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III. The bacterially induced infections have become one of the most important global biological challenges due to rapidly developing resistance to the commonly used antibiotics. In the era of the constantly emerging superbugs speeding up the search for novel antibacterial agents is of great practical importance. Applying the mode of action-based platform devised in our laboratory, we detected antibacterial activity and ribosome stalling induction ability of the molecule (so-called 49252) produced by Actinoplanes sp. VKM Ac-2862. Having observed the concentration-de-pendent effect on translation inhibition in vitro and in vivo we directly proved that protein biosynthesis machinery is the target of 49252. A primary analysis of the resistant mutants provided us with some inklings of the 49252 mechanism of action. The first group of clones carries the mutations in the region of 16SrRNA called loop 560, with C564G substitution providing the highest level of resistance. There are no available literature data on the functional role of most of the identified mutations and these nucleotides do not overlap with binding sites of the existed small subunit inhibitors. This lets us assume that the molecule is likely to have a novel site of action on the ribosome. Another target, revealed in the second group of clones, was the S4 protein, which plays a pivotal role in the decoding process. Interestingly, all of the described mutations belong to the so-called ram or ribosomal ambiguity category, which means that such ribosomes are error-prone. Using in vivo miscoding assay, we succeeded in confirming that the second group of mutants indeed have a higher rate of misreading during translation. Surprisingly, the C564G mutant variant of the 16S rRNA group also appeared to have error-prone ribosomes. All these facts led us to formulate the hypothesis that 49252 is likely to have an impact on translation fidelity. This work was supported by RSF- 20-74-10031grant (I. Osterman).