Genetic manipulation of polyphosphate metabolism affects cadmium tolerance in Escherichia coli.

The polyphosphate metabolic pathways in Escherichia coli were genetically manipulated to test the effect of polyphosphate on tolerance to cadmium. A polyphosphate kinase (ppk) and polyphosphatase (ppx) mutant strain produced no polyphosphate, whereas the same strain carrying multiple copies of ppk on a high-copy plasmid produced significant quantities. The doubling times of both strains increased with increasing cadmium concentrations. In contrast, the mutant strain carrying multiple copies of ppk and ppx produced 1/20 of the polyphosphate found in the strain carrying multiple copies of ppk only and showed no significant increase in doubling time over the same cadmium concentration range.     

Studies on phosphate transport in Escherichia coli. II. Effects of metabolic inhibitors and divalent cations.

1. Study has been made of the effects of a variety of metabolic inhibitors and divalent cations (Ni2+ and Mn2+), normally after 5 min exposure, on the biphasic uptake of inorganic phosphate (Pi) exhibited by phosphate-deprived cells of Escherichia coli, strains AB3311 (Reeves met-) and CBT302 (a (Ca2+ + Mg2+)-ATPase-deficient mutant). 2. In AB3311 cells cyanide (1-10 mM) produced comparable reductions in phosphate uptake to anaerobiosis, but in both instances significant uptake was maintained. Examination of intracellular Pi concentrations showed that, despite these inhibitions, Pi is still concentrated 130 times compared to 394 times under aerobic conditions. Arsenate (100 muM) and iodoacetate (100 muM pre-exposed 15 min) both abolished anaerobic-supported uptake. Under aerobic conditions the former eliminated primary uptake while the latter reduced both phases of uptake 60%. The uncouplers, dinitrophenol (100-1000 muM) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (50muM) produced very significant, but not complete inhibitions of both phases of uptake. Inhibitions by iodoacetate and dinitrophenol were additive while dithiothreitol protected against the effects of 50-250 mum CCCP. N,N'-Dicyclo-hexylcarbodiimide (DCCD), the potent inhibitor of membrane-bound (Ca2+ + Mg2+)-ATPase, at 10(-3) M caused significant inhibitions of aerobic- (approx. 60%) and anaerobic- (approx. 80%) supported uptakes thus suggesting some obligatory requirement for this ATPase. 3. CBT302 cells, like AB3311, supported Pi transport both aerobically and anaerobically. CCCP (50muM) reduced the primary uptake similarly to AB3311 cells, but the secondary uptake was less affected. DCCD (10(-5)-10(-3) M), as expected, showed no effects in contrast to AB3311 cells. 4. In AB3311 cells Ni2+ (10 mM) caused significant but different reductions of secondary (70%) and primary (33%) phases of phosphate uptake. Mn2+ (10 mM) showed a greater differential effect with the primary uptake being minimally affected and the secondary uptake being abolished (97%). Partial relief of these inhibitions by Mg2+ (10 mM), suggested that these ions compete with Mg2+ transport. High voltage electrophoresis studies showed that Ni2+ cause intensification in the labelling from 32Pi (i.e. during Pi uptake) of hexose phosphates and a reduction in the labelling of complex molecules left at the origin. With Mn2+, labelling of fructose 1,6-diphosphate was reduced, the triose phosphate area was intensified and an unknown area (X) was intensely labelled. When Mn2+ was combined with anaerobiosis, phosphate uptake though diminished in rate exceeded after 16 min the plateau level of uptake under aerobic conditions with Mn2+ present.     

Substrate analogues and divalent cations as inhibitors of glutamate decarboxylase from Escherichia coli

To examine the idea that glutamate decarboxylase from E. coli can be a convenient source for the study of the effects of compounds on GABA synthesis in the nervous system, a series of substrate analogues and divalent cations were tested as potential inhibitors of the bacterial enzyme. Those analogues exhibiting inhibitor activity did so in a competitive manner. The most effective inhibitors were 3-mercaptopropionic acid, 4-bromoisophthalic acid and isophthalic acid which exhibited Ki values of 0.13 mM, 0.22 mM and 0.31 mM, respectively. Eight other analogues produced lesser degrees of inhibition. In addition, seven divalent metal cations were tested as inhibitors of the enzyme. However, only Hg2+, Cd2+, Cu2+ and Zn2+ were effective at a concentration of 0.1mM. When these results were compared to the patterns of inhibition of glutamate decarboxylase from mouse brain, certain differences in the manner in which the enzymes responded to the inhibitors, emerged. Consequently, the bacterial decarboxylase may not be a good model for the study of drug action on brain GABA synthesis.     

Kinetics of promoter search by Escherichia coli RNA polymerase. Effects of monovalent and divalent cations and temperature

The rapid mixing/photocross-linking technique developed in our laboratory has been employed in the study of the mechanism of promoter binding by Escherichia coli RNA polymerase (RPase). We have previously reported on the quantitation of the one-dimensional diffusion coefficient (D1) for RPase along the DNA template (Singer, P. T., and Wu, C.-W. (1987) J. Biol. Chem. 262, 14178-14189). In this paper, we describe the effect of salt concentration and temperature on the kinetics of promoter search by RPase using plasmid pAR1319 DNA, which contains the A2 early promoter from bacteriophage T7, as template. Over a range of KCl concentrations from 25 to 200 mM, the apparent bimolecular rate constant (ka) for the association of RPase with the A2 promoter on this DNA template varied approximately 2-fold, achieving a maximal value between 100 and 125 mM KCl. More significantly, the transient distribution of RPase among nonspecific DNA binding sites changed markedly as a function of salt concentration, indicative of gross changes in the average number of base pairs covered by sliding during a nonspecific lifetime. Using the mathematical treatment outlined in our earlier report, the nonspecific dissociation rate constant (koff) was calculated from the binding curves for the nonspecific as well as promoter-containing DNA. The observed variations in ka as a function of monovalent cation concentration ([M+]) were due primarily to changes in koff, as D1 was found to be essentially independent of [M+]. Interestingly, D1 decreased by one-third as the concentration of magnesium was lowered from 10 to 1 mM. In addition, the dependence of koff (and consequently the nonspecific equilibrium association constant, keq) on [M+] agreed qualitatively with the results of deHaseth et al. (deHaseth, P.L., Lohman, T. M., Burgess, R. R., and Record, M. T., Jr. (1977) Biochemistry 17, 1612-1622), though we consistently measure a weaker Keq. The association rate constant was also measured between 4 and 37 degrees C, and was found to vary approximately 2-fold over that range. An activation energy for the bimolecular association of RPase to the A2 promoter was calculated to be 2.2 +/- 0.4 kcal/mol, while the activation energy for one-dimensional diffusion was 4.7 +/- 0.8 kcal/mol.     

Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli.

The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.     

A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells

Reconstitution into planar lipid bilayers of a poly-3-hydroxybutyrate/calcium/polyphosphate (PHB/Ca(2+)/polyP) complex from Escherichia coli membranes yields cationic-selective, 100 pS channels (Das, S., Lengweiler, U.D., Seebach, D. and Reusch, R.N. (1997) Proof for a non-proteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 94, 9075-9079). Here, we report that this complex can also form larger, weakly selective pores, with a maximal conductance ranging from 250pS to 1nS in different experiments (symmetric 150mM KCl). Single channels were inhibited by lanthanum (IC(50)=42+/-4microM, means+/-S.E.M.) with an unusually high Hill coefficient (8.4+/-1.2). Transition to low-conductance states (<250pS) was favored by increased membrane polarization (/V/ >or=50mV). High conductance states (>250pS) may reflect conformations important for genetic transformability, or "competence", of the bacterial cells, which requires the presence of the PHB/Ca(2+)/polyP complex in the membrane.     

Polyphosphates and enzymes of polyphosphate metabolism in Escherichia coli

This review summarizes the results of our study of polyphosphate and enzymes of polyphosphate metabolism in E. coli and their regulation by exogenous orthophosphate and other physiological and genetic factors.     

Systematic analysis of native membrane protein complexes in Escherichia coli

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.     

Cellular incorporation of poly-beta-hydroxybutyrate into plasma membranes of Escherichia coli and Azotobacter vinelandii alters native membrane structure

Under growth-limiting conditions or conditions which mediate genetic transformation, Escherichia coli and Azotobacter vinelandii incorporate poly-beta-hydroxybutyrate into their plasma membranes. Genetic transformation competence of both bacteria increased in proportion to the concentration of membrane poly-beta-hydroxybutyrate. The effects of this lipid polymer on membrane structure were investigated by freeze-fracture electron microscopy. Before poly-beta-hydroxybutyrate incorporation, freeze-fracture revealed a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membrane. As the cells incorporated the lipid polymer into the membrane, transformability developed and small semiregular plaques which possessed shallow particles were seen. These plaques grew in size and frequency as the membrane poly-beta-hydroxybutyrate concentrations and transformability increased.     

Phosphatidylserine synthetase mutants of Escherichia coli. Genetic mapping and membrane phospholipid composition.

Mutants of Escherichia coli K-12 defective in CDP-diglyceride:L-serine phosphatidyltransferase (phosphatidylserine synthetase) can be isolated by a rapid autoradiographic screening assay described previously (Raetz, C. R. H. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2274-2278). Four organisms of this kind have now been characterized. The gene (designated pss) which is altered in these mutants is closely linked to the nadB locus near minute 49 on the E. coli chromosome. Strains carrying the pss-8 mutation do not grow at elevated temperatures and have low levels of an altered synthetase in cell extracts. An analysis of several hundred transductants and temperature-resistant revertants reveals that the pss-8 mutation is responsible both for the enzyme defect and for the phenotype. When a pss-8 mutant is shifted to the nonpermissive temperature, the cells stop dividing and form long filaments. After 3 hours at 44 degrees the level of phosphatidylethanolamine drops from 66 to 32% (percentage of the total lipid phosphorus), while the combined levels of phosphatidylglycerol and cardiolipin rise from 34 to 68%.     

The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.     

Spin-labeling of Escherichia coli membrane by enzymatic synthesis of phosphatidylglycerol and divalent cation-induced interaction of phosphatidylglycerol with membrane proteins.

Escherichia coli membrane particulate fraction has been spin-labeled by incubating with sn-glycerol-3-phosphate, CTP, palmitoyl CoA and 12-nitroxide stearoyl CoA. Incorporation of the spin-labeled acyl chain into phosphatidyl-glycerol was confirmed. ESR spectrum of the spin-labeled phosphatidylglycerol in E. coli membrane consisted at least of two components; one due to the labels undergoing rapid anisotropic motions and the other due to strongly immobilized labels (the overall splitting value, approx. 58 G). The relative intensity of the two components was dependent on the concentration of divalent cations. The immobilized component decreased on treatment of the membrane with EDTA and increased on addition of Mg2+ or Ca2+. The spectrum at 1 mM Mg2+ or Ca2+ consisted almost only of the immobilized component. Spin-labeled phosphatidylglycerol in total lipid membrane showed ESR spectrum due to mobile labels and the spectrum was not affected appreciably by the divalent cations. The results suggest the divalent cation-mediated interaction of phosphatidylglycerol with proteins in E. coli membrane. Phosphoenolpyruvate-dependent uptake of methyl-alpha-D-glucoside was markedly accelerated by Mg2+. Ca2+ was not effective for the enhancement. The divalent cation-induced interaction of phosphatidylglycerol with proteins was discussed in relation to the sugar transport.     

Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations

Abstract Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo' -deficient mutants of Escherichia coli K-12 with the following order of potency: Zn ( II ) > Cd ( II ) > > Co ( II ). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities.     

Permeation and interaction of divalent cations in calcium channels of snail neurons

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.     

Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.     

Voltage-dependent changes in the permeability of nerve membranes to calcium and other divalent cations.

Transmitter release from depolarized nerve terminals seems to be preceded by a rise in the intracellular concentration of ionized calcium. In squid giant axons, depolarization promotes calcium entry by two routes: one that is blocked by tetrodotoxin and one that is insensitive to tetrodotoxin. The TTX-sensitive route seems to be the sodium channel of the action potential; but the TTX-insensitive route seems to be quite distinct from the sodium and potassium channels of the action potential. It is blocked by Mg-2+, Mn-2+ and Co-2+ ions and by the organic calcium antagonist D-600 and has many features in common with the mechanism that couples excitation to secretion.     

Long-chain polyphosphate causes cell lysis and inhibits Bacillus cereus septum formation, which is dependent on divalent cations.

We investigated the cellular mechanisms that led to growth inhibition, morphological changes, and lysis of Bacillus cereus WSBC 10030 when it was challenged with a long-chain polyphosphate (polyP). At a concentration of 0.1% or higher, polyP had a bacteriocidal effect on log-phase cells, in which it induced rapid lysis and reductions in viable cell counts of up to 3 log units. The cellular debris consisted of empty cell wall cylinders and polar caps, suggesting that polyP-induced lysis was spatially specific. This activity was strictly dependent on active growth and cell division, since polyP failed to induce lysis in cells treated with chloramphenicol and in stationary-phase cells, which were, however, bacteriostatically inhibited by polyP. Similar observations were made with B. cereus spores; 0.1% polyP inhibited spore germination and outgrowth, and a higher concentration (1.0%) was even sporocidal. Supplemental divalent metal ions (Mg(2+) and Ca(2+)) could almost completely block and reverse the antimicrobial activity of polyP; i. e., they could immediately stop lysis and reinitiate rapid cell division and multiplication. Interestingly, a sublethal polyP concentration (0.05%) led to the formation of elongated cells (average length, 70 microm) after 4 h of incubation. While DNA replication and chromosome segregation were undisturbed, electron microscopy revealed a complete lack of septum formation within the filaments. Exposure to divalent cations resulted in instantaneous formation and growth of ring-shaped edges of invaginating septal walls. After approximately 30 min, septation was complete, and cell division resumed. We frequently observed a minicell-like phenotype and other septation defects, which were probably due to hyperdivision activity after cation supplementation. We propose that polyP may have an effect on the ubiquitous bacterial cell division protein FtsZ, whose GTPase activity is known to be strictly dependent on divalent metal ions. It is tempting to speculate that polyP, because of its metal ion-chelating nature, indirectly blocks the dynamic formation (polymerization) of the Z ring, which would explain the aseptate phenotype.     

Chromosome partition in Escherichia coli requires postreplication protein synthesis.

After inhibition of protein synthesis, the number of nuclear bodies (nucleoids) visible in cells of Escherichia coli B/rA corresponded closely to the number of completely replicated chromosomes. We calculated that nucleoid partition follows almost immediately after replication forks reach the chromosome terminus. We show that such a partition is dependent on protein synthesis and that this may reflect the requirement that cells must achieve a certain minimum length before partition (and subsequent cell division) can take place.