Fig 1.
A diagrammatic representation of the var operon.
The locality of the β-lactamase, varG, the MDR varABCDEF transporter complex and the divergently transcribed regulatory varR genes are shown. Arrows indicate orientation of transcription. Three intergenic regions varRG, varGA and varBC to which VarR is hypothesised to regulate transcription are also illustrated.
Table 1.
MIC values for E. coli KAM3 cells expressing VarG.
Fig 2.
The steady-state kinetics of β-lactam degradation by the VarG β-lactamase.
The β-lactamase activity of purified VarG was monitored as the decrease in β-lactam absorbance that resulted from opening of the β-lactam ring during hydrolysis. The rate of (A) moxalactam and (B) meropenem β-lactam hydrolysis was measured as a function of the β-lactam concentration and the data fitted to a sigmoidal equation. The data indicated that the β-lactams moxalactam and meropenem are hydrolysed by VarG with values for the Vmax, Km and Hill Coefficient of 2.1 (± 0.22) and 18.6 (+ 1.92) μMmin-1, 0.6 (± 0.06) and 0.35 (± 0.063) mM, and 2.4 (± 0.29) and 1.4 (± 0.17), respectively.
Table 2.
Steady state kinetic parameters for VarG hydrolysis of β-lactam antibiotics.
Table 3.
MIC values for E. coli KAM3 cells expressing VarDEF.
Fig 3.
Accumulation of Hoechst 33342 by E. coli KAM3 and E. coli TG1 cells harboring pQE100-varDEF.
(A) The E. coli cells (150 μl in PBS) were placed into the well of a 96-well plate, D-glucose was added to a final concentration of 25 mM and left to incubate for 3 min, after which 2.5 μM Hoechst 33342 (Sigma) was added and the fluorescence of the cells monitored with time in a (BioTek) fluorescence plate reader (Excitation 360 nm, Emission 460 nm). (B) Effect of the ATPase inhibitor sodium orthovanadate (NaV) on the accumulation of Hoechst 33342 (2.5 μM) by E. coli KAM3 harboring pQE100-varDEF. Hoechst 33342 was incubated for 38 min with E. coli KAM3 harboring pQE100-varDEF and 40 μg/ml NaV. Cells treated with NaV accumulated substantially more Hoechst 33342 consistent with inactivation of the VarDEF ATP-driven efflux pump. All assays were performed in triplicate.
Table 4.
MIC values for E. coli KAM3 cells expressing VarABCDEF.
Fig 4.
Accumulation of Hoechst 33342 (2.5 μM) by E. coli TG1 cells harboring pQE100-VarDEF and pSYC-VarABCDEF.
The E. coli cells (150 μl in PBS) were placed into the well of a 96-well plate, D-glucose was added to a final concentration of 25 mM and left to incubate for 3 min, after which 2.5 μM Hoechst 33342 (Sigma) was added and the fluorescence of the cells monitored with time in a (BioTek) fluorescence plate reader (Excitation 360 nm, Emission 460 nm). Assays were performed in triplicate.
Fig 5.
VarR binds to the varR-varC intergenic region.
(A) The nucleotide sequence of the 111bp varRG intergenic region containing the putative promoter sequences represented by -35 and -10 regions (highlighted red). The Shine-Dalgarno sequence is highlighted blue. The operator site which is hypothesised to act as the binding site for VarR is highlighted purple and/or underlined if overlapping with promoter sites. The start codons and the deduced amino acid for VarR and VarG are highlighted in green. (B) EMSA using increasing titrations of VarR with 30 bp putative operator varR-varG IR, including 30 bp non-specific DNA Lanes 1 to 9. Titrations of VarR (0, 1.25, 2.5, 5, 10, 25, 50, 100, 200ng, respectively) with 0.08ng 30 bp varR-varG IR. Lanes 10 to 15, titrations of VarR (0, 5, 10, 50, 100, 200ng, respectively) with 0.08ng 30 bp non-specific DNA. (C) EMSA of 50 ng VarR/ 0.08ng 30 bp varR-varG IR DNA (labelled) complex with titrations of unlabelled 30 bp varR-varG IR DNA. Lane 1, 0.08ng 30 bp varR-varG IR DNA only. Lanes 2 to 10, competition assay of 50 ng VarR/ 0.08 ng 30 bp varR-varG IR DNA (labelled) complex with titrations of unlabelled 30 bp varR-varG IR DNA (0, 0.125, 0.25, 0.5, 1, 2, 5, 10, 20ng, respectively).
Table 5.
VarR regulation of CmR expression.
Fig 6.
VarR binds to the varG-varA intergenic region.
(A) The nucleotide sequence of the 176bp varG-varA intergenic region containing the putative promoter sequences represented by -35 and -10 regions (highlighted red). The Shine-Dalgarno sequence is highlighted blue. An operator site is highlighted in purple or underlined if overlapping with promoter sites. The terminator site is highlighted in grey. The start and stop codons and the deduced amino acid for VarA and VarG, respectively, are highlighted in green. (B) EMSA of VarR with varG-varA IR DNA. Lanes 1 and 2, 0 and 50 ng of VarR with 0.08 ng 30 bp varR-varG IR DNA (positive control). Lanes 3 to 18, VarR (0 and 50 ng, respectively) with 0.08 ng 30 bp varG-varA IR DNA fragments 1 to 8, respectively. VarR binds specifically to a 30 bp varG-varA1 IR DNA fragment, which incorporates the last 12 bp of the varG gene and the first 18 bp of the varG-varA IR. (C) Competitive EMSA of VarR/ 0.08 ng varG-varA1 DNA complex with unlabelled varR-varG IR DNA. Lane 1 and 2, 0 and 50 ng VarR with 0.08 ng varG-varA1 IR DNA, respectively. Lanes 3 to 12, competitive assay of 50 ng VarR/ 0.08 ng varG-varA1 IR DNA complex with titrations of unlabelled 30 bp varR-varG IR DNA (0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 ng, respectively).
Fig 7.
VarR binds to the varB-varC intergenic region.
(A) The nucleotide sequence of the varB-varC intergenic region containing the putative promoter sequences represented by -35 and -10 regions (highlighted red). The Shine-Dalgarno sequence is highlighted blue. The start and stop codons and the deduced amino acid for VarC and VarB, respectively, are highlighted in green. An operator site is highlighted in purple or underlined if overlapping with promoter sites. (B) EMSA using increasing titrations of VarR with the 25 bp varB-varC IR DNA. Lanes 1 to 10, titrations of VarR (0, 1.25, 2.5, 5, 10, 25, 50, 100, 200, 400 ng, respectively) with 0.08 ng 25 bp varB-varC IR DNA. Lanes 11 to 13, titrations of VarR (0, 50 and 200ng, respectively) with 0.08ng 30 bp non-specific DNA (negative control). Lanes 14 and 15, 0 and 50 ng VarR with 0.08 ng 30 bp varR-varG IR DNA (positive control).