Isolation and Characterization of Nucleases from a Clinical Isolate of Serratia marcescens kums 39581

Yonemura, Kensuke; Matsumoto, Kousuke; Maeda, Hiroshi

doi:10.1093/oxfordjournals.jbchem.a134262

Cited by 33 publications

(39 citation statements)

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“…This was demonstrated first with synthetic homopolymers (Yonemura et al, 1983;Filimonova et al, 1994) and confirmed recently with macromolecular DNA of natural origin and various synthetic oligodeoxynucleotides (Meiss et al, 1995). The products of the reaction are 5'-phosphorylated mononucleotides (in very low yield) plus di-, tri-and tetra-nucleotides (Eaves and Jeffries, 1963 ;Nestle and Roberts, 1969b).…”

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confidence: 81%

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Friedhoff

Meiß

Kolmes

et al. 1996

European Journal of Biochemistry

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The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serrutia nuclease. Most of the natural substrates are cleaved with similar k,,, and K,,, values, the k,,,lK,,, ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA) . poly(dT) or poly(dG) . poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K,,,, respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-R,-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the R,-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the R,-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p4trophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-@-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.

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confidence: 81%

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Friedhoff

Meiß

Kolmes

et al. 1996

European Journal of Biochemistry

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“…Contrary to common belief, extracellular nucleases are rare, and have only been observed in association with a small number of bacterial species (Benedik and Strych, 1998). The S. marcescens extracellular nuclease has been comprehensively studied (Benedik and Strych, 1998;Eaves and Jeffries, 1963;Yonemura et al, 1983). S. marcescens and other Serratia species are readily isolated from soil, water, plants, and animals (Ahrenholtz et al, 1994b;Grimont and Grimont, 1991).…”

Section: Enzymatic Degradation Of Dnamentioning

confidence: 99%

Release and persistence of extracellular DNA in the environment

Nielsen

Johnsen

Bensasson

et al. 2007

Environ. Biosafety Res.

464

382

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The introduction of genetically modified organisms (GMOs) has called for an improved understanding of the fate of DNA in various environments, because extracellular DNA may also be important for transferring genetic information between individuals and species. Accumulating nucleotide sequence data suggest that acquisition of foreign DNA by horizontal gene transfer (HGT) is of considerable importance in bacterial evolution. The uptake of extracellular DNA by natural transformation is one of several ways bacteria can acquire new genetic information given sufficient size, concentration and integrity of the DNA. We review studies on the release, breakdown and persistence of bacterial and plant DNA in soil, sediment and water, with a focus on the accessibility of the extracellular nucleic acids as substrate for competent bacteria. DNA fragments often persist over time in many environments, thereby facilitating their detection and characterization. Nevertheless, the long-term physical persistence of DNA fragments of limited size observed by PCR and Southern hybridization often contrasts with the short-term availability of extracellular DNA to competent bacteria studied in microcosms. The main factors leading to breakdown of extracellular DNA are presented. There is a need for improved methods for accurately determining the degradation routes and the persistence, integrity and potential for horizontal transfer of DNA released from various organisms throughout their lifecycles.

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“…22) and the Anabaena nuclease are by far the best studied enzymes. Both enzymes, Serratia nuclease secreted by the Gram-negative bacterium Serratia marcescens (23)(24)(25) and Anabaena nuclease produced by members of the cyanobacterial genus Anabaena (10) are largely nonspecific endonucleases which cleave double-and single-stranded DNA and RNA and produce 5Ј-phosphorylated oligonucleotides. For the Serratia nuclease the three-dimensional structure is known (5,26,27), and due to a detailed mutational (28,29) as well as a kinetic analysis using natural and synthetic, in part chemically modified, substrates (30 -32) its mechanism of action is well understood.…”

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confidence: 99%

On the Advantage of Being a Dimer, a Case Study Using the DimericSerratia Nuclease and the Monomeric Nuclease fromAnabaena sp. Strain PCC 7120

Franke

Meiß

Pingoud

1999

Journal of Biological Chemistry

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The extracellular endonucleases from Serratia marcescens and Anabaena sp. are members of a family of nonspecific endonucleases. In contrast to the monomeric Anabaena nuclease, the Serratia nuclease is a dimer of two identical subunits. To find out whether the two active sites of the Serratia nuclease function independently of each other and what the advantage of being a dimer for this enzyme might be, we produced (i) dimers in which the two subunits were cross-linked, (ii) heterodimers consisting of a wild type and an inactive mutant subunit which were also cross-linked, and (iii) monomeric variants which are unable to dimerize. The monomeric H184R variant and the cross-linked S140C variant exhibit the same activity as the wild type enzyme, while the cross-linked heterodimer with one inactive subunit shows only half of the activity of the wild type enzyme, demonstrating functional independence of the two subunits of the Serratia nuclease. On the other hand at low enzyme and substrate concentrations dimeric forms of the Serratia nuclease are relatively more active than monomeric forms or the monomeric Anabaena nuclease in cleaving polynucleotides, not, however, oligonucleotides, which is correlated with the ability of dimeric forms of the Serratia nuclease to form large enzyme-substrate networks with high molecular weight DNA and to cleave polynucleotides in a processive manner. We conclude that in the natural habitat of Serratia marcescens where the supply of nutrients may become growth limiting the dimeric nuclease can fulfil its nutritive function more efficiently than a monomeric enzyme.

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Isolation and Characterization of Nucleases from a Clinical Isolate of Serratia marcescens kums 39581

Cited by 33 publications

References 0 publications

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Release and persistence of extracellular DNA in the environment

On the Advantage of Being a Dimer, a Case Study Using the DimericSerratia Nuclease and the Monomeric Nuclease fromAnabaena sp. Strain PCC 7120

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