Table 1.
Bacterial strains and plasmids.
Figure 1.
Influence of ArmZ on spectinomycin-inducible mexXY expression.
mexX expression was assessed in the indicated P. aeruginosa strains in the absence (−) or presence (+) of spectinomycin (SPC) using quantitative RT-PCR. The armZ and mexZ status (−, absent; +, present) of the strains is highlighted. Expression was normalized to rpoD and is reported relative (fold change) to the wild-type P. aeruginosa PAO1 strain K767 not exposed to spectinomycin. Values represent the mean ± SEM from at least three independent determinations, each performed in triplicate.
Figure 2.
A MexZ-ArmZ interaction and the impact of armZ mutations this interaction was assessed using a bacterial 2-hybrid assay, in which the interaction leads to repression of lacZ expression and reduced β-galactosidase activity in the reporter E. coli strain SU202. (a) The β-galactosidase activity of E. coli SU202 harbouring pMS604 derivatives expressing wild type (WT) or no (−) MexZ together with DP804 derivatives expressing wild type (WT), mutant (amino acid substitutions highlighted) or no (−) ArmZ is indicated. The results shown are mean ± SEM from at least three independent determinations, each performed in triplicate. (b) Western immunoblot of whole cell extracts of E. coli SU202 harbouring pMS604 derivatives expressing wild type MexZ and pDP804 derivatives expressing wild type and mutant ArmZ developed with anti-LexA antibodies.
Figure 3.
Location of mutations in ArmZ that compromise its interaction with MexZ.
The 3-dimensional model of ArmZ was created by threading the ArmZ amino acid sequence onto the crystal structure of the PH1602-extein protein from Pyrococcus horikoshii (PDB code: 1UC2, chain A).
Figure 4.
Impact of armZ mutations on spectinomycin-inducible mexXY expression.
mexX expression was assessed in P. aeruginosa strains expressing wild type (WT) or mutated (amino acid substitution highlighted) armZ genes in the absence (−) or presence (+) of spectinomycin (SPC) using quantitative RT-PCR. Expression was normalized to rpoD and is reported relative (fold change) to the wild-type P. aeruginosa PAO1 strain K767 not exposed to spectinomycin. Values represent the mean ± SEM from at least three independent determinations, each performed in triplicate.
Table 2.
Influence of armZ mutations on aminoglycoside resistance in P. aeruginosa.