Genome analysis of two Pseudonocardia phylotypes associated with Acromyrmex leafcutter ants reveals their biosynthetic potential

The attine ants of South and Central America are ancient farmers, having evolved a symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal partner against co-evolved fungal pathogens of the genus Escovopsis. Pseudonocardia mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring secondary metabolites of Pseudonocardia on a genome-wide scale. Here we report a genomic analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 strains revealed that the phylotypes are distinct species and each encodes between 11-15 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. Strains show variations in the arrangement of these BGCs that resemble those seen in gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with Acromyrmex ants and supports the hypothesis that Pseudonocardia species could provide a valuable source of new antimicrobials

DISCOVERY OF THE ParST TOXIN-ANTITOXIN SYSTEM AND ITS PUTATIVE ROLE IN DISRUPTING NUCLEOTIDE METABOLISM

Toxin-antitoxin (TA) systems are two-component operons that control cell growth, bacteriostasis, and death and have been implicated in important clinical issues such as biofilm formation and persister growth. Chapter 2 describes the extensive structural and biochemical elucidation of a TA system first predicted bioinformatically in 2009, here named ParST. We showed that this TA family is exceptionally widespread among bacteria, and a high-resolution crystal structure of the ParT toxin revealed similarity to known mono-ADP-ribosyltransferases, such as diphtheria and cholera toxins. Further experiments confirmed this function and identified phosphoribosyl pyrophosphate synthetase (Prs) as a putative target. Prs is an essential metabolic enzyme in nucleotide synthesis, not previously known to be targeted by cellular toxins, which makes ParST an interesting candidate for further study. While Chapter 2 represents a new area of study in the lab, Chapters 3 through 5 return to the lab’s primary research interest – lasso peptides. Lasso peptides are ribosomally synthesized, post-translationally modified natural products that consist of an 7-9 amino acid ring, with the C-terminal portion of the peptide threaded through. In Chapter 3, for the first time, various noncanonical amino acids (ncAAs) were successfully introduced into lasso peptide microcin J25 (MccJ25). All 16 ncAA-substituted variants we examined were produced and retained antimicrobial activity against Salmonella newport. Work in this chapter has laid the groundwork for applications currently underway probing the interaction of MccJ25 with its target, RNA polymerase. Chapter 4 investigates the dynamics of lasso peptide isopeptidases, enzymes that cleave the characteristic lasso peptide isopeptide bond. We describe the creation of a dual-labeled isopeptidase capable of intramolecular fluorescence resonance energy transfer (FRET) for examining conformational changes, as well as preliminary results of FRET monitoring. Finally, Chapter 5 discusses the Sphingopyxis fribergensis family of lasso peptides, bergenodins 1 through 6. To date, six lasso peptides represents the most produced by a single organism. We confirm the production of bergenodins 1 through 5, though we could not isolate bergenodin 6. We also desired to examine any crosstalk between the many isopeptidase enzymes and lasso peptide substrates, but further optimization to the isopeptidase expression systems remains necessary

Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids

Using the amber suppression approach, four noncanonical amino acids (ncAAs) were used to replace existing amino acids at four positions in lasso peptide microcin J25 (MccJ25). The lasso peptide biosynthesis enzymes tolerated all four ncAAs and produced antibiotics with efficacy equivalent to wild-type in some cases. Given the rapid expansion of the genetically encoded ncAA pool, this study is the first to demonstrate an expedient method to significantly increase the chemical diversity of lasso peptides

Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids

Using the amber suppression approach, four noncanonical amino acids (ncAAs) were used to replace existing amino acids at four positions in lasso peptide microcin J25 (MccJ25). The lasso peptide biosynthesis enzymes tolerated all four ncAAs and produced antibiotics with efficacy equivalent to wild-type in some cases. Given the rapid expansion of the genetically encoded ncAA pool, this study is the first to demonstrate an expedient method to significantly increase the chemical diversity of lasso peptides