Tomato bacterial canker, caused by Clavibacter michiganensis (Cm), is a destructive vascular disease threatening global tomato production and seed trade.

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Source: Heinz USA , Bugwood.org

Bacterial canker and wilt of tomato (Clavibacter michiganensis ssp. michiganensis (Smith 1910) Davis et al. 1984)

Streptomycin, a widely used aminoglycoside antibiotic in both plant disease management and human medicine, inhibits bacterial growth by targeting the ribosome. Known streptomycin resistance mechanisms in Cm primarily involve mutations in ribosomal components, such as rpsL gene, which prevent antibiotic binding.

However, the genetic basis underlying the unusually high-level streptomycin resistance observed in the field-derived strain TX-0702 could not be explained by these mechanisms and remained unknown.

Through transposon insertion mutagenesis and de novo genome sequencing, the authors identified a previously uncharacterized 51,235 bp plasmid, designated pCM3, in TX-0702. Comparative analysis revealed that pCM3 shares high nucleotide identity with plasmid D26b from Curtobacterium species and carries multiple mobile genetic elements, including a MobC-like relaxase and transposases, suggesting that pCM3 was likely acquired from environmental bacteria via horizontal gene transfer (HGT) and has the potential for further dissemination.

Resistance mechanism

Genetic and biochemical analyses further revealed the resistance mechanism mediated by pCM3. The plasmid harbors aph(3), encoding an aminoglycoside 3’-phosphotransferase. Plasmid-curing experiments showed that loss of pCM3 reduced the streptomycin MIC from 128 μg/ml to 4 μg/ml in all cured strains.

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Source: mLife

(A) Physical map of the aph(3) gene derived from de novo sequencing data. (B) Minimum inhibitory concentrations (MICs) of streptomycin against plasmid-cured strains of C. michiganensis. (C) MICs of streptomycin against C. michiganensis strains with different levels of aph(3) expression. (D) LC-MS analysis of streptomycin standard. (E) Extracted ion chromatogram (EIC) of APH(3)-treated samples at m/z 582.2738. (F) LC-MS detection of phosphorylated streptomycin in APH(3)-treated samples. Credit

Knockout of aph(3) similarly abolished resistance, while overexpression further elevated the MIC to 256 μg/ml, confirming aph(3) as the core resistance determinant. In vitro phosphorylation assays combined with LC-MS analysis demonstrated that APH(3) phosphorylates streptomycin to yield a monophosphorylated product, completely abolishing its inhibitory activity against Cm. This represents the first demonstration of an APH(3)-dependent phosphorylation-driven resistance mechanism in a Gram-positive plant pathogen.

Novel mechanism

This study reveals a novel streptomycin resistance mechanism in Cm and fills a gap in the understanding of antibiotic resistance in plant pathogens.

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The high sequence similarity of pCM3 to environmental bacterial plasmids, combined with its mobile genetic elements, suggests that plant pathogens may serve as reservoirs and vectors for resistance genes, bridging soil, plant, and human-associated microbial communities.

These findings carry important implications for resistance gene surveillance under the One Health framework, and underscore the need for strengthened monitoring of resistance gene dissemination in agricultural ecosystems.

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