The unbalance of membrane phospholipid composition of Escherichia coli affects the PPHO promoter activity

Activity of phosphate PPHO or arabinose PBAD promoter of Escherichia coli has been studied depending on the content of zwitter-ionic phosphatidylethanolamine (PE) and anionic phospholipids in membranes. In the absence of PE or under significant decrease in the content of anionic phospholipids, there is a significant decline of PPHO promoter activity but not PBAD promoter. Since the PPHO promoter belongs to the Pho-regulon--a member of the family of two-component regulatory systems of signal transduction having membrane sensors, the regulation of gene expression by phospholipids is presumed to be realized through a membrane sensor.     

Growth,photosynthesis, and metabolism of sugar beet at an early stage of exposure to elevated CO<Subscript>2</Subscript>

The effects of CO₂ concentration (C _a) on growth, photosynthesis, and the activity of enzymes associated with the translocation and assimilation of CO₂ were studied in sugar beet (Beta vulgaris L. subsp. saccharifera, cv. Ramonskaya) plants. The plants were grown in controlled-climate chamber to the stage of 3–4 leaves and then used in experiments. Experimental plants were exposed in boxes to doubled C _a (700 µl/l, 2C plants), whereas control plants were kept in a chamber with ambient atmosphere (350 µl/l, 1C plants). As compared with 1C plants, in 3 and 8 days, the leaf area of 2C plants increased by 14 and 26%, respectively. The rate of their photosynthesis (P _n) measured in 3, 6, and 8 days increased by 85, 47, and 52%, respectively, whereas in normal air, the values of P _n in 2C plants were by 12, 19, and 15% lower than in 1C plants. After 8-day growth, the content of soluble carbohydrates in the leaves of 2C plants attained 7.2%, being by 80% greater than in 1C plants; the content of starch did not exceed 3%. The total content of chlorophylls a and b in the leaves of 2C plants was by 14% greater than in 1C plants, but their ratio was essentially the same. The level of protein in 2C plants was by 13.4% lower than in 1C plants. The activity and content of Rubisco in 1C and 2C plants were similar. As compared with 1C plants, in 2C plants the activity of soluble carbonic anhydrase (sCA) was lower by 34% in 3 days and by 18% in 8 days; the activity of carbonic anhydrase of membrane preparations (mCA), was lower by 24 and 77%, respectively. Catalase activity in 2C plants became by 8% lower than in 1C plants only after 8 days. A reduction in the photosynthetic ability of 2C plants in ambient atmosphere, a decrease in activity of sCA and, especially, of mCA observed together with invariable activity and content of Rubisco in the leaf extracts are interpreted as early symptoms of acclimation of young plants of sugar beet to elevated CO₂.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 184–190.Original Russian Text Copyright © 2005 by Ignatova, Novichkova, Mudrik, Lyubimov, Ivanov, Romanova.This revised version was published online in April 2005 with a corrected cover date.     

Effect of<Emphasis Type="Italic"> p</Emphasis>CO<Subscript>2</Subscript> and temperature on the boron isotopic composition of the zooxanthellate coral<Emphasis Type="Italic"> Acropora</Emphasis> sp.

Culture experiments were carried out with Acropora sp. (a branching scleractinian coral) in seawater at two pCO₂ conditions (438 and 725 µatm) and two temperatures (25 and 28 °C) in order to establish the pH and temperature dependence of the boron isotopic composition of the skeleton. A clear pCO₂ effect, but no temperature effect, on the coral boron isotope composition is seen. For corals cultured at normal pCO₂ (438 µatm), the ¹¹B of the skeleton was 24.0±0.2 at 25 °C, and 23.9±0.3 at 28 °C. The values of ¹¹B measured for corals cultured at higher pCO₂ (725 µatm) were lower: 22.5±0.1, and 22.8±0.1 at 25 and 28 °C, respectively. The ¹¹B of corals cultivated at both high and normal pCO₂ conditions are consistent with a dominant pH control, and are very close to that calculated from theoretical considerations. Thus, the corals do not seem to significantly alter ambient seawater for calcification with respect to pH. Co-variation between boron and carbon isotope values is explored.Communicated by: Guest Editor A. Grottoli     

Exercise and recovery metabolism in the pacific spiny dogfish (<Emphasis Type="Italic">Squalus acanthias</Emphasis>)

We examined the effects of exhaustive exercise and post-exercise recovery on white muscle substrate depletion and metabolite distribution between white muscle and blood plasma in the Pacific spiny dogfish, both in vivo and in an electrically stimulated perfused tail-trunk preparation. Measurements of arterial-venous lactate, total ammonia, -hydroxybutyrate, glucose, and l-alanine concentrations in the perfused tail-trunk assessed white muscle metabolite fluxes. Exhaustive exercise was fuelled primarily by creatine phosphate hydrolysis and glycolysis as indicated by 62, 71, and 85% decreases in ATP, creatine phosphate, and glycogen, respectively. White muscle lactate production during exercise caused a sustained increase (~12 h post-exercise) in plasma lactate load and a short-lived increase (~4 h post-exercise) in plasma metabolic acid load during recovery. Exhaustive exercise and recovery did not affect arterial PO₂, PCO₂, or PNH₃ but the metabolic acidosis caused a decrease in arterial HCO₃^– immediately after exercise and during the first 8 h recovery. During recovery, lactate was retained in the white muscle at higher concentrations than in the plasma despite increased lactate efflux from the muscle. Pyruvate dehydrogenase activity was very low in dogfish white muscle at rest and during recovery (0.53±0.15 nmol g wet tissue^–1 min^–1; n=40) indicating that lactate oxidation is not the major fate of lactate during post-exercise recovery. The lack of change in white muscle free-carnitine and variable changes in short-chain fatty acyl-carnitine suggest that dogfish white muscle does not rely on lipid oxidation to fuel exhaustive exercise or recovery. These findings support the notion that extrahepatic tissues cannot utilize fatty acids as an oxidative fuel. Furthermore, our data strongly suggest that ketone body oxidation is important in fuelling recovery metabolism in dogfish white muscle and at least 20% of the ATP required for recovery could be supplied by uptake and oxidation of -hydroxybutyrate from the plasma.Abbreviations CoA-SH free coenzyme A - CPT-1 carnitine palmitoyltransferase-1 - CrP creatine phosphate - H⁺_m metabolic proton load - Lac lactate load - PDH pyruvate dehydrogenase - PVP polyvinylpyrrolidone - SCFA-carnitine short-chain fatty acyl-carnitine - TAG triacylglycerol - TENS trancutaneous electrical nerve stimulator Communicated by: L.C.-H. Wang     

Ligand Binding Characteristics of the Human Serotonin<Subscript>1<Emphasis Type="Italic">A</Emphasis></Subscript> Receptor Heterologously Expressed in CHO Cells

The serotonin_1A (5-HT_1A) receptors are important members of the superfamily of seven transmembrane domain G-protein coupled receptors. They appear to be involved in various behavioral, cognitive and developmental functions. Mammalian cells in culture heterologously expressing membrane receptors represent convenient systems to address problems in receptor biology. We report here the pharmacological characterization of one of the first isolated clones of CHO cells stably expressing the human 5-HT_1A receptor using the selective agonist 8-OH-DPAT and antagonist p-MPPF. In addition, we demonstrate that agonist and antagonist binding to the receptor exhibit differential sensitivity to the non-hydrolyzable GTP analogue, GTP--S, as was observed earlier with the native receptor from bovine hippocampus. These results show that the human 5-HT_1A receptor expressed in CHO cells displays characteristic features found in the native receptor isolated from bovine hippocampus and promises to be a potentially useful system for future studies of the receptor.These authors have contributed equally to the work     

Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli

Aim: To develop an approach to enhance polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose P_BAD promoter in Escherichia coli. Method and Results: The polyhydroxyalkanoates (PHAs) synthesis operon, (phaCAB), from Ralstonia eutropha was overexpressed under the regulation of the arabinose P_BAD promoter in Escherichia coli, and the vgb gene encoding bacterial haemoglobin from Vitreoscilla stercoraria (VHb) was further cloned at downstream of phaCAB to form an artificial operon. The cell dry weight (CDW), PHB content and PHB concentration were enhanced around 1·23‐, 1·57‐, and 1·93‐fold in the engineered cell harbouring phaCAB–vgb (SY‐2) upon 1% arabinose induction compared with noninduction (0% arabinose). Furthermore, by using a recombinant strain harbouring P_BAD promoter‐vgb along with native promoter‐phaCAB construction, the effect of vgb expression level on PHB biosynthesis was positive correlation. Conclusions: The results exploit the possibility to improve the PHB production by fusing the genes phaCAB–vgb from different species under the arabinose regulation system in E. coli. It also demonstrates that increase in VHb level enhances the PHB production. Significance and Impact of the Study: We were successful in providing a new coexpressed system for PHB synthesis in E. coli. This coexpressed system could be regulated by arabinose inducer, and is more stable and cheaper than other induced systems (e.g. IPTG). Furthermore, it could be applied in many biotechnology or fermentation processes.     

Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of <Emphasis Type="Italic">Penaeus monodon</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the -factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.     

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the P_fr/P ratio present at the beginning of the assay, such that far-red was more efficient at high P_fr/P and red at low P_fr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe Fluram (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.Abbreviations F far-red light - P_r and P_fr phytochrome in the red and far-red absorbing forms - P_tot total phytochrome - R red light - RNP ribonucleoprotein     

Modulation of gene expression from the arabinose-inducible araBAD promoter

The arabinose-inducible P_BAD promoter suffers from all-or-none gene expression in which cells harboring the natively controlled arabinose transport gene (araE) are either induced or uninduced, the relative fraction of which is controlled by the concentration of arabinose. The population-averaged variation in expression from P_BAD as a function of inducer concentration is proportional to the percentage of cells that are fully induced (vs. uninduced) rather than the level of expression in individual cells. Because of its undesirable effects on the expression of heterologous genes, the all-or-none phenomenon was eliminated in Escherichia coli by expression of araE from arabinose-independent (either the Lactococcus lactis constitutive or IPTG-inducible lac) promoters. In these arabinose-transport engineered cells, variation in P_BAD expression with arabinose concentration was a result of variation of the expression level in individual cells with all cells in the population having approximately the same induction level. Journal of Industrial Microbiology & Biotechnology (2002) 29, 34–37 doi:10.1038/sj.jim.7000259 Received 20 November 2001/ Accepted in revised form 29 March 2002     

An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of TnΩP_BAD to place the highly regulable, arabinose inducible P_BAD promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P_BAD promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.     

Experimental and theoretical study on high temperature induced changes in chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence oscillations in barley leaves upon 2 % CO<Subscript>2</Subscript>

Oscillations in many of photosynthetic quantities with a period of about 1 min can be routinely measured with higher plant leaves after perturbation of the steady state by sudden change in gas phase. Among all hypotheses suggested so far to explain the oscillations, an effect of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activation status to control the oscillations is highly probable, at least upon high temperature (HT) treatment when in vivo RuBPCO activity controlled by RuBPCO activase (RuBPCO-A) decreases. Therefore, we measured the oscillations in fluorescence signal coming from barley leaves (Hordeum vulgare L. cv. Akcent) after their exposure for various time intervals to different HTs in darkness. We also evaluated steady state fluorescence and CO₂ exchange parameters to have an insight to functions of electron transport chain within thylakoid membrane and Calvin cycle before initiation of the oscillations. The changes in period of the oscillations induced by moderate HT (up to 43 °C) best correlated with changes in non-photochemical fluorescence quenching (q_N) that in turn correlated with changes in gross photosynthetic rate (P _G) and rate of RuBPCO activation (k_act). Therefore, we suggest that changes in period of the oscillations caused by moderate HT are mainly controlled by RuBPCO activation status. For more severe HT (45 °C), the oscillations disappeared which was probably caused by an insufficient formation of NADPH by electron transport chain within thylakoid membrane as judged from a decrease in photochemical fluorescence quenching (q_P). Suggestions made on the basis of experimental data were verified by theoretical simulations of the oscillations based on a model of Calvin cycle and by means of a control analysis of the model.     

Effects of NaCl or Na<Subscript>2</Subscript>SO<Subscript>4</Subscript> salinity on plant growth,ion content and photosynthetic activity in <Emphasis Type="Italic">Ocimum basilicum</Emphasis> L.

Basil (Ocimum basilicum L., cultivar Genovese) plants were grown in Hoagland solution with or without 50 mM NaCl or 25 mM Na₂SO₄. After 15 days of treatment, Na₂SO₄ slowed growth of plants as indicated by root, stem and leaf dry weight, root length, shoot height and leaf area, and the effects were major of those induced by NaCl. Photosynthetic response was decreased more by chloride salinity than by sulphate. No effects in both treatments on leaf chlorophyll content, maximal efficiency of PSII photochemistry (F _v/F _m) and electron transport rate (ETR) were recorded. Therefore, an excess of energy following the limitation to CO₂ photoassimilation and a down regulation of PSII photochemistry was monitored under NaCl, which displays mechanisms that play a role in avoiding PSII photodamage able to dissipate this excess energy. Ionic composition (Na⁺, K⁺, Ca²⁺, and Mg²⁺) was affected to the same extent under both types of salinity, thus together with an increase in leaves Cl⁻, and roots SO₄ ²⁻ in NaCl and Na₂SO₄-treated plants, respectively, may have resulted in the observed growth retardation (for Na₂SO₄ treatment) and photosynthesis activity inhibition (for NaCl treatment), suggesting that those effects seem to have been due to the anionic component of the salts.     

Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4

Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.     

Pre-treatment with H<Subscript>2</Subscript>O<Subscript>2</Subscript> induces salt tolerance in Barley seedlings

Barley seedlings were pre-treated with 1 and 5 μM H₂O₂ for 2 d and then supplied with water or 150 mM NaCl for 4 and 7 d. Exogenous H₂O₂ alone had no effect on the proline, malondialdehyde (MDA) and H₂O₂ contents, decreased catalase (CAT) activity and had no effect on peroxidase (POX) activity. Three new superoxide dismutase (SOD) isoenzymes appeared in the leaves as a result of 1 μM H₂O₂ treatment. NaCl enhanced CAT and POX activity. SOD activity and isoenzyme patterns were changed due to H₂O₂ pre-treatment, NaCl stress and leaf ageing. In pre-treated seedlings the rate of ¹⁴CO₂ fixation was higher and MDA, H₂O₂ and proline contents were lower in comparison to the seedlings subjected directly to NaCl stress. Cl⁻ content in the leaves 4 and 7 d after NaCl supply increased considerably, but less in pre-treated plants. It was suggested that H₂O₂ metabolism is involved as a signal in the processes of barley salt tolerance.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H₂O₂ on cytosolic as well as mitochondrial calcium (Ca²⁺) concentrations, mitochondrial inner membrane potential (_m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H₂O₂, in the absence of extracellular Ca²⁺ ([Ca²⁺]_o) led to an increase either in cytosolic and in mitochondrial Ca²⁺ concentration. Additionally, H₂O₂ induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H₂O₂ on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H₂O₂. However, the H₂O₂-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca²⁺ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H₂O₂-evoked changes in mitochondrial Ca²⁺ concentration but not those in membrane potential and FAD state. The present results have indicated that H₂O₂ can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H₂O₂. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas. (Mol Cell Biochem 269: 165–173, 2005)     

Characterization of a human coagulation factor Xa-binding site on <Emphasis Type="Italic">Viperidae</Emphasis> snake venom phospholipases A<Subscript>2</Subscript> by affinity binding studies and molecular bioinformatics

Background  

The snake venom group IIA secreted phospholipases A₂ (SVPLA₂), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA₂-inhibitors from snake serum; and (iii) coagulation factors present in human blood.     

Decreased 5-HT<Subscript>1A</Subscript> Receptor Gene Expression and 5-HT<Subscript>1A</Subscript> Receptor Protein in the Cerebral Cortex and Brain Stem during Pancreatic Regeneration in Rats

The present study was to investigate the role of central 5-HT and 5-HT_1A receptor binding and gene expression in a rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content and 5-HT_1A receptor gene expression in the cerebral cortex (CC) and brain stem (BS) of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content significantly increased in the CC (P < 0.01) and BS (P < 0.05) of 72 h pancreatectomised rats. Sympathetic activity was decreased as indicated by the significantly decreased norepinephrine (NE) and epinephrine (EPI) level (P < 0.001 and P < 0.05) in the plasma of 72 h pancreatectomised rats. 5-HT_1A receptor density and affinity was decreased in the CC (P < 0.01) and BS (P < 0.01). These changes correlated with a diminished 5-HT_1Areceptor mRNA expression in the brain regions studied. Our results suggest that the brain 5-HT through 5-HT_1A receptor has a functional role in the pancreatic regeneration through the sympathetic regulation.     

Effects of elevated CO<Subscript>2</Subscript> and/or O<Subscript>3</Subscript> on hormone IAA in needles of Chinese pine

This study examined the effects of season-long exposure of Chinese pine (Pinus tabulaeformis) to elevated carbon dioxide (CO₂) and/or ozone (O₃) on indole-3-acetic acid (IAA) content, activities of IAA oxidase (IAAO) and peroxidase (POD) in needles. Trees grown in open-top chambers (OTC) were exposed to control (ambient O₃, 55 nmol mol⁻¹ + ambient CO₂, 350 μmol mol⁻¹, CK), elevated CO₂ (ambient O₃ + high CO₂, 700 μmol mol⁻¹, EC) and elevated O₃ (high O₃, 80 ± 8 nmol mol⁻¹ + ambient CO₂, EO) OTCs from 1 June to 30 September. Plants grown in elevated CO₂ OTC had a growth increase of axial shoot and needle length, compared to control, by 20% and 10% respectively, while the growth in elevated O₃ OTC was 43% and 7% less respectively, than control. An increase in IAA content and POD activity and decrease in IAAO activity were observed in trees exposed to elevated CO₂ concentration compared with control. Elevated O₃ decreased IAA content and had no significant effect on IAAO activity, but significantly increased POD activity. When trees pre-exposed to elevated CO₂ were transferred to elevated O₃ (EC–EO) or trees pre-exposed to elevated O₃ were transferred to elevated CO₂ (EO–EC), IAA content was lower while IAAO activity was higher than that transferred to CK (EC–CK or EO–CK), the change in IAA content was also related to IAAO activity. The results indicated that IAAO and POD activities in Chinese pine needles may be affected by the changes in the atmospheric environment, resulting in the change of IAA metabolism which in turn may cause changes in Chinese pine’s growth. An erratum to this article can be found at     

Changes in the composition of anionic membrane phospholipids influence protein secretion and cell envelope biogenesis in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD102 and HDL11 with controlled synthesis of the anionic phospholipids, phosphatidylglycerol and cardiolipin. Inactivation of the pgsA gene responsible for the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. An increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of the outer membrane lipopolysaccharides and lipopolyproteins, which determine its barrier properties. The results obtained show that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between protein secretion and biogenesis of cell envelope components.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 179–184.Original Russian Text Copyright © 2005 by Anisimova, Badyakina, Vasileva, Nesmeyanova.     

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans

The -amylase gene (amy) from Streptomyces griseus IMRU 3570 and the -galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P₂ or P₃. The -amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum. Correspondence to: J. F. Martín