The Identification and Validation of Novel Small Proteins in Pseudomonas Putida KT-2440

Small proteins of less than 50 amino acids (s-­proteins) have been understudied and largely overlooked in genome annotations and classical proteomic studies. As the s-proteins represent an untapped pool of bioactive molecules, their investigation will likely uncover novel cellular functions and activities and may lead to the discovery of novel antimicrobial agents. Our project focuses on the identification, validation and characterization of novel s-­‐proteins in the bacterium Pseudomonas putida KT-­2440. As there is virtually no information on s-­‐proteins in pseudomonads, the first step is to establish methods for the identification of short open reading frames (sORFs). A custom bioinformatics tool has been developed to predict sORFs from the genome and sRNA sequencing databases, where sequences containing a Shine-­Dalgarno (SD) sequence and start/stop codon are identified. In a second approach, total protein samples are prepared, fractionated, and analyzed with mass spectrometry (MS/MS). The MS/MS data are compared to a custom database containing >80000 putative sORF sequences to identify candidates for validation. A total of 56 and 22 putative sORFs were obtained from MS/MS data and bioinformatics prediction, respectively, where there is no overlap between the putative sORFs obtained from the two approaches. The sequences encoding the putative sORFs will be integrated onto the Tn7 site on the chromosome as well as on a plasmid expression vector for validation.