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Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.

Zhitnitsky D, Lewinson O - Mol. Microbiol. (2014)

Bottom Line: Of the 38 conserved positions tested, 24 had small effects on metal tolerance.Based on structural modelling, the functionally important residues line a constricted 'channel', tightly surrounded by the residues that were found to be inconsequential for function.In addition, by substituting six trans-membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn(2+)/Cd(2+) to Ag(+).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.

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Growth in liquid media of wild type and mutant variants of the Cu+/ Ag+ pumps pfCopA (A), rrCopA (B), rrCopB (C) in the presence of the indicated AgNO3 concentrations. In A, wild type pfCopA open squares, E254A closed triangles, E270A crosses, empty vector closed circles. In B, wild type rrCopA open squares, E274A closed triangles, E290A crosses, empty vector closed circles. In C, wild type rrCopB open squares, E281A closed triangles, E297A crosses, empty vector closed circles. For each culture, 100% was defined as the growth in the absence of metal. In A–C error bars (shown unless smaller than icons) represent standard deviations of three repeats.
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fig06: Growth in liquid media of wild type and mutant variants of the Cu+/ Ag+ pumps pfCopA (A), rrCopA (B), rrCopB (C) in the presence of the indicated AgNO3 concentrations. In A, wild type pfCopA open squares, E254A closed triangles, E270A crosses, empty vector closed circles. In B, wild type rrCopA open squares, E274A closed triangles, E290A crosses, empty vector closed circles. In C, wild type rrCopB open squares, E281A closed triangles, E297A crosses, empty vector closed circles. For each culture, 100% was defined as the growth in the absence of metal. In A–C error bars (shown unless smaller than icons) represent standard deviations of three repeats.

Mentions: To investigate the role of the two glutamates in Cu+/Ag+ ATPases, mutants were generated in three such pumps: Rhizobium radiobacter CopA and CopB and Pyrococcus furiosus CopA (Lewinson et al., 2009). In all three proteins, Cu+-tolerance was not greatly affected by mutations in either of the glutamates (Table 2). In contrast, Ag+-tolerance was reduced by the mutations (Fig. 6, Table 2). The most pronounced effects were of the E270A mutation in pfCopA and of E297A of rrCopB. In these two cases the growth of the mutant was very similar to the growth of cells transformed with an empty (control) plasmid (Fig. 6, Table 2). A complete failure to grow was observed with an rrZntA mutant where both glutamates were mutated to alanines. The growth of this mutant was indistinguishable from that of the negative control, with both Zn2+ and Cd2+ (Table 1).


Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.

Zhitnitsky D, Lewinson O - Mol. Microbiol. (2014)

Growth in liquid media of wild type and mutant variants of the Cu+/ Ag+ pumps pfCopA (A), rrCopA (B), rrCopB (C) in the presence of the indicated AgNO3 concentrations. In A, wild type pfCopA open squares, E254A closed triangles, E270A crosses, empty vector closed circles. In B, wild type rrCopA open squares, E274A closed triangles, E290A crosses, empty vector closed circles. In C, wild type rrCopB open squares, E281A closed triangles, E297A crosses, empty vector closed circles. For each culture, 100% was defined as the growth in the absence of metal. In A–C error bars (shown unless smaller than icons) represent standard deviations of three repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4285229&req=5

fig06: Growth in liquid media of wild type and mutant variants of the Cu+/ Ag+ pumps pfCopA (A), rrCopA (B), rrCopB (C) in the presence of the indicated AgNO3 concentrations. In A, wild type pfCopA open squares, E254A closed triangles, E270A crosses, empty vector closed circles. In B, wild type rrCopA open squares, E274A closed triangles, E290A crosses, empty vector closed circles. In C, wild type rrCopB open squares, E281A closed triangles, E297A crosses, empty vector closed circles. For each culture, 100% was defined as the growth in the absence of metal. In A–C error bars (shown unless smaller than icons) represent standard deviations of three repeats.
Mentions: To investigate the role of the two glutamates in Cu+/Ag+ ATPases, mutants were generated in three such pumps: Rhizobium radiobacter CopA and CopB and Pyrococcus furiosus CopA (Lewinson et al., 2009). In all three proteins, Cu+-tolerance was not greatly affected by mutations in either of the glutamates (Table 2). In contrast, Ag+-tolerance was reduced by the mutations (Fig. 6, Table 2). The most pronounced effects were of the E270A mutation in pfCopA and of E297A of rrCopB. In these two cases the growth of the mutant was very similar to the growth of cells transformed with an empty (control) plasmid (Fig. 6, Table 2). A complete failure to grow was observed with an rrZntA mutant where both glutamates were mutated to alanines. The growth of this mutant was indistinguishable from that of the negative control, with both Zn2+ and Cd2+ (Table 1).

Bottom Line: Of the 38 conserved positions tested, 24 had small effects on metal tolerance.Based on structural modelling, the functionally important residues line a constricted 'channel', tightly surrounded by the residues that were found to be inconsequential for function.In addition, by substituting six trans-membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn(2+)/Cd(2+) to Ag(+).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.

Show MeSH
Related in: MedlinePlus