Isolation of DNA-dependent RNA polymerase fromStreptomyces granaticolor and its binding to phage ϕ29 DNA

Partially purified DNA-dependent RNA polymerase ofStreptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. These four subunits, represented 93% proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage ϕ29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases fromEscherichia coli andBacillus subtilis.     

Effect of heat shock on DNA-dependent RNA polymerase from the cyanobacteriumSynechococcus sp.

Purification of DNA-dependent RNA polymerase from exponentially growing cells of the cyanobacteriumSynechococcus sp. is described in cultures grown at normal temperature (39°C) and after heat shock (HS) (47°C). Polyethyleneimine precipitation followed by chromatography and gel filtration steps results in a 39% yield. The enzyme has a component of molar mass of 43 kDa, designated σ, in addition to the typical procaryotic β’β∝₂ and γ. The results suggest thatSynechococcus RNA polymerase is similar to that of cyanobacterial andE. coli RNA polymerases. Electrophoresis of the HS preparation showed that the enzyme has a component of 18 kDa. This suggests the existence of a functional relationship between this protein and the HS response ofSynechococcus RNA polymerase, probably in salvaging denatured RNA polymerase or helping to regain its native structure.     

Formation of the ternary complex EF-Tu.GTP.Phe-tRNA in the translation system ofStreptomyces aureofaciens

The efficiency of formation of the ternary complex consisting of the elongation factor Tu and Phe-tRNA’s fromEscherichia coli andStreptomyces aureofaciens was tested to explain the lower activity of thein vitro poly(U) translation system fromS. aureofaciens. Both factors were shown to be functionally interchangeable in the ternary complex formation with Phe-tRNA from eitherE. coli orS. aureofaciens. However, the efficiency of binding ofS. aureofaciens Phe-tRNA to EF-Tu was much lower with both factors.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

Extracellular proteinase and chitinase produced by a culture ofStreptomyces kurssanovii

Extracellular enzymes—a chitinase and a proteinase with molecular weights 22 and 32 kDa, respectively—were isolated fromStreptomyces kurssanovii cells. After purification on modified regenerated chitin, the enzymes were virtually homogeneous according to denaturing PAGE. Both enzymes were found to degrade chitosan.     

Studies on the production of daunomycinonederived glycosides and related metabolites inStreptomyces coeruleorubidus andStreptomyces peucetius

Strains ofStreptomyces coeruleorubiduě ISP 5145,JA 10092 and 39–146, differing mutually in antibiotic activity, were found to produce identical spectrum of metabolites (at least nine antibiotically active glycosides, 13-dihydrodaunomycinone, ε-rhodomycinone and a larger number of unidentified compounds); only trace quantities of daunomycin and daunomycinone could be detected. A fraction of glycosides with a higher R_f (0.4–0.7), isolated from strain 39–146, could be transformed to daunomycin by mild hydrolysis and to daunomycinone by total hydrolysis.Streptomyces peucetius IMI 101 335 differed fromStreptomyces coeruleorubidus in an increased production of ε-rhodomycinone and a lower content of glycosides; the zone of daunomycin was most pronounced among the glycoside spots.Streptomyces coeruleorubidus 39-146 exhibited the highest activity in a medium containing 3.5% soluble starch, 3.0% soybean meal, 0.3% NaCl and 0.3% CaCo₃; glucose was a more useful carbon source for the remaining strains The activity ofStreptomyces coeruleorubidus was inhibited by 1-propanol, Na-propionate,5,5-diethylbarbiturate and bacitracin. Ferrous sulphate stimulated the production of glycosides only in strain JA 10092, decreasing simultaneously the production of aglycones.     

Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability

Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those of Thermomonospora fusca. Genes encoding two different endo-β-1,4-xylanases were cloned from T.␣alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48 456 Da. The protein contains a 38-amino-acid leader peptide with six Arg⁺ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/xylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 °C, and had broad pH stability in the range from pH 4.0 to pH 10.0. Received: 9 January 1997 / Received revision: 27 March 1997 / Accepted: 13 April 1997     

Macromolecular synthesis accompanying the transition from spores to vegetative forms ofStreptomyces granaticolor

The rates of RNA, protein and DNA synthesis were estimated in synchronously germinating spores ofStreptomyces granaticolor. Rapid uptake of labelled precursors of RNA and proteins was observed after 20 s. The germination process took place through a sequence of time + ordered events. RNA synthesis started after 3 min of germination, protein synthesis began at 4 min and net DNA synthesis at 60–70 min of germination. A characteristic feature of germination was the biphasic pattern in the rate of RNA and protein synthesis. Spores ofStreptomyces granaticolor were sensitive to actinomycin D, rifampicin and chloramphenicol even at the start of germination. Protein synthesis during germination was dependent on new mRNA synthesis and was independent during the first 60–70 min on replication of the spore genome.     

Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts

The precursors of the F₁-ATPase -subunits fromNicotiana plumbaginifolia andNeurospora crassa were imported into isolated spinach (Spinacia oleracea L.) leaf mitochondria. Both F₁ precursors were imported and processed to mature size products. No import of the mitochondrial precursor proteins into isolated intact spinach chloroplasts was seen. Moreover, the precursor of the 33 kDa protein of photosynthetic water-splitting enzyme was not imported into the leaf mitochondria. This study provides the first experimental report ofin vitro import of precursor proteins into plant mitochondria isolated from photosynthetic tissue and enables studies of protein sorting between mitochondria and chloroplasts in a system which is homologous with respect to organelles. The results suggest a high organellar specificity in the plant cell for the cytoplasmically synthesized precursor proteins.     

Preliminary characterization of a thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41

A thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 was characterized in this study. The polI was cloned, sequenced and over-expressed in Escherichia coli. The expressed 110 kDa fusion protein of PolI was stable at 70°C for 1 h. Compared with DNA polymerase I of E. coli (TaKaRa), the relative polymerase activity of this PolI was 3.33 ± 0.1 RFU μl⁻¹ at 37°C using fluorescent quantitative analysis. It showed higher polymerase activity than E. coli PolI at higher temperature, with a relative activity of 3.75 ± 0.1 RFU μl⁻¹ at 70°C. The polI sequence analysis and the protein structure prediction indicated that this protein had a high similarly to other PolI from thermophilic micro-organisms. This information is of importance for future study for evolution of the house-keeping gene polI in entomopathogenic bacterium B. sphaericus.     

Purification and characterization of κ-carrageenase from a novel γ-proteobacterium,<Emphasis Type="Italic">Pseudomonas elongata</Emphasis> (MTCC 5261) syn.<Emphasis Type="Italic">Microbulbifer elongatus</Emphasis> comb. Nov.

The phenotypic and carrageenolytic features of a novel halo tolerant marine bacterium, isolated from decayed red algal samples collected along the west coast of India were studied. This gram-negative strain was identified asPseudomonas elongala (MTCC 5261) syn.Microbulbifer elongalus comb. nov according to its morphological, physiological and molecular characterization. The extracellular κ-carrageenase was purified 106.54-fold by a combination of ammonium sulfate precipitation (40∼60%) and successive gel filtration chromatography. The purified protein fraction yielded significantly high activity of 426.19 units/mg protein and migrated as a single band on a sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of ∼128 kDa. For κ-carrageenase activity, optimum temperature was 40°C whereas two pH optimai.e. 5.6 and 7.7 were observed. For κ-carrageenan, the enzyme gave aK _m value of 6.66 mg/mL and aV _max value of 4 μmol/min/mg when the reaction was carried out at 40°C and pH 5.6. Isolated κ-carrageenase could successfully generate protoplasts ofKappaphycus alvarezii. This is the first report on the production of κ-carrageenase by this bacterium isolated from west coast of India. Molecular mass and various characteristics showed that the carrageenase fromP. elongata was much different from those previously reported.     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

Synthesis of ribulose 1,5-bisphosphate carboxylase by isolatedSorghum mesophyll chloroplasts

Chloroplasts isolated fromSorghum vulgare are active in light-dependent, organelle protein synthesis. Intact chloroplasts can use light as an energy source; photosynthetically inactive chloroplasts require the addition of ATP for this protein synthesis. Preincubation of chloroplasts in light at 25°C for 1 h depleted the endogenous templates completely; such preincubated chloroplasts translated exogenously added heterologous templates efficiently. When total cellular RNA fromChlorella protothecoides, a C₃ plant, was used as template for translation in a cell-free light-dependent system of isolated mesophyll chloroplasts fromSorghum vulgare, a C₄ type plant, polypeptides of 55 kDa (large subunit) and 15 kDa (small subunit) were detectable in the fluorographic profile of the newly synthesized proteins; these polypeptides were absent in the products obtained with endogenous RNA. Evidence for the fidelity of the system was obtained by immunological analysis of ribulose 1, 5-bisphosphate carboxylase obtained by the translation ofChlorella cellular RNAs.     

Molecular cloning and characterization of the cDNA encoding the largest subunit of mouse RNA polymerase I

We describe the cloning and analysis of mRPA1, the cDNA encoding the largest subunit (RPA194) of murine RNA polymerase I. The coding region comprises an open reading frame of 5151 bp that encodes a polypeptide of 1717 amino acids with a calculated molecular mass of 194 kDa. Alignment of the deduced protein sequence reveals homology to the β′ subunit of Escherichia coli RNA polymerase in the conserved regions a-h present in all large subunits of RNA polymerases. However, the overall sequence homology among the conserved regions of RPA1 from different species is significantly lower than that observed in the corresponding β′-like subunits of class II and III RNA polymerase. We have raised two types of antibodies which are directed against the conserved regions c and f of RPA194. Both antibodies are monospecific for RPA194 and do not cross-react with subunits of RNA polymerase II or III. Moreover, these antibodies immunoprecipitate RNA polymerase I both from murine and human cell extracts and, therefore, represent an invaluable tool for the identification of RNA polymerase I-associated proteins. Received: 27 January 1997 / Accepted: 1 April 1997     

Ornithine cycle inNostoc PCC 73102: Stimulation of in vitro ornithine carbamoyl transferase activity by addition of arginine

Cells ofNostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycadMacrozamia, were cultured under different conditions and examined for the presence ofin vitro active ornithine carbamoyl transferase (OCT). Cells grown in darkness showed a significant increase ofin vitro OCT activity compared with the activity when grown in light. Addition of external arginine in the growth medium increasedin vitro OCT activity both in light and in darkness. Moreover, the highestin vitro OCT activity was observed in cells grown in darkness and with the addition of external arginine, a sevenfold increase compared with cells grown in light. Native-PAGE in combination with on gel OCT activity stain demonstrated that external arginine induced the presence of twoin vitro active OCT. In addition to the previously described 80 kDa OCT [Physiol Plant 84:275–282, 1992], a secondin vitro active enzyme with a molecular weight of approximately 118 kDa appeared. Western immunoblots, with native cell-free extracts and antibodies directed either against native or denatured OCT purified fromPisum sativum, confirmed that both enzymes were OCT. Moreover, with a denatured cell-free extract only one polypeptide, with a molecular weight of about 40 kDa, was recognized, indicating that the secondin vitro active OCT might be a trimer with three identical subunits.     

Dependence of aminoglycoside 3′-phosphotransferase VIII activity on serine/threonine protein kinases in <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

In Streptomyces rimosus, selection for resistance to the aminoglycoside antibiotic kanamycin triggers the normally silent aminoglycoside 3-phosphotransferase VIII gene (aphVIII). The expression of APHVIII is accompanied by amplification of the chromosomal DNA fragment containing the aphVIII gene. Earlier, S. rimosus aphVIII gene was isolated and sequenced. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibodies, we have demonstrated that endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 actively phosphorylate two serine residues in the APHVIII molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca²⁺ is 1.84-fold greater than that without Ca²⁺. Analysis of ingel autophosphorylation and phosphorylation of the substrate incorporated into the gel matrix has shown that modification of APHVIII is catalyzed by two serine/threonine PKs (74 kDa and 55 kDa). The activity of 55-kDa PK is dependent on Ca²⁺ and calmodulin. The specific kanamycin phosphotransferase activity of exhaustively phosphorylated APHVIII is 3.72 times higher than that of the unmodified enzyme. It is proposed that the above PKs may be involved in the regulation of kanamycin resistance in S. rimosus.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 255–263.Original Russian Text Copyright © 2005 by Elizarov, Sergienko, Sizova, Danilenko.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Identification of phosphoproteins in Escherichia coli

The substrates of ion- and lipld-stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography. Subsequent N-terminal sequencing suggests that these substrates include the following: a novel 80kDa protein co-purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle. The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment. In addition, a calcium-stimulated protein of 70kDa was identified as the heat-shook protein DnaK, and a 17kDa lipid-stimulated phosphoprotein as nucleotide diphosphate kinase.     

Investigation of Mosquito-Specific Larvicidal Activity of a Soil Isolate of Bacillus thuringiensis serovar canadensis

The LC₅₀ value of alkali-solubilized parasporal inclusion proteins of a Diptera-specific strain, belonging to Bacillus thuringiensis serovar canadensis, was 2.4 μg/ml for larvae of the mosquito, Aedes aegypti. A significant loss in larvicidal activity occurred when solubilized inclusion proteins were treated with A. aegypti larval gut extract, silkworm (Bombyx mori) larval gut juice, and the proteinase K. Approximately 90% of the larvicidal activity was destroyed upon treatment with proteases in 30 min. The parasporal inclusion was composed of major proteins of 65, 53, and 28 kDa and some other minor proteins. Proteolysis profiles showed that the 65-kDa major protein is highly sensitive to proteases. Purification experiments with DEAE-Toyopearl column chromatography revealed that the 65-kDa protein is responsible for the mosquitocidal activity of this strain. The LC₅₀ value of the purified protein was 5.4 μg/ml. Received: 2 December 1996 / Accepted: 7 January 1997