Kal/bat, a polyketide, isolated to high purity (>95%) is characterized by strong and selective antibacterial activity against Staphylococcus species (minimum inhibitory concentration, 0.05 mug/mL), and no resistance was observed in strains already resistant to commonly used antibiotics. The kal/bat biosynthesis gene cluster was determined to a 62 kb genomic region of Pseudomonas fluorescens BCCM_ID9359. The kal/bat gene cluster consists of 16 open reading frames (ORF), encoding a hybrid PKS-NRPS system, extended with trans-acting tailoring functions. A full model for kal/bat biosynthesis is postulated and experimentally tested by gene inactivation, structural confirmation (using NMR spectroscopy), and complementation. The structural and microbiological study of biosynthetic kal/bat analogs revealed the importance of the carbamoyl group and 17-keto group for antibacterial activity. The mechanism of self-resistance lies within the production of an inactive intermediate, which is activated in a one-step enzymatic oxidation upon export. The genetic basis and biochemical elucidation of the biosynthesis pathway of this antibiotic will facilitate rational engineering for the design of novel structures with improved activities. This makes it a promising new therapeutic option to cope with multidrug-resistant clinical infections.