Despite many medical advances, antibiotic resistant bacteria increasingly plague the modern world, necessitating discovery of new antibiotics. One area of nature that can provide inspiration for antibiotics is antimicrobial peptides. Many of these peptides exist in nature, with some classes that have not been studied or characterized well. One such class is the defensin-like peptides generated by the plant Medicago truncatula as part of their symbiotic relationship with Sinorhizobium meliloti. Nodule-specific Cysteine Rich (NCR) peptides are defined by the presence of multiple cysteines, and regulate the growth of S. meliloti within plant cells. While some of these NCR peptides have been shown to have antimicrobial properties, hundreds of peptides remain uncharacterized. We have developed an assay for further characterization of these peptides in E. coli. Of the seven peptides that have been tested using this assay, three have exhibited definitive antimicrobial properties against both E. coli and S. meliloti. Additionally, we have developed a system for discovering novel antimicrobial peptides. This platform, called PepSeq, uses the expression of random peptides in E. coli combined with deep sequencing to detect antimicrobial activity. This technology is capable of screening through millions of peptide molecules simultaneously. Using this platform, we have discovered and confirmed six novel antimicrobial peptides, with hundreds of additional predicted antimicrobial peptides. In addition to the peptides we have analyzed using PepSeq, additional peptide scaffolds could be used to discover more potent antimicrobial peptides.

The remarkable metabolic capacity of the soil-dwelling bacterium Sinorhizobium meliloti is encoded on its three circular replicons: the chromosome and two large megaplasmids, pSymA and pSymB. Despite making up 45% of the genome, the pSymA and pSymB megaplasmids can be cured from S. meliloti. This unique attribute provides an opportunity to study the essentiality of chromosomal genes in the presence or absence of nearly half the genome. By interrogating chromosomal genes via massively parallel transposon insertion sequencing (Tn-seq) in the presence and absence of pSymA and pSymB, we identified 307 genes as being essential for viability regardless of the genomic context and 104 genes as being essential specifically when the megaplasmids are absent. We also found that ten percent of genes encoded on the chromosome genetically interact with genes on pSymA and pSymB. In addition, Tn-seq data were utilized to significantly refine a metabolic model of S. meliloti, facilitating more accurate fitness predictions in user-defined nutrient and genetic contexts. Furthermore, the development of a library of barcoded transposon insertion (BarSeq) mutants has enabled us to identify genes that are essential for robust growth in hundreds of nutrient environments simultaneously. This will greatly assist efforts to assign more specific functions to the ~30% of S. meliloti genes that have remained uncharacterized over the years. S. meliloti has been studied for decades as a model organism for symbiotic communication. Its legume host, Medicago truncatula, provides fixed carbon for the bacteria in order to receive fixed nitrogen in return. The molecular dialogue between S. meliloti and M. truncatula, initiates and controls each stage of symbiotic development. When inside host cells, intracellular bacteria are subjected to an arsenal of plant-derived Nodule-specific Cysteine-Rich (NCR) peptides that induce significant morphological changes prior to nitrogen fixation. It was previously shown that a bacterial peptidase, HrrP, present in about 10% of S. meliloti isolates, could degrade host-derived peptides and give the bacterial symbionts greater fitness at the expense of the host. In a screen through peptidases conserved throughout the core S. meliloti genome, we identified one peptidase (sapA) that, when overexpressed, significantly modulates symbiotic outcome. In a manner similar to HrrP, SapA degrades NCR peptides in vitro. Additionally, expression of sapA seems to occur specifically inside the plant host providing compelling evidence that some rhizobial peptidases may have evolved away from housekeeping and toward symbiotic functions.