Pyrite Suspended in Artificial Sea Water Catalyzes Hydrolysis of Adsorbed ATP: Enhancing Effect of Acetate

Origins of Life and Evolution of Biospheres -     

Lytic Water Dynamics Reveal Evolutionarily Conserved Mechanisms of ATP Hydrolysis by TIP49 AAA+ ATPases

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Coupling of Proteolysis to ATP Hydrolysis upon Escherichia coliLon Protease Functioning: II. Hydrolysis of ATP and Activity of the Enzyme Peptide Hydrolase Sites

The absence of direct correlation between the efficiency of functioning of ATPase and peptide hydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptide hydrolase sites is provided by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme ATPase sites. It was revealed that the ADP–Mg complex, an inhibitor of the native enzyme, is an activator of the Lon-K362Q (the Lon protease mutant in the ATPase site). Variants of functional contacts between different sites of the enzyme are considered. It was established that two ways of signal transduction from the ATPase sites to peptide hydrolase ones exist in the Lon protease oligomer--intra- and intersubunit ways. The enzyme ATPase sites are suggested to be located in the areas of the complementary surfaces of subunits. It is hypothesized that upon degradation of protein substrates by the E. coliLon protease in vivoATP hydrolysis acts as a factor of limitation of the enzyme degrading activity.     

Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.     

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis.Faithful segregation of low copy number plasmids in bacteria depends on partition loci, named Par. Such loci are composed of two genes, generically termed parA and parB, encoding an ATPase and a DNA-binding protein, respectively, and a cis-acting centromeric site parS (reviewed in Ref. 1). These three essential elements are sufficient for the partition process. ParBs assemble on parS to form nucleoprotein structures called partition complexes (2–6). ParA ATPases are considered to be motors that direct displacement and positioning of partition complexes inside the cell.Partition systems have been classified into two major types, distinguished by the nature of their ATPase proteins (7). Type I is characterized by Walker box ATPases, which are specified by many plasmids and most bacterial chromosomes. In some (Type Ia) the nucleotide-binding P-loop is preceded by an N-terminal regulatory domain, in the others (Type Ib) it is not. Type II specifies actin-like ATPases and is present on relatively few plasmids. It is presently the best understood system at the molecular level (8–10). However, the underlying mechanism that drives partition still remains elusive for both systems. Our work aims at the understanding of an archetypal representative of Type Ia, namely SopABC of the Escherichia coli plasmid F.The several activities of Type Ia ParA proteins are regulated by binding of adenine nucleotides (11, 12), which induce conformational changes in the proteins (13, 14). In their apo and/or ADP-bound forms these proteins display site-specific DNA binding activity, recognizing their cognate promoters through their N-terminal domains. Such activity is involved in the autoregulation of par operon expression (15, 16). In the ATP-bound form, they specifically interact with cognate partition complexes through contact with ParB proteins. The ATP-bound form of type I ParAs spontaneously forms polymers, which appear as bundled filaments in electron micrographs (12, 17–19). The role of these filaments is not understood but they could be related to the rapid movement of partition complexes in the cell. In vivo, ParA proteins form dynamic assemblies that move back and forth in the cell if the cognate ParB protein and parS centromere are present (20–23). The link between this oscillatory behavior and the segregation of partition complexes is not clear. They both require the ATPase activity of ParA proteins but the role of ATP hydrolysis in the partition process is not understood.It has long been known that ParA partition proteins exhibit low intrinsic ATPase activity (24, 25). ATP hydrolysis is modestly stimulated by either DNA or the cognate ParB alone but is strongly activated (up to 35-fold) when both DNA and ParBs are present (12, 24, 25). The lack of major stimulation of ATPase by DNA in the absence of ParB proteins has been taken to mean that the DNA-bound form of ParB is the effective activator (26). However, incorporation of centromere sites in the DNA added to ParB did not increase stimulation of ATPase (24, 25), leaving doubts as to the role of the partition complex in ATPase activation.The mechanism by which ATP hydrolysis acts in the partition process is not known for type I systems. This is in marked contrast to actin-based partition ATPases whose ATPase activity is stimulated in growing filaments (8), where it provokes the rapid disassembly of filaments unless these are capped by the cognate partition complex (9). Therefore, for the type II partition system, ATP hydrolysis ensures discrimination between unproductive filaments that are rapidly disassembled and productive filaments that drive partition complexes to opposite ends of the cell. This dynamic instability, which ensures elongation of actin-like filaments only between two partition complexes to be segregated, thus provides regulation of the partition process.Recently, it has been shown that two members of the type I ParA family, Soj of Thermus thermophilus and SopA of plasmid F, bind nonspecific DNA in the presence of ATP (12, 26). Two studies revealed that this DNA binding activity is essential for partition (27, 28). Importantly, it has been shown that a SopA mutant deficient in DNA binding no longer stimulates ATP hydrolysis efficiently, suggesting that DNA could play a direct role in the regulation of the ATPase activity (28). This finding raises the issue of the interactions required for activation of the type I partition ATPase activity by cognate proteins and DNA.In this study, we have investigated the mechanism of activation of ATP hydrolysis by SopA. First, we have found that the formation of the F partition complex is required for strong stimulation of the SopA intrinsic ATPase activity. We have also found that the partition complex and DNA stimulate ATP hydrolysis independently but that these two independent interactions act in synergy to amplify SopA ATPase activity. Lastly, we have identified an arginine finger motif in SopB responsible for the stimulation of SopA ATPase activity.     

Coordination of Substrate Binding and ATP Hydrolysis in Vps4-Mediated ESCRT-III Disassembly

ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.     

Orientation and Mobility of Actin in Different Intermediate States of the ATP Hydrolysis Cycle

Using polarization fluorimetry, we have investigated conformational changes of FITC-phalloidin-labeled F-actin in ghost muscle fibers. These changes were induced by myosin subfragment-1 (S1) in the absence and presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes of orientation and mobility of actin monomers during the ATPase cycle. The most pronounced differences in orientation (~4 degrees ) and in mobility (~43%) of actin were found between the actomyosin states induced by MgADP and MgATP.     

Effect of Geometrical and Chemical Constraints on Water Flux across Artificial Membranes

Studies have been made on the temperature dependence of both the hydraulic conductivity, L_p, and the THO diffusion coefficient, ω, for a series of cellulose acetate membranes (CA) of varying porosity. A similar study was also made of a much less polar cellulose triacetate membrane (CTA). The apparent activation energies, E_a, for diffusion across CA membranes vary with porosity, being 7.8 kcal/mole for the nonporous membrane and 5.5 kcal/mole for the most porous one. E_a for diffusion across the less polar CTA membrane is smaller than E_a for the CA membrane of equivalent porosity. Classical viscous flow, in which the hydraulic conductivity is inversely related to bulk water viscosity, has been demonstrated across membranes with very small equivalent pores. Water-membrane interactions, which depend upon both chemical and geometrical factors are of particular importance in diffusion. The implication of these findings for the interpretation of water permeability experiments across biological membranes is discussed.     

Magnetic-Isotope Effects of Magnesium and Zinc in Enzymatic ATP Hydrolysis Driven by Molecular Motors

Biophysics - The effects of different magnesium and zinc isotopes on the enzyme activity of myosin subfragment-1 have been explored. The rate of the enzymatic ATP hydrolysis in reaction media...     

Development of Sea Urchin Embryos in Artificial Sea Water Containing Br−in Place of Cl−

In artificial sea water in which the Cl⁻concentration was reduced to less than 10% of that in normal sea water by its replacement with Br⁻, sea urchin eggs were fertilized and developed into abnormal plutei following almost the same time schedule as in natural sea water. These embryos had poorly developed spicules, short pluteus arms, somewhat jagged embryo-walls and quasi-normal archenterons. Similar embryos were obtained in another artificial sea water in which 90% of the Cl⁻concentration in normal sea water was reduced by Br⁻and 10% by acetate. In artificial sea water, in which either 90% of the Cl⁻was replaced by Br⁻or 10% was replaced by acetate, embryos developed into plutei with quasi-normal spicules, pluteus arms and archenterons. These findings indicate that deficiency of Cl⁻results in somewhat abnormal sea urchin embryos. When cells derived from isolated micromeres, were cultured in these Cl⁻-deficient artificial sea waters, containing Br⁻in place of more than 70% of the normal Cl⁻concentration in sea water, spicule formation was strongly inhibited, but pseudopodial cables were well developed. Thus, external Cl⁻seems to be necessary for at least normal formation of spicule rods.     

Effect of Some Unicellular Algae on Escherichia coli Populations in Sea Water and Oysters

Acrylic acid was detected in sea water in which oysters had been maintained and was shown to be derived from unicellular algae containing dimethyl-β-propiothetin (DMPT). Anticoliform activity was noted in samples containing acrylic acid. Intensification of the effect was achieved in laboratory studies and Phaeodactylum tricornutum (DMPT present) caused a rapid decline of Escherichia coli populations in sea water and in oysters which had been fed this alga. Pavlova lutheri (DMPT absent) was ineffective against E. coli .     

Evaluation of Factors Affecting Survival of Escherichia coli in Sea Water: V. Studies with Heat- and Filter-sterilized Sea Water1

The bactericidal action of sea water was measured as the difference in survival of cells of Escherichia coli in untreated and autoclaved portions of water samples. The beneficial effect of sterilization by heat on the survival of E. coli in sea water varied with season and was most marked during summer months, however, the magnitude of the effect differed greatly from sample to sample. The more obvious and commonly suggested explanations for the bactericidal action of sea water were tested experimentally. The pH and salinity of sea water were changed by autoclaving, but the direction of the former was detrimental rather than beneficial and the significance of the latter was not clarified. The survival of cells of E. coli in filtered portions of some water samples was greater than that in untreated portions and equal to that in autoclaved portions, indicating that predators and competitors removed by filtration had contributed significantly to the rapid death of the bacterium in the untreated water. However, in the majority of samples tested, survival of E. coli in autoclaved water was considerably greater than survival in filtered water.

The possibility that the beneficial effect of autoclaving over and above that of filtration resulted from inactivation or destruction by heat of bacteriophages and thermolabile toxic substances such as antibiotics was considered. Moreover, the suggestion was tested that the increased survival of E. coli in autoclaved sea water was due to the ability of heat to disrupt and degrade microbial cells and thermolabile compounds and, thereby, to cause an increase in concentration of available nutrients in sea water. It was concluded that the bactericidal action of sea water is not explicable in terms of the destruction or inactivation by heat of bacteriophages or antibiotics. Although added organic matter influenced the survival of E. coli, the test organism was not an effective competitor in sea water and the nutrient levels required to offset the bactericidal action were excessive.

Artificial sea water was demonstrated to exert a bactericidal action comparable to that of natural sea water. Low levels of cysteine which favor survival of E. coli in natural sea water had a similar effect in artificial sea water. Nevertheless, it is not at this time possible to conclude that the factors responsible for the bactericidal action of artificial sea water are identical with those responsible in natural sea water.

     

Artificial Regulation of Water Level and Its Effect on Aquatic Macrophyte Distribution in Taihu Lake

Management of water levels for flood control, water quality, and water safety purposes has become a priority for many lakes worldwide. However, the effects of water level management on the distribution and composition of aquatic vegetation has received little attention. Relevant studies have used either limited short-term or discrete long-term data and thus are either narrowly applicable or easily confounded by the effects of other environmental factors. We developed classification tree models using ground surveys combined with 52 remotely sensed images (15–30 m resolution) to map the distributions of two groups of aquatic vegetation in Taihu Lake, China from 1989–2010. Type 1 vegetation included emergent, floating, and floating-leaf plants, whereas Type 2 consisted of submerged vegetation. We sought to identify both inter- and intra-annual dynamics of water level and corresponding dynamics in the aquatic vegetation. Water levels in the ten-year period from 2000–2010 were 0.06–0.21 m lower from July to September (wet season) and 0.22–0.27 m higher from December to March (dry season) than in the 1989–1999 period. Average intra-annual variation (CV_a) decreased from 10.21% in 1989–1999 to 5.41% in 2000–2010. The areas of both Type 1 and Type 2 vegetation increased substantially in 2000–2010 relative to 1989–1999. Neither annual average water level nor CV_a influenced aquatic vegetation area, but water level from January to March had significant positive and negative correlations, respectively, with areas of Type 1 and Type 2 vegetation. Our findings revealed problems with the current management of water levels in Taihu Lake. To restore Taihu Lake to its original state of submerged vegetation dominance, water levels in the dry season should be lowered to better approximate natural conditions and reinstate the high variability (i.e., greater extremes) that was present historically.