The expanding antimicrobial diversity of the genus Pantoea.

With the rise of antimicrobial resistance, there is high demand for novel antimicrobials to combat multi-drug resistant pathogens. The bacterial genus Pantoea produces a diversity of antimicrobial natural products effective against a wide range of bacterial and fungal targets. These antimicrobials are synthesized by specialized biosynthetic gene clusters that have unique distributions across Pantoea as well as several other genera outside of the Erwiniaceae. Phylogenetic and genomic evidence shows that these clusters can mobilize within and between species and potentially between genera. Pantoea antimicrobials belong to unique structural classes with diverse mechanisms of action, but despite their potential in antagonizing a wide variety of plant, human, and animal pathogens, little is known about many of these metabolites and how they function. This review will explore the known antimicrobials produced by Pantoea: agglomerins, andrimid, D-alanylgriseoluteic acid, dapdiamide, herbicolins, pantocins, and the various Pantoea Natural Products (PNPs). It will include information on the structure of each compound, their genetic basis, biosynthesis, mechanism of action, spectrum of activity, and distribution, highlighting the significance of Pantoea antimicrobials as potential therapeutics and for applications in biocontrol.

Distribution and comparative genomic analysis of antimicrobial gene clusters found in Pantoea.

Members of the bacterial genus Pantoea produce a variety of antimicrobial products that are effective against plant, animal, and human pathogens. To date, little is known about the distribution and evolutionary history of these clusters. We surveyed the public databases for the 12 currently known antibiotic biosynthetic gene clusters found across Pantoea strains to determine their distribution. We show that some clusters, namely pantocin B, PNP-3, and PNP-4 are found strictly in Pantoea, while agglomerin, andrimid, AGA, dapdiamide, herbicolin, PNP-1, PNP-2, PNP-5, and pantocin A, are more broadly distributed in distantly related genera within Vibrionaceae, Pectobacteriaceae, Yersiniaceae, Morganellaceae, and Hafniaceae. We evaluated the evolutionary history of these gene clusters relative to a cpn60-based species tree, considering the flanking regions of each cluster, %GC, and presence of mobile genetic elements, and identified potential occurrences of horizontal gene transfer. Lastly, we also describe the biosynthetic gene cluster of pantocin B in the strain Pantoea agglomerans Eh318 more than 20 years after this antibiotic was first described.

A replica plating method for efficient, high-throughput screening of antibiotic gene clusters in bacteria uncovers a holomycin-like cluster in the clinical isolate, Pantoea agglomerans 20KB447973.

Bacterial natural products remain a major untapped source for novel antimicrobial scaffolds. Many of these products are encoded by biosynthetic gene clusters (BGCs), which can be identified using functional genomics. We developed a replica-plating approach to quickly screen for antibiotic production mutants from transposon mutant libraries and identify candidate antibiotic BGCs. In this technique, filter paper is used to transfer up to 200 mutants simultaneously onto a soft agar overlay or spread plate containing a target microbe to identify antibiotic-production mutants. These mutants can then be analyzed to identify disrupted genes and antibiotic BGCs. We first tested and optimized this technique by screening for previously characterized BGCs in Pantoea. We then applied the technique to uncover the gene cluster responsible for the production of an unknown broad-spectrum antibiotic from P. agglomerans 20KB447973, which we call Pantoea Natural Product 5 (PNP-5). Analysis of the predicted gene cluster for PNP-5 showed similarity to previously identified gene clusters for the broad-spectrum dithiolopyrrolone antibiotic, holomycin. Analysis of the spectrum of activity of PNP-5 showed activity against members of the Enterobacteriaceae, Erwiniaceae, and Streptococcaceae, including clinically relevant pathogens such as Klebsiella sp. and Escherichia coli. We also identified the production of a second antibiotic, pantocin A. Our findings demonstrate the utility of our replica-plating mutant transfer method in exploring unknown antibiotic BGCs. Adoption of this technique may accelerate the identification of potentially novel antimicrobial BGCs within strain collections, advancing the search for novel antimicrobials that can be used to treat multi-drug resistant infections.