Biofilm development in the extremely acidophilic archaeon ‘Ferroplasma acidarmanus’ Fer1

‘Ferroplasma acidarmanus’ Fer1 is an iron-oxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA. This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies. Bioinformatic analysis of the ‘F. acidarmanus’ Fer1 genome identified genes annotated as involved in attachment and biofilm formation. No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in ‘F. acidarmanus’ Fer1 biofilm supernatant. Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth. Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations. Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth. This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.     

Characterization of Ferroplasma Isolates and Ferroplasma acidarmanus sp. nov., Extreme Acidophiles from Acid Mine Drainage and Industrial Bioleaching Environments

Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y^T by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate “Ferroplasma acidarmanus” Fer1^T required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42°C and the pH optima were 1.0 to 1.7, and “F. acidarmanus” Fer1^T was capable of growing at pH 0. The optimum yeast extract concentration for “F. acidarmanus” Fer1^T was higher than that for the other three isolates. Phenotypic results suggested that isolate “F. acidarmanus” Fer1^T is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y^T group as a single species. “F. acidarmanus” Fer1^T groups separately, and we propose the new species “F. acidarmanus” Fer1^T sp. nov.     

Extreme zinc tolerance in acidophilic microorganisms from the bacterial and archaeal domains

Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon ??Ferroplasma acidarmanus??, the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of ??F. acidarmanus?? ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, ??F. acidarmanus?? kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.     

Molybdate treatment and sulfate starvation decrease ATP and DNA levels in Ferroplasma acidarmanus

Sulfate is a primary source of sulfur for most microbes and in some prokaryotes it is used an electron acceptor. The acidophile Ferroplasma acidarmanus (strain fer1) requires a minimum of 150 mM of a sulfate-containing salt for growth. Sulfate is assimilated by F. acidarmanus into proteins and reduced to form the volatile organic sulfur compounds methanethiol and dimethyldisulfide. In the absence of sulfate, cell death occurs by an unknown mechanism. In this study, cell viability and genomic DNA and ATP contents of F. acidarmanus were monitored in response to the absence of sulfate or the presence of sulfate and the sulfate analog molybdate ( MoO42- ). Cellular DNA and ATP contents were monitored as markers of cell viability. The absence of sulfate led to a decrease in viable cell numbers of greater than 7 log₁₀ within 5 days, a > 99% reduction in genomic DNA within 3 days, and a > 60% decrease in ATP within 6 h. Likewise, cells incubated with lost viability (decreased by > 2 log₁₀ in 5 days), extractable genomic DNA (reduction of > 60% in 2 days), and ATP (reduction of > 70 % in 2 hours). These results demonstrate that sulfate deprivation or the presence of molybdate have similar impacts on cell viability and essential biomolecules. Sulfate was coupled to cellular ATP content and maintenance of DNA integrity in F. acidarmanus, a finding that may be applicable to other acidophiles that are typically found in sulfate-rich biotopes.     

Towards determining details of anaerobic growth coupled to ferric iron reduction by the acidophilic archaeon ‘<Emphasis Type="Italic">Ferroplasma</Emphasis><Emphasis Type="Italic"> acidarmanus</Emphasis>’ Fer1

Elucidation of the different growth states of Ferroplasma species is crucial in understanding the cycling of iron in acid leaching sites. Therefore, a proteomic and biochemical study of anaerobic growth in ‘Ferroplasma acidarmanus’ Fer1 has been carried out. Anaerobic growth in Ferroplasma spp. occurred by coupling oxidation of organic carbon with the reduction of Fe³⁺; but sulfate, nitrate, sulfite, thiosulfate, and arsenate were not utilized as electron acceptors. Rates of Fe³⁺ reduction were similar to other acidophilic chemoorganotrophs. Analysis of the ‘F. acidarmanus’ Fer1 proteome by 2-dimensional polyacrylamide gel electrophoresis revealed ten key proteins linked with central metabolic pathways ≥4 fold up-regulated during anaerobic growth. These included proteins putatively identified as associated with the reductive tricarboxylic acid pathway used for anaerobic energy production, and others including a putative flavoprotein involved in electron transport. Inhibition of anaerobic growth and Fe³⁺ reduction by inhibitors suggests the involvement of electron transport in Fe³⁺ reduction. This study has increased the knowledge of anaerobic growth in this biotechnologically and environmentally important acidophilic archaeon.     

The effect of the polar moiety of lipids on the ion permeability of bilayer membranes

Summary Bilayer membranes were prepared with the negatively charged lipids phosphatidylglycerol and diphosphatidylglycerol, the positively charged lipid lysyl phosphatidylglycerol, the zwitterionic lipid phosphatidylethanolamine, and an uncharged glycolipid, diglucosyldiglyceride, all isolated from gram-positive bacteria. Bilayer membranes of all these lipids manifested specific resistances of 10⁷ to 10⁹ cm² and capacitances of 0.3 to 0.4 F cm^–2. The membrane potentials of these bilayers were measured as a function of the sodium chloride, potassium chloride, and hydrogen chloride transmembrane concentration gradients (0.01 to 0.10m) and were found to be linear with the logarithm of the salt activity gradients. Membranes made from lysyl phosphatidylglycerol (one net positive charge) were almost completely chloride selective, whereas membranes from phosphatidylglycerol and diphosphatidylglycerol (one and two net negative charges, respectively) were highly cation selective. Membranes prepared with either diglucosyldiglyceride or phosphatidylethanolamine showed only slight cation selectivity. These findings indicate that the charge on the polar head group of membrane lipids plays an important role in controlling the ion-selective permeability of the bilayer.     

Production of methanethiol and volatile sulfur compounds by the archaeon “Ferroplasma acidarmanus”

Acidophiles are typically isolated from sulfate-rich ecological niches yet the role of sulfur metabolism in their growth and survival is poorly defined. Studies of heterotrophically grown “Ferroplasma acidarmanus” showed that its growth requires a minimum of 100 mM of a sulfate-containing salt. Headspace gas analyses by GC/MS determined that the volatile sulfur compound emitted by active “F. acidarmanus” cultures is methanethiol. In “F. acidarmanus” cultures grown either heterotrophically or chemolithotrophically, methanethiol was produced constitutively. Radiotracer studies with ³⁵S-labeled methionine, cysteine, and sulfate showed that all three were used in methanethiol production. Additionally, ³H-labeled methionine was incorporated into methanethiol and was probably used as a methyl-group donor. Methanethiol production in whole cell lysates supplied with SO₃²⁻ indicated that NADPH-dependant sulfite reductase and methyltransferase activities were present. Cell lysates also contained enzymatic activity for methionine-γ-lyase that cleaved the side chain of either methionine to form methanethiol or cysteine to produce H₂S. Since methanethiol was detected from the degradation of cysteine, it is likely that sulfide was methylated by a thiol methyltransferase. Collectively, these data demonstrate that “F. acidarmanus” produces methanethiol through the metabolism of methionine, cysteine, or sulfate. This is the first report of a methanethiol-producing acidophile, thus identifying a new contributor to the global sulfur cycle.     

Influence of thylakoid membrane lipids on the structure of aggregated light‐harvesting complexes of the diatom Thalassiosira pseudonana and the green alga Mantoniella squamata

The study investigated the effect of the thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure of two algal light‐harvesting complexes (LHCs). In contrast to higher plants whose thylakoid membranes are characterized by an enrichment of the neutral galactolipids MGDG and DGDG, both the green alga Mantoniella squamata and the centric diatom Thalassiosira pseudonana contain membranes with a high content of the negatively charged lipids SQDG and PG. The algal thylakoids do not show the typical grana–stroma differentiation of higher plants but a regular arrangement. To analyze the effect of the membrane lipids, the fucoxanthin chlorophyll protein (FCP) complex of T. pseudonana and the LHC of M. squamata (MLHC) were prepared by successive cation precipitation using Triton X‐100 as detergent. With this method, it is possible to isolate LHCs with a reduced amount of associated lipids in an aggregated state. The results from 77 K fluorescence and photon correlation spectroscopy show that neither the neutral galactolipids nor the negatively charged lipids are able to significantly alter the aggregation state of the FCP or the MLHC. This is in contrast to higher plants where SQDG and PG lead to a strong disaggregation of the LHCII whereas MGDG and DGDG induce the formation of large macroaggregates. The results indicate that LHCs which are integrated into thylakoid membranes with a high amount of negatively charged lipids and a regular arrangement are less sensitive to lipid‐induced structural alterations than their counterparts in membranes enriched in neutral lipids with a grana–stroma differentiation.     

Dependence on the F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases 1 and 2 during glucose and glycerol fermentation at high and low pH in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli has four [NiFe]-hydrogenases (Hyd); three of these, Hyd-1, Hyd-2 and Hyd-3 have been characterized well. In this study the requirement for the F₀F₁-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases Hyd-1 and Hyd-2 was examined. During fermentative growth on glucose at pH 7.5 an E. coli F₀F₁-ATP synthase mutant (DK8) lacked hydrogenase activity. At pH 5.5 hydrogenase activity was only 20% that of the wild type. Using in-gel activity staining, it could be demonstrated that both Hyd-1 and Hyd-2 were essentially inactive at these pHs, indicating that the residual activity at pH 5.5 was due to the hydrogen-evolving Hyd-3 enzyme. During fermentative growth in the presence of glycerol, hydrogenase activity in the mutant was highest at pH 7.5 attaining a value of 0.76 U/mg, or ~50% of wild type activity, and Hyd-2 was only partially active at this pH, while Hyd-1 was inactive. Essentially no hydrogenase activity was measured at pH 5.5 during growth with glycerol. At this pH the mutant had a hydrogenase activity that was maximally only ~10% of wild type activity with either carbon substrate but a weak activity of both Hyd-1 and Hyd-2 could be detected. Taken together, these results demonstrate for the first time that the activity of the hydrogen-oxidizing hydrogenases in E. coli depends on an active F₀F₁-ATP synthase during growth at high and low pH.     

Energy flux controls tetraether lipid cyclization in Sulfolobus acidocaldarius

Microorganisms regulate the composition of their membranes in response to environmental cues. Many Archaea maintain the fluidity and permeability of their membranes by adjusting the number of cyclic moieties within the cores of their glycerol dibiphytanyl glycerol tetraether (GDGT) lipids. Cyclized GDGTs increase membrane packing and stability, which has been shown to help cells survive shifts in temperature and pH. However, the extent of this cyclization also varies with growth phase and electron acceptor or donor limitation. These observations indicate a relationship between energy metabolism and membrane composition. Here we show that the average degree of GDGT cyclization increases with doubling time in continuous cultures of the thermoacidophile Sulfolobus acidocaldarius (DSM 639). This is consistent with the behavior of a mesoneutrophile, Nitrosopumilus maritimus SCM1. Together, these results demonstrate that archaeal GDGT distributions can shift in response to electron donor flux and energy availability, independent of pH or temperature. Paleoenvironmental reconstructions based on GDGTs thus capture the energy available to microbes, which encompasses fluctuations in temperature and pH, as well as electron donor and acceptor availability. The ability of Archaea to adjust membrane composition and packing may be an important strategy that enables survival during episodes of energy stress.     

Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl‐constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER‐to‐chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.     

Endogenously produced CO2 induces stationary phase in tetrahymena cultures

Media concentration of total soluble CO₂ increases with culture age of Tetrahymena pyriformis. CO₂ is a weak acid and is capable of acidifying intracellular pH (pHi). Changes in pHi have been demonstrated to affect cell metabolism and growth in many systems. For these reasons, we investigated whether the concentrations of CO₂ produced in vitro were sufficient to affect cell proliferation and pHi in Tetrahymena. In this study, we used DMO to mimic the weak acid properties of CO₂. DMO is freely permeable to membranes in its uncharged form and has a pKa similar to that of CO₂/HCO. In addition, it has the advantages of being metabolically inert and non-volatile. At concentrations similar to endogenously produced CO₂, DMO acidifies pHi and arrests culture growth. In addition, procedures are described which decrease the media CO₂ concentrations in both growing and non-growing cultures. These conditions lead to increased maximum culture density at stationary phase. The data indicate that, under our conditions, accumulation of CO₂ in the culture leads to cessation of growth, probably through elimination of transmembrane pH gradients, which are necessary for regulation of metabolism and growth.     

Polyunsaturated membranes are required for photosynthetic competence in a mutant of Arabidopsis

High levels of polyunsaturation are characteristic of all the membranes of plant and animal cells. For example, the chloroplasts of leaf cells contain about 75–80% polyunsaturated fatty acids. For the extra-chloroplast membranes in leaf cells and the membranes of non-photosynthetic tissues, values of 60–65% are typical. We report here the production of Arabidopsis double mutants that contain negligible levels of polyunsaturated fatty acids. The mutants were not capable of autotrophic growth and produced extremely chlorotic cotyledons and leaves. However, on sucrose media, the double mutants were robust plants showing strong leaf and root development. These observations indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids. By contrast, photosynthesis is one process that does require high levels of membrane polyunsaturation.     

Lipid membranes from halophilic and alkali-halophilic Archaea have a low H+ and Na+ permeability at high salt concentration

The influence of pH and the salt concentration on the proton and sodium ion permeability of liposomes formed from lipids of the halophile Halobacterium salinarum and the haloalkaliphile Halorubrum vacuolatum were studied. In contrast with liposomes formed from Escherichia coli lipids, liposomes formed from halophilic lipids remained stable up to 4 M of NaCl and KCl. The proton permeability of the liposomes from lipids of halophiles was independent of the salt concentration and was essentially constant between pH 7 and pH 9. The sodium ion permeability increased with the salt concentration but was 10- to 100 fold lower than the proton permeability. It is concluded that the membranes of halophiles are stable over a wide range of salt concentrations and at elevated pH values and are well adapted to the halophilic conditions. Received: February 25, 1999 / Accepted: June 11, 1999     

Cu(II)-reduction by <Emphasis Type="Italic">Escherichia coli</Emphasis> cells is dependent on respiratory chain components

Copper is both an essential nutrient and a toxic element able to catalyze free radicals formation which damage lipids and proteins. Although the available copper redox species in aerobic environment is Cu(II), proteins that participate in metal homeostasis use Cu(I). With isolated Escherichia coli membranes, we have previously shown that electron flow through the respiratory chain promotes cupric ions reduction by NADH dehydrogenase-2 and quinones. Here, we determined Cu(II)-reductase activity by whole cells using strains deficient in these respiratory chain components. Measurements were done by the appearance of Cu(I) in the supernatants of cells exposed to sub-lethal Cu(II) concentrations. In the absence of quinones, the Cu(II)-reduction rate decreased ~70% in respect to the wild-type strain, while this diminution was about 85% in a strain lacking both NDH-2 and quinones. The decrease was ~10% in the absence of only NDH-2. In addition, we observed that quinone deficient strains failed to grow in media containing either excess or deficiency of copper, as we have described for NDH-2 deficient mutants. Thus, the Cu(II)-reduction by E. coli intact cells is mainly due to quinones and to a lesser extent to NDH-2, in a quinone-independent way. To our knowledge, this is the first in vivo demonstration of the involvement of E. coli respiratory components in the Cu(II)-reductase activity which contributes to the metal homeostasis.     

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.     

Membrane lipids and soluble sugars dynamics of the alkaliphilic fungus Sodiomyces tronii in response to ambient pH

Alkaliphily, the ability of an organism to thrive optimally at high ambient pH, has been well-documented in several lineages: archaea, bacteria and fungi. The molecular mechanics of such adaptation has been extensively addressed in alkaliphilic bacteria and alkalitolerant fungi. In this study, we consider an additional property that may have enabled fungi to prosper at alkaline pH: altered contents of membrane lipids and cytoprotectant molecules. In the alkaliphilic Sodiomyces tronii, we showed that at its optimal growth pH 9.2, the fungus accumulates abundant cytosolic trehalose (4–10% dry weight) and phosphatidic acids in the membrane lipids, properties not normally observed in neutrophilic species. At a very high pH 10.2, the major carbohydrate, glucose, was rapidly substituted by mannitol and arabitol. Conversely, lowering the pH to 5.4–7.0 had major implications both on the content of carbohydrates and membrane lipids. It was shown that trehalose dominated at pH 5.4. Fractions of sphingolipids and sterols of plasma membranes rapidly elevated possibly indicating the formation of membrane structures called rafts. Overall, our results reveals complex dynamics of the contents of membrane lipids and cytoplasmic sugars in alkaliphilic S. tronii, suggesting their adaptive functionality against pH stress.

     

Time Course and Involvement of Protein Kinase C-Mediated Phosphorylation of F1/GAP-43 in Area CA3 After Mossy Fiber Stimulation

1. Protein kinase C (PKC) activity and phosphorylation of F1/growth associated protein (GAP)-43, a PKC substrate, have been proposed to play key roles in the maintenance of long-term potentiation (LTP) at the synapses of Schaffer collateral/commissural on pyramidal neurons in CA1 (Akers et al., 1986). We have studied in the involvement of PKC and PKC-dependent protein phosphorylation of F1/GAP-3 in in vitro LTP observed at the synapses of mossy fiber (MF) on CA3 pyramidal neurons of rat hippocampus by post hoc in vitro phosphorylation.2. After LTP was induced in CA3 in either the presence or absence of D-2-amino-5-phosphonovaleric acid (AP5), an NMDA receptor antagonist, the CA3 region was dissected for in vitro phosphorylation assay. In vivo phosphorylation of F1/GAP-43 was increased in membranes at 1 and 5 min after tetanic stimulation (TS) but not at 60 min after TS.3. The degree of phosphorylation of F1/GAP-43 in the cytosol was inversely related to that in membranes at each time point after LTP.4. The similar biochemical changes obtained from either control slices or AP5-treated slices indicate that LTP and the underlying biochemical changes are independent of the NMDA receptor. Immunoreactivity of the phophorylated F1/GAP-43 in LTP slices was not significantly different from control, indicating that results from western blotting and post hoc in vitro phosphorylation are consistent.5. Post hoc in vitro phosphorylation of F1/GAP-43 was PKC-mediated since phosphorylation of F1/GAP-43 was altered by the PKC activation cofactors, Ca²⁺, phosphatidylserine and phorbol ester.6. Calmodulin (CaM) at >5 M inhibited phosphorylation, consistent with the presence of CaM-binding activity at the site on F1/GAP-43 acted upon by PKC.7. We conclude that phosphorylation of F1/GAP-43 is associated with the induction but not the maintenance phase of MF-CA3 LTP.     

Identification and characterization of a null-activity mutant containing a cryptic pre-mRNA splice site for cytosolic fructose-1,6-bisphosphatase in <Emphasis Type="Italic">Flaveria linearis</Emphasis>

Cytosolic fructose-1,6-bisphosphatase (cytFBPase) (E.C. 3.1.3.11) catalyzes the first irreversible reaction of daytime sucrose synthesis. A Flaveria linearis (F. linearis) mutant (line 84-9) previously shown to have ~10% wildtype cytFBPase activity contains no cytFBPase activity based on enzymatic and immunoprecipitation analysis. Genetic segregation and Southern analysis of an F2 population shows one gene copy of cytFBPase in F. linearis and linkage of null cytFBPase activity to the cytFBPase structural gene. A point mutation is present in the structural gene coding for cytFBPase in the mutant, causing a cryptic pre-mRNA splice site and a corresponding 24 amino acid deletion spanning the active site of the enzyme. Collectively, these data support the identification of a null-activity mutant for cytFBPase in F. linearis. This is the first report of a null mutant in the daytime sucrose synthesis pathway confirmed by both enzymatic and molecular analysis. Null cytFBPase in F. linearis does not predispose all lines to high starch accumulation due to an epistatic gene interaction; low starch accumulation in null cytFBPase lines segregates with elevated pyrophosphate-dependent phosphofructokinase (PFP) activity when grown in a 16 h photoperiod. Surprisingly, growth of parental lines and F2 progeny having null cytFBPase in continuous light rescued the wildtype growth phenotype. All null cytFBPase lines showed CO₂-insensitivity/reversed sensitivity of photosynthesis, indicating that null cytFBPase causes a reduced total capacity for both photosynthesis and end-product synthesis regardless of starch and PFP phenotype. Collectively, the data indicate that F. linearis, a C3-C4 photosynthetic intermediate, has alternative cytFBPase-independent pathways for daytime sucrose synthesis.     

Permeation of membranes by the neutral form of amino acids and peptides: Relevance to the origin of peptide translocation

The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10⁵ slower than facilitated inward transport across biological membranes. This suggests that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10¹⁰ times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g., for certain signal sequences, toxins and thylakoid proteins) in vivo.Abbreviations LUV large unilamellar vesicle - pH transmembrane pH gradient - PAH polyaromatic hydrocarbon Correspondence to: A.C. Chakrabarti