海岸带生物学与生物资源保护实验室知识库

(1) 中国科学院烟台海岸带研究所

[Objective] c-di-GMP,an important second messenger regulating multiple functions of bacteria,is generally synthesized and hydrolysed by proteins containing GGDEF or EAL domain.In this study,we analyzed the genome-wide GGDEF/EAL domain-containing proteins of Azorhizobium caulinodans ORS571,and selected three GGDEF-EAL composite proteins (AZC_3085,AZC_3226 and AZC_4658) for functional analysis.[Methods] SMART and CLUSTALW were used for prediction and multi-alignment of GGDEF/EAL domain-containing proteins.Mutants were constructed by homologous recombination.Phenotypes including cell motility,exopolysaccharide (EPS) production,biofilm formation and nodulation with legume host were investigated.[Results] There were 37 GGDEF/EAL domain-containing proteins in A.caulinodans ORS571.Mutant Delta4658 showed deficiency in cell motility,while its EPS production and biofilm formation were higher than that of wild type.Mutant Delta4658 showed stronger competitiveness than wild type in competitive nodulation assay.The loss of AZC_4658 led to the increase of intracellular c-di-GMP level.Mutants Delta3085 and Delta3226 did not show obvious difference in comparison with wild type.[Conclusion] The vast number of GGDEF/EAL domain-containing proteins suggested that c-di-GMP may play an important role in signal transduction of ORS571.The GGDEF-EAL composite protein AZC_4658 was involved in cell motility,EPS production,biofilm formation and nodulation of A.caulinodans ORS571.

【目的】环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白(AZC_3085、AZC_3226和AZC_4658)进行功能研究。【方法】利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。【结果】茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株Delta4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株Delta4658的胞内c-di-GMP水平高于野生型。突变株Delta3085和Delta3226的各种表型与野生型相比没有明显差异。【结论】茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。