Evaluation of Antibiotic Production and its Regulation in Pantoea agglomerans Tx10

Abstract

The prevalence of antimicrobial-resistant pathogens, such as methicillinresistant Staphylococcus aureus (MRSA), has prompted numerous efforts to identify new antimicrobials. One untapped source of antimicrobial products is the enterobacterial genus Pantoea. Although antibiotic discovery efforts in Pantoea have focused largely on agricultural applications, Pantoea remains an unexplored reservoir of antibiotics that may have utilization in a clinical setting. Using an agar overlay assay, Pantoea agglomerans Tx10 (Tx10)—an isolate from the sputum of a cystic fibrosis patient—was shown to inhibit the growth of several pathogens, including Gram-positive Staphylococcus and Streptococcus, and the Gram-negative Citrobacter, E. coli, Erwinia, and Salmonella. A genetic screen to identify the genes involved in the production of this antimicrobial natural product identified a predicted 6-gene biosynthetic cluster which, when disrupted, abolished antibiotic production. I called this compound, Pantoea Natural Product 2 (PNP-2). Subsequent assays with mutants deficient in PNP-2 production revealed that they were still able to inhibit Erwinia amylovora, suggesting the production of a second antibiotic, which I identified as the well-known pantocin A. A survey of resistance among the Stavrinides Pantoea collection revealed that the majority of isolates are susceptible to either pantocin A, or PNP-2, and only a minority were resistant to both. Analysis of the expression of PNP-2 revealed it was produced only on minimal media. Using the luciferase reporter gene, PNP-2 expression was shown to be linked to amino acid starvation, with the absence of certain amino acids enabling antibiotic production. Overall, the antibiotic production from this cystic fibrosis isolate, Tx10, shows the potential reservoir of antibiotics from the genus of Pantoea, as well as insight into the production and expression of the antibiotic in the environment.