Effects of Tetracycline Susceptibility and Virulence in an acrR Mutant of Erwinia amylovora.

This study investigated whether AcrR-mediated regulation of the acrAB efflux system is associated with tetracycline susceptibility in Erwinia amylovora. We compared tetracycline susceptibility, acrAB expression, intracellular tetracycline accumulation, intracellular pH, ATP-associated luminescence, envelope-associated phenotypes, virulence, and responses to exogenous acyl-homoserine lactones (AHLs) among the wild type (WT), an acrR mutant (ΔacrR), the same mutant after prior tetracycline exposure (ΔacrRTc), and an acrR-complemented mutant (ΔacrR(acrR)). Relative to WT, ΔacrR showed fourfold increases in tetracycline MIC and MBC, whereas ΔacrRTc showed 16- and 32-fold increases, respectively. ΔacrRTc also showed markedly increased acrA and acrB expression, reduced intracellular tetracycline accumulation, lower intracellular pH, and reduced ATP-associated luminescence, suggesting that prior tetracycline exposure was associated with an enhanced AcrAB response in the acrR-deficient background. ΔacrRTc further displayed altered cell-envelope ultrastructure, increased waaL expression and lipopolysaccharide (LPS)-associated band intensity, reduced biofilm formation, and a weaker cellulose-associated colony phenotype, whereas amylovoran production and levansucrase-associated activity remained unchanged. In the pathogenicity assay, ΔacrRTc produced weaker necrotic symptoms, lower bacterial populations, and reduced in planta dspE expression. Among the AHLs tested, OHHL produced a distinct virulence pattern, with ΔacrR showing reduced disease severity relative to WT and ΔacrR(acrR), whereas this separation was not observed with HHL or OOHL; ΔacrRTc remained less virulent under all AHL conditions. These findings support tetracycline as an AcrAB substrate and suggest that prior tetracycline exposure can strengthen the subsequent AcrAB-associated response. The OHHL-specific phenotype further raises the possibility that OHHL may also serve as a substrate of the AcrAB efflux system.