PMC

Requirement for PA5471 for drug-inducible mexXY expression in MexZ⁺ but not MexZ⁻ P. aeruginosa. The expression of mexX (A), mexZ (B), and rpoD (C) was assessed in strain K767 (PA5471⁺ MexZ⁺) and its derivatives K2413 (PA5471⁻ MexZ⁺), K2415 (PA5471⁺ MexZ⁻), and K2416 (PA5471⁻ MexZ⁻) grown without (−) and with (+) chloramphenicol (Cam; 8 μg/ml), using semiquantitative RT-PCR. The rpoD reaction served as an internal control that ensured that equal amounts of RNA were employed in all of the RT-PCRs shown. The PCR portion of the reactions was carried out for 28 (mexX), 40 (mexZ), or 20 (rpoD) cycles (top) and 30 (mexX), 42 (mexZ), or 22 (rpoD) cycles (bottom). Data are representative of two or three replicates.

PA5471 stimulates mexXY expression in the absence of antibiotics. The expression of PA5471 (A), mexX (B), and rpoD (C) was assessed in P. aeruginosa strain K767 carrying pUCP20T (lane 1) or pYM010 (pUCP20T::PA5471), using semiquantitative RT-PCR. The rpoD reaction served as an internal control that ensured that equal amounts of RNA were employed in all of the RT-PCRs shown. The PCR portion of the reactions was carried out for 15 (top panel) and 17 (bottom panel) cycles for PA5471, 19 (top panel) and 21 (bottom panel) cycles for rpoD, and 30 (top panel) and 32 (bottom panel) cycles for mexX. Data are representative of two or three replicates.

Requirement for PA5471 for antibiotic-inducible mexXY expression. The expression of mexX, PA5471, and rpoD was assessed in P. aeruginosa strains K767 and K2413 (K767 ΔPA5471) grown without antibiotics (lane 1) or with norfloxacin (lane 2), cefotaxime (lane 3), chloramphenicol (lane 4), erythromycin (lane 5), tetracycline (lane 6), and kanamycin (lane 7) by semiquantitative RT-PCR. The rpoD reaction served as an internal control that ensured that equal amounts of RNA were employed in all of the RT-PCRs shown. The PCR portion of the reactions was carried out for 28 (top panel) and 30 (bottom panel) cycles for mexX, 19 (top panel) and 21 (bottom panel) cycles for PA5471 and rpoD, and 31 (top panel) and 33 (bottom panel) cycles for PA5471-PA5470. Data are representative of two or three replicates.