The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI.

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus.

Identification of Protein-Protein Interactions Using Pool-Array-Based Yeast Two-Hybrid Screening.

Array-based yeast two-hybrid (Y2H) screening in combination with a pooling strategy permits identification of complete interaction networks. After discussing the general advantages and drawbacks of Y2H, this chapter provides a detailed protocol for an array-based Gal4p Y2H screen using a pooling strategy to improve efficacy of the experimental work. This includes bait transformation, bait autoactivation testing, pooling of the baits and preys, yeast mating and evaluation of the results.

A Protein Interaction Map of the Kalimantacin Biosynthesis Assembly Line.

The antimicrobial secondary metabolite kalimantacin (also called batumin) is produced by a hybrid polyketide/non-ribosomal peptide system in BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further.

Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach.

Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method.