Spontaneous mutants were 2- to 20-fold more resistant to protaminethan wild type, and displayed cross-resistance to other AMPs (colistin, lactoferricin, human -defensin HNP1). Resistance mutations were identified as deletions, nonsense or missense in haem biosynthesis (hemA, hemB, hemC, hemL) with 14 different mutations in four steps of haem biosynthesis were observed indicating that the lack of an end product in this pathway is responsible for protamine resistance.