Two Essential Arginine Residues in the T Components of Energy-Coupling Factor Transporters

Energy-coupling factor (ECF) transporters, a recently discovered class of importers of micronutrients, are composed of a substrate-specific transmembrane component (S component) and a conserved energy-coupling module consisting of a transmembrane protein (T component) and pairs of ABC ATPases (A proteins). Based on utilization of a dedicated (subclass I) or shared (subclass II) energy-coupling module, ECF systems fall into two subclasses. The T components are the least-characterized proteins of ECF importers, and their function is essentially unknown. Using RcBioN and LmEcfT, the T units of the subclass I biotin transporter (RcBioMNY) of a gram-negative bacterium and of the subclass II folate, pantothenate, and riboflavin transporters of a lactic acid bacterium, respectively, we analyzed the role of two strongly conserved short motifs, each containing an arginine residue. Individual replacement of the two Arg residues in RcBioN reduced ATPase activity, an indicator of the transporter function, by two-thirds without affecting the modular assembly of the RcBioMNY complex. A double Arg-to-Glu replacement destroyed the complex and abolished ATPase activity. The corresponding single mutation in motif II of LmEcfT, as well as a double mutation, led to loss of the T unit from the subclass II ECF transporters and inactivated these systems. A single Arg-to-Glu replacement in motif I, however, abolished vitamin uptake activity without affecting assembly of the modules. Our results indicate that the conserved motif I in T components is essential for intramolecular signaling and, in cooperation with motif II, for subunit assembly of modular ECF transporters.Energy-coupling factor (ECF) transporters are a recently discovered novel class of importers of micronutrients in prokaryotes (9, 12, 13, 20). They are composed of a conserved energy-coupling module consisting of a transmembrane protein (T component) and pairs of ATP-binding cassette-containing proteins (A proteins), as well as an S unit (S component) through which substrate specificity is conveyed. S components represent a group of highly diverse small integral membrane proteins with predicted or experimentally established individual specificity for transition metal ions, B vitamins or their precursors, biotin, lipoate, and intermediates of salvage pathways. A link between substrate specificity and S components has been experimentally demonstrated in the case of CbiMN (Co²⁺) and NikMN (Ni²⁺) (13), RibU (riboflavin) (4, 6, 19), BioY (biotin) (9), FolT (folate) (8, 12), and ThiT (thiamine) (8, 12, 14). Based on utilization of a dedicated or shared AAT module, ECF transporters fall into two subclasses. Members of subclass I are encoded by operons containing one or two ABC ATPase genes, a T-component gene, and an S-component gene (12). RcBioMNY, the biotin transporter of Rhodobacter capsulatus, is the prototype of these systems. Previous work has established that the solitary BioY (S component) can function as a low-affinity transporter. It is converted into a high-affinity system in the presence of the AAT module BioMN (9). Notably, the majority of ECF transporters belong to subclass II. These promiscuous systems are widespread in the Firmicutes and also occur in members of the Thermotogales lineage and in archaea (12). In general, the cells contain a single ecfA1A2T operon and a number of genes for S units that are scattered around the genome. Cooccurrence of subclass I and subclass II ECF transporters is found in archaeal and bacterial species. The bioinformatic prediction for subclass II systems suggesting that several diverse S components interact with the same EcfA1A2T module has recently been confirmed experimentally for folate, riboflavin, and thiamine transporters of Bacillus subtilis, Lactobacillus casei, and Leuconostoc mesenteroides and for the hypothetical pantothenate transporter of L. mesenteroides (12).The modular composition of ECF transporters poses questions about their oligomeric structure, the specificity of subunit recognition, and the intersubunit signaling that couples substrate uptake to ATP hydrolysis by the ABC ATPase domains. These issues are essentially unsolved for any ECF transporter.In the present study, we confirm the predicted role of L. mesenteroides PanT (LmPanT) as a pantothenate-specific S component that functionally interacts with the LmEcfA1A2T module. The main focus is on the role of the T components, which are the least-characterized proteins of ECF importers. T proteins have moderately similar primary structures. Two 3-amino-acid signatures with Ala-Arg-Gly as the consensus sequence in the C-terminal part are the most conserved feature in the T units. Using RcBioMNY as a member of subclass I and LmEcfA1A2T plus LmFolT, LmPanT, or LmRibU as representatives of subclass II ECF transporters, we obtained evidence that replacement of either of the Arg residues in the T proteins strongly reduces or abolishes activity of the systems. While double mutations interfered with complex stability in each case, single replacements of the Arg residue in motif I did not have a marked impact on stability of the complexes, suggesting that this residue is important primarily for intramolecular signaling in both subclasses of ECF transporters.     

ENaC, renal sodium excretion and extracellular ATP

Sodium balance determines the extracellular fluid volume and sets arterial blood pressure (BP). Chronically raised BP (hypertension) represents a major health risk in Western societies. The relationship between BP and renal sodium excretion (the pressure/natriuresis relationship) represents the key element in defining the BP homeostatic set point. The renin–angiotensin–aldosterone system (RAAS) makes major adjustments to the rates of renal sodium secretion, but this system works slowly over a period of hours to days. More rapid adjustments can be made by the sympathetic nervous system, although the kidney can function well without sympathetic nerves. Attention has now focussed on regulatory mechanisms within the kidney, including extracellular nucleotides and the P2 receptor system. Here, we discuss how extracellular ATP can control renal sodium excretion by altering the activity of epithelial sodium channels (ENaC) present in the apical membrane of principal cells. There remains considerable controversy over the molecular targets for released ATP, although the P2Y₂ receptor has received much attention. We review the available data and reflect on our own findings in which ATP-activated P2Y and P2X receptors make adjustments to ENaC activity and therefore sodium excretion.     

Coordination of opposing sex-specific and core muscle groups regulates male tail posture during Caenorhabditis elegans male mating behavior

Background  

To survive and reproduce, animals must be able to modify their motor behavior in response to changes in the environment. We studied a complex behavior of Caenorhabditis elegans, male mating behavior, which provided a model for understanding motor behaviors at the genetic, molecular as well as circuit level. C. elegans male mating behavior consists of a series of six sub-steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. The male tail contains most of the sensory structures required for mating, in addition to the copulatory structures, and thus to carry out the steps of mating behavior, the male must keep his tail in contact with the hermaphrodite. However, because the hermaphrodite does not play an active role in mating and continues moving, the male must modify his tail posture to maintain contact. We provide a better understanding of the molecular and neuro-muscular pathways that regulate male tail posture during mating.     

Redox state of glutathione in human plasma

Thiol and disulfide forms of glutathione (GSH) and cysteine (Cys) were measured in plasma from 24 healthy individuals aged 25-35 and redox potential values (E(h)) for thiol/disulfide couples were calculated using the Nernst equation. Although the concentration of GSH (2.8 +/- 0.9 microM) was much greater than that of GSSG (0.14 +/- 0.04 microM), the redox potential of the GSSG/2GSH pool (-137 +/- 9 mV) was considerably more oxidized than values for tissues and cultured cells (-185 to -258 mV). This indicates that a rapid oxidation of GSH occurs upon release into plasma. The difference in values between individuals was remarkably small, suggesting that the rates of reduction and oxidation in the plasma are closely balanced to maintain this redox potential. The redox potential for the Cys and cystine (CySS) pool (-80 +/- 9 mV) was 57 mV more oxidized, showing that the GSSG/2GSH and the CySS/2Cys pools are not in redox equilibrium in the plasma. Potentials for thiol/disulfide couples involving CysGly were intermediate between the values for these couples. Regression analyses showed that the redox potentials for the different thiol/disulfide couples within individuals were correlated, with the E(h) for CySS-mono-Gly/(Cys. CysGly) providing the best correlation with other low molecular weight pools as well as protein disulfides of GSH, CysGly and Cys. These results suggest that E(h) values for GSSG/2GSH and CySS-mono-Gly/(Cys. CysGly) may provide useful means to quantitatively express the oxidant/antioxidant balance in clinical and epidemiologic studies.     

Conservation rules, their breakdown, and optimality in Caenorhabditis sinusoidal locomotion

Undulatory locomotion is common to nematodes as well as to limbless vertebrates, but its control is not understood in spite of the identification of hundred of genes involved in Caenorhabditis elegans locomotion. To reveal the mechanisms of nematode undulatory locomotion, we quantitatively analysed the movement of C. elegans with genetic perturbations to neurons, muscles, and skeleton (cuticle). We also compared locomotion of different Caenorhabditis species. We constructed a theoretical model that combines mechanics and biophysics, and that is constrained by the observations of propulsion and muscular velocities, as well as wavelength and amplitude of undulations. We find that normalized wavelength is a conserved quantity among wild-type C. elegans individuals, across mutants, and across different species. The velocity of forward propulsion scales linearly with the velocity of the muscular wave and the corresponding slope is also a conserved quantity and almost optimal; the exceptions are in some mutants affecting cuticle structure. In theoretical terms, the optimality of the slope is equivalent to the exact balance between muscular and visco-elastic body reaction bending moments. We find that the amplitude and frequency of undulations are inversely correlated and provide a theoretical explanation for this fact. These experimental results are valid both for young adults and for all larval stages of wild-type C. elegans. In particular, during development, the amplitude scales linearly with the wavelength, consistent with our theory. We also investigated the influence of substrate firmness on motion parameters, and found that it does not affect the above invariants. In general, our biomechanical model can explain the observed robustness of the mechanisms controlling nematode undulatory locomotion.     

Genetic labelling and application of the isoproturon-mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere

AIMS: To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory systems. METHODS AND RESULTS: We inserted the mini-Tn5-luxAB marker into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the ability to mineralize isoproturon and was therefore selected for monitoring colonization of barley roots. CONCLUSIONS: We successfully constructed a genetically labelled isoproturon-mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction of a luxAB-labelled strain SRS2 maintaining the degradative ability, provides a powerful tool for ecological studies serving as the basis for evaluating SRS2 as a bioremediation agent.     

Savanna–forest hysteresis in the tropics

A simple dynamic model relating forest area in a region, its contribution to dry season precipitation and the effect on its own establishment was developed. The model equation shows hysteresis between forest and savannas as a function of imported dry season precipitation. Regions are either dominated by forests or savannas, with each ecosystem showing stability despite changes in imported dry season precipitation. Deforestation beyond a certain threshold value, however, could cause a collapse of forest ecosystems and replacement by savannas in marginal areas. The predictions of this model corroborate pollen core analysis in the Amazon basin, where historical stability of tropical forest cover has been shown despite global climate change.     

Developmental Control of CAM in Peperomia scandens

Experiments were conducted to examine the development of photosynthetic carbon metabolism in Peperomia scandens, a tropical epiphyte. Leaves were sampled during a 10-day period when they were between 30 to 165 days old. P. scandens exhibits a C₃ to CAM-cycling to CAM shift during maturation with the magnitude of CAM increasing with age. Initially, during both day and night, no significant CO₂ uptake or diurnal acid flux was evident. C₃ gas exchange was detected at 41 days of age with a gradual shift towards CAM gas exchange maximized thereafter. An acidity flux of 130 to 150 microequivalents per gram fresh weight was evident by 41 days. Between 40 and 90 days, the leaves shifted their CO₂ uptake pattern from a daytime to a nighttime peak. After 90 days, the leaves remained in CAM. The δ¹³C values became progressively less negative as the leaves matured. In the 30-day-old leaves, the δ¹³C value was −21.1% while in the 165-day-old leaves the δ¹³C value was −18.3%. The time-dependent shift from C₃ to CAM-cycling to CAM in P. scandens does not appear to result from changes in water, light, or temperature regimes since these variables were constant for all leaves sampled.