An exodeoxyribonuclease from Streptomyces coelicolor: Expression, purification and biochemical characterization

Brnakova, Zuzana; Godány, Andrej; Timko, Jozef

doi:10.1016/j.bbagen.2006.11.017

Cited by 5 publications

(8 citation statements)

References 28 publications

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“…In any case, it is very hard to characterize these enzymes in natural hosts, so it is easier to describe the function of separate genes in vitro in heterologous E. coli expression system. Recently, we cloned and characterized the recombinant exonuclease recExoSc from S. coelicolor A3(2) (Brnáková et al 2007) which was, according to the genome sequence, proposed to be a secreted protein. With regard to the hypothesis that all streptomycetes possess nucleases with similar activities and the fact that S. aureofaciens B96 produces several intra-and extracellular nonspecific nucleases (Brnáková et al 2005), we used primers designed for exoSc also at S. aureofaciens genomic DNA as a template.…”

Section: Resultsmentioning

confidence: 99%

“…Assays of nuclease activity and optimum reaction conditions. Activities of nuclease, phosphomonoesterase and phosphodiesterase, and optimum conditions in reactions were defined according to Brnáková et al (2007). During the purification steps, the enzyme activity was monitored using  DNA in activity buffer (in mmol/L: NaCl 25, Tris-HCl 20, MgCl 2 10, CaCl 2 5; pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

“…ExoSa-6 × His-tagged (recExoSa) protein was isolated and purified according to Brnáková et al (2007). Assays of nuclease activity and optimum reaction conditions.…”

Section: Methodsmentioning

confidence: 99%

“…However, scarce information is available on the function of nucleases in the biology of these microorganisms, their roles having been only predicted. Recently, the cloning and partial characterization of S. coelicolor A3(2) exonuclease recExoSc (Brnáková et al 2007) was published; here, its homologue from S. aureofaciens B96, exonuclease exoSa, was amplified. The main aim of this work was cloning of the exoSa gene, its expression in E. coli, characterization of its protein product and inactivation of exoSa and exoSc genes in their natural hosts.…”

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confidence: 99%

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Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2)

2009

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Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5'-phosphomonoesterase activities at optimum temperature 37 degrees C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…ExoSa-6 × His-tagged (recExoSa) protein was isolated and purified according to Brnáková et al (2007). Assays of nuclease activity and optimum reaction conditions.…”

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2)

2009

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“…RNase from Pleurotus tuber regium and Pleurotus eryngii mushroom showed the optimum activity at pH 6.2 [29,38]. In case of exo-deoxy ribonuclease from Streptomyces coelicolor recExoSc exonuclease was active in a broad pH from 6.5 to 10.5 with a neutral maximum at pH 7.5 [7]. Nuclease from Neurospora crassa (mitochondria) also exhibited different pH optima for the hydrolysis of ssDNA (pH 6.5 -7.5) and dsDNA (5.5 -6.5) [11] and this observation strongly supported the present study.…”

Section: Discussionmentioning

confidence: 96%

Purification and Characterization of Extracellular Nuclease from Bacillus Firmus VKPACU-1

Ashokkumar¹

2018

IJRASET

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An extracellular nuclease from Bacillus firmus VKPACU-1 was purified by ammonium sulfate precipitation, phenylsepharose followed by gel filtration chromatography. The overall yield was 4.09%. Molecular weight of the purified enzyme was determined by SDS/PAGE, it was 17.1 kDa. The specific pH (6.5) and temperature (35°C) was optimized for the nuclease activity. Stability of the enzyme was pH 6.5 to 8.0 and temperature upto 40°C. Without NaCl concentration showed maximum nuclease activity. Enzyme was not affected by dithiothreitol, β-mercaptoethanol and PMSF. The enzyme was inhibited by Mg 2+ , Ca 2+ , Ba 2+ , Zn 2+ , Cu 2+ and Co 2+ , inorganic phosphate, pyrophosphate, Triton X-100, Tween-80 and metal chelator EDTA. It was highly stable to high concentrations of organic solvents and Urea, SDS and guanidine hydrochloride. This enzyme showed the substrate specificity is in the order of RNA, ssDNA and dsDNA. This may be the first report on the extracellular nuclease from B. firmus VKPACU1.

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Contractile injection systems facilitate sporogenic differentiation of Streptomyces davawensis through the action of a phage tapemeasure protein-related effector

Nagakubo,

Nishiyama,

Yamamoto

et al. 2024

Nat Commun

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Contractile injection systems (CISs) are prokaryotic phage tail-like nanostructures loading effector proteins that mediate various biological processes. Although CIS functions have been diversified through evolution and hold the great potential as protein delivery systems, the functional characterisation of CISs and their effectors is currently limited to a few CIS lineages. Here, we show that the CISs of Streptomyces davawensis belong to a unique group of bacterial CISs distributed across distant phyla and facilitate sporogenic differentiation of this bacterium. CIS loss results in decreases in extracellular DNA release, biomass accumulation, and spore formation in S. davawensis. CISs load an effector, which is a remote homolog of phage tapemeasure proteins, and its C-terminal domain has endonuclease activity responsible for the CIS-associated phenotypes. Our findings illustrate that CISs can contribute to the reproduction of bacteria through the action of the effector and suggest an evolutionary link between CIS effectors and viral cargos.

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An exodeoxyribonuclease from Streptomyces coelicolor: Expression, purification and biochemical characterization

Cited by 5 publications

References 28 publications

Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2)

Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2)

Purification and Characterization of Extracellular Nuclease from Bacillus Firmus VKPACU-1

Contractile injection systems facilitate sporogenic differentiation of Streptomyces davawensis through the action of a phage tapemeasure protein-related effector

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