Sequence analysis of a 3.4-kb region Streptomyces peucetius daunorubicin (DNR) gene cluster established the presence of the dnrH and dnmT genes. In dnrH mutants, DNR production increased 8.5-fold, compared with that in the wild-type strain, while dnmT mutants accumulated -rhodomycinone (RHO), which normally becomes glycosylated in daunorubicin biosynthesis. Hence, dnmT may be involved in the biosynthesis or attachment of daunosamine to RHO or in the regulation of this process. Since the DnrH protein is similar to known glycosyl transferases, this protein may catalyze the conversion of DNR to its polyglycosylated forms, known as baumycins. Overexpression of dnmT in the wild-type and dnrH mutant strains resulted in a major decrease in RHO accumulation and increase in DNR production.Daunorubicin (DNR) and its C-14 hydroxylated derivative doxorubicin (DXR) are important antitumor anthracycline antibiotics (26) isolated from Streptomyces peucetius ATCC 29050 (2, 5, 7). Studies of DNR and DXR production ( Fig. 1) in this organism have elucidated the organization and regulation of many of the biosynthetic genes (11,12,21,23,29,31), as well as the mechanism of antibiotic resistance (14, 19) and the regulation of DNR and DXR biosynthesis (20,28,34).Here we report the characterization of the dnrH and dnmT genes, which are situated between the dpsH (formerly dnr-ORF7) and dnrE (formerly dnrH) genes in the cluster of DXR biosynthesis genes ( Fig. 2 and 3). Dickens et al. (10) have described homologs of these genes in the Streptomyces sp. strain C5 (dau-ORF2 and -ORF3) but were only able to suggest a possible function for dnrH, as deduced from the DNA sequence. From the results of the expression of the dnrH and dnmT genes in the wild type and a dnrH mutant strain, plus the effects of insertional inactivation of both genes, it seems likely that these two genes govern steps subsequent to the formation of the aglycone ε-rhodomycinone (RHO), a key intermediate of DNR biosynthesis (Fig. 1). Moreover, introduction of dnmT on a high-copy-number plasmid into the dnrH mutant resulted in an 8.5-fold increase in DNR production with a concomitant large decrease in the level of RHO, a useless by product of DNR-producing strains.Sequence analysis of the dnrH and dnmT genes. DNA sequencing of both strands of a 2,162-nucleotide (nt) SphIBamHI fragment by previously reported methods (29) followed by CODONPREFERENCE analysis (9) revealed the C-terminal end of dpsH (the rest of this gene has been sequenced in other work [30]), the complete dnmT gene, and the N-terminal part of dnrH. The remainder of dnrH extends 931 nt into the adjacent 1.3-kb BamHI-AlwNI fragment described by Grimm et al. (13) (Fig. 3). The first ATG codon for dnmT is likely to be located at nt 237 (Fig. 3), even though a probable ribosome binding site is not evident near the 5Ј end of this open reading frame (ORF), and a stop codon (TGA) is located at position 1,752, which suggests that dnmT encodes a 505-amino-acid polypeptide with an M r of 55,433. The dnrH gene has a pr...
Aims: To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. Methods and Results: Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and DNA sequencing. The isolates were characterized as motile, rod‐shaped, endospore forming, catalase positive, Gram‐positive bacteria. ITS‐PCR allowed typing mainly at species level. Rep‐PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). Conclusions: Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. Significance and Impact of the study: Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria.
Sequence around the 159' region of theThe nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined. Six ORFs were recognized, one of which extended for 13 341 nucleotides. Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism. A t the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans WA gene, responsible for the synthesis of a polyketide pigment, were recognized. The observed similarities suggest that the 20 kb region and the previously reported 13-6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.
We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nd€F operons of intembacferiscese. The sequencing of the conditional lethal mutation ts-Al3, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of n d € and ndF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia d i m The operon was induced by thymidine starvation and i t s expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria.
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