Multiple Paths for Nonphysiological Transport of K ⁺ in Escherichia coli

Abstract

Mutants of Escherichia coli lacking all of the known saturable K+ transport systems, “triple mutants,” require elevated K+ concentrations for growth. K+ transport activity in such mutants, called TrkF activity, has low substrate specificity and a low rate that increases with increasing external pH. Attempts to isolate mutants requiring even higher concentrations of K+ failed, implying that either TrkF is essential or is composed of multiple minor K+ transport activities. Instead, we sought mutations that allowed triple mutants to grow at lower K+ concentrations. Mutations so identified include ones altering MscL, the large mechanosensitive channel, or Opp, the oligopeptide permease. However, a possible contribution of wild-type Opp and MscL to TrkF activity was not proven. In contrast, expression of wild-type ProP, TrkG, and TrkH proteins increased uptake when encoded on multicopy plasmids. In all of these situations, the driving force for K+ appeared to be the transmembrane electric potential, and in most cases substrate specificity was low; these are characteristics of TrkF activity. These results support the view that TrkF is composed of multiple, “aberrant” K+ transport activities, i.e., paths that, regardless of their physiological function, allow K+ to cross the cell membrane by a uniport process

YbdG in Escherichia coli is a threshold-setting mechanosensitive channel with MscM activity

“…To isolate mutant YbdG channels with altered gating frequencies, we randomly mutagenized the ybdG gene and screened for mutant channels that suppressed the potassium transport deficiency of a strain deleted for the major K + transport systems. This selection operates on the basis that the channel gates frequently enough to open the nonselective MS pore and allow K + ions to move into the cell down the membrane potential; through frequent cycles of transient openings, followed by rapid closure, the cell can acquire the K + needed for growth (31). Strain MJF622 is deficient for both potassium uptake and YbdG protein (Table S2) and requires ≥30 mM K + for rapid growth.…”

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

“…alanine, concomitant with adaptations that retain the kink and its function in the protein (57). Interestingly, Buurman et al (58) suggested that multiple "aberrant" K + fluxes function as electrogenic uniport activities in E. coli and may together comprise an observed low affinity K + uptake capacity that has been termed TrkF. Perhaps some of these fluxes represent evolving transporters that are in the process of closing up small K + leaks created as membrane-embedded proline residues were replaced.…”

The Implication of YggT ofEscherichia coliin Osmotic Regulation

“…Such nonphysiological K þ uptake activity was reported for Prip and TrkG proteins of E. coli. 7) Of course, we cannot rule out the possibility that the observed K þ uptake partially contributes to the compensation for the growth defect of the E. coli TK2420 cells caused by concentrated Na þ . E. coli has a transport system for Na þ but not for mannitol.…”

“…E. coli strain TK2420 (F À , thi, rha, lacZ, nagA, Á(kdpFAB)5, Á(trk-mscL 0 ) trkD1), 7) which is defective in the three major K þ transport genes, was the gift of Professor, Wolfgang Epstein of the University of Chicago. E. coli TO114 (ÁnhaA, ÁnhaB, ÁchaA), lacking the three Na þ /H þ antiport systems, NhaA, NhaB and ChaA, was the gift of Professor, Hiroshi Kobayashi of Chiba University, and was used in the detection of Na þ export activity.…”

“…These mutant cells take in K þ , probably through multiple minor K þ transport activities. 7) Under hyper-osmotic conditions, these mutant cells require a high concentration of K þ for growth. For example, E. coli TK420 cells lacking the three K þ uptake systems grow in a synthetic medium containing 10 mM K þ and 11.5 mM Na þ , but not in one containing 10 mM K þ and 100 mM Na þ .…”