Figure 1.
Sequence analysis of MATE pumps from various organisms and expression of these genes in parent strains.
A. Multiple alignment of the amino acid sequences of VCD, VFD, VCH and VFH with those of representative homologs in Vibrio parahaemolyticus (NorM) and Escherichia coli (DinF and YdhE) using CLUSTAL W2. *, identical residues. B. Phylogenetic analysis of the above mentioned proteins.
Figure 2.
2D and 3D structure predictions of H- and D- type MATE pumps.
A and C. Schematic representation of the predicted secondary structure of VFD and VFH respectively. The topology was predicted based on the algorithm TMHMM Server 2.0. B and D. Schematic representation of the predicted 3D structure of VFD and VFH respectively. The topology was predicted based on the algorithm I-TASSER.
Table 1.
Minimum inhibitory concentrations (MICs) for recombinant clones carrying MATE-type efflux pump genes from V. fluvialis and V. cholerae.
Figure 3.
Presence of energy-dependent and Sodium-driven transporters in parent strain of V. fluvialis.
A. Level of intracellular concentration of ethidium bromide in parent Vibrio fluvialis strain L12387. Reserpine was added as membrane decoupler leading to disruption in efflux activity and an increase in intracellular concentration of the dye. This effect was reversed on addition of glucose that energized the cells and the efflux activity resulting in a sharp decline in drug concentration inside the Vibrio cells. B. Decrease in the levels of intracellular concentration of ethidium bromide in the parent strain in the presence of 100 mM NaCl. Shown are the levels of accumulated dye in the presence and the absence of the salt.
Figure 4.
Involvement of recombinant efflux pumps in the transport of various compounds.
A. VCD-induced efflux of norfloxacin from recombinant E. coli harbouring vcd gene. pBR322: Accumulation of norfloxacin in KAM32 cells transformed with pBR322 empty vector, VCD: Accumulation of norfloxacin in KAM32 cells transformed with pBR322-vcd. *, the values of fluorescence for VCD were significantly different for as compared to pBR322 control (P<0.05). B. Accumulation of ethidium bromide in cells transformed with pBR322 empty vector (pBR), pBR322-vcd (VCD), pBR322-vfd (VFD), pBR322-vch (VCH) and pBR322-vfh (VFH) *, the values of fluorescence for VFH were significantly different as compared to pBR322 control (P<0.05). For other recombinants it was significantly different at either of indicated time points (P<0.05).
Figure 5.
Sodium-ion dependence of recombinant efflux pumps
Induction of norfloxacin efflux in the presence of 100 mM sodium chloride (+NaCl) or 100 mM potassium chloride (+KCl) in the E. coli KAM32 cells transformed with A. pBR322 or B. pBR322-vfh. C. Effect of increase in Sodium ion concentration on the efflux pump activity of VFH. *, The activity of VFH in the presence of 100 mM and 200 mM NaCl was significantly different from that of the control without NaCl (P<0.05).