Interactions between bacterial membranes and peptidolipids: lysis of micrococcus luteus protoplasts by derivatives of peptidolipidic antibiotics from bacillus subtilis.

The lysis of protoplasts of Micrococcus luteus has been tested with various derivatives of three peptidolipidic antibiotics: iturin A, mycosubtilin and bacillomycin L. The lytic activity is dependent to the nature of the substituting group and to the position of the substituted aminoacid residue. The acetylation of OH groups leads to a decrease of the lytic activity of the natural antibiotics. The methylation of aspartyl residues of bacillomycin L gives a strong lytic activity while natural bacillomycin L has no lytic activity. The methylation of the tyrosyl residue enhances the lytic activities of iturin A and of bacillomycin L-dimethyl ester and reduces that of mycosubtilin. Correlations between the structures of derivatives and their lytic action on M. luteus protoplasts are discussed.     

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.     

Interactions between model membranes and lignin-related compounds studied by immobilized liposome chromatography

In order to elucidate the modes of interaction between lignin precursors and membranes, we have studied the influence of temperature, lipid composition and buffer composition on the partitioning of monolignol and dilignol model substances into phospholipid bilayers. The partitioning was determined by immobilized liposome chromatography, which is an established method for studies of pharmaceutical drugs but a new approach in studies of lignin synthesis. The temperature dependence of the retention and the effect of a high ammonium sulfate concentration in the mobile phase demonstrated that the interaction involved both hydrophobic effects and polar interactions. There was also a good correlation between the partitioning and the estimated hydrophobicity, in terms of octanol/water partitioning. The partitioning behavior of the model substances suggests that passive diffusion over the cell membrane is a possible transport route for lignin precursors. This conclusion is strengthened by comparison of the present results with the partitioning of pharmaceutical drugs that are known to pass cell membranes by diffusion.     

Zeta potential of protoplasts from gravireacting maize roots

Protoplasts were isolated from cortical cells of the elongating zone of maize (Zea mays L. cv. LG 11) roots and submitted to microelectrophoresis. Significant and transient differences in zeta potential between protoplasts from upper and lower root sides were compared with the gravireaction and the differential elongation of these roots. The maximum difference in the zeta potential was obtained between protoplasts from the upper and lower cortical cells after 90 min, exactly the time of gravipresentation for which the maximum rate of gravireaction was observed. In addition, this almost corresponded to the time for which the difference between the elongation rates of upper and lower sides of the extending zone began to increase. Consequently, the changes in the charges of the plasmalemma of the cortical cells from the growing part of roots could be more or less directly related to the root graviresponse.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

NMR field-cycling relaxation spectroscopy of bovine serum albumin,muscle tissue,micrococcus luteus and yeast: 14N1H-quadrupole dips

The frequency dependence of the proton spin lattice relaxation time of bovine serum albumin, muscle tissue, Micrococcus luteus and yeast has been measured by the aid of the field-cycling technique. In all systems ¹⁴N¹H-quadrupole dips have been observed. The conclusion is that amide groups are the dominating relaxation centers up to approx. 10⁷Hz. This finding can be understood by the fact that protein backbone fluctuations and, if possible, tumbling of the whole molecule rather than side group motions are the relevant mechanisms in this frequency range. A proton relaxation scheme for cells and tissue is presented.     

Conformational aspects of drug-DNA interactions: Studies on anthracycline antibiotics and psoralen derivatives

The interaction of anticancer agents, analogues of adriamycin and of photo-chemotherapeutic compounds of the psoralen structural type with DNA was investigated using spectroscopic, hydrodynamic and chiroptical techniques. The nucleic acid may undergo conformational changes from the B form to more compact structures as a result of the binding process to charged compounds. Different complex geometries adopted byvarious drugs were observed and discussed in terms of intercalation into the polynucleotide double helix and orientation of the ligand in the base-pair pocket. The binding of chemotherapeutic agents to functionally organized DNA was also studied. Lower binding affinities and modified spectral responsesareindicativeofdifferent drug-DNA complexation patternsinthiscase. The results of these studies allow a better understanding of drug-nucleic acid interactions at a molecular level.     

Purification and Characterization of Novel Transglutaminase from Bacillus subtilis Spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as α_s-casein and BSA.     

Bioreduction of Cr(VI) by alkaliphilic Bacillus subtilis and interaction of the membrane groups

Detoxification of Cr(VI) under alkaline pH requires attention due to the alkaline nature of many effluents. An alkaliphilic gram-positive Bacillus subtilis isolated from tannery effluent contaminated soil was found to grow and reduce Cr(VI) up to 100% at an alkaline pH 9. Decrease in pH to acidic range with growth of the bacterium signified the role played by metabolites (organic acids) in chromium resistance and reduction mechanism. The XPS and FT-IR spectra confirmed the reduction of Cr(VI) by bacteria into +3 oxidation state. Chromate reductase assay indicated that the reduction was mediated by constitutive membrane bound enzymes. The kinetics of Cr(VI) reduction activity derived using the monod equation proved (K_s = 0.00032) high affinity of the organism to the metal. This study thus helped to localize the reduction activity at subcellular level in a chromium resistant alkaliphilic Bacillus sp.     

Regulation of blue-light-induced proton pumping by Vicia faba L. guard-cell protoplasts: Energetic contributions by chloroplastic and mitochondrial activities

An initial response during signal transduction in guard cells, following absorption of blue light, is the extrusion of protons. Translocation of protons across the guard-cell plasmalemma is an energy-requiring activity. The present study has investigated the energetic contribution from guard-cell chloroplasts and mitochondria to blue-light-induced proton pumping by Vicia faba guard-cell protoplasts. The addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea to the protoplast suspension had a minimal effect on rates of acidification when oxygen concentrations of the medium were maintained close to near-saturating levels. Under the same conditions, oligomycin reduced both the rates of blue-light-induced acidification and total proton efflux. Lowering the oxygen concentration of the suspending medium to approximately 20 M resulted in complete inhibition of blue-light-induced acidification activity. Swelling of protoplasts induced by blue light was also inhibited by low oxygen levels. Levels of ATP from whole-protoplast extracts were reduced by about 64% when exposed to low levels of oxygen. Increasing oxygen levels to near-saturating levels restored both blue-light-induced acidification rates and swelling of the protoplasts within a 60-min recovery period. Levels of ATP also increased during the recovery period. Addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or oligomycin to the suspending medium prior to increasing the oxygen concentration caused a reduction in acidification rates after the recovery period by 40 and 80%, respectively. Levels of ATP in guard-cell protoplasts were also reduced by both inhibitors after a 60-min recovery period. The results demonstrate that both guard-cell chloroplasts and mitochondria contribute energetically to blue-light-induced proton pumping by guard-cell protoplasts. Furthermore, both energy sources are inhibited by low oxygen concentrations, suggesting coordinated metabolic regulation between photo- and oxidative phosphorylation in guard cells.Abbreviations BL blue light - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - GCPs guard-cell protoplasts This research was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada and a University Research Grant from The University of Calgary. Dr. L. Gedamu (University of Calgary) is thanked for providing access to the bioluminometer. Technical assistance by C. Chmielewski, C. Turnnir, S. Ham and K. Meyer is gratefully acknowledged.     

Modulation of photosynthesis and respiration in guard and mesophyll cell protoplasts by oxygen concentration

Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O₂ mg^?1 chl h^?1, while guard cell protoplasts respired at rates of 200–300 μmoles O₂ mg chl^?1 h^?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m^?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents.     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

Interaction between the delta-endotoxin produced by Bacillus thuringiensis ssp. entomocidus and liposomes

The δ-endotoxin produced by Bacillus thuringiensis ssp. entomocidus induced the release of encapsulated [¹⁴C]sucrose from reverse-phase vesicles composed of phosphatidylcholine and cholesterol. No such release was detected when the phospholipid component of the vesicles was either phosphatidylethanolamine, phosphatidylglycerol, or sphingomyelin. The toxin-induced release was competitively inhibited by negatively charged organic ions while positively charged organic ions, apart from choline chloride, had no such effect. The existence of a polar head group in the phospholipid as well as intermolecular hydrogen bonding at the membrane surface, was found to be of major importance in the toxin-liposome interaction.     

Cytotoxicity of mitochondria-targeted resveratrol derivatives: Interactions with respiratory chain complexes and ATP synthase

We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low μM range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H₂O₂ produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. “Capping” free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca^2 +] “spikes” in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F₀F₁ ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore.     

Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21(WAF1/Cip1), p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.     

The redox levels and subcellular distribution of pyridine nucleotides in illuminated barley leaf protoplasts studied by rapid fractionation

The redox level and compartmentation of pyridine nucleotides was studied under photorespiratory and non-photorespiratory conditions using rapid fractionation of barley ( Hordeum vulgare L. cv. Gunilla, Svalöv) leaf protoplasts. From comparative measurements of the NADPH/NADP⁺ ratio and the ATP/ADP ratio one acidic and one alkaline extraction medium was chosen which quenched the metabolism very efficiently. The mitochondrial NADH/NAD⁺ was higher under photorespiratory conditions than under non-photorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, lowered the mitochondrial NADH/NAD⁺ ratio. This supports the hypothesis that glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol. The chloroplastic NADPH/NADP⁺ as well as the NADH/NAD⁺ ratios were quite stable in saturating and limiting CO₂ as well as in the presence of aminoacetonitrile, although the triosephosphate/phosphoglycerate ratios changed. Thus, the redox level in the stroma seems to be tightly regulated.     

Identification of the catalytic nucleophile in Family 42 beta-galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis

The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 beta-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.