Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure

The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUalpha(2) and EcHUbeta(2), and a heterodimer, EcHUalphabeta. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E.coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N(2)<-->2D), this present thermodynamic study shows that the three E.coli dimers melt according to a two-step mechanism (N(2)<-->I(2)<-->2D). The native dimer, N(2), melts partially into a dimeric intermediate, I(2), which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUalpha(2) dimer has been solved. Comparative thermodynamic and structural analysis between EcHUalpha(2) and the HU homodimer from Bacillus stearothermophilus suggests that the E.coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I(2), corresponds to an HU dimer having partly lost its alpha-helices. The partially unfolded dimer I(2) is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N(2)<-->I(2) equilibrium plays a central role in the physiology of E.coli HU. The I(2) molecular species seems to be the EcHUbeta(2) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation. Besides, I(2) might be required in E.coli for the HU chain exchange, which allows the heterodimer formation from homodimers.     

Expression and regulation of pOb24 and lipoprotein lipase genes during adipose conversion

Lipoprotein lipase (LPL) and pOb24 mRNAs are known to be early markers of adipose cell differentiation. Comparative studies of the expression of pOb24 and LPL genes during adipose conversion of Ob1771 preadipocyte cells and in mouse adipose tissue have shown the following: 1) the expression of both genes takes place at confluence; this event can also be triggered by growth arrest of exponentially growing cells at the G1/S stage of the cell cycle; 2) In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 and lipoprotein lipase mRNAs requires neither growth hormone or tri-iodothyronine as obligatory hormones nor insulin as a modulating hormone; 3) in mouse adipose tissue, pOb24 mRNA is present at a high level in stromal-vascular cells and at a low level in mature adipocytes, and in contrast LPL mRNAs are preferentially expressed in mature adipocytes. Thus, these two genes do not appear to be regulated in a similar manner, as also shown by the differential inhibition of their expression by tumor necrosis factor (TNF) and transforming growth factor-beta (TGF-beta).     

Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-beta-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-beta-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h on glucose, 0.13 h on xylose, and 0.10 h on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g h on arabinose to 0.11 g g h on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of beta-hydroxybutyric and beta-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter. The beta-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter.     

A 2H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate ²H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. ²H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences.     

Calcium-induced gelation of low methoxy pectin solutions--thermodynamic and rheological considerations

Rheological changes occurring during the gelation of low methoxy pectins in the presence of calcium ions were investigated. The onset of gelation was found to correspond to the same macroscopic rheology (i.e. value of G') whatever the gelling conditions. Sol-gel diagrams for calcium pectin systems were mapped out in terms of calcium level, temperature and pectin concentration. The thermodynamic parameters for the cross-linking process were derived.     

Influence of availability of perches on the behavioral well-being of captive,group-living mangabeys

Environmental enrichment is expected to increase the well-being of animals. Changes in well-being can be measured by variations in behavioral patterns. This study reports on behavioral changes induced, in arboreal monkeys, by progressively increasing the number of perches, from none to five, in an “experimental cage.” A cage equipped with five perches was used as the control cage. The behaviors of a group of seven gray-cheeked mangabeys in the control cage and in the “experimental” cage were compared. A total deprivation of perches yielded an increase in aggressive behaviors and locomotion, and a decrease in cohesiveness. Placing perches progressively in the experimental cage restored the level of all the variables to levels found in the control cage. This restoration to control levels actually occurred only when the number of perches in the experimental cage was close or equal to that in the control cage. Therefore perches constitute a necessary feature of an adequate environment for mangabeys. We suggest that this restoration is a consequence of providing appropriate structure of the utilizable space for the monkeys. This structure might increase the control and the predictability that monkeys should have over social events. © 1996 Wiley-Liss, Inc.     

Structure of the human glycogen-associated protein phosphatase 1 regulatory subunit hGM: homology modeling revealed an (alpha/beta)8-barrel-like fold in the multidomain protein

Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.     

Type III secretion in plant growth-promoting Pseudomonas fluorescens SBW25

In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20-kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS-secreted protein AvrE. P. fluorescens SBW25 is non-pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp-specific sigma factor RspL elicit an AvrB-dependent HR in Arabidopsis thaliana ecotype Col-0, and a host-specific HR in Nicotiana clevelandii. The inability of wild-type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant-colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere-expressed TTSS of P. fluorescens SBW25 remains unclear.     

Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases

(1) Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [¹⁴C]arachidonate/[³H]arachidonoylphosphatidylcholine or free [¹⁴C]oleate/[³H]oleoylphosphatidylcholine. Whereas [¹⁴C]arachidonate was incorporated at a 10–15 times higher rate than [¹⁴C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free ³H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A₂ hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [³H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [¹⁴C]choline-labelled dipalmitoyl- and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.