茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究

Prediction and functional analysis of GGDEF/EAL domain-containing proteins in Azorhizobium caulinodans ORS571

摘要:

[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△4658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△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226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。

Abstract:

[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 △4658 showed deficiency in cell motility, while its EPS production and biofilm formation were higher than that of wild type. Mutant △4658 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 △3085 and △3226 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.