蛋白具有更近的亲缘关系。BacA蛋白失活使华癸中生根瘤菌丧失了与宿主植物建立有效共生固氮的能力。
与华癸中生根瘤菌野生型菌株相比,bacA基因突变株M1对细胞膜完整性检测试剂(酸性pH,乙醇,SDS和结晶紫)更加敏感,但对一种糖肽类抗生素博来霉素的抗性却略有上升。质谱分析表明:BacA蛋白决定了华癸中生根瘤菌LPS结构类脂A组分中超长链脂肪酸(如27-OHC-28:0和29-OHC-30:0等)的特异性修饰。
本研究结果表明:在华癸中生根瘤菌bacA基因突变株中,细胞膜结构已发生了改变。细胞膜性质的改变能降低根瘤菌对释放到宿主细胞内后环境变化的适应性,但目前尚无证据表明这种细胞膜缺陷与类脂A的特异性VLCFA修饰的改变相关。
Mesorhizobium huakuii is capable of fixing atmospheric nitrogen in symbiosis with the legume Astragalus sinicus,which is an economically important forage and green manure growing in winter throughout Eastern Asia.So far,little is known about the molecular basis of events after the bacteria are released from infection threads into the nodule cells and before nitrogen fixation begins.
In this study,a bacA gene homologue of S.meliloti was isolated from M.huakuii. Alignment of BacA amino acid sequences and topological prediction for cytoplasmic membrane-spanning region both demonstrated that the BacA protein of M.huakuii showed high homology to S.meliloti.Furthermore,phylogenetic analysis illustrated that BacA of M.huakuii was much closer to BacA of S.meliloti rather than SbmA of E.coli, which is functionally interchangeable with BacA of S.meliloti.Inactivation of the bacA gene abolished the ability of M.huakuii to establish a successful symbiosis with its host.
Compared with wild-type M.huakuii,the bacA mutant M1 was more sensitive to cell envelope-disrupting agents(acid pH,ethanol,SDS,and crystal violet) and showed increased resistance to bleomycin,a glycolpeptide antibiotic.Mass spectrometry analysis revealed that BacA affected the very-long-chain fatty acids(27-OHC-28:0 and 29-OHC-30:0) contents of lipid A in M.huakuii.
The evidences from this study suggest that the cell envelope was altered in the bacA mutant of M.huakuii.This alteration of cell envelope may also frustrate bacterial attempts to survive the environmental chang encountered within host cells,while there is no evidence whether this change of cell envelope is related to the unusual lipid A modification.
引文
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