The KdpC subunit of the Escherichia coli K+-transporting KdpB P-type ATPase acts as a catalytic chaperone

In Bacteria and Archaea, high-affinity potassium uptake is mediated by the ATP-driven KdpFABC complex. On the basis of the biochemical properties of the ATP-hydrolyzing subunit KdpB, the transport complex is classified as type IA P-type ATPase. However, the KdpA subunit, which promotes K(+) transport, clearly resembles a potassium channel, such that the KdpFABC complex represents a chimera of ion pumps and ion channels. In the present study, we demonstrate that the blending of these two groups of transporters in KdpFABC also entails a nucleotide-binding mechanism in which the KdpC subunit acts as a catalytic chaperone. This mechanism is found neither in P-type ATPases nor in ion channels, although parallels are found in ABC transporters. In the latter, the ATP nucleotide is coordinated by the LSGGQ signature motif via double hydrogen bonds at a conserved glutamine residue, which is also present in KdpC. High-affinity nucleotide binding to the KdpFABC complex was dependent on the presence of this conserved glutamine residue in KdpC. In addition, both ATP binding to KdpC and ATP hydrolysis activity of KdpFABC were sensitive to the accessibility, presence or absence of the hydroxyl groups at the ribose moiety of the nucleotide. Furthermore, the KdpC subunit was shown to interact with the nucleotide-binding loop of KdpB in an ATP-dependent manner around the ATP-binding pocket, thereby increasing the ATP-binding affinity by the formation of a transient KdpB/KdpC/ATP ternary complex.     

Rapid inactivation of the Escherichia coli Kdp K+ uptake system by high potassium concentrations

The Kdp K+ uptake system of Escherichia coli is induced by limitation for K+ and/or high osmolarity. In the present study, the regulation of the activity of the Kdp system has been investigated in E. coli mutants possessing only the Kdp system as the mechanism of K+ accumulation. Cells grown in the presence of low K+ (0.1-1 mM) exhibit normal growth. However, growth inhibition results from exposure of cells to moderate levels of external K+ (> 5 mM). Measurement of the cytoplasmic pH, of K+ pools and of transport via the Kdp system demonstrates that the Kdp system is rapidly and irreversibly inhibited by moderate external K+. Concentrations of K+ greater than 2 mM are sufficient to cause inhibition of Kdp. At pH 6, this results in rapid lowering of the capacity for pH homeostasis, but at pH 7 the intracellular pH is unaffected. Parallel analysis of the expression of the Kdp system in a Kdp+/kdpFABC-lacZ strain shows that levels of K+ that are sufficient to inhibit Kdp activity also repress expression. As a result, growth inhibition of strains solely possessing Kdp arises jointly from inhibition of Kdp activity and repression of Kdp gene expression. These data identify an important aspect of the regulation of potassium transport via the Kdp system and also provide support for a model of regulation of Kdp expression via at least two mechanisms: sensing of both turgor and external K+ concentration.     

Modulation of gastric H+,K+-transporting ATPase function by sodium

Gastric H+,K+-ATPase activity is not affected by Na+ at pH 7.0 but is significantly stimulated by Na+ at pH 8.5. For the stimulation at the latter pH, the presence of both Na+ and K+ were essential. Contrary the H+,K+-ATPase, the associated K+-pNPPase was inhibited by Na+ at both pH values. Sodium competes with K+ for the K+-pNPPase reaction. Also, unlike the H+, K+-ATPase activity the ATPase-mediated transport of H+ within the gastric microsomal vesicles was inhibited by Na+. For the latter event only the extravesicular and not the intravesicular Na+ was effective. The data suggest that the K+-pNPPase activity does not represent the phosphatase step of the H+,K+-ATPase reaction. In addition, the observed inhibition of vesicular H+ uptake by Na+ appears to be due to the displacement by Na+ of a cytosolic (extravesicular) H+ site responsible for the vectorial translocation of H+.     

A K+ transport ATPase in Escherichia coli.

A K+ -stimulated ATPase in membranes of Escherichia coli has been identified as an activity of the Kdp system, and ATP-driven K+ transport system. Three characteristics support association of the ATPase with the Kdp system: (i) ATPase and Kdp transport are both repressed by growth in media containing high concentrations of K+; (ii) the ATPase and Kdp system accept only K+ as substrate, neither requires Na+ nor accepts Rb+ as a substrate; (iii) the affinity of the ATPase and that of th Kdp system for K+ is similar and is altered by mutations in the structural genes of the Kdp system. Discovery of an ATPase associated with a bacterial transport system suggests functional similarities with the ATP-driven transport systems of animal cells.     

Wide distribution of homologs of Escherichia coli Kdp K+-ATPase among gram-negative bacteria.

We used Southern blotting to screen a variety of bacterial genes for homology to the kdp genes of Escherichia coli, genes that encode an ATP-driven K+ transport system. We found that most enterobacteria have sequences homologous to those of the three kdp structural genes and the kdpD regulatory gene. A number of distantly related species, including some cyanobacteria, have sequences homologous to those of the structural genes but not the regulatory gene. In all cases only a single region of homology was found. These results suggest that ATP-driven transport systems similar to the Kdp system in structure and regulation are found in many enteric organisms. In other gram-negative organisms, the ATPase is more divergent, retaining good homology at the DNA level only to the highly conserved phosphorylated subunit of the ATPase.     

Inhibition of H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase by strophanthidin.

Studies of the effect of strophanthidin on H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase activities are reported. Inhibition observations and kinetic results suggest the existence of a common digitalis aglycone binding site located on the extracellular surface of the enzyme, which is affected competitively by the binding of potassium to H(+)-transporting ATPase, Ca(2+)-transporting ATPase, as well as H+/K(+)-transporting ATPase and Na+/K(+)-transporting ATPase. This may lead to a better understanding of the mechanism of the pharmacological action of cardiac glycosides and imply the possibility that the positive inotropic effect may result from the inhibition of both Ca(2+)-transporting ATPase and Na+/K(+)-transporting ATPase.     

The activity of the high-affinity K+ uptake system Kdp sensitizes cells of Escherichia coli to methylglyoxal.

Expression of the Kdp system sensitizes cells to methylglyoxal (MG) whether this electrophile is added externally or is synthesized endogenously. The basis of this enhanced sensitivity is the maintenance of a higher cytoplasmic pH (pHi) in cells expressing Kdp. In such cells, MG elicits rapid cytoplasmic acidification via KefB and KefC, but the steady-state pHi attained is still too high to confer protection Lowering pHi further by incubation with acetate increases the sensitivity of cells to MG.     

N,N'-dicyclohexylcarbodiimide-sensitive K+-dependent ATPase activity in Escherichia coli

ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide and dependent on K+ content in medium is observed only in anaerobically grown Escherichia coli and as the analysis of mutants with defects in different subunits of (F0F1) H+-ATPase and in potassium transport shows only under the structural integrity of both F0F1 and K+-ionophore (the Trk system). The obtained results confirm the data on the H+/K+-exchange and indicate that the F0F1 and Trk systems in anaerobically grown bacteria unite into the same membrane supercomplex inside which the direct energy transfer occurs without a mediation of delta-mu H+.     

ATP binding properties of the soluble part of the KdpC subunit from the Escherichia coli K(+)-transporting KdpFABC P-type ATPase

P-Type ATPases catalyze the transport of cations across the cell envelope via site-specific hydrolysis of ATP. Modulation of enzyme activity by additional small subunits and/or a second regulatory nucleotide binding site is still a subject of discussion. In the K(+)-transporting KdpFABC complex of Escherichia coli, KdpB resembles the catalytic P-type ATPase subunit, but ATP binding also occurs in the essential but noncatalytic subunit, KdpC. For further characterization, the soluble portion of KdpC (KdpC(sol), residues Asn39-Glu190) was synthesized separately and purified to homogeneity via affinity and size exclusion chromatography. Protein integrity was confirmed by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy, which revealed an alpha-helical content of 44% together with an 8% beta-sheet conformation consistent with the values deduced from the primary sequence. The overall protein structure was not affected by the addition of ATP to a concentration of up to 2 mM. In contrast, labeling of KdpC(sol) with the photoreactive ATP analogue 8-azido-ATP resulted in the specific incorporation of one molecule of 8-azido-ATP per peptide. No labeling could be observed upon denaturation of the protein with 0.2% sodium dodecyl sulfate, which suggests the presence of a structured nucleotide binding site. Labeling could be inhibited by preincubation with either ATP, ADP, AMP, GTP, or CTP, thus demonstrating a low specificity for nucleotides. Following 8-azido-ATP labeling and tryptic digestion of KdpC(sol), mass spectrometry showed that ATP binding occurred within the Val144-Lys161 peptide located within the C-terminal part of KdpC, thereby further demonstrating a defined nucleotide binding site. On the basis of these findings, a cooperative model in which the soluble part of KdpC activates catalysis of KdpB is suggested.     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

High-efficiency, temperature-sensitive suppression of amber mutations in Escherichia coli.

We have constructed a high-copy-number plasmid carrying an allele of the supD gene (supD43,74). The plasmid conferred temperature-sensitive suppression of amber mutations. Strains carrying the plasmid exhibited 50 to 60% suppression at 30 degrees C but little or no suppression at 42 degrees C. After a temperature shift from 30 to 42 degrees C the efficiency of suppression decreased gradually over a 60- to 90-min period before reaching the 42 degrees C steady-state level of suppression.     

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.     

Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli McrA endonuclease

McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates. We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system. We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein. Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified lambda and T4gt, respectively) and for induction of the E. coli SOS response in the presence of M.HpaII methylation, indicative of DNA damage. Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a linker region largely tolerant of amino acid insertions. DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4gt. Disruption of the N-terminal domain abolishes restriction of both substrates. Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified lambda in vivo. We suggest a common explanation for this "restriction without damage" and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions. Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the lambda genome disrupts the phage development program at an early stage. We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict lambda but did not restrict T4gt at all. This mutant may have a selective impairment in substrate recognition, distinguishing methylated from hydroxymethylated substrates. The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.     

Optimal conditions for mutagenesis by ozone in Escherichia coli K12

A procedure for the quantitative determination of ozone-induced maltose and methionine mutants in Escherichia coli K12 was defined. High yields of induced mutants with over 90% survival have been obtained when washed cells, taken from logarithmic phase of growth, were aerated by a stream of ozone (0.05–1.0 ppm) in buffer at pH 5.0 for 30 to 60 min. It is postulated that ozone itself is probably exerting directly both lethal and mutagenic effects on the cells via a primary effect on the permeability of the cellular membrane.     

An amber dna mutant of Escherichia coli K12 affecting DNA ligase

We have isolated an amber mutant (dnaL321) of Escherichia coli K12, which affects DNA ligase and which is lethal unless it is suppressed. DNA is degraded under the restrictive conditions. The mutation also affects the sensitivity of the cell to ultraviolet light irradiation, and the capacity to support the growth of phage λ that is deficient in general recombination. This pleiotropy is considered to be due to a single mutation, and is suppressed by supD^?_Isu+ and by supF^?su_III⁺). The mutation is cotransducible with dapE(2%), and with ptsI(85%), by phage Plvir.