<jats:title>ABSTRACT</jats:title> <jats:p> The increasing prevalence of antibiotic resistance in bacterial pathogens has renewed focus on natural products with antimicrobial properties. Lantibiotics are ribosomally synthesized peptide antibiotics that are posttranslationally modified to introduce (methyl)lanthionine bridges. Actinomycetes are renowned for their ability to produce a large variety of antibiotics, many with clinical applications, but are known to make only a few lantibiotics. One such compound is planosporicin produced by <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Planomonospora alba</jats:named-content> , which inhibits cell wall biosynthesis in Gram-positive pathogens. Planosporicin is a type AI lantibiotic structurally similar to those which bind lipid II, the immediate precursor for cell wall biosynthesis. The gene cluster responsible for planosporicin biosynthesis was identified by genome mining and subsequently isolated from a <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. alba</jats:named-content> cosmid library. A minimal cluster of 15 genes sufficient for planosporicin production was defined by heterologous expression in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Nonomuraea</jats:named-content> sp. strain ATCC 39727, while deletion of the gene encoding the precursor peptide from <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. alba</jats:named-content> , which abolished planosporicin production, was also used to confirm the identity of the gene cluster. Deletion of genes encoding likely biosynthetic enzymes identified through bioinformatic analysis revealed that they, too, are essential for planosporicin production in the native host. Reverse transcription-PCR (RT-PCR) analysis indicated that the planosporicin gene cluster is transcribed in three operons. Expression of one of these, <jats:italic>pspEF</jats:italic> , which encodes an ABC transporter, in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Streptomyces coelicolor</jats:named-content> A3(2) conferred some degree of planosporicin resistance on the heterologous host. The inability to delete these genes from <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. alba</jats:named-content> suggests that they play an essential role in immunity in the natural producer.