The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis

Summary The oligopeptide permease is encoded by at least four genes which are transcribed as a single operon. We cloned and characterized this operon from Salmonella typhimurium, as well as the flanking genes, tonB, ana and a new gene, cwd, which affects cell wall synthesis. We correlated the physical map of opp DNA with a detailed genetic map of the opp operon and the individual opp genes were accurately located with respect to various restriction sites by Southern blotting. The region of the chromosome near opp was found to be highly unstable with deletions arising at a high frequency. The operon also contains hot-spots for IS1 and IS5 insertions.     

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

Summary The wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.Abbreviations bp base pairs - EDTA ethylenediaminetetraacetic acid - kb kilobases' - TES n-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid     

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.     

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

Cloning and nucleotide sequence of a carbomycin-resistance gene from Streptomyces thermotolerans

Two plasmids (pOJ158 and pOJ159) containing DNA fragments from the carbomycin(Cb)-producing strain Streptomyces thermotolerans were identified in Streptomyces griseofuscus based on their ability to confer resistance to Cb. The Cb-resistance determinants on pOJ158 and pOJ159 were designated carA and carB, respectively. In S. griseofuscus, pOJ159 also confers resistance to spiramycin, rosaramicin, lincomycin, and vernamycin B, but not to tylosin; in Streptomyces lividans, pOJ159 additionally confers resistance to erythromycin and oleandomycin. The carB gene was localized on pOJ159 to a 1.25-kb region whose nucleotide sequence was determined. The sequence has a G + C content of 68% and contains the coding sequence for carB and portions of the 5' and 3' untranslated regions. A comparison of the amino acid sequence of the protein encoded by carB (as deduced from the nucleotide sequence) with the deduced amino acid sequence of the RNA methylase from Streptomyces erythraeus (encoded by ermE) revealed extensive homology, suggesting that carB also encodes an RNA methylase. The region 5' to the coding sequence does not contain a small ORF or regions of complementarity that are commonly associated with translationally regulated macrolide-lincosamide-streptogramin B resistance genes. The 3' untranslated region contains an inverted repeat sequence that potentially can form a stable RNA stem-loop structure with a calculated delta G of -70 kcal.     

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene.

Streptomyces griseus trypsin (E.C. 3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.     

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.