Molecular charcterization of tatD DNAse gene from Ralstonia paucula RA4T soil bacterium

Ralstonia paucula strain RA4^T, a gram negative, non-spore forming, motile bacterium having positive catalase and oxidase test, was isolated from surface soil. Twin arginine translocation protein type D (TatD) is shown to be located in cytoplasm and exhibits magnesium-dependent DNase. A tatD DNase gene was isolated and cloned from Ralstonia paucula RA4^T genome. Nucleotide sequence analysis of the gene revealed 813 nucleotides encoding a protein of 270 amino acid residues. The tatD gene showed a high similarity to homolog gene from Ralstonia pickettii strain 12D. The deduced polypeptide sequence of TatD DNase from R. paucula RA4^T had a typical catalytic site, HHPLDEHRHDP, and its calculated molecular mass and predicted isoelectric point were 29616 Da and 5.33, respectively. The deduced amino acid sequence showed a high degree of similarity to TatD DNase isoforms from Ralstonia genus and other sources. Predicted three-dimensional structure of TatD confirmed the presence of active site and theoretical function as DNase.     

An activating amino acid substitution in the c-abl oncogene protein fails to produce a local conformational change

Thebcr-abl chimeric gene of Philadelphia chromosome positive chronic myelogenous leukemias is only weakly transforming. This transformation activity is greatly enhanced by a Lys-for-Glu substitution at position 832 in the c-abl gene, as occurs in the highly transforming v-abl genes. It has been suggested that this mutation results in a significant structural change in the encoded protein product. Using conformational energy analysis, we have determined the allowed low-energy conformations for residues 828–836 of this protein with Lys and Glu at position 832. In both cases, the overwhelmingly preferred conformation for this region is a bend-helix motif. The helix terminates at residue 836, and there are no discernible differences in conformation between the Lys- and Glu-containing sequences. These results suggest that the activating amino acid substitution at position 832 in the c-abl protein product does not produce its effect via a local conformational change.     

Streptosporangium subfuscum sp. nov., isolated from the rhizosphere of marigold (Tagetes erecta L.)

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13^T, was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13^T belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847^T (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847^T, Streptosporangium jomthongense NBRC 110047^T (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849^T (97.6 % 16S rRNA gene similarity). A combination of DNA–DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13^T can be distinguished from S. fragile DSM 43847^T and S. jomthongense NBRC 110047^T. Moreover, strain NEAU-TWSJ13^T can also be differentiated from S. violaceochromogenes DSM 43849^T and other Streptosporangium species showing high 16S rRNA gene sequence similarity (>98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13^T represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13^T ( = CGMCC 4.7146^T = DSM = 46724^T).     

Crystal structure of tRNA m1A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-l-methionine

The N ¹-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m¹A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.     

Synthesis and characterization of iodopropyl derivatives of adenosine 3′,5′-cyclic-monophosphate: Potential affinity labels of cAMP binding sites in enzymes

The syntheses of two potential cAMP affinity lables, 1,N ⁶-(3-iodopropyleno)adenosine 3′,5′-cyclic-monophosphate and 2′-O-(2-iodo-3-hydroxypropyl) adenosine 3′,5′-cyclic-monophosphate, by a two-step chemical procedure are described. TheN ⁶- and 2′-O-allyl intermediates were prepared selectively by alkylation of cAMP in organic and alkaline aqueous solutions, respectively. Treatment of theN ⁶-allyl derivative withN-iodosuccinimide resulted in iodine addition to the double bond and cyclization to theN ¹ position of the purine ring. The iodohydrin analog was synthesized by reaction of 2′-O-allyl-cAMP with potassium iodide and thallium trichloride in acetate buffered solution. The products were isolated by column chromatography and characterized by thin-layer chromatography, elemental analysis, and ultraviolet,¹³C, and¹H NMR spectroscopy. The cAMP analogs were found to react with lysine and cysteine. Both cAMP derivatives were tested for their reaction with the low-K _m cAMP phosphodiesterase of human platelets. The ribose-substituted analog functioned as a competitive inhibitor (K _I =0.72 μM) and caused a time-dependent irreversible inactivation of the phosphodiesterase. In contrast, the purine-substituted derivative acted neither as a reversible competitive inhibitor nor as an irreversible inactivator of the enzyme. These results indicate the specificity of these potential cAMP analogs in their interaction with the phosphodiesterase.     

Paenibacillus abyssi sp. nov., isolated from an abyssal sediment sample from the Indian Ocean

A Gram-positive, rod-shaped bacterium, designated strain SCSIO N0306^T, was isolated from an abyssal sediment sample collected from the Indian Ocean. The isolate was found to grow optimally at 0–2 % (w/v) NaCl, pH 7.0 and 30 °C. Comparative analysis of the 16S rRNA gene sequence showed that the isolate SCSIO N0306^T belongs phylogenetically to the genus Paenibacillus, and to be most closely related to P. algorifonticola XJ259^T (with 95.47 % sequence similarity), sharing less than 95.0 % sequence similarity with all other taxa of this genus. Chemotaxonomic analysis revealed MK-7 as the major isoprenoid quinone, the DNA G+C content was determined to be 45.5 mol%, and anteiso-C_15:0, C_16:0, and iso-C_15:0 were identified as the major fatty acids. On the basis of this polyphasic taxonomic data, isolate SCSIO N0306^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus abyssi sp. nov. is proposed. The type strain is SCSIO N0306^T (= DSM 26238^T = CGMCC 1.12987^T).     

Paenibacillus nicotianae sp. nov., isolated from a tobacco sample

A Gram-stain positive, facultative anaerobic endospore-forming bacterium, designated strain YIM h-19^T, was isolated from a tobacco sample. Cells were observed to be motile rods by means of peritrichous flagella and colonies were observed to be convex, yellow, circular and showed catalase-positive and oxidase-negative reactions. Strain YIM h-19^T was able to grow at 4–45 °C, pH 6.0–8.0 and 0–3 % NaCl (w/v). The predominant respiratory quinone was identified as MK-7. Major fatty acids were identified as anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The polar lipids were found to be phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unidentified polar lipids. The genomic DNA G+C content was determined to be 54 mol%. 16S rRNA gene sequence analysis showed the strain YIM h-19^T was most closely related to Paenibacillus hordei RH-N24^T and Paenibacillus hunanensis FeL05^T with similarities of 98.30 and 94.64 % respectively. However, DNA–DNA hybridization data indicated that the isolate represented a novel genomic species with the genus Paenibacillus. All data from genotypic and phenotypic analyses support the conclusion that strain YIM h-19^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nicotianae sp. nov. is proposed. The type strain is YIM h-19^T (=CGMCC1.12819^T = NRRL B-59112^T).     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3′,5′-AMP for the utilization of various carbohydrates

Adenine requiring mutants of Serratia marcescens SM-6-F'lac ⁺ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5′AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5′AMP at a rate of 1.6–2.0 μmoles min⁻¹ mg⁻¹ protein. The loss of the phosphodiesterase activity in S. marcescens cpd W1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257^pc-1 of Escherichia coli. The induced synthesis of β-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd ⁺ strain. Starting from S. marcescens cpd W1181 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40–60% of that of the parental strain which is about 4×10⁻⁴ M. However, the adenylate cyclase in cell extracts of the mutants W1237 and W1270 was like that of the corresponding cya ⁺ strain (about 2×10⁻² μmoles min⁻¹ mg⁻¹ protein).     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Transcription and processing of rRNA in higher plant cells (Daucus carota,Petroselinum crispum,Acer pseudoplatanus)

Actively dividing cells from parsley (Petroselinum crispum) and carrot (Daucus carota) (bothApiaceae) andAcer pseudoplatanus (Aceraceae) were used to detect the primary gene product of the rDNA in plant cells. Parsley and carrot cells were labelled with [³²P] orthophosphate. In both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels under non-denaturing conditions. Its molecular weight did not exceed 2.5 × 10⁶ daltons. The component emerged from the heterogenous material after a labelling period of 5–10 min. In parsley cells 45 min after onset of incubation labelled mature rRNA (25S and 18S) arrived in the cytoplasm. InAcer pseudoplatanus (incubation period 60 min) two rapidly labelled components did emerge from polyacrylamide gels; their molecular weights were 2.3 and 3.2^? 3.4 × 10⁶ daltons. After electrophoresis under denaturing conditions the larger component was no longer present, thus indicating that it was an aggregate of different RNA molecules. The molecular weights of the rRNA precursors ofD. carota andP. crispum determined after electrophoresis in formamide gels were about 2.1 × 10⁶ daltons. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 × 10⁶ daltons.     

Biological activity of 125iodine labelled globin mRNA in an Ehrlich ascites cell-free system

Globin mRNA was isolated from avian erythroblasts and labelled with ¹²⁵iodine to a specific activity of 4.5×10⁶ dpm μg^-1 mRNA. The labelling procedure was modified as compared to common techniques in order to protect the mRNA against degradation. When this RNA is translated in an Ehrlich ascites cell-free system globin chains are synthesized as is demonstrated by the analysis of the in vitro synthesized products. The biological activity of ¹²⁵iodine labelled mRNA has proven to be 30% of that of unlabelled mRNA.     

Characterization of a class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate from Sinorhizobium fredii

5-Enopyruvylshikimate-3-phosphate synthase (EPSP synthase) is an important enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants and microorganisms. A novel class II EPSP synthase AroA _{S. fredii} from Sinorhizobium fredii NGR234 was overexpressed in Escherichia coli BL21. It was purified to homogeneity and its catalytic properties were studied. The enzyme exhibited optimum catalytic activity at pH 8.0 and 50 °C. It was stable below 40 °C, and over a broad range of pH 5.0–9.0. The EPSP synthase was increasingly activated by 100 mM of the chlorides of NH₄ ⁺, K⁺, Na⁺ and Li⁺. Kinetic analysis of AroA _{S. fredii} suggested that the enzyme exhibited a high glyphosate tolerance and high level of affinity for phosphoenolpyruvate, which indicates the enzyme with a high potential for structural and functional studies and its potential usage for the generation of transgenic crops resistant to the herbicide.     

Characteristics of (Na++K+)-ATPase inBoergesenia forbesii

(Na⁺+K⁺)-ATPase proved to be present in the vegetative thalli ofBoergesenia forbesii (Harvey) Feldmann. The ATPase was extracted with Triton X-100 and partially purified by Sephadex G-150 gel filtration. The enzyme was activated with Mg²⁺ and further stimulated by the addition of K⁺ and Na⁺. It was observed thatp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), iodoacetoamide, copper sulfate, zinc sulfate, lead nitrate and cadmium chloride inhibited the enzyme activity, but ouabain was ineffective, andN,N′-dicyclohexylcarbodiimide (DCCD) did not apparently inhibit the activity, but rather promoted it slightly. The ATPase activity was also shown in the isolated cell wall ofBoergesenia thalli, and the enzyme activity was detected in the wall itself by using electron microscopic methods.     

Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei

Protoplast fusion was used to obtain a higher production of lignocellulolytic enzymes with protoplast fusion in Trichoderma reesei. The fusant strain T. reesei JL6 was obtained from protoplast fusion from T. reesei strains QM9414, MCG77, and Rut C-30. Filter paper activity of T. reesei JL6 increased by 18 % compared with that of Rut C-30. β-Glucosidase, hemicellulase and pectinase activities of T. reesei JL6 were also higher. The former activity was 0.39 Uml^?1, while those of QM9414, MCG77, and Rut C-30 were 0.13, 0.11, and 0.16 Uml^?1, respectively. Pectinase and hemicellulase activities of JL6 were 5.4 and 15.6 Uml^?1, respectively, which were slightly higher than those of the parents. The effects of corn stover and wheat bran carbon sources on the cellulase production and growth curve of T. reesei JL6 were also investigated.