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Endophytes induce plant growth and protect host plants against abiotic and biotic stresses, such phytopathogenic bacteria and fungi. Here we explored the genome of plant growth-promoting, intracellular endophyte Methylobacterium extorquens DSM13060, isolated from bud meristems of Pinus sylvestris L., for antimicrobial peptides. A defensinlike antimicrobial peptide MB1533 was identified using computational and prediction models from the genome sequence. MB1533 was produced in Escherichia coli with a molecular weight of 6160.26 Da. The antimicrobial activity was tested against several strains of bacteria and yeasts, and the peptide had activity against the grampositive Staphylococcus aureus and Bacillus subtilis at 128 μg/ml. The potency of the peptide can be improved further by rational-design-techniques. MB1533 is the first defensin-like peptide identified from Methylobacterium genus and from bacterial endophytes, in general.