Investigation of small arginine-rich proteins of the DUF1127 family from Agrobacterium tumefaciens

在任何给定的生物体中，大约三分之一的蛋白质具有一种未知的功能。一个广泛分布的未知功能结构域是DUF1127，一个富含精氨酸的结构域，在大约4000种不同的细菌中发现。DUF1127蛋白的很大一部分是小的，长度在45到75个氨基酸之间。我们系统地鉴定和鉴定了植物病原菌根癌农杆菌中的7个DUF1127成员。它们都能产生真正的蛋白质，并有不同的表达。根据它们的长度、序列和LysR型转录因子的相互调节，这七种蛋白质分为两个亚类。三个短的DUF1127蛋白(47或48个氨基酸)的缺失导致了显著的生长表型和生物膜形成的增加。RNA-seq分析揭示了三个突变体中的大量差异调控基因，表明DUF1127蛋白在营养获取和中心碳和能量代谢中起着关键作用。在限定的介质中的生长实验将有助于提炼导致三重突变体中生长缺陷和生物膜表型的成分。我们目前的数据表明，不同的DUF1127亚类具有不同的功能，我们将通过在突变体中的互补实验来解决这个问题。考虑到DUF1127蛋白的小尺寸，它们本身不太可能具有酶功能。我们假设这些带正电的蛋白质通过与细胞因子，很可能是蛋白质和/或RNA的相互作用来发挥它们的活性。对功能标记的DUF1127蛋白质进行下拉实验，然后进行质谱分析和RNA-SEQ，将揭示这种相互作用伙伴。最后，DUF1127蛋白中精氨酸残基的贡献将通过定点突变和功能分析来检验。本申请中提出的实验将进一步深入了解这一功能未知的广泛分布的细菌结构域的生物学功能。

In any given organism, about 1/3 of all proteins have a yet unknown function. A widely distributed domain of unknown function is DUF1127, an arginine-rich domain found in about 4,000 different bacteria. A large fraction of DUF1127 proteins is small with a length between 45 and 75 amino acids. We systematically identified and characterized seven DUF1127 members in the plant pathogen Agrobacterium tumefaciens. They all give rise to authentic proteins and are differentially expressed. The seven proteins fall into two subclasses on the basis of their length, sequence and reciprocal regulation by a LysR-type transcription factor. The absence of the three short DUF1127 proteins (47 or 48 amino acids) caused a striking growth phenotype and increased biofilm formation. RNA-seq analysis revealed a large number of differentially regulated genes in the triple mutant and suggested a critical role in nutrient acquisition and central carbon and energy metabolism.Based on this solid foundation, we plan to unravel the biological function of the small DUF1127 proteins. Growth experiments in defined media will help refine the components that elicit the growth defect and biofilm phenotype in the triple mutant. Our current data suggests that different DUF1127 subclasses have different functions and we will address this question by complementation experiments in the mutant. Given the small size of the DUF1127 proteins, they are unlikely to have an enzymatic function on their own. We hypothesize that these positively charged proteins exert their activity by the interaction with cellular factors, most likely proteins and/or RNAs. Pulldown experiments with functional tagged DUF1127 proteins followed by mass spectrometry and RNA-seq will reveal such interaction partners. Finally, the contribution of the arginine residues in the DUF1127 proteins will be examined by site-directed mutagenesis followed by functional assays. The experiments proposed in this application are going to provide further insights into the biological function of this widely distributed bacterial domain of unknown function.