Meticillin-resistant Staphylococcus aureus (MRSA) is a serious public health threat causing outbreaksof clinical infection around the world. Mupirocin is a promising anti-MRSA drug, however mupirocin-resistant strains of S. aureus are emerging at an increasing rate. The newly discovered antibiotic batuminmay contribute to anti-MRSA therapy. The objective of this work was to identify possible molecular targetsfor batumin as well as mechanisms of its antistaphylococcal activity using computational moleculardocking and by analysing the complete genome sequence of the batumin-producer Pseudomonas batumiciUCM B-321. It was found that batumin acted very similarly to mupirocin by inhibiting aminoacyl tRNAsynthetases. A previous hypothesis considering the trans-enoyl-CoA reductase FabI as a prime moleculartarget of batumin was rejected. However, indirect inhibition of fatty acid biosynthesis in sensitive bacteriadoes take place as a part of stringent response repression triggered by accumulation of uncharged tRNAmolecules. Paralogues of diverse leucine-tRNA synthetases in the genome of P. batumici indicated that thisprotein might be the prime target of batumin. A batumin biosynthesis operon comprising 28 genes wasfound to be acquired through horizontal gene transfer. It was hypothesised that, in contrast to mupirocin,batumin could inhibit a broader range of aminoacyl tRNA synthetases and that acquired resistance tomupirocin might not endow S. aureus strains with resistance against batumin.