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ABSTRACT Streptomycin (Sm) is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogenAgrobacterium fabrumis a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants ofA. fabrumare commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency inA. fabrum, and we attribute this trait to the presence of a chromosomal gene (strB) encoding a putative aminoglycoside phosphotransferase. We show howstrB, along withrpsL(encoding ribosomal protein S12) andrsmG(encoding a 16S rRNA methyltransferase), modulates streptomycin sensitivity inA. fabrum. IMPORTANCEThe plant pathogenAgrobacterium fabrumis a widely used model bacterium for studying biofilms, bacterial motility, pathogenesis, and gene transfer from bacteria to plants. Streptomycin (Sm) is an aminoglycoside antibiotic known for its broad efficacy against gram-negative bacteria.A. fabrumexhibits endogenous resistance to somewhat high levels of streptomycin, but the mechanism underlying this resistance has not been elucidated. Here, we demonstrate that this resistance is caused by a chromosomally encoded streptomycin-inactivating enzyme, StrB, that has not been previously characterized inA. fabrum. Furthermore, we show how the genesrsmG,rpsL, andstrBjointly modulate streptomycin susceptibility inA. fabrum.