Identification and Characterization of Antibiotic Biosynthetic Gene Clusters in Pantoea

Abstract

Antimicrobial resistance is a global health crisis for which new antibiotics are needed. Natural products (secondary metabolites) produced by microbes have been our primary source of antibiotics; however, the search has been predominantly limited to a few genera. One underexplored bacterial genus is Pantoea, which produces a variety of compounds with antimicrobial activity. Chapter one describes a general overview of secondary metabolites, antibiotic resistance, strategies to overcome resistance, and Pantoea. Chapter two describes the identification and characterization of the biosynthetic gene cluster for Pantoea Natural Product 3 (PNP-3), an antibiotic produced by P. agglomerans 3581r and SN01080r. The cluster contains putative enzymes (pnp3b, pnp3e–h), MFS transporters (pnp3a, pnp3c), and an ArsR family regulator (pnp3d). Transposon mutagenesis and heterologous expression of pnp3e–h showed that the four genes are sufficient for antibiotic production. Disruption by homologous recombination of the putative hydrolase pnp3b increased the PNP-3 zone of inhibition, suggesting Pnp3b may act as a resistance mechanism. PNP-3 activity was demonstrated to be dependent upon low nutrient levels and was broad-spectrum, inhibiting the growth of drug-resistant bacterial strains including Acinetobacter baumannii and Pseudomonas aeruginosa. Further, the PNP-3 gene cluster has a limited distribution across bacterial species, and genome mining tools were unable to identify the cluster, suggesting the metabolite may be novel. Chapter 3 describes the characterization of the two predicted MFS transporters, Pnp3a and Pnp3c, using genetic, phylogenetic, and bioinformatic approaches. Disruption via homologous recombination of the putative transporters in Pantoea suggests a role of Pnp3a in the export of the metabolite. This was supported by experiments where heterologous expression of Pnp3a in E. amylovora conferred resistance to PNP-3 and facilitated the export of the metabolite produced by Pnp3e–h. The role of Pnp3c, however, is unclear as it appears to be dispensable under certain conditions. Comparative genomic analyses identified pnp3a in additional Pantoea strains, several of which carry complete or nearly complete PNP-3 biosynthetic clusters. Two Pantoea strains with PNP-3 clusters (P. vagans C9-1, P. agglomerans SS03231r) were able to inhibit the growth of P. aeruginosa, suggesting the PNP-3 biosynthetic clusters are functional, and all strains carrying a pnp3a homolog, regardless of genomic context, were tolerant to PNP-3. These results suggest Pnp3a plays an essential role in PNP-3 export and resistance. Chapter four describes an antibiotic production survey of 116 Pantoea strains targeting 12 bacterial strains, including several human pathogens. Of the Pantoea strains tested, 59 were antibiotic producers. Among the antibiotic producers was P. agglomerans B025670, which was antagonistic toward Kosakonia, E. coli, Salmonella, Enterobacter, and Pseudocitrobacter species. To identify the antibiotic biosynthetic gene cluster responsible for antibiotic production in B025670, an integrated approach of comparative genomics, genome mining, and functional genetics was employed. The approach identified a 14-gene cluster in the genome of B025670, designated cluster 675. Disruption of the gene cluster via single-integration homologous recombination led to a loss of antibiotic production, confirming a role in the manufacturing of the antimicrobial product. Chapter 5 describes the general thesis conclusions and future directions. Overall, this research has sought to characterize antibiotic biosynthetic gene clusters in Pantoea and highlights the genus as a promising source of antibiotics for further exploration.