The melibiose permease system of Escherichia coli K12

The melibiose permease system of E. coli K12 has been explored using a strain deficient in lactose permease: 300 P. The accumulation of 1-S-methyl-beta-D-thiogalactopyranoside (TMG) was observed. The uptake system was inducible by melibiose and a number of analogs at 30 degrees C. At higher temperatures the differential rate of synthesis decreases until becoming negligible at 42 degrees C. The uptake tends toward a steady state which corresponds to an accumulation several hundredfold over the sugar concentration in the medium. At a given temperature the steady state level was proportional to the initial rate of uptake whatever the degree of induction and the substrate concentration. Lowering the temperature decreased the initial rate of uptake but increased the steady state level. This uptake system was pH dependent with better efficiency at pH 8. It was also dependent on the presence of sodium or lithium ions active at 5 mM whereas potassium at 170 mM enable only about half maximal uptake. The uptake in a medium with choline chloride was less than one fifth of optimal activity. Addition of Li+ brought about half maximal activation at approximately 0.5 mM. The activation consists mainly in a decrease of apparent Km. The emphasis of this study was put on the similarities and differences with lactose permease which is able to transport the same sugar to approximately the same extent. Inducer specificities and substrate specificities were compared and a method of measuring the two activities in the same cells was devised.     

Arabidopsis KEA2, a homolog of bacterial KefC, encodes a K(+)/H(+) antiporter with a chloroplast transit peptide

KEA genes encode putative K(+) efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K(+) efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na(+)(K(+))/H(+) antiporter Nhx1p to confer hygromycin resistance and tolerance to Na(+) or K(+) stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H(+) exchange with preference for K(+)=Cs(+)>Li(+)>Na(+). When a conserved Asp(721) in transmembrane helix 6 that aligns to the cation binding Asp(164) of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu(835) between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K(+)/H(+) antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids.     

The bacterial Kdp K(+)-ATPase and its relation to other transport ATPases, such as the Na+/K(+)- and Ca2(+)-ATPases in higher organisms

The Kdp system is a three-subunit member of the E1-E2 family of transport ATPases. There is sequence homology of the 72 kDa KdpB protein, the largest subunit of Kdp, with the other members of this family. The predicted structure of the 21 kDa KdpC subunit resembles that of the beta subunit of the Na+,K(+)-ATPase, suggesting that these subunits may have a similar function. The 59 kDa KdpA subunit has no known homologue; it is very hydrophobic and is predicted to cross the membrane 10-12 times. Genetic studies implicate this subunit in the binding of K+. As the binding site must be close to the beginning of the transmembrane channel, we suggest that KdpA also forms most or all of the latter. KdpA may have evolved from a K+/H+ antiporter that was recruited by the KdpB precursor to achieve the high affinity and specificity for K+, and the activation of transport by low turgor pressure characteristic of Kdp. Turgor pressure controls the expression of Kdp. This action is dependent on the 70 kDa KdpD and 23 kDa KdpE proteins. We are in the process of sequencing these genes. KdpE is homologous to the smaller protein of other members of a family of pairs of regulatory proteins implicated in control of a variety of bacterial processes such as porin synthesis, phosphate regulon expression, nitrogen metabolism, chemotaxis and nodule formation.     

Analysis of KdpC of the K(+)-transporting KdpFABC complex of Escherichia coli.

The Kdp complex, a high affinity ATP-driven K(+) transport system of Escherichia coli, is composed of the four membrane-bound subunits KdpF, KdpA, KdpB and KdpC. Whereas the role of KdpB (catalytical subunit), KdpA (K(+)-translocating subunit) and KdpF (stabilizing peptide) is well understood, the function of KdpC is still unknown. Therefore, a kdpC deletion strain was constructed and complementation experiments were performed using different kdpC constructs. Truncations of the kdpC gene revealed that only one derivative, which lacks base pairs coding for the four C-terminal amino acids, was able to complement the chromosomal deletion of kdpC. Furthermore, complementation was also observed with kdpC of Mycobacterium tuberculosis, but not with kdpC from Clostridium acetobutylicum or Synechocystis sp. PCC6803. Sequence alignment of 17 different KdpC proteins led to the construction of hybrids between kdpC of E. coli and that of C. acetobutylicum. Complementation revealed that the N-terminal transmembrane segment as well as the C-terminal-third of the protein can be exchanged between both species, but only one after the other. A simultaneous substitution of both regions was not possible.     

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

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.     

Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

Two routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cell's primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.     

The nature of the transformation process in Escherichia coli K12

Summary The nature of the transformation process in E. coli was studied by investigating various factors which affect the efficiency of transformation. CaCl₂ treatment of the recipient cells is absolutely necessary for transformation and the optimum concentration was found to be 30 mM. The efficiency of transformation is dependent upon temperature during incubation of the recipient cells with DNA. The efficiency is also affected by the molecular weight of donor DNA used. Sheared DNA with molecular weights ranging from 10 to 30x10⁶ daltons was most efficient, increasing the number of transformants by a factor of 5 to 10 as compared to unsheared DNA. The intracellular status of recB-recC DNase (ATP-dependent DNase) is another important factor which determines the transformability of E. coli K12. This was shown by demonstrating that a recB ^- recC ^- sbcA ^- strain was transformable as well as the previously demonstrated recB ^- recC ^- sbcB ^- strain. Therefore, it seems reasonable to conclude that the E. coli K12 strain is transformable if the ATP-dependent DNase is absent or diminished in function and a state of recombinational proficiency exists.