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

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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.