Secretion of tyrosinase in Streptomyces glaucescens

In Streptomyces glaucescens, the intracellular and the extracellular enzyme forms of tyrosinase were found to be indentical in molecular weight (29 000), in copper content (0.21%), in the 19 amino acids at the amino-terminal end and in the ratio of cresolase to catecholase activity (0,005). The tyrosinase secretion process exhibited a constant rate of 0.15 units h-1 (mg protein)-1. Under highly induced conditions intracellular tyrosinase was accumulated. Mutations responsible for the non-melanogenic, tyrosinase-positive non-secretor mutant type are located chromosomally on the upper right arc of the S. glaucescens map near the ade-1 marker.     

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.     

Isolation of tetracenomycin C-nonproducing Streptomyces glaucescens mutants

We analyzed the properties of tetracenomycin C (TcmC)-nonproducing mutants of Streptomyces glaucescens to establish the nature of pathway intermediates and to provide some information about the genetics of antitumor anthracycline antibiotic production. Using cosynthesis properties and metabolite accumulation data, we classified a collection of 34 TcmC-nonproducing strains into seven different groups. From this information, we deduced the positions of the tcm mutations in relation to a hypothetical TcmC biosynthetic pathway and suggest which pathway enzymes are affected by the different mutations.     

Chromosomal deletion and rearrangement in Streptomyces glaucescens.

The Streptomyces glaucescens genome frequently undergoes gross genomic rearrangement events which result in the deletion of extremely large segments of chromosomal DNA. The structure and origin of the DNA forming the novel junctions arising from five of these deletion events are described. Only one junction proved to be the result of a relatively simple event; the remainder were more complex, with one involving DNA which originated from at least five distinct loci. In three of the investigated cases, DNA sequences present in the junctions appeared to have resulted from the duplication of previously unique sequences, suggesting that duplication of chromosomal segments may be an important factor in genetic instability. The nucleotide sequences surrounding these junctions and their respective wild-type termini were determined.     

Primary structure of tyrosinase from Streptomyces glaucescens

The complete amino acid sequence of Streptomyces glaucescens tyrosinase is reported. The molecule consists of 273 amino acids and has a Mr of 30 900 including two copper atoms. The primary structure was determined by a combination of amino acid and DNA sequence analysis. Peptide sequence information was derived from the cyanogen bromide, tryptic, and thermolytic fragments of apotyrosinase by automated Edman degradation and aminopeptidase M and carboxypeptidase C digestions. The nucleotide sequence of the tyrosinase gene cloned into the PvuII site of pBR322 was determined. The enzyme contains no apparent leader peptide despite the fact that it is secreted into the culture medium. As observed for a number of different Streptomyces genes, the tyrosinase gene shows a strong preference (97%) for codons ending in G or C. A comparison of the amino acid sequence of Streptomyces glaucescens tyrosinase with that of Neurospora crassa tyrosinase reveals an overall sequence homology of only 24.2%. However, the sequence homology is much higher in those regions thought to be involved in metal binding of the binuclear active site copper of this monooxygenase.     

Glaucescin, a bacteriocin-like substance from Streptomyces glaucescens

Streptomyces glaucescens ETH 22794 produced a variety of antibiotic substances. Besides low molecular weight antibiotics like hydroxystreptomycin and the tetracenomycins, this strain excreted glaucescin, a high molecular weight product with bacteriocin-like properties. In plate tests the antagonism of glaucescin against Streptomyces canadiensis was masked by the large inhibition zone caused by the tetracenomycins. Glaucescin activity was revealed when a tetracenomycin-resistant mutant of S. canadiensis NRRL 3155 was used as an indicator. Glaucesin was produced on complex and minimal solid and liquid media. It was not inducible by mitomycin C. The killing activity of glaucescin was thermolabile and resistant to DNAase, RNAase, various proteinases, and lipase. Its apparent molecular weight was estimated as 196000 by gel filtration and glycerol gradient centrifugation. Glaucescin preferentially killed outgrowing spores of S. canadiensis. Resting spores and mycelium were considerably less sensitive to the inhibitor, and adsorption of glaucescin by S. canadiensis paralleled sensitivity. The activity spectrum of the bacteriocin was restricted to spore-forming Actinomycetales. Non spore-forming nocardiae and a variety of Gram-positive and Gram-negative bacteria were resistant to glaucescin.     

Identification of two histidines as copper ligands in Streptomyces glaucescens tyrosinase

The physiochemical properties of wild type and two mutants of Streptomyces glaucescens tyrosinase are reported. The native enzyme contains two coppers at the active site which are EPR nondetectable. The two coppers react stoichiometrically with one hydrogen peroxide molecule giving rise to oxytyrosinase. Its optical features are similar to those reported earlier for a molluscan hemocyanin. The two mutants in which histidine-62 and -189 were changed to asparagine by site-directed mutagenesis have lost their enzymatic activity and their ability to bind oxygen and contain only one copper ion which is fully EPR detectable. The EPR parameters indicate that the remaining copper is in a tetragonally distorted ligand environment. These data are in agreement with His-62 and His-189 serving as copper ligands in S. glaucescens tyrosinase.     

Anthracycline metabolites of tetracenomycin C-nonproducing Streptomyces glaucescens mutants.

Mutants of Streptomyces glaucescens GLA.0 which are blocked in the production of tetracenomycin C (compound 1), an anthracycline antibiotic having significant antitumor activity, accumulated several new anthracycline metabolites structurally related to compound 1 and to intermediates of its biosynthetic pathway. Through chemical and spectroscopic comparisons with the known anthracycline metabolites of the wild-type strain, we identified the two regioisomers of tetracenomycin B2 (compounds 7a and 7b), 8-demethyltetracenomycin C (compound 12), tetracenomycin D2 (compound 11), tetracenomycin E (compound 13), and the 12-naphthacenone forms of compounds 7a, 7b, and 2 (tetracenomycin D1). A hypothetical biosynthetic pathway to compound 1 is presented that is consistent with the occurrence of compounds 7b, 13, and 5 (tetracenomycin A2) and with the cosynthetic behavior of tetracenomycin C-nonproducing mutants (H. Motamedi, E. Wendt-Pienkowski, and C. R. Hutchinson, J. Bacteriol. 167:575-580, 1986).     

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.     

Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions

Summary Streptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens. The repeated DNA units were 2.9 to 35 kb in lenght. No two sequences were identical. The amplified sequences occupied up to 45% of the total genomic DNA. Structural analysis of the cloned repeated DNA stretches by means of restriction enzymes and by cross hybridization revealed the presence of two chromosomal areas rich in DNA reiterations. In some cases reiterated regions were accompanied by nearby rearrangements.     

Extracellular deoxyribonuclease production by a thermophile, Streptomyces thermonitrificans

A thermophilic bacterial strain, Streptomyces thermonitrificans, produced high levels of extracellular deoxyribonuclease (DNase) when grown on NBG medium (containing 1% peptone, 0.3% beef extract, 1% glucose and 0.5% NaCl). Maximum DNase activity (140 U ml⁻¹) was obtained, in 24 h, when the culture was grown on modified NBG medium (containing 1.3% beef extract, 1% glucose, 0.5% NaCl and 50 μM Mn²⁺ at 45°C. The crude enzyme showed higher activity on native DNA than on sonicated and heat denatured DNA. Moreover, addition of Mn²⁺ in the assay mixture resulted in a significant stimulation (10–15 fold) of the enzyme activity. Received 24 November 1998/ Accepted in revised form 25 April 1999     

Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans

Summary Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408. 1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.     

A new restriction endonuclease from Streptomyces albus G.

A restriction endonuclease, SalI, has been partially purified from Streptomyces albus G. This enzyme cleaves adenovirus-2 DNA at three sites, bacteriophage λ DNA at two sites, but does not cleave simian virus 40 DNA or φX174 DNA. It recognizes the sequence

and cuts at the sites indicated by the arrows. An endonuclease (XamI) with similar specificity has also been isolated from Xanthomonas amaranthicola.     

A comparative study on proteins released from metaphase chromosomes after digestion with restriction endonucleases and deoxyribonuclease I

Extensive digestion of Chinese hamster metaphase chromosomes with Alu I, Hae III and Hinf I released up to 40 distinct chromosomal proteins. Some of the proteins released by Hae III or Hinf I were enriched in the protein moiety liberated by Alu I but several proteins released by Hae III were not released by Alu I digestion. The amount of chromosomal protein released by deoxyribonuclease I (DNase I) was comparable to that liberated by the three restriction enzymes so far tested, while only four abundant protein species were detectable in the protein moiety released by DNase I. Two of them with molecular weights of 58,000 and 50,000 were also released by the three restriction enzymes and are similar in size to those found previously in the core-like structure of histone-depleted chromosomes.     

Heterogeneous genomic amplification in Streptomyces glaucescens: structure, location and junction sequence analysis

Summary Certain chromosomal markers inStreptomyces glaucescens behave unstably, being lost at high frequency as a result of extensive genomic deletion. Additionally, mutant strains possessing such deletions frequently display intense DNA amplification. With the help of a wild-type cosmid library we investigated the structure of the amplified DNA sequences (ADS) and the corresponding wild-type amplifiable units of DNA (AUD). The reiterations were heterogeneous in location, copy number and sequences involved and originated predominantly from a single 100 kb region of the chromosome called the AUD locus. All strains bearing reiterations possessed associated deletions which terminated either close to or at the ADS. The termini of four AUDs were sequenced in order to gain more knowledge about these heterogeneous amplifications. In three of the four cases investigated small, interrupted homologies were found bordering the AUDs. With the help of orthogonal-field-alternation gel electrophoresis (OFAGE) we were able to visualize a tandem reiteration of more than 1500 kb in length.