Isolation of a CK2α Subunit and the Holoenzyme from the Mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> and Construction of the <Emphasis Type="Italic">CK2α</Emphasis> and <Emphasis Type="Italic">CK2β</Emphasis> cDNAs

Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α′) and two regulatory (β) subunits, forming a differently assembled tetramer. The free and catalytically active α/α′ subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2β subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2β subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2β subunits have similar features to their mammalian orthologs.     

Responses of the ciliates <Emphasis Type="Italic">Tetrahymena</Emphasis> and <Emphasis Type="Italic">Paramecium</Emphasis> to external ATP and GTP

The unicellular ciliates Paramecium and Tetrahymena are the simplest eukaryotic cells to show reliable depolarizing responses to micromolar concentrations of external ATP and GTP. Their simplicity allows for combined analysis of swimming behavior, electrophysiology, receptor binding, behavioral mutant and drug screens as well as molecular genetic approaches such as RNAi and gene knockouts experiments. ATP and GTP are depolarizing chemorepellents in both ciliates, producing measurable receptor potentials and Ca²⁺-based action potentials that are correlated with jerking behaviors called avoiding reactions (AR). GTP also causes repetitive continuous ciliary reversals (CCR) and oscillating plateau depolarizations in Paramecium. Both ciliates show high affinity, saturable external binding of ³²P-GTP and ³²P-ATP but GTP does not compete for ATP binding and vice versa. Chemosensory adaptation occurs after continued exposure (15 min) to these ligands, producing a loss of external binding and forward swimming. However, cells adapted to ATP still bind and respond to GTP and GTP-adapted cells still bind and respond to ATP. This, combined with pharmacological analyses, suggests that there are two separate receptor systems: A metabotropic ATP receptor pathway and a different, novel GTP receptor pathway. A Paramecium mutant (ginA) lacks the GTP-induced oscillating depolarizations but does show AR in GTP, unveiling isolated GTP-receptor potentials for study. An ecto-ATPase is also present that may be involved in inactivation of ATP and GTP signals. Gene knockout experiments are currently underway to determine the roles of the ecto-ATPase and a putative 7-transmembrane spanning receptor in these responses.     

Strong Expression and Conserved Regulation of <Emphasis Type="Italic">ACT2</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Arabidopsis ACT2 represents an ancient class of vegetative plant actins and is strongly and constitutively expressed in almost all Arabidopsis sporophyte vegetative tissues. Using the beta glucuronidase report system, the studies showed that ACT2 5′ regulatory region was significantly more active than CaMV 35S promoter in Arabidopsis seedlings and gametophyte vegetative tissues of Physcomitrella patens. Its activity was also observed in rice and maize seedlings. Thus, the ACT2 5′ regulatory region could potentially serve as a strong regulator to express a transgene in divergent plant species. ACT2 5′ regulatory region contained 15 conserved sequence elements, an ancient intron in its 5′ un-translated region (5′ UTR), and a purine-rich stretch followed by a pyrimidine-rich stretch (PuPy). Mutagenesis and deletion analysis illustrated that some of the conserved sequence elements and the region containing PuPy sequences played regulatory roles in Arabidopsis. Interestingly, mutation of the conserved elements did not lead a dramatic change in the activity of ACT2 5′ regulatory region. The ancient intron in ACT2 5′ UTR was required for its strong expression in both Arabidopsis and P. patens, but did not fully function as a canonical intron. Thus, it was likely that some of the conserved sequence elements and gene structures had been preserved in ACT2 5′ regulatory region over the course of land plant evolution partly due to their functional importance. The studies provided additional evidences that identification of evolutionarily conserved features in non-coding region might be used as an efficient strategy to predict gene regulatory elements.     

Veratrol-<Emphasis Type="Italic">O</Emphasis>-demethylase of <Emphasis Type="Italic">Acetobacterium dehalogenans</Emphasis>: ATP-dependent reduction of the corrinoid protein

The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co^I] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E _SHE) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be −290 mV (pH 7.5), whereas E _SHE at pH 7.5 was lower than −450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP, 2–4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM).     

Functional characterization of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with alterations in the <Emphasis Type="Italic">atpE</Emphasis> gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular Detection of <Emphasis Type="Italic">Xenostrobus securis</Emphasis> and <Emphasis Type="Italic">Mytillus Galloprovincialis</Emphasis> Larvae in Galician Coast (Spain)

The mussel species Xenostrobus securis from New Zealand was detected in the Spanish coast recently, in the mouth of the Verdugo River into the Vigo Ria. In view of the great importance of the farm mussel sector in this region, the presence of this alien species greatly concerned producers and administration authorities, because of its potential medium- or long-term effects on the autochthonous species, Mytilus galloprovincialis, an important marine resource widely exploited in this location. The goal of this study was to develop a DNA-based technique to identify X. securis and M. galloprovincialis larvae in plankton samples, which would allow monitoring for the presence of X. securis in different points of the Vigo Ria. The techniques used were simplex and multiplex polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and fragment analysis. The application of this system to planktonic samples could be an effective means to assess the presence of the alien species, allowing monitoring if its dispersion is increasing, or on the contrary, if its distribution is restricted to the mouth of the Verdugo River, where X. securis was first detected. In addition, the application of this system at different times could be useful to assess the presence of larvae of these two species in the plankton.     

Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.     

Analysis of two promoters that control the expression of the <Emphasis Type="Italic">GTP cyclohydrolase I</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5′-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between −98 and +31, and between −73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between −98 and −56 and between −73 and −41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilis GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.     

Identification of the <Emphasis Type="Italic">bkdAB</Emphasis> gene cluster,a plausible source of the starter-unit for virginiamycin M production in <Emphasis Type="Italic">Streptomyces virginiae</Emphasis>

The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain α-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for virginiamycin production. Gene disruption of bkdA completely abolished the production of virginiamycin M (a polyketide-peptide antibiotic), while the production of virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the virginiamycin M production, indicating that the bkdAB cluster is essential for virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the α and β subunits, S. virginiae bkdA seemed to encode the fused form of the α and β subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli. Supply of an additional bkdA gene under the strong and constitutive promoter ermE* in the wild-type strain of S. virginiae resulted in enhanced production of virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of virginiamycin M.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Natural occurrence of soil-borne entomopathogenic fungi in the Moroccan Endemic forest of <Emphasis Type="Italic">Argania spinosa</Emphasis> and their pathogenicity to <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The occurrence and abundance of entomopathogenic fungi were analysed in 203 soil samples of the Moroccan endemic forests of Argania spinosa, the world main refuge of the Mediterranean fruit fly, Ceratitis capitata. Using the Galleria baiting method and selective media, entomopathogenic fungi were isolated from 186 of the 203 (91.62%) soil samples, with only three species found: Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin and Paecilomyces lilacinus (Thom.) Samson. B. bassiana was the most widespread entomopathogenic fungi (90.64%) in the Argan forest whereas M. anisopliae was less common (15.27%) and P. lilacinus was very rare (1.48%). This is the first report of natural occurrence of M. anisopliae and P. lilacinus in Morocco. Furthermore, 118 Moroccan B. bassiana isolates were studied for their pathogenicity to C. capitata and thermotolerance. Most of these autochtonous B. bassiana isolates were virulent (86.44%) to Medfly pupae and tolerant (55.08%) to temperature stress at 45°C for 2 h. Only 60.17% of Moroccan B. bassiana isolates might be considered as highly entomopathogenic and will serve as a source of potential biological control agents to C. capitata. The percentage of thermotolerant and pathogenic B. bassiana to C. capitata were shown to decrease significantly at winter time characterized by low temperatures and absence of any noticeable medfly in the Argan forest. The occurrence, thermotolerance and virulence of B. bassiana isolates to C. capitata seemed to be related to the sampling periods and location. Our data are discussed with respect to fungal ecology and biocontrol potential of B. bassiana isolates in relation to their habitat.     

Thymidine and thymidylate kinases from the scallop <Emphasis Type="Italic">Mizuhopecten yessoensis</Emphasis> gonads

Thymidine and thymidylate kinases were isolated from the gonads of scallop Mizuhopecten yessoensis. The enzymes were purified 537-and 100-fold, respectively, and were free of phosphatase and ATPase impurities. Ions of bivalent metals and ATP were necessary for both the nucleoside and nucleotide kinase activities; the pH optimum fall into the range of 7.5–8.5. KCl and NaCl at a concentration of up to 100 mM had no inhibiting effect on the activities of these scallop enzymes. Thymidine kinase catalyzed thymidine, and, at a lower rate, deoxycytidine phosphorylations did not utilize ribo-and deoxyribonucleosides, as well as pyrimidine ribonucleosides, as a phosphate acceptor. Thymidylate kinase phosphorylated TMP and dCMP with an efficiency of about 30%. In addition to ATP, these enzymes can also utilize with different efficiencies dATP, dGTP, GTP, UTP, and CTP as a donor of phosphate groups. Thymidine kinase activity was inhibited by TMP, TTP, and dCTP.     

Horizontal Transfer of the Tetracycline Resistance Gene <Emphasis Type="Italic">tetM</Emphasis> Mediated by pCF10 Among <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in the House Fly (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) Alimentary Canal

The house fly (Musca domestica L.) alimentary canal was evaluated for the potential of horizontal transfer of tetM on plasmid pCF10 among Enterococcus faecalis. Two sets of experiments were conducted: (1) house flies without surface sterilization and (2) surface-sterilized flies. Both sets of flies were exposed to E. faecalis OG1RF:pCF10 as donor for 12 h and then E. faecalis OG1SSp as recipient for 1 h. Another group of flies received the recipient first for 12 h followed by exposure to the donor strain for 1 h. House flies were screened daily to determine the donor, recipient, and transconjugant bacterial load for up to 5 days. In addition, the sponge-like mouth parts used for food uptake (labellum) of surface-sterilized house flies were removed and analyzed for donors, recipients, and transconjugants, separately. In both groups of flies (n = 90 flies/group), transfer occurred within 24 h after exposure with a transconjugant/donor rate from 8.6 × 10⁻⁵ to 4.5 × 10¹. Transconjugants were also isolated from the house fly labellum. Our data suggest that the house fly digestive tract provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among enterococci. Our results emphasize the importance of this insect as a potential vector of antibiotic-resistant bacterial strains.     

A MOFRL family glycerate kinase from the thermophilic crenarchaeon, <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis>, with unique enzymatic properties

A glycerate kinase gene (ST2037) from the hyperthermophilic crenarchaeon Sulfolobus tokodaii was cloned and expressed in Escherichia coli. The purified homodimeric protein (45 kDa) specifically catalyzed the formation of 2-phosphoglycerate with d-glycerate as substrate. The thermostable enzyme displayed maximum activity (over 20 min) at 90°C and pH 4.5. The maximal activity was in the presence of Co²⁺. The MOFRL family glycerate kinase used AMP as phosphate donor with maximal activity towards GTP. These characteristics of the enzyme suggested its potential in the catalytic production of 2-phosphoglycerate.     

Enhanced uridine diphosphate <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine production using whole-cell catalysis

Uridine diphosphate N-acetylglucosamine (UDPAG) can be produced by chemical, enzymatic, chemoenzymatic, and fermentative methods. In this study, we used whole-cell catalysis method to produce UDPAG for the first time by Saccharomyces cerevisiae. In order to increase the ATP utilization efficiency and UDPAG conversion yield, the response surface methodology was applied to optimize the whole-cell catalytic conditions for UDPAG production. Firstly, effects of uridine 5′-monophosphate (5′-UMP), glucosamine, vitamin B1, glycerol, magnesium chloride, potassium chloride, temperature, sodium dihydrogen phosphate, sodium acetate, fructose, and pH on UDPAG production were evaluated by a fractional factorial design. Results showed that UDPAG production was mainly affected by sodium dihydrogen phosphate, temperature, and vitamin B1. Then, the concentrations of sodium dihydrogen phosphate and vitamin B1 and temperature were further investigated with a central composite design and response surface analysis. The cultivation conditions to obtain the optimal UDPAG production were determined: sodium dihydrogen phosphate, 31.2 g/L; temperature, 29°C, and vitamin B1, 0.026 g/L. This optimization strategy led to an enhancement of UDPAG production from 2.51 to 4.25 g/L, yield from 44.6% to 75.6% based on the initial 5′-UMP concentration, and ATP utilization efficiency from 7.43% to 12.6%.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.