We attempted colony PCR without prior extraction of DNA as the template for rapid and simple detection of specific DNA sequences in actinomycetes. Microscopic amounts of mycelia were picked up with toothpicks and directly transferred to PCR mixtures (20 μl) which were immediately subjected to heating (95°C, 3 min) followed by 30 cycles of PCR (95°C, 30 sec → 60°C, 30 sec → 72°C, 60 sec). Specific and clear amplification of the target region (518 bp) of an aminoglyocoside acetyltransferase (AAC) gene, kan, coding for an AAC(3) in Streptomyces griseus strains was observed by colony PCR using originally designed 20mer oligonucleotide primers (kanl and kan2). Stable amplification was not obtainable by the addition of spore-bearing aerial mycelia or visible amounts of mycelia. Good amplification of 16S rDNA sequence (1.5 kb) was also detected by using known primers of 9F (19mer) and 1541R (17mer). Employing a higher annealing temperature (65°C) provided good amplification with better specificity of the target sequences of kan and 16S rDNA. Findings confirm that colony PCR will definitely be useful for characterization and manipulation of genes in actinomycetes.

View full abstract

A xylanase-encoding gene from lignocellulolytic Streptomyces viridosporus T7A was isolated, expressed, cloned and sequenced. The gene was isolated by screening an Escherichia coli cosmid genomic library of S. viridosporus T7A genomic using a substrate overlay method. One xylanase expressing cosmid clone contained a 30 kb DNA insert. The xylanase gene was subsequently subcloned on a 2 kb PstI fragment and sequenced. Analysis of the nucleotide sequence revealed a 915 bp open reading frame coding for a 329 amino acid protein with a calculated molecular weight of 33,550 Da. This gene, designated svxA (Genbank accession number AF198618), shows high sequence homology to genes belonging to xylanase family G.

View full abstract

A recombinant clone, pBLP, containing a 4.1kb fragment of Streptomyces viridosporus T7A chromosomal DNA was shown to confer endoglucanase activity in Escherichia coli cells. Further subcloning and sequence analysis revealed two co-transcribed Open Reading Frames (ORF) with high sequence similarities to other Streptomyces endoglucanase-encoding genes. A signal peptide, a catalytic domain and cellulose binding domain were identified within ORF1 (celS1). The amino acid sequence of ORF2 (celS2) showed similarity with cellulose binding protein (p40) of Streptomyces halstedii. The hydrophobic analysis of CelSl revealed that it belongs to the family H cellu-lase catalytic domain. The rare TTA codon encoding leucine was found in the signal sequence of both the cellu-lase ORFs, indicating translational dependence on the bldA gene product, a cognate tRNA responsible for translating the TTA codons. The presence of 14 bp inverted repeats in the 5’-end of these genes, was consistent with the highly conserved positive regulatory structure found in other endoglucanase genes.

View full abstract

The expression of the obg gene of S.griseus, which codes for a GTP-binding protein and controls morphological development of this organism, was studied by Western and Northern blot analyses. Western analysis revealed that Obg protein level sharply decreases just after the onset of aerial mycelium development or at the end of vegetative growth on agar plate cultures, indicating that expression of obg is regulated in a growth phase-dependent manner. The obg gene was found to be transcribed as a mRNA of 1.7 kb and its transcription was cut off just prior to the decrease of Obg protein, indicating that obg gene expression is controlled at the transcription level.

View full abstract

Since the discovery of penicillin by Fleming and the establishment of the general screening method for the useful antimicrobial substances by Waksman, antibiotics have been playing a very important role in human medicines. In this paper, some of our screening programs for novel drugs from microbial secondary metabolites are described.

View full abstract