Abstract
In any given organism, approximately one-third of all proteins have a yet-unknown function. A widely distributed domain of unknown function is DUF1127. Approximately 17,000 proteins with such an arginine-rich domain are found in 4,000 bacteria. Most of them are single-domain proteins, and a large fraction qualifies as small proteins with fewer than 50 amino acids. We systematically identified and characterized the seven DUF1127 members of the plant pathogen Agrobacterium tumefaciens They all give rise to authentic proteins and are differentially expressed as shown at the RNA and protein levels. The seven proteins fall into two subclasses on the basis of their length, sequence, and reciprocal regulation by the LysR-type transcription factor LsrB. The absence of all three short DUF1127 proteins caused a striking phenotype in later growth phases and increased cell aggregation and biofilm formation. Protein profiling and transcriptome sequencing (RNA-seq) analysis of the wild type and triple mutant revealed a large number of differentially regulated genes in late exponential and stationary growth. The most affected genes are involved in phosphate uptake, glycine/serine homeostasis, and nitrate respiration. The results suggest a redundant function of the small DUF1127 paralogs in nutrient acquisition and central carbon metabolism of A. tumefaciens They may be required for diauxic switching between carbon sources when sugar from the medium is depleted. We end by discussing how DUF1127 might confer such a global impact on cell physiology and gene expression.IMPORTANCE Despite being prevalent in numerous ecologically and clinically relevant bacterial species, the biological role of proteins with a domain of unknown function, DUF1127, is unclear. Experimental models are needed to approach their elusive function. We used the phytopathogen Agrobacterium tumefaciens, a natural genetic engineer that causes crown gall disease, and focused on its three small DUF1127 proteins. They have redundant and pervasive roles in nutrient acquisition, cellular metabolism, and biofilm formation. The study shows that small proteins have important previously missed biological functions. How small basic proteins can have such a broad impact is a fascinating prospect of future research.