Bacterial resistances to toxic metal ions - a review

Bacterial plasmids encode resistance systems for toxic metal ions, including Ag⁺, AsO₂^-, AsO₄^3-, Cd²⁺, Co²⁺, CrO₄^2-, Cu²⁺ Hg²⁺, Ni²⁺, Pb²⁺, Sb³⁺, TeO₃^2-, Tl⁺ and Zn²⁺. The function of most resistance systems is based on the energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The Cd²⁺-resistance ATPase of Gram-positive bacteria (CadA) is membrane cation pump homologous with other bacterial, animal and plant P-type ATPases. CadA has been labeled with ³²P from [α-³²p]ATP and drives ATP-dependent Cd²⁺ (and Zn²⁺) uptake by inside-out membrane vesicles (equivalent to efflux from whole cells). Recently, isolated genes defective in the human hereditary diseases of copper metabolism, namely Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to bacterial CadA than to other ATPases from eukaryotes. The arsenic resistance efflux system transports arsenite [As(III)], alternatively using either a double-polypeptide (ArsA and ArsB) ATPase or a single-polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. The triple-polypeptide Czc (Cd²⁺, Zn²⁺ and Co²⁺) chemiosmotic efflux pump consists of inner membrane (CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell.     

Testing the polar auxin transport model with a selective plasma membrane H+-ATPase inhibitor

Auxin is unique among plant hormones in that its function requires polarized transport across plant cells. A chemiosmotic model was proposed to explain how polar auxin transport is derived by the H⁺ gradient across the plasma membrane (PM) established by PM H⁺-adenosine triphosphatases (ATPases). However, a classical genetic approach by mutations in PM H⁺-ATPase members did not result in the ablation of polar auxin distribution, possibly due to functional redundancy in this gene family. To confirm the crucial role of PM H⁺-ATPases in the polar auxin transport model, we employed a chemical genetic approach. Through a chemical screen, we identified protonstatin-1 (PS-1), a selective small-molecule inhibitor of PM H⁺-ATPase activity that inhibits auxin transport. Assays with transgenic plants and yeast strains showed that the activity of PM H⁺-ATPases affects auxin uptake as well as acropetal and basipetal polar auxin transport. We propose that PS-1 can be used as a tool to interrogate the function of PM H⁺-ATPases. Our results support the chemiosmotic model in which PM H⁺-ATPase itself plays a fundamental role in polar auxin transport.     

Active transport of l-sorbose and 2-deoxy-d-galactose in Saccharomyces fragilis

Sorbose and 2-deoxy-d-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars.The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy-d-galactose and chemiosmotic coupling in the case of sorbose and 2-deoxy-d-galactose. The difference between the two mechanisms are discussed.Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport.The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.     

Inhibition of anion transport across the red cell membrane by dinitrophenylation of a specific lysine residue at the H2DIDS binding site of the band 3 protein

The mechanism of free energy coupling in active transport is discussed with special reference to the sarcoplasmic reticulum Ca²⁺-ATPase. In the current working schemes for cation transport ATPases, free energy transduction is nearly always based on enzyme conformational changes. The principal objective of the present article is to examine whether recent experimental results on Ca²⁺-ATPase may in fact be better explained by assuming the existence of a direct chemiosmotic process. In the scheme proposed, free energy transduction between ATP and calcium is based on a transfer of solvation water between the acylphosphate bond and the bound calcium ions.     

Chemiosmotic concept of the membrane bioenergetics: What is already clear and what is still waiting for elucidation?

The present state of the chemiosmotic concept is reviewed. Special attention is paid to (i) further progress in studies on the Na⁺-coupled energetics and (ii) paradoxical bioenergetic effects when protonic or sodium potentials are utilized outside the coupling membrane (TonB-mediated uphill transports across the outer bacterial membrane). A hypothesis is put forward assuming that the same principle is employed in the bacterial flagellar motor.     

Orphan enzyme or patriarch of a new tribe: the arsenic resistance ATPase of bacterial plasmids

The plasmid-determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F₀F₁-type H⁺-translocating ATP synthases, the related vacuolar H⁺-translocating ATPases, the P-type cation-translocating ATPases, and the superfamily which includes the periplasmic binding-protein-dependent systems in Gram-negative bacteria, the human multidrug resistance P-glycoprotein, and the cystic fibrosis transport regulator). The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins. New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases. Furthermore, fundamental questions of energy-coupling (ATP-driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system.     

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V_max only with no apparent change in K_m. Sucrose transport rates driven by membrane potential only, i.e. in the absence of ΔpH, were comparable to ΔpH-driven flux. Both membrane potential and ΔpH-driven sucrose transport were used to examine proton binding to the symport and the apparent K_m for H⁺ was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.     

Genes for all metals—a bacterial view of the Periodic Table

Bacterial chromosomes have genes for transport proteins for inorganic nutrient cations and oxyanions, such as NH₄ ⁺, K⁺, Mg²⁺, Co²⁺, Fe³⁺, Mn²⁺, Zn²⁺ and other trace cations, PO₄ ^3-, SO₄ ^2- and less abundant oxyanions. Together these account for perhaps a few hundred genes in many bacteria. Bacterial plasmids encode resistance systems for toxic metal and metalloid ions including Ag⁺ AsO₂ ^-, AsO₄ ^3-, Cd²⁺, Co²⁺, CrO₄ ²⁻, Cu²⁺, Hg²⁺, Ni²⁺, Pb²⁺, TeO₃ ²⁻, TI⁺ and Zn²⁺. Most resistance systems function by energy-dependent efflux of toxic ions. A few involve enzymatic (mostly redox) transformations. Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. The Cd²⁺-resistance cation pump of Gram-positive bacteria is membrane P-type ATPase, which has been labeled with ³²P from [γ-³²P]ATP and drives ATP-dependent Cd²⁺ (and Zn²⁺) transport by membrane vesicles. The genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson’s disease, encode P-type ATPases that are similar to bacterial cadmium ATPases. The arsenic resistance system transports arsenite [As(III)], alternatively with the ArsB polypeptide functioning as a chemiosmotic efflux transporter or with two polypeptides, ArsB and ArsA, functioning as an ATPase. The third protein of the arsenic resistance system is an enzyme that reduces intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. In Gram-negative cells, a three polypeptide complex functions as a chemiosmotic cation/protein exchanger to efflux Cd²⁺, Zn²⁺ and Co²⁺. This pump consists of an inner membrane (CzcA), an outer membrane (CzcC) and a membrane-spanning (CzcB) protein that function together. Received 08 August 1997/ Accepted in revised form 01 November 1997     

Active transport of L-sorbose and 2-deoxy-D-galactose in Saccharomyces fragilis.

Sorbose and 2-deoxy-D-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars. The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy-D-glucose and chemiosmotic coupling in the case of sorbose and 2-deoxy-D-galactose. The differences between the two mechanisms are discussed. Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport. The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.     

Chemiosmotic coupling of ion transport in the yeast vacuole: Its role in acidification inside organelles

Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H⁺-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl^– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl^– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl^– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK _m for Cl^– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail.     

Molecular modeling of the bacterial outer membrane receptor energizer, ExbBD/TonB, based on homology with the flagellar motor, MotAB

The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B₁₂ across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque.     

Millisecond Delayed Light as an Indicator of Electrical Gradients across Chloroplast Thylakoids

DELAYED light emission from photosynthetic organisms was discovered by Strehler and Arnold¹. The emitted light has a spectrum similar to that of chlorophyll a fluorescence and can often persist for minutes after terminating the illumination. In recent years it has been found that the intensity of emission during the first few milliseconds of the decay is sensitive to the high energy state of the chloroplasts². Wraight and Crofts³ have suggested that this sensitivity is due to the establishment of electrical and pH gradients across the thylakoids during the illumination stage. The linking of the high energy state with these gradients is an essential feature of Mitchell's chemiosmotic hypothesis⁴. Wraight and Crofts³ have argued that the light-induced pH and electrical gradients act in such a way as to decrease the activation energy necessary to lift electrons from the metastable state, created during the preillumination, to the first singlet of chlorophyll. If this hypothesis is correct, the establishment of an electrical gradient across the thylakoid membranes by some means other than light-induced electron transport should change the intensity of millisecond delayed light emission. One possible way to create membrane potentials is to subject chloroplasts to salt gradients. The magnitude of the potentials developed will be a function of the concentration gradients and the relative rates of penetration of the ions across the thylakoid membranes^5,6.     

Effect of lysolecithin treatment on the structure and functions of the mitochondrial inner membrane

Lysolecithin treatment of electron transport particles (ETP) generated membrane fragments capable of catalyzing ATP-³²P_i exchange, which was resistant to the uncoupling action of Valinomycin plus Nigericin or Valinomycin plus Monensin A in the presence of K⁺. Electron micrographs of ultrathin, positively stained sections of lysolecithin treated ETP were virtually devoid of circular patterns characteristic of closed vesicles. The results suggest that the closed vesicular structure of the mitochondrial inner membrane demanded by the chemiosmotic hypothesis of energy transduction (1) may not be essential for the ATP-³²P_i exchange reaction.     

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag⁺, AsO ⁻₂ , AsO ³⁻₄ , Cd²⁺, Co²⁺, CrO ²⁻₄ , Cu²⁺, Hg²⁺, Ni²⁺, Pb²⁺, TeO ²⁻₃ , Tl⁺ and Zn²⁺. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd²⁺-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd²⁺, Zn²⁺, and Co²), others are known that efflux Ag⁺, Cu⁺, Ni²⁺, and Zn²⁺. Resistance to inorganic mercury, Hg²⁺ (and to organomercurials, such as CH₃Hg⁺ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.

     

Molecular modeling of the bacterial outer membrane receptor energizer,ExbBD/TonB,based on homology with the flagellar motor,MotAB

The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B12 across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque.     

The association of proton movement with galactose transport into subcellular membrane vesicles of Escherichia coli.

1. Subcellular membrane vesicles were prepared from a strain of Escherichia coli constitutive for the GalP galactose-transport system. 2. The addition of substrates of the GalP transport system to vesicle suspensions promoted alkaline pH changes, which provided direct evidence for the coupling of sugar and proton transport. 3. Respiration-energized galactose transport was progressively inhibited at pH values above 6.0, and was abolished by agents that render the membrane permeable to protons. 4. The combined effects of valinomycin, the nigericin-like compound A217 and pH on galactose transport suggested that both delta pH and delta psi components of the protonmotive force contributed to energization of galactose transport. 5. These results substantiate the conclusion that the GalP transport system operates by a chemiosmotic mechanism.     

Electrophysiology of Turgor Regulation in Marine Siphonous Green Algae

We review electrophysiological measures of turgor regulation in some siphonous green algae, primarily the giant-celled marine algae, Valonia and Ventricaria, with particular comparison to the well studied charophyte algae Chara and Lamprothamnium. The siphonous green algae have a less negative plasma membrane potential, and are unlikely to have a proton-based chemiosmotic transport system, dominated by active electrogenic K⁺ uptake. We also make note of the unusual cellular structure of the siphonous green algae. Hypertonic stress, due to increased external osmotic pressure, is accompanied by positive-going potential difference (PD), increase in conductance, and slow turgor regulation. The relationship between these is not yet resolved, but may involve changes in K⁺ conductance (G _K) or active K⁺ transport at both membranes. Hypotonic turgor regulation, in response to decreased external osmotic pressure, is ∼3 times faster than hypertonic turgor regulation. It is accompanied by a negative-going PD, although conductance also increases. The conductance increase and the magnitude of the PD change are strongly correlated with the magnitude of hypotonic stress.     

The kinetic mechanism of action of an uncoupler of oxidative phosphorylation.

The chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study.     

Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter,to a Plasma Membrane H+ Gradient

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na⁺-Li⁺ counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H⁺ gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na⁺ electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H⁺-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na⁺ efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H⁺ gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H⁺-coupled transport is more widespread. The coexistence of Na⁺ and H⁺-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.     

TonB-dependent receptors—structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel β-barrel traverses the outer membrane and adjacent β-strands are connected by extracellular loops and periplasmic turns. Located inside the β-barrel is the plug domain, composed primarily of a mixed four-stranded β-sheet and a series of interspersed α-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.