The erythromycin-producing polyketide synthase.
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Erythromycin↗
Biomedical subjects
J F Aparicio
Publications and source records attributed to J F Aparicio.
A Streptomyces glaucescens endodeoxyribonuclease which shows a strong preference for CC dinucleotide.
We have characterized a deoxyribonuclease from Streptomyces glaucescens that cleaves double-stranded DNA preferably between the dinucleotide 5'-CC-3'. The cleavage specificity was demonstrated by both analysis of the terminal nucleotides of the generated fragments and DNA sequencing of partially digested DNA. Digestion of lambda DNA with this enzyme resulted in the production of double-stranded fragments with 5' and/or 3'-protruding single-stranded tails. DNase I footprinting experiments indicated that the nuclease specifically binds to its cleavage sites on the DNA under non-catalytic conditions. The enzyme is not affected by cytosine methylation in hemimethylated DNA.
Bacteriophage lambda↗
Purification and characterization of a nutritionally controlled endodeoxyribonuclease from Streptomyces glaucescens.
Streptomyces glaucescens has a DNAase whose synthesis is under nutritional control. We have purified this enzyme to apparent homogeneity by phosphocellulose chromatography followed by heparin-agarose, Cibacron Blue F3-GA-Sepharose and Sephadex G-75 chromatography and MonoQ f.p.l.c. The enzyme had an apparent Mr of 39,600 and a pI of approx. 8.15. The Mr of the native enzyme estimated by gel chromatography was 49,000. The DNAase had a pH optimum of 7.5 and an absolute requirement for bivalent cations in the reaction buffer. It was inhibited by high salt concentrations, chelating agents or phosphate-containing compounds and was stimulated by dimethyl sulphoxide. The activity was greatly diminished unless dithiothreitol or 2-mercaptoethanol was included in the reaction mixture. Reagents such as Hg2+ or iodoacetate strongly inhibited the enzyme. The nuclease hydrolysed both double-stranded and single-stranded DNA, showing greater affinity for double-stranded DNA, and no detectable hydrolysis of RNA. The enzyme produced nicks in double-stranded DNA, generating 3'-hydroxy and 5'-phosphate termini, and degraded circular DNA.
Chromatography, Affinity↗
Characterization of Rrh4273I, a restriction-modification system of Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) which recognizes the same sequence as the Streptomyces albus G SalI restriction-modification system.
Rhodococcus rhodochrous ATCC 4275 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements of activity similar to SalI, and a native Mr of about 46,000. The DNA-MTase is a protein with an Mr of about 67,000. No DNA homology was detected between the cloned salI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.
Base Sequence↗
Sensitivity to phages of Streptomyces coelicolor strains harbouring type II restriction endonucleases.
The role of type II restriction endonucleases in phage development in two different strains of Streptomyces coelicolor has been analyzed. Two of ten phages tested (phi A4 R4c 1) presented a low efficiency of plating (e.o.p.) in the studied strains. The isolation of host-range mutants of phi A4 and R4c 1, with improved e.o.p. and higher adsorption capability in these two bacterial strains, suggests that the presence of host endonucleases is not the main barrier for these phages, but rather adsorption inability.
Adsorption↗
A non-specific deoxyribonuclease with restriction function in Streptomyces glaucescens.
A non-specific deoxyribonuclease with a possible role in the restriction of some actinophages was detected in Streptomyces glaucescens ETHZ 22794. Production of this enzyme activity was influenced by the medium composition, indicating nutritional control of enzyme synthesis. Restriction was confirmed when phage adsorption and efficiency of plating in nuclease-productive and non-productive media were investigated, and also by analysis of a mutant which lacked exonucleolytic activity. In vivo escape from restriction in nuclease-productive media is mainly related to the ability of phages to adsorb in a growth phase earlier than that in which enzyme synthesis occurs.
Actinomycetales↗
An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.
Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction.
Bacteriophages↗