Identification of an ancillary protein, YabF, required for activity of the KefC glutathione-gated potassium efflux system in Escherichia coli

Abstract

A new subunit, YabF, for the KefC K(+) efflux system in Escherichia coli has been identified. The subunit is required for maximum activity of KefC. Deletion of yabF reduces KefC activity 10-fold, and supply of YabF in trans restores activity. IS2 and IS10R insertions in yabF can be isolated as suppressors of KefC activity consequent upon the V427A and D264A KefC mutations.

FIG. 1

Diagram showing the organization of the carB and kefC region in K. aerogenes and E. coli (a) and the overlap between yabF and kefC in E. coli (b). (a) The bar indicates the 4.2-kb fragment cloned and sequenced from pASRB1, showing the position of the 3′ end of carB (hatched), the carB-yabF intergenic region (open), yabF (diagonally striped), and kefC (filled). The inverted triangle above the block indicates the approximate position of the 13-kb insert found in the E. coli genome sequence (23). (b) DNA sequence of the E. coli yabF-kefC junction. The arrows indicate the position of the 3′ end of yabF (TAG stop codon underlined) and the 5′ end of kefC (putative Shine-Dalgarno sequence and start codon double underlined).

FIG. 2

YabF and YheR are required in trans for the activity of KefC and KefB. Potassium efflux was measured in mutant strains transformed with the appropriate plasmids to allow the contribution of YabF and YheR to be analyzed. Potassium efflux was measured according to methods in previous publications (21), and each experiment has been repeated at least three times. The data shown are representative. The arrow indicates the time of addition of 0.5 mM NEM. (A) KefC activity. Symbols: ▵, strain MJF276 (YheR⁺ KefB⁻ YabF⁺ KefC⁻); ●, MJF276/pkC952; ○, MJF276/pkC11; ■, MJF366/pkC952; □, MJF366/pkC11. (B) KefB activity. Symbols: ▵, strain MJF276; ●, MJF276/pΔYheR; ○, MJF276/pKefB; ■, MJF370/pΔYheR; □, MJF370/pKefB. Strain MJF276 expresses YabF and YheR, MJF366 expresses only YheR, and MJF370 expresses only YabF. Plasmids pkC952 (YabF⁻ KefC⁺), pkC11 (YabF⁺ KefC⁺), pΔYheR (YheR⁻ KefB⁺), and pKefB (YheR⁺ KefB⁺) are described in Table 2. Data obtained with MJF374 (YabF⁻ KefC⁻ YheR⁻ KefB⁻) transformed with the above plasmids were identical to the data obtained with either MJF366 (A) or MJF370 (B) (data not shown).

FIG. 3

Analysis of KefCV427A suppressor mutants. Potassium efflux was determined as described previously (10, 12). (A) Strains MJF116#22, (KefCV427A, IS2 5′ to yabF) and MJF527 (MJF116#22, GshA⁻) were transformed with pCWYabF (YabF⁺) to create strains that possess different combinations of YabF and GSH biosynthesis (Table 2). The strains were incubated in potassium-free medium (18), and either spontaneous K⁺ efflux (no NEM) or NEM-elicited K⁺ efflux (NEM added 3 min after resuspension in K⁺-free medium) was measured. Open columns, YabF⁻ strains; filled columns, YabF⁺ strains. The percentage of K⁺ retained 15 min after resuspension in K⁺-free medium is shown. Low figures indicate high KefC activity. One hundred percent is set for MJF527 immediately after resuspension in K⁺-free medium. (B) Details are as for panel A, substituting strains MJF116#52 and MJF526 (MJF116#52, GshA⁻) for strains MJF116#22 and MJF527, respectively. Similar data were obtained with MJF116#5 and MJF116#34 and their GshA⁻ derivatives (data not shown). (C) Potassium efflux from GSH-deficient strains: MJF335 (MJF276, GshA⁻) (▵), MJF532 (MJF116, GshA⁻) (□), MJF526 (MJF116#52, GshA⁻) (○), and MJF526/pCWYabF (●). Spontaneous K⁺ efflux was measured as described previously (18), and the experiments have been repeated at least three times. The data shown are representative.