Data Sheet 1_Two zinc ABC transporters contribute to Rhizobium leguminosarum symbiosis with Pisum sativum and Lens culinaris.docx

The establishment of the rhizobium-legume symbiosis requires adjusting the behavior of both partners to nodule conditions in which transition metals are delivered to the bacteria, as many rhizobial metalloenzymes are essential for bacteroid functions and symbiotic performance. A previous proteomic analysis revealed the existence of a relevant number of proteins differentially expressed in bacteroids induced by Rhizobium leguminosarum bv. viciae (Rlv) UPM791 in pea and lentil nodules. Among these proteins, a metal-binding protein (RLV_3444) component of an ABC-transporter system (RLV_3442-3444) was shown to be overexpressed in pea bacteroids, suggesting that metal provision to the bacteroid is more restrictive in the rhizobium-pea symbiosis. In this work, protein sequence analysis and structural modelling have revealed that RLV_3444 is highly similar to the functionally characterized zinc-binding protein ZniA from Klebsiella pneumoniae, so the host-dependent binding protein was renamed as ZniA and the transporter system as ZniCBA. The genome of Rlv UPM791 also encodes the conserved high-affinity ZnuABC transporter system. We demonstrate that at least one of the two systems must be present for Rlv to grow under zinc-limiting conditions and for optimal symbiotic performance with pea and lentil plants. The three conserved histidine residues present in multiple Zn2+-binding proteins have been shown as essential for the function of Rlv ZniA, and in-silico modelling suggests that they might participate in metal coordination. We also demonstrate that both ZniCBA and ZnuA are regulated by zinc in a Zur-dependent manner, consistent with the presence of a Zur box in their regulatory region. The expression patterns revealed that ZniCBA is expressed at lower levels than ZnuA, and its expression increased in a znuA mutant under both free-living and symbiotic conditions. These results, along with the observed increment in the expression of ZniCBA in pea versus lentil bacteroids, suggest that the host-dependent transporter system might play an auxiliary function for zinc uptake under zinc starvation conditions and might play a relevant role in the adaptation of rhizobia to the legume host.

根瘤菌-豆科植物共生（rhizobium-legume symbiosis）的建立，需要双方调整自身行为以适配根瘤（nodule）内的环境：根瘤会向类菌体（bacteroid）递送过渡金属（transition metals），因为多数根瘤菌金属酶（metalloenzyme）对类菌体功能与共生效能至关重要。既往蛋白质组学分析（proteomic analysis）显示，豌豆与扁豆根瘤中，由豌豆根瘤菌生物型豌豆变种（Rhizobium leguminosarum bv. viciae，简称Rlv）UPM791诱导形成的类菌体内，存在大量差异表达蛋白（differentially expressed protein）。其中，ABC转运蛋白系统（ABC-transporter system，RLV_3442-3444）的锌结合蛋白（zinc-binding protein）组分RLV_3444在豌豆类菌体内呈现过表达特征，这提示在根瘤菌-豌豆共生体系中，向类菌体供应金属的过程更为受限。本研究通过蛋白质序列分析与结构建模（structural modelling）发现，RLV_3444与功能已得到表征的肺炎克雷伯菌（Klebsiella pneumoniae）锌结合蛋白ZniA具有高度同源性，因此将该宿主依赖性结合蛋白重命名为ZniA，对应的转运系统则命名为ZniCBA。豌豆根瘤菌UPM791的基因组同时编码保守的高亲和力（high-affinity）ZnuABC转运系统。研究证实，若要在锌限制条件（zinc-limiting conditions）下存活并实现与豌豆、扁豆植株的最优共生效能，Rlv至少需要保留这两套转运系统中的一套。存在于多种Zn²⁺结合蛋白中的三个保守组氨酸残基，已被证明对Rlv ZniA的功能至关重要；计算机模拟建模（in-silico modelling）表明，这些残基可能参与金属配位过程。本研究还证实，ZniCBA与ZnuA均受Zur依赖型（Zur-dependent）锌调控，这与其调控区域（regulatory region）中存在Zur box序列相符。表达模式分析显示，ZniCBA的表达水平低于ZnuA，且在znuA突变体（znuA mutant）的自由生长与共生状态下，其表达量均有所上调。上述结果，连同豌豆根瘤内ZniCBA的表达量高于扁豆根瘤的观测现象，提示这套宿主依赖性转运系统可能在锌饥饿条件下辅助完成锌摄取，并在根瘤菌适应豆科宿主的过程中发挥关键作用。